【作者】 夏勇；

【导师】 雷建军；

【作者基本信息】 西南农业大学 ， 蔬菜学， 2001， 硕士

【摘要】 芜菁花叶病毒（Turnip Mosaic Virus，TuMV）病是危害甘蓝生产的主要病害之一。而采用化学防治很难收到满意的效果。目前，筛选抗（耐）病材料，培育抗（耐）病品种是防治芜菁花叶病毒病最经济，最有效的手段。由于现有的甘蓝抗TuMV材料常伴随有一些不良的经济性状，用常规育种方法难以打破它们之间的连锁关系。因此，甘蓝抗TuMV育（?）进展缓慢。但利用基因工程技术，分离甘蓝抗TuMV基因，将其导入到受体材料中后既可增强受体材料的抗病性又能保持优良的经济性状。 根据抗病基因的保守序列设计简并引物，然后用PCR法从抗病材料的基因组或cDNA中扩增应用于分离抗病基因或抗病基因同源序列的探针，对用含抗病基因的材料构建的cDNA文库进行筛选，进而克隆抗病基因，已成为植物抗病基因分离的一个最经济、最快捷的途径。本研究中，我们在国内外首次以高抗TuMV的甘蓝84075为材料，利用基于同源序列的PCR法从甘蓝基因组和cDNA第1链中扩增出2个抗病基因同源片段，经测序后，研究了它们在甘蓝基因组中的组织结构，并对甘蓝cDNA文库进行了初步筛选，获得如下结果： 1．甘蓝抗病基因同源片段的分离 参照烟草的抗花叶病毒基因N，拟南芥的抗丁香假单孢杆菌基因RPS2及亚麻的抗叶锈病基因L6的保守区域设计简并引物，其中5’引物为：5’-GGIGGIGTIGGIAAIACIAC-3’，3’端引物为：5’-A（A/G）IGCTA（A/G）IGGIA（A/G）ICC-3’。采用PCR热启动方法，分别从高抗TuMV甘蓝材料84075幼苗的cDNA第1链及基因组中扩增出1条约510bp左右的DNA条带。 2．PCR产物的克隆 采用T/A克隆法，用T₄连接酶连接pUCm-T载体与经琼脂糖回收分离并纯化的PCR片段。转化大肠杆菌DH5α，利用α—互补筛选重组子，进一步通过PCR及酶切验证表明目的片段已克隆进入质粒载体中。 3．克隆片段的序列测定与分析 分别从RT—PCR产物及基因组DNAPCR产物克隆成功的重组子中挑取一个单菌落，委托大连宝生物工程公司进行序列测定，结果表明，所克隆的目的片段均为513bp，编码171个氨基酸，二者核苷酸的一致性为71.3％，氨基酸的一致性为61.4％。将二者暂命名为Bor1和Bor2。同源性分析后发现，这2个基因片段与所有的NBS—LRR类抗病基因均有一定的同源性，与拟南芥抗丁香假单孢杆菌基因RPS2同源性最高。 4．限制性片段长度多态性（RFLP） 以Bor1基因片段为探针对甘蓝材料84075幼苗基因组进行了RFLP多型性分析，结果Bor1基因在甘蓝基因组中除有2条强杂交带外，还有若干条弱杂交带，说明Bor1基因在甘蓝基因组中是以较小的基因家族的形式存在。由于Bor1与Bor2同源性较高，说明它们可能就是该基因家族中的成员。5．甘蓝C＊NA文库的初步筛涉 以 Borl基因片段为探针，用o—’‘P同位素进行标记，对用甘蓝材料 84075构建的人噬菌体CDNA文库进行了初步筛选，获得了几个阳性克隆。并对其中的一个阳性克隆进行了序列分析。结果表明该基因片段全长为 828hp，包含有 1个 504hp的由起始密码子 ATG和终止密码于 TGA组成的开放阅读框架，编码 168个氨基酸，前端含有 85hp的非编码区，末端含有236hp的非编码区。与基因库的已知序列的比较表明，该基因片段与许多动、植物转译调控肿瘤蛋白（Translationally controlled tUmor pfotin，TCTh）在氨基酸水平上具有较高的同源性，其中与拟南芥的 TCTh类似物同源性最高为 90．5％；其次与豌豆、马铃薯、甜瓜、水稻和烟草的TCTh类似物的同源性分别为77．4％、77．4％、77．4％、76，2％、73．2％；而与人类、鸡、酵母的 TCTh类似物的同源世较低，分别为 39．3％、36．9％和 37．5％。更多还原

【Abstract】 The sequences of cloned plant disease resistance genes show that majority , whether conferring resistance to viral, bacterial, fungal pathogens or nematode, contain similar sequences and structure motifs . According to the similarities among resistance genes to design the primers, large quantity of resistance gene homologous sequences(RGHSs) have been obtained by polymerase chain reaction from many plant species . It is possible to use these amplification products as probes for the isolation of genes which conferring resistance to different types pathogens from the cDNA or genomic DNA library.In this paper, resistance gene homologous sequences were amplified by PCR from first cDNA strand and genomic DNA in cabbage 84075 which conferring highly resistance to TuMV (Turnip Mosaic Virus). The product of PCR were cloned and sequenced. Using the products of RT-PCR as probe, the genome orgnization of these RGHSs on cabbage was studied, and cDNA library was screened .The main results are as follows: 1.Isolation of resistance gene homologous sequences in cabbageRegion of amino acid identity in the N gene from tobacco, the RPS2 gene from Arabidopsis and the L6 gene from flax were used to design degenerate primers. Primer sequences were as follow:5’ primer,5’-GGIGGIGTIGGIAAIACIAC-3’; 3’ primer,5’-A(A/G)IGCTA(A/G)IGGIA(A/G)ICC-3’. Two fragments of 513bp were amplified by RT-PCR and genomic DNA PCR, then cloned into multi-clone site of pUCm-T. The cloned vectors were named as pBorl (RT-PCR products) and pBor2(genomic DNA PCR products), respectively. 2.Seqnencing and analysis of Borl and Bor2The results of DNA sequencing show that the identity between Borl and Bor2 are 71.3% at nucleic acid level and 61.4% at amino acid level, respectively. Homologous analysis using BLAST software showed that this two RGHSs were homologous not only to the resistance genes RPS2 and N but also to the genes 12 and L6, all of which are member of the same structural class of plant resistance genes containing NBS-LRR. 3.Restriction fragment length polymorphismsUsing Borl as a probe, genomic Southern blots on cabbage 84075 were detected . The result exhibited that except for two strong bands, many weak polymorphic bands were observed .So we think Borl fragment belong to a multigene family of cabbage . Moreover, they are so homologous between Borl and Bor2 that they should be describe as member of the same multigene family.4.Screening of the cDNA library of cabbageBorl fragment was used as probe for screening of the cDNA library of cabbage. Through two successive screenings, several positive cDNA clones were achieved. One of them was sequenced. The full length of this cDNA cloned is 828bp. It contained an open reading frame of 504bp encoding a sequence of 168 amino acids. Homologous analysis show that it is highly homologous at the amino acid level to several plant analogs of TCTP(Translationally controlled tumor protein). The overall amino acid sequence of this clone shares 90.5%, 77.4%, 77.4% 77.4%, 76.2% and 73.2% identity with TCTP analogs from Arabidopsis, pea, potato, melon, rice and tobacco, respectively. Amino acid homology score of 39.3%, 36.9% and 37.5% were obtained for the TCTP analog of cabbage and proteins from human ,chicken and yeast, respectively.更多还原