可定点整合的无筛选标记LacS基因表达载体的构建与鉴定

试验旨在构建一个能够定点整合且无筛选标记基因的半乳糖苷酶基因LacS表达载体.本研究以pEGFP-N1质粒为原始框架,通过PCR及限制性酶切位点在pEGFP-N1质粒的标记基因两端添加两个同向LoxP序列,在多克隆位点上游添加能够定点整合的attB序列,最后再将目的基因BC promoter-LacS-PolyA通过限制性酶切位点插入多克隆位点.每一步都进行PCR、酶切及测序鉴定.PCR产物及酶切片段大小符合预期结果,测序结果也与相应寡核苷酸序列一致,证明各个基因片段正确地连接到载体相应位置.本试验成功构建了可定点整合的无筛选标记半乳糖苷酶基因表达载体pEGFP-N1-LacS.     

Emergence and early evolution of fungicide resistance in North American populations of Zymoseptoria tritici

Although fungicide resistance in crop pathogens is a global threat to food production, surprisingly little is known about the evolutionary processes associated with the emergence and spread of fungicide resistance. Early stages in the evolution of fungicide resistance were evaluated using the wheat pathogen Zymoseptoria tritici, taking advantage of an isolate collection spanning 20 years in Oregon, USA, and including two sites with differing intensity of fungicide use. Sequences of the mitochondrial cytb protein conferring single‐mutation resistance to QoI fungicides and the nuclear CYP51 gene implicated in multiple‐mutation resistance to azole fungicides were analysed. Mutations associated with resistance to both fungicides were absent in the 1992 isolates, but frequent in the 2012 collection, with higher frequencies of resistance alleles found at the field site with more intensive fungicide use. Results suggest that the QoI resistance evolved independently in several lineages, and resulted in significant mitochondrial genome bottlenecks. In contrast, the CYP51 gene showed signatures of diversifying selection and intragenic recombination among three phylogenetic clades. The findings support a recent emergence of resistance to the two fungicide classes in Oregon, facilitated by selection for mutations in the associated resistance genes.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

重组植物乳杆菌生物学特性比较研究

试验对植物乳杆菌(Lactobacillus plantarum,Lp)出发菌与携带绿色荧光蛋白(green fluorescent protein,GFP)标记基因的重组植物乳杆菌(Lp-GFP)生物学特性进行了详细的比较研究,以便于深入开展益生菌植物乳杆菌在动物肠道内分布和定植规律的研究。结果显示,Lp和Lp-GFP生长曲线的变化趋势基本相同,其中Lp-GFP的生长速度略有滞后;Lp和Lp-GFP最适培养温度均为37 ℃,最适初始pH均为6.5;Lp和Lp-GFP耐高温、耐人工胃液、耐人工肠液和耐胆盐的特性趋势一致;Lp和Lp-GFP的抑菌特性无显著差异(P>0.05);除了红霉素,Lp和Lp-GFP对其他药物的敏感性基本没有发生变化。综上所述,植物乳杆菌的重组对其生物学特性没有显著的影响,这为重组标记菌株的后续培养和机理研究奠定了重要基础。     

Mapping and Comparative Analysis of QTL for Rice Plant Height Based on Different Sample Sizes within a Single Line in a RIL Population

To clarify the most appropriate sample size for obtaining phenotypic data for a single line, we investigated the main-effect QTL (M-QTL) of a quantitative trait plant height (ph) in a recombinant inbred line (RIL) population of rice (derived from the cross between Xieqingzao B and Zhonghui 9308) using five individual plants in 2006 and 2009. Twenty-six ph phenotypic datasets from the completely random combinations of 2, 3, 4, and 5 plants in a single line, and five ph phenotypic datasets from five individual plants were used to detect the QTLs. Fifteen M-QTLs were detected by 1 to 31 datasets. Of these, qph7a was detected repeatedly by all the 31 ph datasets in 2006 and explained 11.67% to 23.93% of phenotypic variation; qph3 was detected repeatedly by all the 31 datasets and explained 5.21% to 7.93% and 11.51% to 24.46% of phenotypic variance in 2006 and 2009, respectively. The results indicate that the M-QTL for a quantitative trait could be detected repeatedly by the phenotypic values from 5 individual plants and 26 sets of completely random combinations of phenotypic data within a single line in an RIL population under different environments. The sample size for a single line of the RIL population did not affect the efficiency for identification of stably expressed M-QTLs.     

牛环形泰勒虫GST-Tams1融合蛋白间接ELISA检测方法的建立

[目的与方法]以纯化的牛环形泰勒虫GST-Tams1融合蛋白作为检测抗原,通过优化ELISA反应条件,建立检测牛环形泰勒虫血清特异性抗体的间接ELISA方法.[结果]方阵试验确定的GST-Tams1抗原的最适包被浓度为10 μg/mL,血清最佳稀释倍数为80倍,ELISA阳性反应的临界值为OD<,450>≥0.282,批内和批间重复试验的变异系数均小于15;.Tams1间接ELISA方法能排除GST的干扰,与其它梨形虫病无交叉反应,与环形泰勒虫病巢式PCR检测方法的阳性符合率为96;.[结论]建立的Tams1间接ELSIA检测法重复性好、特异性强、灵敏度高.这是国内首次利用重组蛋白建立的牛环形泰勒虫病血清学诊断方法,为大规模地进行牛环形泰勒虫病的流行病学调查和血清学诊断提供有效的技术手段.     

利用Cre/lox重组系统建立番茄基因工程雄性不育恢复系

 【目的】利用Cre/lox重组系统具有的重组删除特性建立番茄工程恢复系,特异删除F1中的雄性不育基因恢复其育性。【方法】将TA29-Barnase雄性不育基因表达盒置于两个同向lox位点之间并与NPTⅡ基因、Bar基因融合后获得植物表达载体pBinBarloxTABn,转化番茄获得雄性不育转基因植株。Cre基因在 CaMV35S启动子的驱动下转入番茄获得工程恢复系。两者在开花时进行杂交,利用Cre重组酶删除F1中的TA29-Barnase不育基因表达盒使育性恢复。【结果】利用NPTⅡ作为转化筛选标记基因获得了番茄雄性不育转基因植株。转基因植株中的Bar基因能正常表达,真叶叶盘在含PPT 3 mg?L-1（phosphinothricin）分化培养基上能分化愈伤组织及芽;真叶具有抗PPT 20 mg?L-1浓度以上的能力。获得的TA29-Barnase转基因植株表现雄蕊退化、无花粉产生或产生少量形状畸形且无生活力的花粉。雄性不育植株自花授粉不能坐果,用非转基因保持系花粉授粉后,果实正常膨大结籽,杂交后代对除草剂Basta的抗性按1﹕1分离。不育植株与Cre转基因工程恢复系杂交后,果实也正常膨大结籽。对不育植株与Cre转基因工程恢复系杂交后代进行分子检测,结果发现同时含Bar 基因和Cre基因F1植株中的TA29-Barnase雄性不育基因被精确删除。TA29-Barnase基因删除植株育性被恢复,能正常开花结果。【结论】利用Cre/lox重组系统建立的Cre工程恢复系成功将番茄F1代中的不育基因删除,恢复了 F1的育性。该研究结果为植物基因工程雄性不育系的育性恢复提供了一条新的途径。     

一种快速高效构建植物表达载体的方法

植物表达载体是将目的基因转化宿主细胞的媒介,载体构建是通过遗传转化方法开展基因功能鉴定与应用等研究的重要环节。以构建玉米谷氨酰胺合成酶基因(GS1)表达载体pCUbi1390-Ubi-GS1-35S-EPSPS为例,研究简单、通用、高效的构建载体的方法。该方法目的片段和载体不需要酶切产生互补的黏性末端,只需通过在目的基因PCR上下游引物的5’端设计与线性载体两端有15个碱基的同源序列,根据序列的同源性,将目的基因插入载体中,通过PCR程序实现DNA重组。结果表明,正确构建了设计的转化载体,该方法大大简化了载体构建的过程,也适用于任何一个基于单酶切位点把外源DNA重组插入目标序列。     

黄瓜花叶病毒分子变异分析

利用不同生物信息学软件及算法对黄瓜花叶病毒(Cucumber mosaic virus,CMV)的分子变异进行分析.结果表明:不同CMV分离物外壳蛋白基因的核苷酸序列同源性为76.1%～99.2%.系统进化分析显示不同CMV分离物分为3个大的类群,不同分离物呈现一定的地域相关性,未见明显寄主相关性.重组分析显示澳大利亚...     

尼罗罗非鱼整胚原位杂交技术的建立和初步应用

为了研究尼罗罗非鱼胚胎发育和器官形成过程中基因功能和基因表达图式,本研究建立了尼罗罗非鱼的整胚原位杂交流程。尼罗罗非鱼的胚胎具有卵黄大、不透明、色素出现早等特点,因此现有鱼类整胚原位杂交方法不能完全适用于尼罗罗非鱼的胚胎,故本研究做了相应的调整和优化:通过提高H2O2的浓度和添加KOH,改良了尼罗罗非鱼胚胎的色素去除方法;使用冷丙酮代替蛋白酶K在提高胚胎通透性的同时保持胚胎完整;减少了探针回收和抗体回收后的洗涤次数,完善了结果的图像采集和胚胎保存方案。使用尼罗罗非鱼的重组激活基因Rag1作为探针基因,整胚原位杂交结果显示Rag1基因表达的位置与已报道的斑马鱼和日本青鳉的Rag1基因在胚胎中的表达位置高度保守,胚胎完整,基因表达位置清晰可见,表明此套尼罗罗非鱼的整胚原位杂交技术流程成功有效。