【作者】 李强；

【导师】 曹孟良；

【作者基本信息】 中南大学 ， 植物学， 2008， 硕士

【摘要】 野生稻含有许多栽培稻中已消失的有利基因,特别是对各种生物逆境和非生物逆境的抗性基因。但由于野生稻的遗传背景复杂,利用传统的克隆方法存在着很多问题和不足。候选抗性基因克隆策略是最近十年发展出来的克隆抗病基因的新方法,利用抗病基因的保守序列从而获得抗病基因同源序列并最终克隆获得新基因。本研究首先从野生稻中提取总RNA,反转录以获得cDNA;根据NBS-LRR类和STK激酶类抗病基因的保守序列设计引物,PCR扩增野生稻cDNA得到了预计大小的目的条带,再经克隆和测序。共分别获得了11条NBS-LRR类序列和26条STK激酶类序列。经过分析将NBS-LRR类抗病基因同源序列分为3类,而STK激酶类抗病基因同源序列可分为13类。利用BLAST比对分析发现三类NBS-LRR类序列均与水稻抗病基因RPR1有很高的同源性,经电子定位分析将其定位到水稻11号染色体RPR1基因座位上,推测为RPR1基因家族在进化过程中复制与分化重组的结果。STK激酶类抗病基因同源序列除一类序列在水稻基因组上无法找到相似性极高的序列外,其余均可以找到90%以上的同源序列且有10类序列可电子定位于RAP-DB预测基因内。这些序列可为进一步的基因克隆提供探针以及发展成为分子标记,或者作为电子克隆的初始序列,为克隆野生稻的新抗性基因提供支持。更多还原

【Abstract】 Wild rice contains a large number of favorable gene which have disappeared in the cultivated rice,especially the resistance genes to biotic stesses and abiotic stresses. Because the wild rice genetic background is complex, there are many problems and shortcomings in the use of traditional methods to clone them. The strategy of cloning by the candidates of Resistance gene is a new method to clone the resistance gene that developed in the last decade years, which using the conserved resistance gene sequence to gain resistance gene analogs and ultimately clone the new genes. We extracted total RNA from Wild rice at first, reverse transcript to obtain cDNA; then designed PCR primers by the NBS-LRR and STK kinase gene conserved domains and amplified cDNA of the wild rice.the expected size of the zone is obtained, and clone into the vector,then sequenced it,finally we obtained 11 NBS-LRR resistance gene sequence homology and 26 STK kinase resistance gene homologous. the NBS-LRR resistance gene homology sequences can be divided into three categories, and STK kinase resistance gene homologous sequences can be divided into 13 categories. using BLAST alignment to analyse the NBS-LRR sequences,we found all three types of NBS-LRR sequences have high homologous to the resistance gene RPR1 of rice, and mapped them preliminary into the region near the RPR1 in the chromosome 11 of the rice genome by the method of electronic mapping. we infered it is the result of replication and differentiation of the RPR1 gene family in the process of Evolutionary. in addition to a category of sequence could not find the high sequence similarity in the rice genome , STK kinase resistance gene analogs could be found more than 90% homologous to the rice genome, and 10 categories can be mapped to hypothetical gene. These sequences may provide as probes in the further gene cloning、develop to be molecular markers, or as an initial sequence of the silico cloning,which can provide support to the cloning of new resistance gene from wild rice.更多还原