Identification of Tremella fuciformis strains Using SRAP, ISSR and RAPD markers

Three types of molecular markers （SRAP, ISSR and RAPD） were used to identify four Tremella fuciformis strains, T6 （white）, T7 （white）, T8 （yellow） and T9 （light yellow）. Twelve SRAP primer pairs, ten ISSR primers and eight RAPD primers were screened, and identification data obtained using the three molecular markers were consistent in that the four T. fuciformis strains were divided into three groups with T7 and T9 clustered together in a single group. Each RAPD primer generated a higher average number of polymorphic bands than either the SRAP or ISSR primers, and the average similarity between the four strains was 81.34%. SRAP markers reflected more genetic information compared with the two other markers, and the average similarity was 68.98%. Genetic information reflected by ISSR markers was intermediate between SRAP and RAPD, and the average similarity was 77.48%.     

SRAP、ISSR和RAPD分子标记技术在银耳菌株鉴别上的应用

Three types of molecular markers (SRAP, ISSR and RAPD) were used to identify four Tremella fuciformis strains, T6 (white), T7 (white), T8 (yellow) and T9 (light yellow). Twelve SRAP primer pairs, ten ISSR primers and eight RAPD primers were screened, and identification data obtained using the three molecular markers were consistent in that the four T. fuciformis strains were divided into three groups with T7 and T9 clustered together in a single group. Each RAPD primer generated a higher average number of polymorphic bands than either the SRAP or ISSR primers, and the average similarity between the four strains was 81.34%. SRAP markers reflected more genetic information compared with the two other markers, and the average similarity was 68.98%. Genetic information reflected by ISSR markers was intermediate between SRAP and RAPD, and the average similarity was 77.48%.     

Sequence analysis,prokaryotic expression and antiserum preparation of coat protein gene of Actinidia virus A in Sichuan province北大核心CSCD

【Objective】The purpose of the research was to study the occurrence and molecular diversity of Actinidia virus A (AcVA), so as to provide scientific basis for the rapid detection, scientific prevention and control of AcVA and the variety breeding of kiwifruit in China.【Methods】Total RNA was extracted from kiwifruit leaves by CTAB (Cetyl Trimethyl Ammonium Bromide). Nested- PCR was used to amplify AcVA CP gene sequence. The first round PCR amplification was performed by using primers AcVA- 1F (5’- ATGAATCGTTCGAGCA TAGGT- 3’) and AcVA- 1R (5’- TGCGAACATGGTCCCACACTTA-3’), and the pair of primers was designed according to the full length of AcVA sequence, and the amplified fragment was 888 bp that included the complete CP gene sequence. The reaction was conducted under conditions of initial 5 min denaturation at 94 ℃, 34 cycles of 94 ℃ for 60 s, 54 ℃ for 60 s, 72 ℃ for 60 s and extension for 10 min at 72 ℃. Then, 1 μL PCR product was used as template for the second round of PCR amplification with the primers AcVA- 2F (5’- ATGGCAAAGAATATCTCAAG-3’) and AcVA-2R (5’-CTATATTTCAACAGCCTGC-3’). PCR was performed using the following parameters: one cycle at 94 ℃ for 5 min, 34 cycles at 94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 40 s, and extension for 10 min at 72 ℃. The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene. DNAMAN was used to analyze the AcVA CP gene sequence, and MegAlign was used to analyze the sequence identity. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA 6.0. The restriction enzymes Bam HⅠ and Sal Ⅰ were added to the corresponding end of the AcVA CP gene sequence, respectively. The PCR purified products and pET28a vector were digested by Bam HⅠ and Sal Ⅰ, after that they were ligated with T4 DNA ligase (TaKaRa) and transferred into E. coli (Escherichia coli) strains DH5á (TaKaRa), and finally plated onto LB (Luria-Bertani) agar containing Kana (Kanamycin). The expression strain BL21 containing the recombinant plasmid was cultured at 37 ℃ overnight, and transferred to a new medium at a 10% inoculum on the second day, until OD600 reached 0.4-0.6, IPTG (Isopropyl β-D-Thiogalactoside) was added to a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mM, and incubated at 16 ℃ overnight. The expressed protein was purified and then anti-AcVA-CP antibody was obtained from a rabbit. The optimal titer of the antiserum was tested by Western blot.【Results】The complete CP gene sequences of AcVA Sichuan isolates were cloned by nested-PCR and named as AcVA-DJY4, AcVA-HYZ1 and AcVA-WBS12, respectively. In the study the frequency of AcVA in Sichuan province was also counted. The full length of the CP gene sequence was 597 bp, encoding 198 amino acids. Based on the comparison of the nucleotide sequence and the deduced amino acid sequence of CP gene of the AcVA isolates, the nucleotide identity between the AcVA isolates from Sichuan province ranged from 86.9% to 88.3%, and the identity of the encoded amino acids ranged from 96.0 to 96.5%. The CP genes of three AcVA Sichuan isolates shared 87.9%-90.8% nucleotide identity and 94.9%-97.5% amino acid identity with the Actinidia virus A isolate TP7-93A (AcVA-TP7-93A) from New Zealand and the Actinidia virus A isolate Haenam (AcVA-Haenam) from South Korea. Phylogenetic analysis based on nucleotide sequence of AcVA CP gene showed that AcVA-HYZ1 was clustered with AcVA-Haenam (Group I), and AcVA-WBS12 was clustered with AcVA-TP7-93A (Group II), but AcVA-DJY4 was an independent branch. Phylogenetic analysis based on amino acid sequence of AcVA CP gene showed that AcVA-WBS12 was a single branch, AcVA-DJY4 and AcVA-HYZ1 were clustered into one group (Group I), and the AcVA-Haenam reported in South Korea and the AcVA-TP7-93A reported in New Zealand were clustered into another group (Group II). The results of the phylogenetic analyses indicated that there were significant differences among the AcVA isolates. The prokaryotic expression plasmid pET28a-AcVA-DJY4-CP was successfully constructed. The target fusion protein (27 kDa) was highly expressed in E. coli induced by 0.6 mmol · L- 1 IPTG at 16 ℃. The expressed protein was purified and retrieved and then used to immune rabbits and the corresponding specific antiserum was prepared. Western blot analysis confirmed that the antiserum reacted strongly and specifically with the CP of AcVA-DJY4, with the optimal titer of the antiserum being up to 1:5 000.【Conclusion】Three AcVA Sichuan isolates were obtained for the first time, enriching the sequence diversity of the AcVA CP gene sequences. In the study, the optimal conditions were explored for prokaryotic expression and specific antisera was prepared for detection of AcVA-DJY4-CP. Western blot analysis showed that the antiserum could be used for the detection of AcVA in the kiwifruit producing districts. These results can also provide a technical support for the rapid detection, scientific prevention and control of virus disease in kiwifruit plant and the breeding of kiwifruit varieties. © 2019 Journal of Fruit Science     

Cloning and Sequencing of a Putative Mitochondrial Intermediate Peptidase Gene Fragment from the Edible Fungus Lentinula edodes

A 531 bp fragment of a putative mitochondrial intermediate peptidase (mip) gene from Lentinula edodes was amplified by PCR using a pair of degenerate primers designed according to conserved sequences of mip genes reported in other Basidiomycetes. Alignment analysis revealed that this putative partial mip gene from L. edodes exhibited high homology with corresponding segments of mip genes from the other Basidiomycetes. Since the gene encoding MIP appears to be very closely linked with the A type mating gene of Basidiomycetes, identification of mip may prove a useful tool for locating and cloning the A type mating gene in L. edodes and other edible fungi.     

Identification of resistance to anthracnose in leaves of grape resources and QTL localization of disease resistance genes北大核心CSCD

【Objective】 Grape anthracnose is one of the main diseases of grape, which mainly infects the fruits, young leaves and new branches of grape, causing fruit rot, shedding, or water loss and shrinkage into stiff fruits. Breeding disease-resistant grape varieties is the most economical, effective, and environmentally friendly long-term control strategy for the prevention of the anthracnose disease. There have been a few studies on the identification of the resistance to anthracnose and the QTL localization of relative disease resistant genes so far. The study aimed to identify the resistance to the anthracnose disease of different grape germplasms and seek for new QTL locus for the resistance and provide germplasm materials and basis for the breeding and research on the disease resistance mechanism. 【Methods】 The indoor ex vivo leaf inoculation method was used to identify and evaluate the resistance to anthracnose in 60 strains of Chinese wild grapes, 122 accessions of V. vinifera and 76 accessions of V. vinifera-V. labrusca, and the hybrid offsprings of Manicure Finger (susceptible)×Ciputao0940 (resistant), and the QTL localization of grape anthracnose resistance was carried out using the genetic map constructed by SNP markers.【Results】 A total of 1 germplasm with high resistance, 43 germplasms with resistance and 75 germplasms with medium resistance were screened out, accounting for 0.39%, 16.67% and 29.07% of the total identified accessions, respectively. There were great differences in the resistance to anthracnose in different germplasm populations. 63.34% of the East Asian population 56.57% of the V. vinifera-V. labrusca population, and 31.15% of the Eurasian population had the resistance to anthracnose. In the identification of anthracnose resistance in Chinese wild grape germplasms, it was found that the resistance of different strains in the same population varied greatly, and the species with strong resistance on the whole included V. davidii, V. pseudoreticulata, V. amurensis, and V. piasezkii. The strong resistance of the strains include Shanputao &, Longyuwanfuye1 &, Duolieye-yingyu, Shanputao- shanyang1807, Mianmaoputao-chayu1955, and Ciputao0940. The resistance of the hybrid offsprings of Manicure Finger (susceptible)×Ciputao0940 (resistant) were distributed in five grades. According to the normality test and single-sample Kolmogorov-Smirnov test based on the resistance level distribution of the F1 population, it was found that the hybrid offsprings of Finger (susceptible) × Ciputao0940 (resistant) showed continuous variation in anthracnose resistance, which was a typical quantitative trait controlled by polygens, and the phenotypic distribution of disease resistance identification results showed a trend of partial normal distribution, which could be analyzed by QTL localization. In the positioning of QTL related to grape resistance to anthracnose, the interval or site of LOD≥3.0 was used as the threshold value for QTL, and when the above conditions were met, the site corresponding to the highest LOD value in the interval was considered to be one QTL of grape anti-anthracnose. In this experiment, a QTL interval associated with grape anthracnose resistance was detected on the 8th linkage group, and the locus with the highest LOD value in this interval was located at 140.682 cM, and the tightly linked label was Maker1675910, which could explain the 14.7% of the phenotypic variation. Based on the gene annotation results of the QTL locus interval, 15 resistance- related genes were screened out, and they were hypothesized to play a role in grape anthracnose resistance. 【Conclusion】 The resistance level of different grape germplasms to anthracnose was clarified, one QTL site against anthracnose was located and 15 resistant-related genes were screened. © 2023 Journal of Fruit Science. All rights reserved.     

A new pest in vineyard under grass management model: study on the biological characteristics and control of Smaragdina nigrifrons北大核心CSCD

【Objective】For many years, our team have been working on organic cultivation management and pest control of wine grapes. Ground cover management model has been a new green management model currently implemented in the northern orchards. However, under the grass-covering system, new or secondary pests and diseases may increase in the orchards if the pasture type and planting and management models are not carefully chosen. These pests and diseases might become a new problem in fruit production. For example, a serious insect pest of wine grapes, Smaragdina nigrifrons (Hope) (Coleoptera: Eumolpidae) was found in the vineyard in Changli, China for the first time. This pest is highly harmful and occurs continuously in grass-covered orchards. In order to understand the occurrence and damage of the pest, the authors conducted field and lab investigations for three consecutive years. Its morphological characteristics were described, and its living habits, damage, and occurring pattern were observed, and the control methods were suggested.【Methods】Systematic field investigation was combined with indoor feeding and observation. Morphological characteristics of the beetles were observed under Primo Star microscope, and their taxonomic status was determined. The population dynamics and occurrence pattern were monitored by yellow cards trapping technique. 【Results】 The results showed that the adult of S. nigrifrons was long ovate. Female adults were 4.5 to 5.9 mm in length and 2.2 to 3.0 mm in width; males were 4.0 to 5.6 mm in length and 1.9 to 2.6 mm in width. Their head was black, the top was highly convex, and the leading edge had a wavy fold. There was no obvious boundary between the lip base and the forehead, which was slightly raised with numerous deep points. The tentacles were short and thin. The basal 4 knots of the tentacle were yellowish brown, the other sections were brown or black, and the sections after the 5th section were jagged. The chest was red brown or yellow brown. The scutellum and the elytra were yellow brown or red brown, and the elytra had wide black transverse band. Male's abdomen penultimate web was raised and female’s was sag. The adults of S. nigrifrons do damage on the tender new leaves. They had pseudo- lethality, coming out during the day and hiding at night. Generally it damaged tender leaves on the top of the canopy, with irregular notches in the injured leaves. The damage rate of local wine grape reached 100%. The adults usually appeared in July and August and might have one generation a year. They were active during 8:00 to 11:00 and 15:00 to 18:00. They had no phototaxis but had feigned death. In addition to harming the top leaves of wine grape trees, they also harmed the inter-row weeds, and the damage in the grass growing area was significantly more serious than in the weeding area. 【Conclusion】It is speculated that the occurrence and harm of S. nigrifrons in the wine grape vine of Changli Langes Winery is closely related to the implementation of the new orchard management mode with grass cover, indicating that it has potential risk. Therefore, it is extremely important to carry out a risk assessment analysis of S. nigrifrons. The occurrence and damage of S. nigrifrons in other fruit orchards in the north, especially in slopes, semi-mountainous areas and mountainous areas, needs further investigation. © 2019 Journal of Fruit Science     

A Critical Note on the Scientific Name of the Cultivated Edible Fungus,Zhudugu

Consumer demand for food diversity ,andtechnological developments in the domesti-cation and cultivation of edible mushrooms ,have ledto a continuous increaseinthe numberof cultivated mushroom varieties . Zhudugu(Chinese commercial name) is one of severalnewly cultivated species that has gainedpopularity in both domestic and overseasmarkets . The cultivated strain was originallyisolated froma wild mushroomby the SanmingInstitute of Fungi ,Fujian Province , initiallymisidentified asClitocybe i…     

Effects of Fruit Thinning Severity on Yield and Quality in ‘Sensation’ Mango （Mangifera indica）

Four different fruit thinning severities were tested.The thinning treatments were carried out in October before the occurrence of physiological fruit drop.Different parameters were measured(both qualitative and quantitative) and the results compared among treatments as well as to that of the control.The results showed that wher fruit on ‘Sensation’ mango pani-cles were thinned to two fruit per panicle,a significant difference was obtained for most of the quantitative parameters.The fruits of the treatment where one fruit per panicle was retained and 50% panicles removed,produced the best results for most of the qualitative parameters.     

Risk assessment of dietary intake of pesticide residues in kiwifruit北大核心CSCD

【Obiective】Risk assessment of dietary exposure to pesticide residues in kiwifruit and quantifiction of the dietary risk levels of commonly used pesticides provide references for safe production and guidance for consumption of the fruit, food safety supervision, and revision of the Maximum Residue Limits (MRLs) of kiwifruit.【Methods】Based on detection of the residues of 66 pesticides in 61 kiwifruit samples from a major producing area, chronic dietary intake risk (%ADI) and acute dietary intake risk (%ARfD) of pesticide residues in these samples were assessed. Based on the veterinary drug residue risk ranking matrix constructed by the British Veterinary Drug Residues Committee, the risk of th epesticides and samples was ranked by integrating kiwifruit consumption and pesticide toxicity, frequency of use and residue level, and maximum residue limit estimates (eMRL) were calculated using allowable daily intake (ADI) values, large portion consumed (LP), and body weight (bw). The study will provide a reference for the regulation of the maximum residue limit (MRL) for the corresponding pesticides.【Results】(1)Among the 62 pesticides, a total of 21 pesticides were detected, and they were low-toxic pesticides except for chlorpyrifos, deltamethrin, cypermethrin and cyhalothrin, which were moderately toxic; (2) Using the pesticide toxicology data, acceptable daily intake (ADI) and acute reference does (ARfD), residual data and kiwifruit consumption data, the risk assessment of the 21 pestcides detected showed that the chronic dietary intake (%ADI) of each pesticide inkiwifruitranged from 0.000 01% to 0.016 04%. All the 21 pesticides detected but acetamiprid and chlorpyrifos without ARfD information had an acute dietary intake (%ARfD) ranging from 0.01% to 26.20%. The acute dietary intake risk difference between different pesticides was significantly. (3) According to the residual risk score, the 21 pesticides detected were medium- to low-risk pesticides. The risk scores for deltamethrin and chlorpyrifos were 16.5 and 16.1 ie(15≤S<20), respectively, which were in the medium-risk range. The risk scores of cypermethrin, pyrimidine, buprofezin, cyhalothrin and difenoconazole were between 12.1 and 12.4, and the risk scores of 14 pesticides including procymidone and chlorpyrifos ranged from 8.0 to 9.3 (S< 15). All of them were low-risk pesticides. Among the 61 kiwifruit samples, 45.9% of the samples were in a very low risk area with a pesticide residue risk index (RI) below 5; 34.4% of the samples were in a low risk range with a pesticide residue RI between 5 and 10; and 18.0% of the samples were in a medium risk range with a pesticide residue RI of 10 and 15; (4) At present, there were few standard pesticides in kiwifruit. Among the 21 pesticides detected, only carbendazim, acetamiprid, chlorpyrifos, deltamethrin, and cyhalothrin had maximum residue limit (MRL) in kiwifruit or berries and other small fruits in GB 2763—2016, while 76.2% the pesticides had not a limit value. Compared with eMRL, the MRL values of the five pesticides was a more strict parameter related to limitation of quantities. For example, the eMRL of carbendazim was 6.6 times that of MRL; the eMRL of acetamiprid was 3.8 times that of MRL; the eMRL of cyhalothrin was 10.9 times that of MRL; the eMRL of deltamethrin 21.9 times that of MRL; and the eMRL of chlorfenuron was 153 times that of MRL. Among the 21 pesticides detected, carbendazim, acetamiprid, cyhalothrin, deltamethrin and chlorpyrifos had MRLs, and azoxystrobin, chlorfenapyr, malathion, and procymidone had no necessecity to formulate MRL. 12 pesticides including thiamethoxam, imidacloprid, thiophanate- methyl, tebuconazole, propiconazole, prochloraz, difenoconazole, buprofezin, trifloxystrobin, chlorpyrifos, cypermethrin and chlorothalonil had no MRLs.【Conclusion】The pesticides detected were all middle- risk or low- risk pesticides, and the acute and chronic dietary risks of the 21 pesticides were acceptable. At the same time, 98.4% of the 61 samples were at medium, low or very low risk. Therefore, the kiwifruit in the major producing area was relatively safe. It was recommended to develope the MRLs of 12 pesticides including thiamethoxam and imidacloprid for in kiwifruit. © 2019 Journal of Fruit Science     

Binding characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages

AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using labeled CCK-8S as ligand. The specificity of -CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of -CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity[Kd=(0.68士0.28) nmol/L] and low binding capacity [Bmax=( 32.50±12.70) pmol·g^-1in PIMs. By means of competitive inhibition studies, the specific binding of [³H]-CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S[IC₅₀=(3.20士1.13)nmol/L],CCK-AR specific antagonist CR 1 409 and CCK-BR specific antagonist CR 2945[IC₅₀=(2.30士0.08)nmol/L].CONCLUSION:These results suggest the presence of two subtypes of CCK一AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.     

Detection and differential analysis of fruit organic acids among different local wrinkled papaya varieties(Chaenomeles speciosa ) by GC-MS北大核心CSCD

【Objective】Organic acid plays a key role in affecting fruit flavor by changing acid-sugar ratio. And GC-MS is also an important detecting platform to inspect fruit organic acids due to its stable, sensitive and accurate features. With methyl ester derivatization, the objective organic acids could be effectively detected for their reduction of ingredient polarity. To provide the basic data for Chaenomeles speciosa (Sweet) Nakai fruit quality improvement, the the organic acid inspection of the fruit sampleswas carried out from 10 main producing areas in China and their compositions and contentswere disclosed by extracting with methal, derivating with methyl esterification and detecting by means of GC-MS.【Methods】In this study, 10 local varieties were used as materials that were collected from 10 main producing areas in China. After methanol extraction and methyl-ester derivatization, the compositions and contents of organic acids with each sample were comprehensively determined by GC- MS. The derivatives were analyzed by a Shimadzu GC-MS 2010 Qplus with a Rxt-5MS weak polar capillary MS column(30 m × 0.25 mm, 0.25 μm, Shimadzu Technology). Helium was used as the carrier gas at 0.87 mL · min- 1 with a split ratio of 53:1 for the testing solution. The GC-MS detecting time was 35 min. Qualitative retrieval was conducted with similarity searching in NIST08 and NIST08S coupled with Kovats Reservation Index(RI value) matching and quantitative analysis was performed by an external standard method and the ingredient peak responding value was adjusted according to the n-alkanes mixed standards that came from USA O2Si calibration standards company. The difference in objective organic acids of all the 10 local variety samples was done through the Excel 2007 software and the Hierarchical Cluster Analysis(HCA) of organic acid composition among different samples was completed with SPSS20.0.【Results】Six standards for organic acids including malic acid, citric acid, oxalic acid, succinic acid, fumaric acid and oleic acid as well as their gradient regression equations showed that there was a high correlation between the standard concentration and their component peak area(R2 ≥ 92.9%). All baselines of the TICs were stable and all the component peaks were evenly distributed during the detecting period and their resolution was high. So the method for extracting by methanol, derivating by methyl ester and detecting by GC-MS was stable and reliable. A total of 43 organic acids including 9 short-chain carboxylic acids, 22 long-chain fatty acids, 4 aromatic dicarboxylic acids, 4 mono-basic phenol acids and 2 amino acids were identified from the 10 fruit samples of different producing areas in China. The top 10 organic acids with the highest contents were dl-malic acid, citric acid, hexadecanoic acid, 9,12-octadecadienoic acid, 9-octadecenoic acid, (+/-)-10-hydroxy-octadecanoic acid, levulinic acid, stearic acid, 9,10-dihydroxy-octadecanoic acid and benzoic acid, respectively. There were 33 common ingredient peaks among the total 10 local varieties and their total contents of organic acids were between 85.02-170.76 mg·g-1. From high to low content, it showed like this: Linyi of Shandong > Jinghong of Yunnan > Zheng’an of Guizhou > Chun’an of Zhejiang > Qijiang of Chongqing> Changyang of Hubei > Xuancheng of Anhui > Yilong of Sichuan > Nanning of Guangxi > Baihe of Shaanxi. The total organic acid content had an extremely significant positive correlation with the long-chain fatty acids, mainly including hexadecanoic acid, 9,12-octadecadienoic acid and 9-octadecenoic acid; a significant positive correlation with the short-chain carboxylic acids, mainly consisting of dl-malic acid and citric acid; a negative correlation with the aromatic carboxylic acids, mainly containing benzoic acid. Analysis of the cluster according to 33 common components showed that all the fruit samples were classified into 4 categories when their clustering distance was 5: Yilong of Sichuan, Xuancheng of An’hui, Baihe of Shaanxi, Nanning of Guangxi, and Changyang of Hubei were clustered into the first group; Qijiang of Chongqing and Chun’an of Zhejiang were clustered into the second branch. Zheng’an of Guizhou and Linyi of Shandong were clustered into the third group, and Jinghong of Yunnan was alone clustered into the fourth group. The Hierarchical Cluster Analysis (HCA) coupled with the total organic acid content showed that Zheng’an of Guizhou and Linyi of Shandong belonged to the high-acid varieties, Qijiang of Chongqing and Chun’an of Zhejiang were the middle-acid varieties, and Yilong of Sichuan, Baihe of Shaanxi and Nanning of Guangxi belonged to the low-acid varieties.【Conclusion】Compared with the acid-base titration method and HPLC, GC-MS method not onlywas more stable, sensitive and accurate, but also could realize a qualitative identification of more chemical components. So it was much better to be used in the determination of total organic acid contents in fruits and their derived products. The results showed that there was a small difference among the different local varieties and an obvious difference existed in organic acid compositions and contents among the samples from different producing areas in China. And dl-malic acid and citric acid were the main components in most of C. speciosa fruit samples from 10 producing areas in China. Therefore, it belonged to the fruit type of malic acid accumulating mode. © 2019 Journal of Fruit Science     

Synthesis of Human Insulin Gene and Transformation into Tremella fuciformis

A human insulin gene was synthesized using PCR-based methodology, cloned into plasmid pUC18, and the sequence of the inserted fragment was confirmed. A Tremella fuciformis expression vector was constructed and used to transform T. fuciformis yeast-like conidia employing Restriction Enzyme-Mediated DNA Integration (REMI). A total of 21 antibiotic resistant colonies were selected at random of which 18 tested positive for β-glucuronidase (GUS) activity. Genomic DNA was extracted from 10 of the 21 positive transformants and PCR performed using genomic DNA as the template and specific primers for amplifying the human insulin gene, the GUS gene and the Tnos sequence. PCR data showed that the expected fragment was amplified from all 10 isolates demonstrating that they were true human insulin gene transformants.     

Effects of nitrogen application rates on the leaf qualityof‘Cabernet Gernischet’in desert areas北大核心CSCD

【Objective】As an essential mineral element in plant growth and development, nitrogen has irreplaceable roles in organ construction, nutritive metabolism, biochemical processes as well as fruit yield and quality. The quality and yield of grapes are greatly influenced by the amount of nitrogen applied. In recent years, the industry of China's wine grape has developed rapidly, but there are problems with unreasonable fertilization in production. The Gansu Corridor has the climatic conditions and geographical resources suitable for producing high-quality wine grapes. However, wine grapes are extensively planted on a sandy loam soil with low organic matter. In the actual production, chemical fertilizers are often applied to ensure the normal growth and development of the grapes. At the same time, water-saving agriculture has become the demand for agricultural development in this area. The aim of our study is to investigate the effects of nitrogen application rates on key enzyme activities and related gene expression during nitrogen metabolism, so as to further provide a theoretical reference for the application of nitrogen in wine grape growing in the Gansu Corridor. 【Methods】The experiment was carried out in a 10-year-old‘Cabernet Gernischet’vineyard of the Mo Gao located in Wuwei City from 2014 to 2015. All the grapevines were planted from west to east with simple hedge-shape. The planting spacing was 0.7 m×3.0 m. The area of the plot was about 240 m2, and each plot contained 40 plants. During the test period, the irrigation was carried out according to the“Wuwei Mo Gao Brewing Grape Drip Irrigation Water Distribution Quota”, which formulated by the research team. The irrigation amount in the bud break stage was 675 m3·hm-2, the flowering stage was 900 m3·hm-2, the first berry swell period was 900 m3·hm-2, the secondary shoot growth period was 150 m3·hm-2, and the second berry swell period was 600 m3 · hm- 2, and overwintering water was 1 275 m3 · hm- 2. The total irrigation amount during the whole growth period was 4 500 m3·hm-2. Five different application amounts of nitrogen (0 kg·hm-2, 150 kg·hm-2, 300 kg·hm-2, 450 kg·hm-2 and 600 kg·hm-2, characterized as CK、N1、N2、N3 and N4, respectively) were applied with a completely randomized block. Urea was used as external nitrogen and applied with water. The 750 kg·hm-2 of calcium superphosphate was applied before the first irrigation during the soil excavation. The 825 kg·hm-2 of potassium sulfate was applied in fruit color-changing period. Effects of nitrogen application rates on total nitrogen content, soluble protein content, key enzyme activities including nitrate reductase(NR), glutamine synthetase(GS), glutamate synthase(GOGAT) and glutamate dehydrogenase(GDH) as well as the related gene expression levels in leaves (VvNR1, VvGS1, VvGOGAT1 and VvGDH1) were determined at different developmental stages (5th day before anthesis, 20th day after anthesis, 50th day after anthesis, 80th day after anthesis). Ten to fifteen leaves were collected from each treatment. Fresh leaves were punched out with a punch and 1.0 g was weighed for determination of soluble protein content and enzyme activity assay with 3 replicates. The new leaves without pests and diseases on the top of the current shoots were selected to extract RNA. 2.0 g of grape leaves wrapped in tin foil were placed in a liquid nitrogen tank and stored in arefrigerator at -80℃ for RNA extraction. 【Results】The results showed that nitrogen application significantly increased the net photosynthetic rate in leaves. The treatment of N2 significantly increased leaf area and the total nitrogen content in leaves in comparison with CK 5th day before anthesis, 20th day after anthesis and 80th day after anthesis. The soluble protein content increased by nitrogen application in leaves on 80th day after anthesis. The NR activity in leaves showed an increasing trend with the increase of nitrogen application in the range of 0 to 300 kg on 5th day before anthesis, 20th day after anthesis and 80th day after anthesis. The GS activity in leaves increased significantly by N2 treatment on 5th day before anthesis, 50th day after anthesis and 80th day after anthesis, which were 1.66, 1.25 and 1.34 times more than the control, respectively. The GOGAT activity in leaves increased significantly by N2 treatment on 5th day before anthesis and 80th day after anthesis, 0.53 and 0.42μmol·g-1·min-1, respectively. Compared with CK, N2 and N3 treatments could improve the GDH activity in leaves during the whole growing period. The expression level of VvNR1 in N2 treatment was significantly higher than CK and other nitrogen treatments on 5th day before anthesis, 20th day after anthesis and 80th day after anthesis. VvGS1 was significantly up-regulated by N1 and N2 on 5th day before anthesis, which was up-regulated by 142.33% and 283.47% in comparison with CK, respectively. VvGDH1 was up-regulated during the whole growing period after nitrogen application. With N2 treatment, fruit soluble sugar, the ratio of sugar to acid, tannins, anthocyanin content reached the maximum, the titratable acid content was the lowest, the yield was at a medium level. 【Conclusion】The results suggested that different nitrogen application rates affected the expression of VvNR1, VvGS1, VvGOGAT1 and VvGDH1 genes in leaves, thereby improving leaf nitrogen metabolism. In addition, the nitrogen application with 300kg·hm-2 (N2) among all the treatments significantly promoted the accumulation of nitrogen in leaves during the late growth period. At the same time, it promoted the increase of leaf area and net photosynthetic rate, and improved the fruit quality. © 2019 Journal of Fruit Science     

Breeding of a new pear cultivar‘Xinli No.11’北大核心CSCD

‘Xinli No.11’is a new pear cultivar,which was bred by crossing‘Kuerlexiangli pear’as the female parent and‘Yali’as the male parent. Five single plant was initially selected in 1989 for theirs strong adaptability,good quality and different maturity charateristics,and then a single plant number‘75-4-1’was finally selected for its better comprehensive quality,early fruiting,high yield,strong cold resistance and stable agronomic traits charateristics,After regional adaptability testing at three sites (including Korla area,Tarim area and Yanqi area) over five years from 2014 to 2018,it was finally selected in 2018. We applied for the registration as a new cultivar in April 2018 and got identification certificate from Trees Variety Approval Committee of Xinjiang Uygur Autonomous Region in November 2019 and named‘Xinli No.11’. The canopy was conical under natural conditions,the young trees growth potential was flourishing. Leaves were ovoid and eiiipse with average 11.30 cm length and 9.08 cm width,petiole was 3.95 cm in length. Leaf color was dark green. Flowers were pale pink,the relative position of petals was separated. The petal shape was oval,pollen was abundant.It was mainly on the short branch in the fields with ratio of 64%. The average setting rate of single flower was 35.4% and the setting rate of single inflorescene was 86.2% under the condition of natural pollination. The fruit was gourd shape and regular,shed calyx. The average single fruit weights was 181.2 g. The ground col- or of fruit skin was green yellow. The fruit skin was smooth and shining,the peel was thin,the fruit dot was middle-small and was not obvious. The fruit flesh was creamy white,fine and crisp. It was juicy,sour-sweet flavor and tasted delicious along with aromas. The fruit quality was excellent. The content of soluble solid,soluble sugar,titratable acid and vitamin C was 13.42% ,10.41% ,0.108% and 41.9 mg · kg- 1 fresh,respectively. The sarcocarp hardness was 4.66 kg · cm- 2. The vigor of growth of‘Xinli No.11’was middle,germinating capability was high,branch development capability was middle. The fruit development period was about 140 to 150 days,the growth period per year was 210 days,and the fruit was ripening in early or middle of September in Korla,China. It can be stored in cold storage conditions for 5 to 6 months. It has the characters of early fruiting and high yield,the production of 3-4 year old tree was 2-3 times compared to‘Kuerlexiangli pear’. It began flowering after 2 years of planting and the average yield of individual plant was 9.7 kg at 4 years after planting,and it had better adaptability and strong cold resistance (can survive minus 28 ℃ low temperature). The orchard should with mild saline-alkaline and well drained soil. The suitable planting space in the rows and plants are (4-5) m×(3-4) m and 4 m×1.5 m,respectively. The suitable tree shape was evacuation and thin-long spindle. ‘Xinli No.11’has high rate of fruit setting under natural conditions,fruit thinning should be carried out within three weeks after fallen petal with a standard of leaving fruit with good shape and bright color without any diseases and pests and mechanical injuries. The fertilizer should be provided properly according to the period of growth and development and the tree age,and the water should be provided properly according to the soil moisture,both drip irrigation and flood irrigation are available. Grapholitha molesta,Cydia pomonella and Valsa canker should be regard as the most important pests and diseases and controled by scientific using physical and chemical technology according to their occurrence rule. © 2019 Journal of Fruit Science     

Efficient exploration and SSR identification of autotetraploids from the seedlings of thirteen apomictic Citrus genotypes北大核心CSCD

【Objective】Citrus is a crucial part of Chinese fruit crops. There are abundant citrus germplasm resources in China, but many excellent local varieties are gradually eliminated by the market due to the problem of numerous seeds within the fruit. The fruits of triploid plants are generally seedless because of their sterile male and female gametes. Therefore, triploid production is a promising strategy to breed seedless cultivars in citrus. Triploids can be obtained by interploidy crossing between diploids and tetraploids. However, the tetraploid germplasm is rare, which limits the application of this strategy. Exploration of tetraploids is an important prerequisite for triploid production with the aim to improve the seedy local cultivars in our country. For the rootstock improvement, tetraploid plants are also valuable resources because of their higher metabolite content, and better resistance than their diploid parents. In this study, we planned to explore tetraploid plants from 13 local cultivars in our country by using the traits of spontaneous doubling of the nucellar cells in polyembryonic citrus varieties. The exploration of tetraploids from the above 13 local cultivars will not only provide excellent tetraploid parents for the production of triploid plants, but also lay the foundation for the basic research about the effect of genome duplication on some important trait change, such as dwarfing, extensive adaptivity and higher medicinal value in tetraploids.【Methods】After the mature fruits were harvested, the seeds were extracted and the seed coats were peeled off, and then they were placed in a thermostat to accelerate germination. When the seeds germinated, they were sown in pots and cultivated in a plant growth chamber. After the seedlings grew up with three or more leaves, putative polyploids were screened according to the morphological feature showing lower height, shorter taproots, less lateral roots, thicker and rounder leaves and declined oil gland density. The ploidy levels of these putative polyploids were further confirmed by flow cytometric analysis and the observation on root tip chromosome numbers. After determination of the ploidy level, some morphological traits, including plant height, root length and diameter, lateral root number, stem diameter, leaf thickness and shape index of the tetraploids and their corresponding diploid parents were measured at the same developmental stage. SSR analysis was used to identify the genetic origin of the explored tetraploids with at least three pairs of SSR primers selected for each cultivar.【Results】The polyembryonic degree of seeds from each cultivar was firstly determined and it showed that the seeds of all 13 cultivars were polyembryonic. Among them, Qu tangerine had the highest number of embryos with an average of 9.4 embryos per seed and Bingtang sweet orange had the lowest number of embryos with an average of 2.2 embryos per seed. Based on the morphological trait screening, we identified 2, 1, 3, 2, 7, 3, 1, 3, 1, 3, 17, 1 and 2 putative polyploids respectively from 343, 499, 892, 385, 519, 290, 457, 241, 119, 690, 828, 114 and 129 seedlings of Qianshanhong tangerine, Bayue tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Shitougan, Bingtang sweet orange, Jinmi sweet orange, Moping Xiangcheng, Japanese Xiangcheng, Zhique and Youpi kumquat. After further confirmation of ploidy levels concerning above putative tetraploids, we obtained 45 tetraploids and one hexaploid plant from Qu tangerine, with an average occurrence rate of 0.85%, among which the rate of Japanese xiangcheng was the highest with 2.05% and the rate of Bayue tangerine was the lowest with 0.20%. The exploration time from seed germination to obtaining tetraploid seedlings varied among cultivars, with the longest time (42 days) used in Youpi kumquat and the shortest time (23 days) in Shitougan. The morphological traits of tetraploids and their corresponding diploid seedlings from nine cultivars of Qianshanhong tangerine, Qu tangerine, Zao tangerine, Bianping tangerine, Ougan tangerine, Bingtang sweet orange, Moping Xiangcheng, Japanese Xiangcheng and Youpi kumquat were measured. For plant height, tap root length, lateral root numbers and leaf thickness, the tetraploid seedlings of seven cultivars showed significant differences with their diploid parents. For taproot and stem diameter, only the tetraploid seedlings explored from Bingtang sweet orange and Japanese Xiangcheng had significant difference with their diploid parents. For leaf shape index, the tetraploid seedings from Bianping tangerine and Moping Xiangcheng exhibited significant differences with their diploid seedlings. In conclusion, most tetraploid seedlings of all nine cultivars showed lower plant height, shorter and thicker taproot, less lateral root number, thicker and rounder leaves than those of their diploid parents. These results provide supports for the screening of putative tetraploids based on morphological trait observation. For analyzing the genetic origin of the tetraploids obtained in this study, at least three SSR markers were used in each genotype. The results showed that the bands of all 45 tetraploids were identical with those of their corresponding diploids, indicating that all the 45 tetraploids might originate from the spontaneous chromosome doubling of nucellar cells of their corresponding diploids. In addition, the bands of the hexaploid from Qu tangerine were also identical with their diploid parent. We speculated that it might derive from chromosome doubling of a triploid zygotic cell, which formed by selfing of a FDR-type 2n gamete with a normal n gamete, and both gametes were produced by Qu tangerine.【Conclusion】This study verified that morphological screening combined with flow cytometry ploidy determination and SSR analysis is an efficient approach to exploring polyploid seedlings from apomictic citrus. Using this method, 45 autotetraploid and one hexaploid plants were obtained from 13 apomictic citrus genotypes. These newly discovered tetraploids are potentially valuable for not only genetic improvement of some elite local citrus cultivars with seeds produced by triploids using interploidy hybridization, but also selection of the promising rootstocks with dwarf, multi-resistance and broad adaptability characteristics to improve the ability to resist various abiotic and biotic stresses. © 2023 Journal of Fruit Science. All rights reserved.     

Analysis the efficiency of inter and intra-specific hybridization of new cultivars of sweet cherry and chinese cherry北大核心CSCD

【Objective】To explore the rules of intra-specional and inters-pecific cross breeding of some new sweet cherry cultivars and improve the efficiency of cross breeding of sweet cherry in the area where winter is warm and summer is hot and moist.【Methods】The intraspecial and interspecifichybridization test of sweet cherry was carried out by using sweet cherry as the mother,using sweet cherry and chinese cherry as the father. The rate of fruit set,embryo,embryo abort of the cross and quality of the F0 fruit were investigated in order to analysis the efficiency of the cross.【Results】The four cultivars of sweet cherry‘Jiangnanhong’‘Chaoyang-1’‘Changfeng-1’and‘Van’were taken as the mother. Five sweet cherry cultivars or selections‘04-8’‘04-11’‘Chaoyang-1’‘Van’‘Lapins’and five cultivars of chinese cherry‘Chaozaohong’‘Duanbing’‘Gejiawu’‘Heizhenzhu’‘Zijing’were taken as the male parent. Among them‘Jiangnanhong’and‘Zijing’are newly identified cultivars in Zhejiang province,and‘Chaoyang-1’and‘Changfeng-1’are new selections. After pollination for 1 week,the fruit setting rate was relatively high and significant decrease in the 4 weeks and mature stages in intra and inter- species with the development of fruits. In the mature stage,all the fruit setting of the other cross of intra-species were higher than 5%,except that of‘Jiangnanhong’ב04-11’was 1.85% lower than 3%,and that of‘Changfeng-1’בLapins’was 4.35% lower than 5%,which indicates the basic affinity between the cross of intra-specific sweet cherry. The fruit setting rate of intraspecific cross was higher than that of interspecific cross. The fruit setting rate of‘Chaoyang-1’was the highest in both intraspecific and interspecific hybridization. The interspecific hybrid combination of‘Changfeng-1’had the lowest fruiting rate. The embryo rate and abortion rate of the intraspecific cross were higher than those of interspecific cross when the female parent is same. The abortion rate is high when hybrid embryos mature,and only 4 crosses group can obtain non-abortion embryos,which are the hybrid cross of‘Jiangnanhong’with‘04-8’‘Lapins’‘Chaozaohong’,and the cross of‘Changfeng-1’with‘Van’. The abortion is lower when the hybrid embryos immature. The embryo rate was lowest and the abortion degree was the highest of the cross of‘Jiangnanghong’in immature embryo stage. The cross with the mother of‘Chaoyang-1’was the best with high setting rate,high embryo rate and low abortion rate in immature embryo stage,so‘Chaoyang-1’is the best female parent in this test.‘Chaoyang-1’and‘Zijing’were the better fathers in the ten test male parents for the cross with them had a high embryo rate (65.84% and 75.21%,respectively),and most of the embryos were bright white and full with good development in the immature embryos stage. ‘Chaoyang-1’is the best female and male parent in this test. The cross between‘Chaoyang-1’and‘Van’shows‘Chaoyang-1’is the better female than male parent. The ripening stage of F0 fruit was delayed,and the color and shape of F0 fruit showed the same col- or and shape of open pollination of parent fruit. The soluble solid of interspecific hybrid fruit was lower than that the intra-specific hybrid,and they all lower than that of the open pollinated female fruit. In order to obtain hybrid offspring,the author suggests three methods could be used in the area where is warm in winter,hot and moist in summer: one is selecting hybrid offspring which comes from the main producing areas,which means making cross and getting hybrid offspring in the main producing areas but select it in future planting area. The other is using embryos rescue technology after getting immature fruit; the third one is using mixed pollen for open pollination.【Conclusion】The fruit set and embryo development have significant differences among the different cross group in the area where winter is warm and summer is hot and moist. The fruit set is higher and the embryo abort is lower of intraspecific cross than that of interspecific cross when the mother is the same sweet cherry.‘Chaoyang-1’is the best female and male parent in this test when making cross. © 2019 Journal of Fruit Science     

整合农艺性状和分子标记数据构建新疆野苹果核心种质

AIM: To explore the effect of traditional Chinese medicine Qiliqiangxin on cardiomyocyte apoptosis after myocardial infarction (MI) in a rat model. METHODS: MI was induced in rats by ligation of the anterior descending coronary artery. The survivors were randomly divided into 3 groups: sham operation group, MI group and Qiliqiangxin treatment group (4 g穔g^-1穌^-1). After 28 days, the infarction size was measured. Apoptotic index (AI) was determined by TUNEL. The expression of Fas was detected by the method of immunohistochemistry in non-infarcted zones (NIZ), and the expression of xanthine oxidase (XO) and caspase-3 in myocardial tissue from NIZ was detected by Western blotting. In addition, XO activity, and O₂ ^-? and OH? scavenging activity of myocardial tissues in the NIZ were measured by colorimetry. RESULTS: Compared with MI group, AI and the expression of Fas and caspase-3 in the NIZ were significantly depressed in Qiliqiangxin treatment group. Moreover, the activity of XO was significantly decreased while O₂ ^-? and OH穝cavenging activity was significantly increased in Qiliqiangxin treatment group. Ventricular remodeling was attenuated. No significant difference in infarct size and XO expression level between Qiliqiangxin treatment group and MI group was observed. CONCLUSION: Qiliqiangxin may inhibit apoptosis of cardiomyocytes in the NIZ of rats by reducing reactive oxygen species and depressing the expression of Fas and caspase-3.     

龙眼TIR1和ABP1基因的克隆及其在体胚发生过程中的表达分析

AIM: To investigate the expression and the role of histone deacetylase 8 (HDAC8) in cardiac hypertrophy and the effects of valproic acid sodium (VPA, a histone deacetylase inhibitor) on the expression of HDAC8 and cardiac hypertrophy in two-kidney two-clip (2K2C) renovascular hypertensive rats. METHODS: Male SD rats were randomly divided into sham operation group, 2K2C group, high dose VPA (400 mg穔g^-1穌^-1) treatment group, low dose VPA (200 mg穔g^-1穌^-1) treatment group and candesartan (10 mg穔g^-1穌^-1) treatment group.Four weeks after surgery,the rats were intraperitoneally injected with VPA for 4 weeks. Sham operation and 2K2C rats were given vehicle for 4 weeks. All animals were sacrificed 8 weeks after surgery. The ratio of left ventricular weight to body weight (LVW/BW) was calculated and pathological changes of the myocardium were observed with HE staining. The mRNA expression of atrial natriuretic factor (ANF) and HDAC8 was examined by RT-PCR. The protein level of HDAC8 was also measured by Western blotting analysis. RESULTS: Compared with the control rats, the mRNA and protein expression of HDAC8 significantly increased in the myocardium in 2K2C rats while the mRNA and protein expression of HDAC8 was significantly decreased by VPA treatment in 2K2C rats. The mass index (as measured by LVW/BW) and cardiomyocyte cross areas were markedly increased and myocardial fibers were disordered in 2K2C rats, but these parameters were markedly reversed after treated with VPA for 4 weeks, indicating that VPA attenuated cardiac hypertrophy. Moreover, VPA also decreased the mRNA expression of ANF. CONCLUSION: HDAC8 may play an important role in cardiac hypertrophy in 2K2C renovascular hypertensive rats. VPA inhibits the expression of HDAC8 and prevents the development of cardiac hypertrophy.     

两种砧木对年橘果实品质与产量的影响

AIM: To investigate the effect of ovariectomy (OVX) and simvastatin (SVS) on the expression of osteoprotegerin (OPG) in bone callus of rats. METHODS: Simvastatin (10 mg穔g^-1穌^-1) was injected subcutaneously to tissue overlying the site of fractured tibiae of ovariectomized rats for a treatment period of 5 days. Vehicle reagent was used as control in sham and OVX rats. Bone callus were harvested at time points of 1, 2 and 4 weeks after fracture and the expression of proliferating cell nuclear antigen (PCNA) and OPG was measured by the method of immunohistochemistry. RESULTS: Compared with OVX+vehicle group, the expression of PCNA decreased by 17.3%, 32.1% and 26.1% in OVX+vehicle group than that in sham+vehicle group (P<0.01), and increased by 60.1%, 67.7% and 67.7% in OVX+SVS group (P<0.01) at the time points of 1, 2 and 4 weeks after fracture, respectively. The expression of OPG was the lowest in OVX+vehicle group. The significant differences of OPG expression between OVX+SVS group and OVX+vehicle group at the 3 time points (P<0.01), and significant differences of OPG expression between sham+vehicle group and OVX+vehicle group at 1st week and 2nd week after fracture (P<0.05) were observed. CONCLUSION: Ovariectomy decreases the expression of OPG in bone callus of rats, while simvastatin increases it, indicating that simvastatin can indirectly inhibit the function of osteoclasts.