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1.
  目的  通过对松材线虫效应因子基因的克隆和功能研究,揭示Bx-Hh-grl对松材线虫致病性的作用,为防治松材线虫提供理论依据。  方法  用松材线虫Bx1022株系接种黑松,20 d后提取线虫总RNA进行转录组测序,将该转录组设为松材线虫植食阶段转录组。提取由灰葡萄孢(Botrytis cinerea)培养的Bx1022的总RNA进行转录组测序,将该转录组设为菌食阶段转录组。比较分析两个转录组,筛选到差异表达基因Bx-Hh-grl。对Bx-Hh-grl编码蛋白质的跨膜结构域和信号肽进行预测。利用原位杂交检测Bx-Hh-grl表达部位。应用RNAi技术干扰Bx-Hh-grl表达以探究Bx-Hh-grl对松材线虫致病性的作用。通过Q-PCR验证,确定RNAi效果显著。将经RNAi处理的松材线虫接种3年生黑松枝条,以未经处理的松材线虫(CK组)和ddH2O(Mock组)处理的黑松作为对照,比较黑松发病情况进而比较不同处理后松材线虫的致病性差异。  结果  筛选出植食阶段与菌食阶段转录组差异表达基因Bx-Hh-grl,其编码蛋白质具有跨膜结构域和信号肽。原位杂交试验结果显示Bx-Hh-grl在松材线虫食道腺表达,符合效应因子基因特征。Q-PCR结果显示经RNAi处理后的松材线虫Bx-Hh-grl表达量下调,RNAi处理效果显著。接种实验表明,Bx-Hh-grl-RNAi组显症的时间明显晚于CK组,Mock组黑松一直保持健康状态。  结论  Bx-Hh-grl是与松材线虫致病相关的效应因子基因。明确Bx-Hh-grl功能有利于进一步了解松材线虫致病机理,为降低松材线虫危害,防治松材线虫奠定理论基础。   相似文献   

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Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province, China.  In order to establish a rapid, reliable and specific molecular detection method for Mvitis, the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target.  The reaction system was optimized and the reliability, specificity and sensitivity of primer were testified, therefore, a rapid PCR detection method for Mvitis was established.  The result showed that the optimal annealing temperature of the primers was 53°C, which was suitable for the detection of different life stages of Mvitis.  Specificity test showed that the specific fragment size of 174 bp was obtained from Mvitis, but other five non-target nematodes did not have any amplification bands, thus effectively distinguish Mvitis and the other five species, and could specifically detect the Mvitis from mixed populations.  Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J2) and 10–4 female.  Futhermore, this PCR technique could be used to detect directly M. vitis from soil samples.  The rapid, sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J2 of Mvitis and the detection of Mvitis in mixed nematode populations and the detection of two J2s or one male in 0.5 g soil samples, which will provide technical support for the investigation of the occurrence and damage of Mvitis and the formulation of efficient green control strategies.  相似文献   

4.
Ininsects,ecdysteroidsaresynthesizedbygenesoftheHalloweenfamilyandplayimportantrolesinseveralkey developmentalevents,includingmoltingandmetamorphosis.However,therolesofthesegenesinAgasicles hygrophila are still largely unknown.In this study, the expression patterns of the two Halloween genesAhCYP307A2andAhCYP314A1weredeterminedbyquantitativePCR(qPCR)atdifferentdevelopmentalstages.Moreover,the functions of these two genes were explored using RNA interference (RNAi), and ovarian development was ob...  相似文献   

5.
Artemisia annua is an important preferred host of the mirid bug Apolygus lucorum in autumn.  Volatiles emitted from Aannua attract Alucorum.  Volatile artemisinic acid of Aannua is a precursor of artemisinin that has been widely investigated in the Chinese herbal medicine field.  However, little is known at this point about the biological roles of artemisinic acid in regulating the behavioral trends of Alucorum.  In this study, we collected volatiles from Aannua at the seedling stage by using headspace solid phase microextraction (HS-SPME).  Gas chromatography-mass spectrometry (GC-MS) analysis showed that approximately 11.03±6.00 and 238.25±121.67 ng h–1 artemisinic acid were detected in volatile samples and milled samples, respectively.  Subsequently, a key gene for artemisinic acid synthesis, the cytochrome P450 gene cyp71av1, was expressed in engineered Saccharomyces cerevisiae to catalyze the production of artemisinic acid.  After the addition of exogenous artemisinic alcohol or artemisinic aldehyde, artemisinic acid was identified as the product of the expressed gene.  In electroantennogram (EAG) recordings, 3-day-old adult Alucorum showed significant electrophysiological responses to artemisinic alcohol, artemisinic aldehyde and artemisinic acid.  Furthermore, 3-day-old female bugs were significantly attracted by artemisinic acid and artemisinic alcohol at a concentration of 10 mmol L–1, whereas 3-day-old male bugs were attracted significantly by 10 mmol L–1 artemisinic acid and artemisinic aldehyde.  We propose that artemisinic acid and its precursors could be used as potential attractant components for the design of novel integrated pest management strategies to control Alucorum.  相似文献   

6.
【背景】条锈病是小麦上的重大病害,由条形柄锈菌小麦专化型(Puccinia striiformis f. sp. tritici,Pst)侵染引起。条锈菌是活体营养型寄生真菌,在侵染过程中形成吸器,通过吸器从寄主植物汲取营养。同时,吸器分泌效应蛋白调控寄主免疫,促进侵染过程。【目的】明确条锈菌效应蛋白的功能及其作用机理,为揭示条锈菌的致病机制打下基础。【方法】比较分析条锈菌夏孢子、芽管和吸器转录组,获得在吸器诱导表达的分泌蛋白基因Hasp83,在本氏烟叶片细胞中瞬时表达观察是否能抑制由BAX引起的细胞坏死;利用qRT-PCR分析该基因在条锈菌侵染小麦不同阶段的表达水平。借助荧光假单胞菌Ⅲ型分泌系统和寄主诱导的基因沉默(host-induced gene silencing,HIGS)分析Hasp83在条锈菌侵染过程中的功能。利用酵母双杂交系统筛选小麦中与Hasp83互作的蛋白,免疫共沉淀技术进一步在烟草细胞中共表达验证Hasp83及其候选靶标蛋白的互作。【结果】Hasp83开放阅读框全长522 bp,编码173个氨基酸,蛋白N端1—29位氨基酸为信号肽,无保守结构域,在本氏烟叶片细胞中...  相似文献   

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Sugar content is a determinant of apple(Malus×domestica Borkh.) sweetness. However, the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive. Herein, this study reported the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apples. The MdSUT2.1 gene encoded a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts.MdSUT2.1 was highly expressed in fruit and was ...  相似文献   

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Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.  In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.  At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated.  From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal.  When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst.  Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L–1.  The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05).  The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal.  Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells.  The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.  They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells.  The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05).  Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration.  This study references the IVF embryo production and embryonic stem cell research of domestic animals.   相似文献   

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The interaction between myocytes and intramuscular adipocytes is a hot scientific topic.  Using a co-culture system, this study aims to investigate the regulation of intramuscular fat deposition in chicken muscle tissue through the interaction between myocyte and adipocyte and identify important intermediary regulatory factors.  Our proteomics data showed that the protein expression of tissue inhibitor of metalloproteinases 2 (TIMP2) increased significantly in the culture medium of the co-culture system, and the content of lipid droplets was more in the co-culture intramuscular adipocytes.  In addition, TIMP2 was significantly upregulated (P<0.01) in muscle tissue of individuals with high intramuscular fat content.  Weighted gene co-expression network analysis revealed that TIMP2 was mainly involved in the extracellular matrix receptor interaction signaling pathway and its expression was significantly correlated with triglyceride, intramuscular fat, C14:0, C14:1, C16:0, C16:1, and C18:1n9C levels.  Additionally, TIMP2 was co-expressed with various representative genes related to lipid metabolism (such as ADIPOQ, SCD, ELOVL5, ELOVL7, and LPL), as well as certain genes involved in extracellular matrix receptor interaction (such as COL1A2, COL4A2, COL5A1, COL6A1, and COL6A3), which are also significantly upregulated (P<0.05 or P<0.01) in muscle tissue of individuals with high intramuscular fat content.  Our findings reveal that TIMP2 promotes intramuscular fat deposition in muscle tissue through the extracellular matrix receptor interaction signaling pathway.  相似文献   

10.
In mammals, microRNAs (miRNAs) play key roles in multiple biological processes by regulating the expression of target genes.  Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors; however, its function remains unclear.  In this study, we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation (P<0.01), tyrosinase activity (P=0.001) and significantly reduced (P<0.001) melanin production.  Functional prediction revealed that the 3´-untranslated region (UTR) of MAP3K8 has a putative miR-370-5p binding site, and the interaction between these two molecules was confirmed using luciferase reporter assays.  In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.  The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits (P<0.01) MAP3K8 expression via direct targeting of its 3´ UTR.  Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition (P<0.01) of melanocyte proliferation and significant reduction (P<0.001) in melanin production, which is consistent with our observations for miR-370-5p.  Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA (containing sites for the targeted binding to miR-370-5p) was significantly rescued (P≤0.001), which subsequently promoted significant increases in cell proliferation (P<0.001) and melanin production (P<0.01).  Collectively, these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.    相似文献   

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Brassica napus is an important cash crop broadly grown for the vegetable and oil values.  Yellow-seeded Bnapus is preferred by breeders due to its improved oil and protein quality, less pigments and lignin compared with the black-seeded counterpart.  This study compared the differences in flavonoid and fatty acid contents between yellow rapeseed from the progenies of BnapusSinapis alba somatic hybrids and the black-seeded counterpart using RNA-seq analysis.  Through HPLC-PDA-ESI(−)/MS2 analysis, it was found that phenylpropanoids and flavonoids (i.e., isorhamnetin, epicatechin, kaempferol, and other derivatives) in yellow seed were significantly lower than those in black seed.  The fatty acid (FA) content in yellow rapeseed was higher than that in black rapeseed due to the variation of C16:0, C18:0, C18:1, C18:2, and C18:3 contents.  RNA-seq analysis of seeds at four and five weeks after flowering (WAF) indicated that differentially expressed genes (DEGs) between black and yellow rapeseeds were enriched in flavonoid and FA biosynthesis, including BnTT3, BnTT4, BnTT18, and BnFAD2.  Also, genes related to FA biosynthesis, desaturation and elongation (FAD3, LEC1, FUS3, and LPAT2) in yellow seed were up-regulated compared to those in black seed, while genes involved in beta-oxidation cycle (AIM1 and KAT2) of yellow seed were down-regulated compared to those in black seed.  The DEGs related to the variation of flavonoids, phenylpropanoids, and FAs would help improve the knowledge of yellow seed character in Bnapus and promote rapeseed improvement.  相似文献   

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The mechanisms that regulate the specificity and maintenance of chicken muscle fiber types remain largely unknown. In mammals, CSRP3 has been shown to play a vital role in the maintenance of typical muscle structure and function. This study investigated the role that CSRP3 plays in chicken skeletal muscle. First, the antibody against chicken CSRP3 protein was prepared, and the expression levels of the mRNA and protein of the CSRP3 gene in four chicken skeletal muscles with different myofiber com...  相似文献   

14.
Aegilops umbellulata (UU) is a wheat wild relative that has potential use in the genetic improvement of wheat.  In this study, 46 Aeumbellulata accessions were investigated for stripe rust resistance, heading date (HD), and the contents of iron (Fe), zinc (Zn), and seed gluten proteins.  Forty-two of the accessions were classified as resistant to stripe rust, while the other four accessions were classified as susceptible to stripe rust in four environments.  The average HD of Aeumbellulata was significantly longer than that of three common wheat cultivars (180.9 d vs. 137.0 d), with the exception of PI226500 (138.9 d).  The Aeumbellulata accessions also showed high variability in Fe (69.74–348.09 mg kg–1) and Zn (49.83–101.65 mg kg–1) contents. Three accessions (viz., PI542362, PI542363, and PI554399) showed relatively higher Fe (230.96–348.09 mg kg–1) and Zn (92.46–101.65 mg kg–1) contents than the others.  The Fe content of Aeumbellulata was similar to those of Aecomosa and Aemarkgrafii but higher than those of Aetauschii and common wheat.  Aegilops umbellulata showed a higher Zn content than Aetauschii, Aecomosa, and common wheat, but a lower content than Aemarkgrafii.  Furthermore, Aeumbellulata had the highest proportion of γ-gliadin among all the species investigated (Aeumbellulata vs. other species=mean 72.11% vs. 49.37%; range: 55.33–86.99% vs. 29.60–67.91%).  These results demonstrated that Aeumbellulata exhibits great diversity in the investigated traits, so it can provide a potential gene pool for the genetic improvement of these traits in wheat.  相似文献   

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Transgenic cotton carrying the Cry1Ac gene has revolutionized insect pest control since its adoption, although the development of resistance in insect pests has reduced its efficacy.  After 10 years of cultivating Bacillus thuringiensis (Bt) cotton with a single Cry1Ac gene, growers are on the verge of adopting Bt cotton that carries the double gene (Cry1Ac+Cry2A) due to its better effectiveness against insect pests.  Thus, the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.  The expression levels of the Cry1Ac and Cry2A genes were evaluated in the leaves of 10 genotypes (2 parents and 8 F1 hybrids) at 30 days after sowing (DAS), while samples of leaves, bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.  The F1 hybrids were developed through reciprocal crosses between two Bt (CKC-1, CKC-2) and two non-Bt (MNH-786, FH-942) parents.  The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay (ELISA).  The results showed that the MNH786×CKC-1 hybrid had the highest concentrations of Cry1Ac gene at 30 DAS (3.08 µg g–1) and 110 DAS (1.01 µg g–1) in leaves.  In contrast, the CKC-2×MNH-786 hybrid showed the lowest concentrations of Cry1Ac gene at 30 DAS (2.30 µg g–1) and 110 DAS (0.86 µg g–1).  The F1 hybrid FH-942×CKC-2 showed the highest concentrations of Cry2A gene at 30 DAS (8.39 µg g–1) and 110 DAS (7.74 µg g–1) in leaves, while the CKC-1×MNH-786 hybrid expressed the lowest concentrations of Cry2A gene at 30 DAS (7.10 µg g–1) and 110 DAS (8.31 µg g–1).  A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS, whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.  When the expression pattern was compared between various plant parts in genotype CKC-2, it was found that leaves had higher concentrations of Cry1Ac (3.12 µg g–1) and Cry2A (8.31 µg g–1) at 70 DAS, followed by bolls (Cry1Ac (1.66 µg g–1) and Cry2A (8.15 µg g–1)) and flowers (Cry1Ac (1.07 µg g–1) and Cry2A (7.99 µg g–1)).  The genotype CKC-2 had higher concentrations of Cry1Ac (3.12 µg g–1) and Cry2A (8.31 µg g1) in the upper canopy but less accumulation (2.66 µg g–1 of Cry1Ac, 8.09 µg g–1 of Cry2A) in the lower canopy at 70 DAS.  Similarly, at 110 DAS, the expression levels of Cry1Ac and Cry2A in upper and lower canopy leaves were 1.52 and 7.92 µg g–1, and 0.99 and 7.54 µg g–1, respectively.  Hence, the current study demonstrates that different genotypes showed variable expression for both of the Cry1Ac and Cry2A genes during plant growth due to different genetic backgrounds.  The Cry2A gene had three-fold higher expression than Cry1Ac with significant differences in expression in different plant parts.  The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1Ac and Cry2A for sustainable cotton production worldwide.  相似文献   

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The rice cultivars carrying dep1 (dense and erect panicle 1) have the potential to achieve both high grain yield and high nitrogen use efficiency (NUE).  However, few studies have focused on the agronomic and physiological performance of those cultivars associated with high yield and high NUE under field conditions.  Therefore, we evaluated the yield performance and NUE of two near-isogenic lines (NILs) carrying DEP1 (NIL-DEP1) and dep1-1 (NIL-dep1) genes under the Nanjing 6 background at 0 and 120 kg N ha–1.  Grain yield and NUE for grain production (NUEg) were 25.5 and 21.9% higher in NIL-dep1 compared to NIL-DEP1 averaged across N treatments and planting years, respectively.  The yield advantage of NIL-dep1 over NIL-DEP1 was mainly due to larger sink size (i.e., higher total spikelet number), grain-filling percentage, total dry matter production, and harvest index.  N utilization rather than N uptake contributed to the high yield of NIL-dep1.  Significantly higher NUEg in NIL-dep1 was associated with higher N and dry matter translocation efficiency, lower leaf and stem N concentration at maturity, and higher glutamine synthetase (GS) activity in leaves.  In conclusion, dep1 improved grain yield and NUE by increasing N and dry matter transport due to higher leaf GS activity under field conditions during the grain-filling period.  相似文献   

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The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB), which leads to considerable crop losses. Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection. However, how the regulatory mechanism works was still largel...  相似文献   

18.
Diamide derivatives are biologically active molecules that have been widely applied in recent years in research on pesticides, especially insecticides.  Using a simple and environmentally friendly scheme, a series of new diamide derivatives containing a trifluoromethylpyridine skeleton was designed, synthesized, and confirmed by 1H, 19F and 13C NMR, and HR-MS.  Their insecticidal activities against Plutella xylostella and Helicoverpa armigera were measured and the relationship between structure and activity was investigated.  Eight of the title compounds (D2, D5, D10, D21, D28, D29, D30 and D33) showed 100% activity against Pxylostella at 500 mg L–1.  One compound, D33, still showed 100% activity against Pxylostella at 100 mg L–1 and had the lowest LC50 (lethal concentration 50%, 3.7 mg L–1) among the synthesized compounds.  Molecular docking analysis revealed that D33 could be thoroughly embedded in the active pocket of the ryanodine receptor via hydrogen bonding in a manner similar to the commercial insecticide chlorantraniliprole.  相似文献   

19.
Arabinogalactan proteins (AGPs) are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.  In this study, we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.  Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes, 38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.  The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.  We further tested their expression patterns using RT-PCR and RT-qPCR.  Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads (RPKM) values during pollen tube growth, implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.  We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.  Therefore, 19 PbrAGPs (PbrAGP1 to PbrAGP19) were selected to test their influences on pollen tube growth.  Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen, and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.  Additionally, pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.  PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.  In summary, this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.  相似文献   

20.
Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality. Therefore, developing crops with higher drought and salt tolerance is highly desirable. This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice. The OsMas1 protein was localized to the cytoplasm. The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, ...  相似文献   

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