Effect of microenvironments and exogenous substance application on 5′-nucleotidase activities in apple peel

The present experiment was conducted to examine the effect of microenvironments and exogenous substance application on 5′-nucleotidase activity in apple peel tissue. By enclosing apple fruits in bags, treating them with exogenous active oxygen species and regulative agents or placing them under controlled conditions at different fruit temperatures or relative humidity, the 5′-nucleotidase activities were compared with the corresponding controls. The results indicated that, a considerable effect of the microenvironments was found on 5′-nucleotidase activities in fruit peel tissue. The highest enzymatic activity appeared in fruits on the southwest exposure of canopy, regardless of bagged or non-bagged fruits, significantly higher than those from any other exposures. Fruits with bags had a significantly higher 5′-nucleotidase activity than the exposed ones. A variation in enzymatic activities was observed in fruits enclosed with different types of bags, which were supposed to alter the microenvironments around them. Within a certain range, gradual or fluctuating rise of fruit temperatures could favor the increase of 5′-nucleotidase activities as a result of heat adaptation, whereas the activity would be inhibited if the temperature-rising period was too short or temperature differential was too large. No matter what temperatures fruits were subjected to, high relative humidity was favorable for stimulating the 5′-nucleotidase activities, which might partly explain why fruit sunburn would not happen in humid climates. Treatments with four kinds of exogenous active oxygen species could reduce the 5′-nucleotidase activities significantly but spraying with CaCl₂ was able to enhance 5′-nucleotidase activities by 55.39%, reaching a 5% significant level. __________ Translated from Acta Horticulturae Sinica, 2005, 32(2): 191–196 [译自: 园艺学报]     

Cloning and sequence analysis of a mutation-type cinnamate 4-hydroxylase gene from Brassica oleracea L. var. acephala DC.

A 2431-bp full-length cinnamate 4-hydroxylase gene, BoC4H, was cloned from Brassica oleracea L. var. acephala DC.. It contains 2 introns. Its mRNA is 1715 bp, encoding a deduced 481-amino-acid polypeptide with wide homologies to C4Hs from other plants. It possesses cytochrome P450 conserved domains and motifs such as the haem-iron binding motif, the E-R-R triad, the T-containing binding pocket motif and the hinge motif necessary for optimal orientation of the enzyme. It also has most of the canonical C4H/CYP73A5-featured substrate-recognition sites (SRSs) and active site residues. However, owing to a single-base deletion at C₂₂₄₂ and subsequent frame shift within the 3′ coding region as compared with C4H genes from Arabidopsis thaliana and other plants, BoC4H shows a 36-aa deletion/variation at its C-terminus and the SRS6 motif together with active site residues therein are absent. Thus BoC4H may be of no function or low activity. BoC4H is a membrane protein and is probably associated with the endoplasmic reticulum. Its secondary structure is dominated by alpha helices and random coils. The Swiss-Model could not predict its tertiary structure. B. oleracea contains a C4H gene family with at least 5 members. __________ Translated from Acta Horticulturae Sinica, 2007, 34(4): 915–922 [译自 : 园艺学报]     

Cloning and bioinformatics analysis of cDNA encoding cattle Smad4 gene

The cDNA of cattle Smad4 gene was cloned by RT-PCR, 3′ RACE and 5’t RACE and got a 3503-bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got an accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99%, 96%, 95%, 91% and 91% similarity in nucleic acid sequences, and 99%, 98%, 98%, 99% and 98% similarity in amino acid sequences with sheep, pig, human, rat and mouse, respectively. Smad4 cDNA was found in the testes, pancreas, liver, small intestine, ovary, lymph, cardiac muscle, skeleton muscle and thymus gland, which indicated that Smad4 was broadly expressed in cattle. __________ Translated from Acta Veterinaria et Zootechnica Sinica, 2007, 38(4): 321–325 [译自: 畜牧兽医学报]     

Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear

To inhibit the browning process in fruits of Yali pear, in this paper, antisense gene techniques were used to reduce the expression of PPO gene. A cDNA fragment of 450 bp, which is located at the 3′ terminal of the polyphenol oxidase (PPO) gene, was amplified from Yali pear using the RT-PCR method, then the antisense expression vector was constructed by inserting the fragment of the Yali pear PPO gene between the CaMV promoter and NOS terminator of the expression vector pBI121 in a reverse orientation. After that, with the agrobacterium-mediated method, the PPO antisense gene was transformed into Yali pear shoots. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased, compared with the non-transformed Yali pear shoots. This lays a good foundation for breeding new varieties of pears with browning resistance in the future.     

Initial function determination for the open reading frame (ORF) region of Pib gene via rice transformation

Three plant binary expression vectors—pNAR501, pNAR502 and pNAR503—were constructed, carrying fragments of exon2-exon3, 5′partial deletion exon1 and 5′partial deletion exon1-exon2-exon3 of Pib gene driven by 35S. These three vectors were transformed into the japonica rice variety Nipponbare through agrobacterium-mediated transformation. More than 30 transgenic rice plants were obtained and confirmed by polymerase chain reaction (PCR), Southern hybridization and the hygromycin resistance test in seed germination of their progeny. A rice blast resistance test for in vitro leaves of T_o transgenic plants in the tillering stage showed higher resistance to the races of E1, F1 and G1 of rice blast than that of the control Nipponbare. However, results of rice blast resistance test for seedlings of T₁ transgenic plants in the 3-to 4-leaf stage were different. All T₁ transgenic seedlings had a lower level of resistance to E1, F1 and G1 races than that of the control Nipponbare. Translated from Journal of Nanjing Agricultural University, 2006, 29(3): 1–5 [译自: 南京农业大学学报]     

双须叶须鱼5种组织乳酸脱氢酶和苹果酸脱氢酶的 组织特异性分析

为了解双须叶须鱼的生化遗传特性,采用聚丙烯胺凝胶垂直板电泳对双须叶须鱼5种组织(心脏、眼睛、肌肉、肝脏和肾脏)的乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)进行了分析。结果表明,双须叶须鱼LDH同工酶和MDH同工酶具有组织特异性和个体差异性,双须叶须鱼的MDH分为上清液型(s-MDH)和线粒体型(m-MDH)。     

Deletion of TTTTA in 5′UTR of goat MSTN gene and its distribution in different population groups and genetic effect on bodyweight at different ages

A total of 436 goat samples from twenty-six populations were scanned for single nucleotide polymorphisms (SNPs) in 5′UTR of goat MSTN gene using sequencing and the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP). The deletion of TTTTA found in 5′UTR of goat MSTN gene was conserved in different species and might be a unique mutation in goats. The Foreign Group and the Boer Goat backcrossed offspring had the highest and smallest genetic diversity of this mutation respectively, and also with the smallest and highest heterozygosity deficiency respectively. The genetic diversity of this mutation for the South Group of Chinese indigenous goat breeds was higher than that of the North Group, and the South Group had the smaller heterozygosity deficiency than the North Group. The estimated marginal means based on the modified population marginal mean and pairwise comparison between significant pairs showed that there was a trend that genotype AA had a significant effect on body weight and size from birth to four-month old (P<0.05 or P<0.01). It could be inferred that the deletion of TTTTA in 5′UTR of goat MSTN gene had a significant effect on body weight and size.     

Genomic organization and sequence polymorphism of a farnesyl diphosphate synthase gene in apples (Malus domestica Borkh.)

Primer pairs were designed to amplify the genomic DNA sequence of the farnesyl diphosphate synthase (FPPS) gene by PCR. The PCR products were sequenced, spliced and compared to the cDNA sequence in the GenBank (accession No. AY083165). The genomic sequence and intron-exon organization of FPPS1 gene in the apple cultivar ‘Fuji’ were thus obtained. The FPPS1 genomic sequence has been registered in the GenBank (accession No. HM545312). It has 11 introns and 12 exons. The sizes of 11 introns were 559 bp, 108 bp, 144 bp, 114 bp, 84 bp, 690 bp, 373 bp, 168 bp, 87 bp, 91 bp and 97bp, and their phases were 0, 1, 0, 0, 0, 2, 0, 0, 0, 0 and 0, respectively. The sizes of 12 exons were 111 bp, 25 bp, 116 bp, 87 bp, 117bp, 89 bp, 52 bp, 96 bp, 45 bp, 90 bp, 72 bp and > 12 bp, respectively. Gene sequence comparison results of five apple cultivars indicated that the development of apple superficial scald was not influenced by the mutations in the exon sequence of FPPS1 gene. A 6-bp repeat unit deletion mutation and many SNP mutations in the introns, mainly in the introns of one allele, were identified in the apple scald-resistant cultivar ‘Golden Delicious’. This is the first report on the genomic organization and coding region polymorphism of FPPS gene in apples and other fruit trees.     

Molecular cloning and characterization of nitrate reductase gene cDNA from non-heading Chinese cabbage

Four non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivars, Suzhouqing, Xuekeqing, Huangxinwu, Aijiaohuang, were planted to investigate the activity of nitrate reductase (NRA) in leaves. After being induced by KNO₃ at 50 mmol/L for different hours the NRAs of the four cultivars were determined in vivo. The results showed that the NRAs changing trends of these four cultivars were similar. The highest NRAs in leaves reached their maximum at the 4th, 4th, 6th, and the 6th hour of induction, respectively. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. Then, the total RNA was isolated from the leaves of Suzhouqing that was induced by KNO₃ at 50 mmol/L for four hours, and two fragments of NR cDNA were obtained through RT-PCR using specific primers. The products of PCR were cloned and sequenced. They are 1 125 and 438 base pairs, which were named nr₁₁₂₅ and nr₄₃₈, encoding 374 and 135 amino acids, respectively. Finally, nr₁₁₂₅ was accepted and released by GenBank (accession number DQ001901). Translated from Journal of Nanjing Agricultural University, 2006, 29(2): 15–19 [译自: 南京农业大学学报]     

刺儿菜性别连锁的同工酶标记分析

[目的]利用刺儿菜进行同工酶分析,确定适合作为遗传标记的同工酶类型。[方法]采用聚丙烯酰胺不连续垂直平板电泳(PAGE)技术,对刺儿菜雌雄株花和叶片中过氧化物酶(POD),苹果酸脱氢酶(MDH),苹果酸酶(ME)及酯酶同工酶(EST)的差异进行比较分析。[结果]POD、MDH及EST同工酶在刺儿菜雌雄株的花和叶中存在明显的性别差异。在花和叶片中,POD同工酶均发现了性别特异的酶带,MDH同工酶仅发现在花中具有1条雄性特异带,苹果酸酶同工酶(ME)没有明显的性别差异。除花中的酯酶酶谱外,两种同工酶在同一性别的不同个体间,酶谱无明显差异。[结论]同一种酶在刺儿菜叶片和花中的雌雄株酶谱有明显差异。     

Integration of C4-specific PPDK gene of maize to C3 rice and its characteristics in relation to photosynthesis

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in photosynthesis in some plants that exploit the C₄ photosynthetic pathway for the fixation of CO₂. The C₄-specific PPDK encoding pyruvate orthophosphate dikinase was introduced into C₃ plant, a rice (Oryza sativa L. cv. indica IR64) mediated by biolistic and Agrobacterium transformation. The C₄-PPDK gene of maize was integrated to indica IR64 with polymerase chain reaction (PCR)-Southern blotting. The total nitrogen of flag leaves of transgenic IR64 was analyzed with Kjeldahl method for quantitative determination of nitrogen, indicating that the total nitrogen of flag leaves of most transgenic IR64 was higher than that of non-transgenic control IR64 formants in the greenhouse. The maximum value of total nitrogen of flag leaves was 3.61% among transgenic IR64 plants, 1.07% higher than that of non-transgenic control IR64 formants. The total nitrogen of flag leaves of transgenic IR64 was increased by 42.1%. The factors for yield of transgenic IR64 plants were analyzed, indicating there was a greater difference in yield-forming factors among transgenic IR64 plants in the greenhouse, i.e. dried plant weight, harvested index and so on. Thus, it could help rice breeders select different materials for breeding. Translated from Molecular Plant Breeding, 2006, 4(6): 797–804 [译自: 分子植物育种]     

Cloning of endo-��-glucanase I gene and expression in Pichia pastoris

Total RNA of Thermoascus aurantiacus was isolated from its mycelium and acted as template for RT-PCR. The full-length cDNA encoding an endo-β-glucanase I was cloned via RACE-PCR method and the cDNA contained an ORF of 1005 bp encoding 305 amino acids. A recombinant plasmid, pPIC9k-egI, was constructed by inserting the ORF sequence of endo-β-glucanase I gene (egI) into the yeast expression vector pPIC9k and transformed to Pichia pastoris GS115. The results showed that the recombinant endo-β-glucanase I was excreted into the fermentation medium. The highest activity of endo-β-glucanase I and the protein content were up to 45.42 U/mL and 788.26 μg/mL at incubation time of 144 h. The optimal temperature and pH for the recombinant endo-β-glucanase I were found to be 70°C and 3.5, respectively.     

Cloning and functional analysis of the genes involved in signal transduction in tomato Cf-4-Avr4 pathosystem

Hypersensitive response (HR) is one of the most efficient and common resistance mechanisms in plants. Cloning signaling genes are very important to elucidate the resistance mechanisms. A gene in tomato homologous to several resistance proteins in plant was involved in HR and named as RGL (Resistance Gene Like). RGL protein was used as a bait to screen interacting protein(s) from tomato cDNA library through the yeast two-hybrid system. Two interacting proteins were found, which were called as RGLIP-1 and RGLIP-2 (RGL Interacting Protein), respectively. RGLIP-1 is a protein of 291 amino acids with significant homology with thylakoid lumen protein. RGLIP-2 is a protein of 248 amino acids with significant homology with transducin protein. Virus-Induced Gene Silencing (VIGS) of the two genes results in a partial and complete suppression of Avr4-induced HR, which indicates that both genes are involved in hypersensitive response. __________ Translated from Acta Horticulturae Sinica, 2006, 33(1): 52–57 [译自: 园艺学报]     

苹果砧木富平楸子嫁接高度对抗旱性的影响

以常规低位嫁接为对照,研究富平楸子高位嫁接对植株抗旱性的影响,为生产中该砧木资源的应用提供参考。以一年生富平楸子组培盆栽苗为砧木,分别在距地面10cm和60cm处嫁接‘秦脆’,采用土壤含水量75%～80%为对照,土壤含水量45%～55%为处理,进行为期60 d的中度干旱胁迫处理,测定相关指标,比较两种嫁接高度下植株的抗旱性强弱。结果表明：长期干旱胁迫后,与对照相比,2种类型苗木的各指标均显著降低,其中高位嫁接植株的生物量、根系指标、叶片相对含水量(RWC)、净光合速率(Pn)、蒸腾速率(Tr)、瞬时水分利用效率(WUEi)及总叶绿素含量的降幅均小于低位嫁接植株,且差异显著;叶片内游离脯氨酸含量与抗氧化酶(SOD、POD)活性的增幅大于低位嫁接植株,其中游离脯氨酸含量增幅差异显著;叶片相对电导率与MDA含量的增幅小于低位嫁接植株,差异显著。利用以上指标进行隶属函数计算,结果显示高位嫁接植株抗旱性更强,且差异显著。富平楸子采用高位嫁接可显著提高植株抗旱性,因此实际生产中以高位嫁接的利用形式对旱地苹果产区是可行的。     

The application of asymmetric PCR-SSCP in gene mutation detecting

The advantages and disadvantages of asymmetric PCR-SSCP and the traditional PCR-SSCP were compared in this study. The mutations in 3′UTR of Smad4 gene of Luxi cattle and the Holstein cow were analyzed by asymmetric PCR-SSCP and one insert “T” mutation and one G/A mutation in this region were found. The G/A mutation created a HhaI restriction enzyme digestion position and the frequencies studied by asymmetric PCR-SSCP and HhaI-RFLP in 116 Luxi cattle and 75 Holstein cows were all the same. The asymmetric PCR-SSCP had fewer, clearer and more stabile bands than traditional PCR-SSCP. This indicates that the asymmetric PCR-SSCP is suited for mutation detection. __________ Translated from Journal of Northwest A & F University (Natural Science Edition), 2007, 35(6): 15–18, 23 [译自: 西北农林科技大学学报(自然科学版)]     

Sequence of a Cashmere goat type I hair keratin gene and its expression in skin

The keratin family includes the epithelial (soft) keratins and the hair (hard) keratins, which can be divided into two subfamilies—the acidic type I and the basic or neutral type II. A cDNA is isolated and sequenced from a cDNA library constructed with poly (A) +RNA derived from the goat skin. Because the gene sequence of the known type I hair keratins 8C1 is in accordance with that of the hHa1, this keratin can be identified as the goat hair keratin gHa1 cDNA (GenBank Accession No. AY510110.1). The 413 amino acid sequence derived is compared with the Sheep 8C1 type I mRNA and they exhibit the highest homology (97.8%). In in situ hybridization, it has revealed a strong expression in the cortex of both primary and secondary hair follicles. __________ Translated from Acta Veterinaria et Zootechnica Sinica, 2006, 37(1): 18–22 [译自: 畜牧兽医学报]     

Analysis of the apple fruit acid/low-acid trait by SSR markers

It is necessary to find out the genetic characteristics of malic acid in the course of apple genomic research and breeding. In this study, the SSR marker linked to the acid/low-acid trait in apple fruit was identified from 140 SSR primer pairs, using 91 F1 population hybrids from the intra-specific cross between apple cultivar ‘Dongguang’ and ‘Fuji’ as the experimental materials. Of 140 SSR primer pairs, only primer SDY085 produced a polymorphic band linked to acid trait, and the linkage distance was 8.89 cM. Also, the titrated acid and malic acid in different developmental stages were determined. The SSR marker analysis, coupled with the change of the total acid and malic acid contents, revealed that the acid/low-acid trait was governed by a major gene and acid trait was completely dominant. __________ Translated from Acta Horticulturae Sinica, 2006, 33(2): 244–248 [译自 : 园艺学报]