大豆对大豆胞囊线虫侵染的应答机制研究

大豆胞囊线虫病(Heterodera glycines,soybean cyst nematode,SCN)是大豆生产上的重要病害,其特点为危害重、分布广、难防治,每年对大豆生产造成极大的损失。种植大豆抗性新品种是防治SCN目前最为有效的措施,研究大豆对SCN侵染的应答机制,是培育大豆持久抗病品种的前提,对加快抗线虫品种选育及SCN的防控具有重要的意义。本文综述了大豆对SCN侵染的组织细胞学应答机制;介绍了大豆在SCN侵染后酶系变化及酚类代谢的生理生化应答机制;从分子水平阐明了SCN侵染后大豆的基因转录变化,差异蛋白及DNA甲基化的应答机制,以期为大豆胞囊线虫病害的进一步研究与防治提供参考。     

一种基于RPA的番茄褪绿病毒检测方法

番茄褪绿病毒Tomato chlorosis virus(ToCV)引起番茄褪绿病毒病,给番茄生产造成严重危害。开发快速准确的检测方法对该病害的防控具有重要意义。利用番茄褪绿病毒外壳蛋白(CP)基因序列,设计特异性引物,建立了ToCV的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,同时分析了该方法的灵敏度和特异性。结果表明,建立的ToCV-RPA方法在38℃恒温下40 min可从ToCV阳性的番茄样品中扩增出246 bp的特异性条带。扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于ToCV的快速检测。     

鸡的微卫星DNA标记与胴体性状的相关分析

测定了青脚麻鸡与隐性白鸡杂交后自交的F2代个体的全净堂重、全净堂率、脂肪宽带、腹脂重和腹脂率6个胴体性状,检测10个微卫星DNA标记,对标记基因型胴体性状值进行方差分析和多重比较,结果表明:MCW328对于10周龄体重和全净膛重两性状各基因型均有显著的差异(P<0.05);ADL292对于腹脂重性状各基因型有极显著的差异(P<0.01),对于腹脂率有显著的差异(P<0.05)。MCW328标记基因与控制10周龄体重和全净膛重QTL连锁,ADL292标记基因与控制腹脂重和腹脂率QTL连锁。     

S1核酸酶突变检测法的优化

优化了S1核酸酶突变检测体系,结果表明(1)扩增法比杂交法产生异源双链DNA更节省时间;(2)PCR产物可以直接作为突变检测的底物;(3)适当降低反应温度、适当增加反应缓冲液中的NaCl浓度、适当缩短反应时间可提高突变检测效果。     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

T7 RNA多聚酶基因的克隆、序列测定及其在E.coli中的表达

从BL21(DE3)E．coli菌株中以PCR的方法扩增得到了与T7RNA多聚酶(T7RNApolymerase，T7pol)基因大小一致的DNA片断。将PCR产物纯化后直接克隆到pGEM—T载体中，经酶切鉴定和DNA序列分析表明克隆得到了正确的T7pol基因。将T7pol基因亚克隆入pET-28b( )中，构建得到原核表达质粒pET28T7。该质粒的BL21(DE3)pLysS转化菌在IPTG的诱导下可表达约98800的蛋白，这与T7pol的相对分子质量一致。将该质粒转化DH5α、JMl09、HBl01、BL21(DE3)和BL21(DE3)pLysS等5种不同的宿主菌，仅有转化T7pol酶活性受到抑制的宿主菌BL21(DE3)pLysS才能得到转化子，而其余4种T7T7pol酶活性不受抑制的E．coli宿主菌不能得到转化子。pET28T7原核表达质粒这种仅能在T7pol酶活性受到抑制的宿主菌中才能存活的现象说明本试验所克隆的T7pol基因能正确表达出具有RNA转录酶活性的蛋白。     

Genetic diversity of herbicide-resistant and -susceptible Avena fatua populations in North Dakota and Minnesota

Genetic diversity within and among 20 herbicide-resistant (HR) and 16 herbicide-susceptible (HS) Avena fatua multi-field populations was determined using 82 polymorphic loci resulting from two intersimple sequence repeat (ISSR) primers and one long-primer random amplified polymorphic DNA (LP-RAPD) primer. Collections from the Red River Valley of North Dakota and Minnesota, sampled in 1964 and 2000, represented A. fatua populations before and after intensive exposure to herbicides. A 1995 collection from south-west North Dakota represented A. fatua exposed to low herbicide selection. Despite differences in years of herbicide exposure among collections, both HR and HS populations from every collection maintained nearly similar levels of ISSR and RAPD diversity. Genetic differentiation among populations (G_ST) varied from 11% to 13% among HR populations and from 9% to 16% among HS populations, indicating that 84–91% of total variation remained within HS or within HR populations. Minimal difference in gene diversity between HR and HS is consistent with multiple origins of resistance, where HR A. fatua most likely evolved from diverse founding individuals.     

花生DNA导入大豆育种效果的研究

利用花粉管通道在大豆自花授粉后将花生ＤＮＡ导入栽培大豆受体中，引起受体的结荚习性、株高、成熟期，主茎节数、分枝数、产量性状和化学品质等性状的广泛变异。对变异株进行选择，获得了产量比受体提高１１．９％～２５．１％，蛋白质含量提高３．９％～５．３％的变异株系。实验结果表明：利用外源ＤＮＡ导入技术进行生态性状、产量性状和化学品质方向的育种是可能的。     

Feline Ubiquitin Fusion Protein Genes

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.     

鸡新城疫病毒F基因和鸡IL—2重组DNA疫苗的构建

利用已克隆到的新城疫D26株F基因和鸡IL－2基因，经过载体改建，将他们共同克隆于真核表达质粒pCDNA3上，经酶切分析、PCR鉴定证实成功构建了共表达鸡新城疫病毒F基因和鸡IL－2的重组质粒，为探讨禽类重组基因疫苗的构建及鸡IL－2在基因疫苗中的作用奠定了基础。