小麦蓝矮病植原体转运蛋白secY基因片段序列分析

 Wheat blue dwarf(WBD) is a disease caused by phytoplasma and only reported from China. A fragment about 1.3 kb in protein translocation gene, secY was amplified by PCR from the total DNA of di-seased wheat sample with primer pair secYF/secYR, which was designed based on secY gene sequence of known 16SrI group members. Nucleotide acid sequence analysis of amplified fragment indicated that the length was 1 240 bp. A phylogenetic tree based on secY gene sequences was constructed and showed that wheat blue dwarf phytoplasma was clustered into the Candidatus Phytoplasma asteris, subgroup 16SrI-C. Wheat blue dwarf phytoplasma showed high homology with clover phyllody phytoplasma strains based on sequence comparison and phylogenetic analysis.     

藜草花叶病毒外壳蛋白基因的克隆与RT-PCR检测

 The nucleotide sequence of coat protein (cp)gene of Sowbane mosaic virus (SoMV)was determined. The cp gene of SoMV consists of 726 nucleotides and encodes a putative protein of 241 amino acid re-sidues. Sequence comparison showed that SoMV was most closely related to Rubus chlorotic mottle virus compared to other sobemoviruses. A primer pair was designed for the detection of SoMV based upon the determined cp nucleotide sequence. An expected fragment with 510 bp could be obtained from SoMV, while no specific band was observed from the healthy control. The result showed that the RT-PCR assay with the primer pair was suitable for the specific detection of SoMV.     

马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

一个二粒小麦LRR类受体蛋白激酶基因的克隆和鉴定

 Near the HMW-glutenin gene of wheat (Triticum aestivum), there is a locus (temporarily named TaXa) encoding LRR-receptor-like protein kinase, which is homologous to disease resistance protein Xa21 of rice (Oryza sativa). Through RT-PCR approach, a cDNA clone of ZS2002 was isolated from the orthologous locus of TaXa in Triticum turgidum. ZS2002 was 3 081 bp long and encoding a peptide composed of 1 026 amino acid. The protein included N-terminal conserved sequence, LRR domains, a transmembrane region and a serine/threonine protein kinase domain. ZS2002 was expressed in root, stem, leaf and spike. The transcribing in seedling leaves was significantly enhanced by Blumeria graminis f.sp. tritici. TaXa gene might play a role in powdery mildew resistance reaction in Triticum.     

来自安徽不同寄主PVY分离物的血清学鉴定及其cp基因分子进化分析

 Tobacco and potato samples showing symptoms of PVY were collected from different regions in Anhui Province, and the ELISA results of partial samples were positive. The total RNA was extracted from the positive samples by TRIZOL methods. Specific primer pair was designed to amplify cp gene of PVY by RT-PCR. Sequencing results indicated that the full length of cp gene of PVY from tobacco (PVY-CP-4) and pota-to (PVY-CP-7) is 801 nts, and each of them encodes 266 amino acids. A phylogenetic tree based on alignment of cp nucleotide sequences was constructed and the sequence comparing of cp gene was conducted. The results showed that PVY-CP-4 could be grouped into one branch with PVY^O and PVY^N:O and shared the highest sequence similarity (99.4%) with PVY^O (EF026074). It was suggested that PVY-CP-4 derived from tobacco in Hefei might belong to PVY^O. PVY-CP-7 clustered together with PVYN and PVYNTN and formed another branch. Furthermore, PVY-CP-7 shared the highest sequence similarity (98.3%) with PVY^NTN(AJ890347), PVY^NTN (EF026075) and PVY^NTN(AJ585342). It was supposed that PVY-CP-7 derived from potato in Wuhe probably belonged to PVY^NTN.     

甘蔗花叶病毒福建分离物外壳蛋白基因的克隆及序列分析

 A fujian isolate of Sugarcane mosaic virus named SCMV-FJ was isolated from infected sugarcane. Cloning and sequence analysis of the coat protein gene of this isolate was carried out. A pair of primers was designed and synthesized based on the nucleotide sequences of coat protein genes of sugarcane mosaic viruses reported. The coat protein gene of SCMV-FJ was amplified from the extracted total RNA of the infected sugarcane by using RT-PCR, and cloned into the pMD18-T vector. The sequencing result indicated that the cloned segment included a 1137 bp open reading frame(ORF) and a 228 bp 3' untranslated region, in which the ORF comprised the whole coat protein and part of the nuclear inclusion b. The nucleotide and the deduced amino acid sequences of the coat protein gene were compared with those of the other isolates or strains of SCMV subgroup reported in GenBank. The result showed that it shares 56.8%-97.1% and 55.3%-99.4% homology in nucleotide and the putative amino acid sequences, respectively, with the highest amino acid homology of 99.4% with SCMV-D. Thus it was identified as a SCMV-D. This experiment provided a rapid, sensitive and relatively inexpensive method for RT-PCR detection of SCMV. At the same time, the cloning of SCMV-FJ coat protein gene provided the foundation for plant gene engineering against SCMV.     

小麦抗白粉病基因Pm6的微卫星标记鉴定

 Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is a prevalent disease worldwide. Breeding and planting resistance cultivars have been proved effective and environmental friendly for control of the disease. To develop easily used PCR-based markers in marker assisted selection (MAS) for Pm6, a dominant powdery mildew resistance gene in wheat, 25 microsatellite markers on chromosome 2BL in wheat were screened between susceptible parent Yumai13 and resistance parent Timgalen carrying Pm6. F₂ population derived from Yumai13 and Timgalen was further analyzed by the marker Xgwm501. The results indicated that Xgwm501 was a co-dominant marker linked to Pm6 gene at a distance of 14.8 cM. 29 Pm-carrying varieties were tested by the marker Xgwm501 and only those carrying Pm6 showed 117 bp resistance specific band. This marker is proved to have high practicability and can be used in MAS of Pm6 gene in wheat breeding programs.     

大麦条纹花叶病毒中国株三基因盒(TGB)序列对比分析

 The tripe gene block (TGB)genes of Barley stripe mosaic virus China strain (BSMV-CH)were amplified from cDNA of BSMV-CH RNAβ (GenBank accession No:AY789694)by PCR with special primer pairs, and cloned into pMD18-T vector for sequencing. Analysis of the sequences showed that the full length of BSMV-CH TGB1, TGB2 and TGB3 were 1 539, 396 and 468 bp, with deduced 512, 131 and 155 amino acids, respectively. BSMV-CH TGB1 shared 94.1%-95.4% nucleotide identities and 91.0%-94.5% amino acid identities with that of other BSMV strains, BSMV-CH TGB2 shared 96.5%-97.2% nucleotide identities and 98.5%-99.2% amino acid identities with that of other BSMV strains, and BSMV-CH TGB3 shared 95.7%-96.6% nucleotide identities and 94.2%-96.8% amino acid identities with that of other BSMV strains. Phylogenetic tree based on the amino acid of Hordeiviruses TGB genes showed that BSMV-CH was relatively closed to CV17 in genetic relationship. Therefore, BSMV-CH was deduced to be a recombined strain of CV17 and CV42.     

1个小麦NBS类抗病基因同源cDNA序列的克隆与鉴定

 利用cDNA末端快速扩增技术对小麦抗叶锈病近等基因系TcLr35中所获得的抗病基因同源片段S2A2 5'端和3'末端序列进行扩增,并根据拼接序列设计特异性引物,进行全长基因的扩增,获得了1个通读的NBS类抗病同源基因S2A2 cDNA序列,该序列全长为3476 bp,编码866个氨基酸序列。经BLASTp比较,该片段含有NB-ARC保守结构域和多个LRR结构域,与已知植物抗病基因I2C-1、L6、RPS2等相应区域相一致。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr35小麦中成功获得了抗病同源基因,这为最终克隆小麦抗叶锈病基因Lr35奠定了基础。     

番茄叶霉病高抗基因Cf-9、Cf-11和Cf-19的分子标记

 本研究以9个含不同叶霉病抗病基因的番茄品种为试材,通过接种鉴定表明,Cf-5、Cf-9、Cf-11和Cf-19基因对我国目前的2个叶霉菌优势生理小种均具有较强的抗性。根据Cf-9基因设计引物,扩增Cf-9基因的片段,含Cf-9、Cf-11和Cf-19基因的3种番茄均获得了2.7kb的扩增片段。但用限制性内切酶TaqⅠ对PCR产物酶切可以将3种材料明显区分开来,Cf-9的2个差异酶切片段为1170和460bp;Cf-11的2个差异酶切片段为1100和410bp;Cf-19的2个差异酶切片段为1210和300bp,从而建立了3个基因的分子标记。在F₂分离群体中验证表明,3个基因的分子标记鉴定结果与抗性接种鉴定结果是一致的,用这些标记可以进行分子标记辅助选择。     

以Ypt1基因序列为靶标的辣椒疫病菌快速分子检测

PCR primers were designed based on the sequence of Ras-related protein gene (Ypt1) of P. capsici. According to the multiple sequence alignment, Ypt1 has the sufficiently polymorphic intron region for the development of P. capsici-specific primers (PcYpt1F/PcYpt1R). One primer pair was developed which can amplify one P. capsici-specific fragment of 156 bp. Using the primer pair, the P. capsici infected plants and soils were detected. Additionally, Ypt1 has an appropriate region for the development of Phytophthora genus-specific primers (Ypt1F/Ypt1R), which can amplify a fragment of about 540 bp from 14 different Phytophthora specices and a fragment of about 350 bp in Pythium species, with no amplification from fungal species. By PCR optimization using P. capsici genomic DNA, the detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (PcYpt1F/PcYpt1R) and nested PCR (Ypt1F/Ypt1R and PcYpt1F/PcYpt1R), respectively. The developed primers were proved to be efficient in detection of Phytophthora pathogens from diseased plant tissues and residues in soils.     

爪哇根结线虫Mj-1-1基因的克隆、鉴定及RNAi表型分析

根据爪哇根结线虫食道腺内表达的EST序列,结合反式剪接序列SL1,从爪哇根结线虫中克隆了一个假定寄生基因Mj-1-1(KU358725),该基因的c DNA全长为573 bp,包含450 bp的开放阅读框(ORF),编码149个氨基酸。DNA全长为740 bp,包含2个内含子,长度分别为20 bp和111 bp。NCBI BLASTn比对表明,Mj-1-1与南方根结线虫的M.incognita zk1236.5(JQ284068)基因相似性最高,为97%。原位杂交表明Mj-1-1基因在爪哇根结线虫的背食道腺中表达,qRT-PCR结果表明Mj-1-1基因在爪哇根结线虫寄生性3龄幼虫阶段表达量最高。对爪哇根结线虫侵染前2龄幼虫的Mj-1-1基因进行沉默,调查发现,沉默Mj-1-1后,爪哇根结线虫对番茄的侵染力显著下降,表明Mj-1-1对爪哇根结线虫侵染和寄生具有重要的调控作用。     

烟草花叶病毒丁香分离物的分离与鉴定

 从表现花叶症状的丁香病株上获得一病毒分离物,其在电镜下为约300 nm×18nm的杆状粒子;电泳分析表明感病组织中ds RNA大约为6.4kbp,而其外壳蛋白分子量约为17.6k Da。以上实验结果初步将该病毒分离物鉴定为烟草花叶病毒属(Tobamovirus)。根据该属病毒复制酶基因序列设计通用引物,进行RT-PCR检测,扩增出约1000 bp的预期特异片段(Gen Bank AY566703)。将PCR产物克隆后测序,序列分析表明,与从蚕豆中分离的TMV-B株系序列(Gen Bank AJ011933.1)同源性为99.90%。根据烟草花叶病毒(Tobacco mosaic virus,TMV)的RNA CP基因序列设计引物,进行RT-PCR,扩增出约800 bp的预期特异片段(Gen Bank AY56672),序列分析表明,与TMV-B株系序列(Gen Bank AJ011933.1)同源性达99%,上述实验结果表明,该病毒分离物为TMV。由于该分离物与TMV-B在指示植物上的症状存在明显差异,所以,作者把该分离物暂命名为TMV-S。     

齿兰环斑病毒RdRp基因的原核表达及多克隆抗体制备

 采用RT-PCR法从感染齿兰环斑病毒(Odontoglossum ringspot virus, ORSV)贵州分离物的苋色藜叶片中扩增出病毒的依赖RNA的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)基因保守序列。测序结果显示,该保守片段长516 bp,编码171个氨基酸残基。构建了该片段的原核表达载体pET32a-ORSV RdRp并转化BL21(DE3)菌株,在25℃以0.4 mmol·L^-1 IPTG诱导表达重组蛋白。SDS-PAGE分析表明,重组蛋白分子量约为36 kDa,与预测相符合。将该重组蛋白作为抗原免疫BABL/C小鼠,制备的多克隆抗体效价达1∶102 400。间接ELISA结果表明,该多抗具有较强的特异性,能检测到ORSV感染的病叶汁液中的RdRp,而与其它感染4种同属或不同属病毒的病叶汁液不发生血清学交叉反应,本实验为进一步研究RdRp的结构和功能以及从分子水平上探讨该病毒的致病机制奠定基础。     

长岭发垫刃线虫(Trichotylenchus changlingensis)双重PCR检测方法研究

正长岭发垫刃线虫(Trichotylenchus changlingensis)是一种迁移性植物外寄生线虫,该线虫于2011年首次在我国吉林省长岭县发现,并根据其形态特征及最新分类系统将其归为发垫刃属[1,2]。该线虫能引起玉米叶片黄化,植株矮化,茎基部开裂,茎节缩短等症状,该病发生率普遍在21%～67%,给玉米生产造成了严重影响[3]。     

桃蚜电压门控钠离子通道基因cDNA克隆及其生物信息学分析

克隆获得桃蚜电压门控钠离子通道基因cDNA序列，明确钠离子通道的典型特征，为研究桃蚜抗性分子机理奠定基础。采用实验技术主要有RT-PCR和PCR，克隆桃蚜钠离子通道基因cDNA序列，利用相关软件对其序列进行生物信息学分析。克隆得到两段cDNA序列MpNa_v-1（NCBI登录号：MN124170）和MpNa_v-2（NCBI登录号：MN176136）。MpNa_v-1长度为2945 bp，包括2877 bp的完整开放阅读框，共编码958个氨基酸；MpNa_v-2长度为3546 bp，包括3486 bp的完整开放阅读框，共编码1161个氨基酸。MpNa_v-1和MpNa_v-2共同组成桃蚜的钠离子通道α亚基，MpNa_v-1包含同源结构域Ⅰ和同源结构域Ⅱ，MpNa_v-2包含同源结构域Ⅲ和同源结构域Ⅳ。同源比对发现，桃蚜与豌豆蚜和高粱蚜钠离子通道基因相似度分别高达97.67%和97.65%，所克隆序列包含昆虫钠离子通道α亚基典型特征，具有MFM模块，并含有蚜虫类钠通道特有模块DENS。成功地克隆桃蚜钠离子通道基因，为阐明其对拟除虫菊酯类药剂产生靶标抗性的分子机制奠定基础。     

大豆蚜Aghsp75基因对吡虫啉及温度胁迫的应激表达

热激蛋白在昆虫抵御温度胁迫和药剂胁迫反应中具有重要作用。本试验通过高通量测序获得一条大豆蚜热激蛋白基因的cDNA序列，该序列全长2982 bp，含1个长度为2052 bp的开放阅读框，编码683个氨基酸组成的多肽，多肽等电点约为6.30，分子质量约为77.8 kDa；推导的氨基酸序列与棉蚜Aphis gossypii HSP75同源性高达99.12%，属于hsp 75家族。我们把该基因定名为Aghsp75，已提交至GenBank（登录号为MN068810）。Aghsp75通过不同浓度吡虫啉药剂和不同温度胁迫成蚜后发现，经LC₅₀吡虫啉浓度胁迫3、6和24 h时以及在LC₃₀浓度吡虫啉胁迫12 h时该基因表达量显著升高；经6℃处理6 h时以及36℃处理3 h和6 h时该基因表达量显著升高。本研究表明该基因可能参与大豆蚜的抗逆过程，为利用分子生物技术手段防治大豆蚜提供理论保障。