马铃薯S病毒外壳蛋白基因的克隆与原核表达

依据马铃薯S病毒(Potato virus S,PVS)外壳蛋白(CP)基因序列(885bp)设计合成了两对 引物,通过RT-PCR扩增得到长0.8kb的目的片段,将目的片段转入大肠杆菌,酶切鉴定证明 得到了含有目的片段的重组子,测定序列结果与其他PVS分离物CP基因的序列比较,发现其核苷酸同源性达95%左右;构建了含PVS CP基因的融合蛋白原核表达载体,并在大肠杆菌中得到表达,SDS-PAGE测定融合蛋白的分子量为58kD.     

一步RT-PCR检测水仙黄条病毒

目的:建立用于快速检测水仙黄条病毒(Narcissus yellow stripe virus,NYSV)的一步RT-PCR方法。方法:根据已报道的NYSV基因序列设计特异性引物,采用一步RT-PCR建立了快速检测水仙黄条病毒的方法。结果:该方法具有良好的特异性,能够从感染水仙黄条病毒的水仙样品上扩增出预期大小的特异性目的片段,而与其他病毒无交叉反应;一步RT-PCR产物序列测定结果表明,该产物的序列与NYSV序列高度同源,同源性达99%;灵敏度测定结果显示,一步法RT-PCR与两步法RT-PCR的灵敏度相当,可以检测稀释到10-4倍的RNA。结论:应用建立的一步RT-PCR对一批水仙样品进行NYSV检测,结果与两步法RT-PCR完全相同,说明该方法可准确用于NYSV的检测。     

应用RT-PCR检测流产胎儿组织中猪繁殖与呼吸综合征病毒

根据猪繁殖与呼吸综合征病毒(PRRSV)美洲型膜蛋白和核衣壳蛋白基因序列,设计了一对含有EcoR I和BamH I酶切位点的引物,用RT-PCR对四个流产猪场的病料进行了检测,扩增出约918 bp的基因片段.通过病毒分离、酶切鉴定和序列分析证实为PRRSV感染.结果说明应用所设计的引物进行RT-PCR快速检测PRRS是可行的,为我国快速特异诊断PRRS和PRRSV强毒株的深入研究奠定了基础.     

侵染落葵的黄瓜花叶病毒分离物CP基因序列分析

通过间接酶联免疫法(ID-ELISA)检测到染病落葵病样中存在黄瓜花叶病毒（Cucumber Mosaic Virus,CMV）。从病叶中提取总RNA,用RT-PCR方法扩增得到657bp的CMV CP基因片断,将扩增产物与T载体连接并进行测序。用DNA MAN将得到的CP基因序列与GenBank收录的黄瓜花叶病毒两亚组部分株系或分离物的CP基因序列进行比较,结果表明该CP基因与CMV亚组Ⅰ、亚组Ⅱ之间的核苷酸序列同源性分别为91.17~95.43%和75.30~75.76%,推导氨基酸序列同源性分别为95.41~97.71%和81.28~81.74%,表明CMV-Ba与亚组Ⅰ同源关系密切。     

中国大豆花叶病毒SC7外壳蛋白基因的序列测定及其与美国株系的比较

为了探索大豆花叶病毒(Soybean mosaic virus,SMV)SC7外壳蛋白(Coat protein,CP)基因序列与病毒致病性作用及其与美国株系的对应关系,本研究通过反转录-聚合酶链式反应(RT-PCR),克隆了我国SMV SC7株系的CP基因,并测定了其全序列。结果表明:CP基因长度为795bp,编码的CP蛋白为265个氨基酸。在致病性反应上,SC7的毒力比美国的毒株更强些。在分子水平上,CP基因核苷酸序列差异为4%~5%,编码的氨基酸序列差异为1%~2%。美国株系N端都存在与蚜传有关的保守基序DAG(Asp-Ala-Gl),但在SC7为DAD(Asp-AlaAsp)。将序列信息与SMV株系在鉴别寄主上的症状反应进行综合分析发现,CP基因的同源性与SMV在鉴别寄主上的致病性反应没有明显关系。     

用PCR和SDS-PAGE两种方法对转基因大豆的检测

采用PCR和SDS-PAGE电泳两种方法对转基因大豆(美国)和非转基因大豆(国内3个不同样品)进行了检测．结果显示：转基因大豆可以检测出195bp的花椰菜花叶病毒启动子(CaMV35S)序列片段和320bp的抗草甘膦基因(EPSPS)片段;SDS-PAGE蛋白质电泳中有一约40kDa的蛋白带出现;而非转基因的3个国内大豆品种中均没有CaMV35S启动子序列片段和抗草甘膦基因片段,SDS-PAGE蛋白质电泳检测也没有发现转基因大豆中存在的40kDa的蛋白带．     

芜菁花叶病毒的3’末端序列克隆与CP基因序列分析

目的:克隆芜菁花叶病毒(Turnip mosaic virus,TuMV)的3'末端序列,并进行CP基因序列分析.方法:以TuMV杭州榨菜分离物(TuMv-HZZC)接种病叶为材料,利用病毒粒子吸附法制备病毒RNA模板,经RT-PCR扩增获得了TuMV-HZZC 3'末端序列,将其克隆到PMD 18-T质粒上进行序列分析.结果:TuMV-HZZC分离物3'末端序列包括部分的Nib基因、完整的TuMVCP基因和3'-UTR,CP基因为864bp,分别编码288个氨基酸,3'-UTR序列(不包括PolyA尾巴)为213bp.经过与其他TuMV分离物的CP基因核苷酸和氨基酸比较,同源性分别达到88.0%～97.6%和91.0%-96.5%.结论:TuMV的系统进化具有典型的地域和寄主关联性.     

马铃薯S病毒外壳蛋白基因的克隆与原核表达

依据马铃薯S病毒 (PotatovirusS ,PVS)外壳蛋白 (CP)基因序列 (885bp)设计合成了两对引物 ,通过RT PCR扩增得到长 0 .8kb的目的片段 ,将目的片段转入大肠杆菌 ,酶切鉴定证明得到了含有目的片段的重组子 ,测定序列结果与其他PVS分离物CP基因的序列比较 ,发现其核苷酸同源性达 95 %左右 ;构建了含PVSCP基因的融合蛋白原核表达载体 ,并在大肠杆菌中得到表达 ,SDS PAGE测定融合蛋白的分子量为 5 8kD。     

抗草甘膦转基因大豆PCR的检测

目的:建立快速、有效的鉴别转基因作物与非转基因作物及其产品的检测方法体系。方法:用抗草甘膦转基因大豆中的外源CaMV35S启动子和CP4-EPSPS基因引物,应用PCR方法,从中扩增出预期大小的DNA片段,将扩增产物回收后测序。结果:经同源性分析,扩增产物为CaMV35S启动子和CP4-EPSPS基因的一部分序列。结论:初步建立了转基因大豆的检测方法,同时讨论了PCR检测过程中假阴性和假阳性的原因。     

抗草甘膦转基因大豆PCR检测及问题探讨

转基因植物的检测具有重要的意义。用抗草甘膦转基因大豆中的外源CaMV35S启动子、CP4 EPSPS和巢式PCR引物,应用PCR方法,从中扩增出预期大小的DNA片段。将扩增产物回收后测序,经同源性分析扩增产物为CaMV35S启动子和CP4 EPSPS的一部分序列。与常规PCR相比,巢式PCR在检测转基因大豆中具有更高的特异性。讨论了PCR检测过程中假阴性和假阳性的原因。     

Bean leaf beetle (Coleoptera: Chrysomelidae) management for reduction of bean pod mottle virus

Bean pod mottle virus (BPMV) is a management concern for soybean, Glycine max (L.), producers in the North Central states because it can cause yield loss and reduce seed quality by induction of seed coat mottling. The main vector of BPMV is the bean leaf beetle, Cerotoma trifurcata (Forster). An experiment was conducted in 2000 and 2001 at two locations in northwestern and central Iowa to test three insecticide treatments for suppression of bean leaf beetles, and subsequently, BPMV. Treatments of insecticide applications with lambda-cyhalothrin were 1) a single early-season application (23 g [AI] /ha) (2.5 oz/acre) at the VE-VC soybean developmental stage; 2) two early-season applications, the first the same as treatment 1 and a second at the same rate 9-13 d later; 3) a single early-season application the same as treatment 1, followed by a mid-season application (28 g [AI] /ha (3.2 oz/acre) at approximately R2 (flowering, near 15 July); and 4) an unsprayed control. Application of lambda-cyhalothrin after soybean emergence and again as first-generation bean leaf beetles emerged in northwestern Iowa in 2000 (treatment 3) significantly reduced beetle densities through mid-season, BPMV field incidence by 31.5%, and seed coat mottling by 31.2%, compared with the unsprayed control. Similar effects were measured at the same location when insecticide was applied twice at early season (treatment 2). Yield was 453.7 kg/ha (6.74 bu/acre) greater in treatment 2 and 525.20 kg/ha (7.80 bu/acre) greater in treatment 3 than in the unsprayed control at the northwestern site in 2000. At both locations in 2001 fewer treatment effects were observed, which was likely related to lower beetle populations in that year. Early-season insecticide sprays targeted at overwintered beetles on VC-VE reduced the initial population of vector insects and may have contributed to a lower first-generation population because of reduced overwintered beetle oviposition. In 1 year at one location there was a benefit to an additional mid-season insecticide spray, although effectiveness of spraying at this time could vary based on the magnitude of the vector population.     

褐色种皮大豆与其黄色种皮衍生亲本的表型及基因型比较

大豆种皮色在从野生大豆到栽培大豆的选择过程中逐渐由黑色变成黄色,是重要的形态标记,因此,大豆种皮色相关基因的研究无论是对进化理论研究还是育种实践都具有非常重要的意义。利用褐色种皮J1265-2大豆及其衍生亲本黄色种皮大豆J1265-1为材料,通过SSR引物扩增片段,检验遗传背景的异同,同时对控制种皮的候选基因GmF3’H进行扩增和测序分析。结果表明,褐色种皮和黄色种皮材料不仅用161对SSR分子标记检测没有发现差异,其褐色种皮候选基因GmF3’H的编码区及起始密码子上游1465 bp序列也是一致的。因此,证明褐色种皮J1265-2大豆与其衍生亲本黄色种皮大豆J1265-1为近等基因系,其控制褐色种皮的基因型与已报道的基因型不同。     

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.     

Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively.     

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

A transgenic,visual screenable marker for soybean seeds

Most soybean cultivars produce buff colored seeds due to a seed coat specific siRNA mechanism. This phenomenon is specifically limited to the seed coat and produces a strong visual effect, thus, a strategy to evade the silencing was used to produce a maternal transgenic marker for soybeans. Expression of a rice chalcone synthase transgene with little DNA sequence homology to the soybean siRNAs resulted in dark colored seed coats. This phenotype is the result of anthocyanin pigment production and does not appear to affect other tissues. This novel approach for producing an easily scored transgenic marker for soybean will facilitate high-throughput screening and analysis of transgenic soybean.     

A soybean cell wall protein is affected by seed color genotype.

The dominant I gene inhibits accumulation of anthocyanin pigments in epidermal cells of the soybean seed coat. We compared saline-soluble proteins extracted from developing seed coats and identified a 35-kilodalton protein that was abundant in Richland (genotype I/I, yellow) and much reduced in an isogenic mutant line T157 (genotype i/i, imperfect black seed coats). We purified the 35-kilodalton protein by a novel procedure using chromatography on insoluble polyvinylpolypyrrolidone. The 35-kilodalton protein was composed primarily of proline, hydroxyproline, valine, tyrosine, and lysine. Three criteria (N-terminal amino acid sequence, amino acid composition, and sequence of a cDNA) proved that the seed coat 35-kilodalton protein was PRP1, a member of a proline-rich gene family expressed in hypocotyls and other soybean tissues. The levels of soluble PRP1 polypeptides and PRP1 mRNA were reduced in young seed coats with the recessive i/i genotype. These data demonstrated an unexpected and novel correlation between an anthocyanin gene and the quantitative levels of a specific, developmentally regulated cell wall protein. In contrast, PRP2, a closely related cell wall protein, was synthesized later in seed coat development and was not affected by the genotype of the I locus.     

Duplications That Suppress and Deletions That Restore Expression from a Chalcone Synthase Multigene Family

Seed coat color in soybean is determined by four alleles of the classically defined / (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the / locus, we demonstrated that the / locus is a region of chalcone synthase (CHS) gene duplications. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the / locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silencing and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant ii allele (yellow seed coats with pigmented hila) to the recessive i allele (fully pigmented) can be associated with the absence of a 2.3-kb Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutations exhibit deletions in the CHS4 promoter region. The dominant / allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridizes with both the CHS coding region and CHS1 promoter-specific probes. Mutations of the dominant / allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-kb Hindlll CHS fragment associated with the dominant / allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed in seed coats.