【作者】 吕启明；

【导师】 李仕贵； 朱立煌；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 稻瘟病是水稻最为严重的病害之一,利用含有抗病基因的水稻品种是一种有效且环保的防治稻瘟病危害手段。迄今已经定位的抗稻瘟病基因有70多个,其中24个已被克隆。目前克隆的抗稻瘟病基因绝大多数都属于NBS-LRR(Nucleotide Binding-site Leucine-rich Repeat)类型,并且很多都是处于同一位点的等位基因或复等位基因。抗稻瘟病基因Pid3是利用假基因化分子标记在籼稻品种地谷中克隆的典型CC-NBS-LRR基因,该基因在粳稻和杂草稻中的同源基因普遍提前终止,但提前终止位点在野生稻与籼稻中没有检测到。鉴于目前已经克隆的这些抗稻瘟病基因特点,本研究利用等位发掘的方法在野生稻和栽培稻中共克隆了41个Pid3同源基因,并将其中11个进行了以感稻瘟病水稻品种TP309为背景的基因转化。对转基因植株抗性鉴定发现来自多年生普通野生稻A4、38-1、40-1和籼稻品种9311.kasalath中的Pid3同源基因具有抗稻瘟病功能：在检测过的一百多个稻瘟病菌小种里面,来自籼稻品种9311中的9311-Pid3抗谱与Pid3相同,其他4个同源基因的抗谱则各不相同,并且来自多年生普通野生稻A4中的A4-Pid3在检测过的稻瘟病菌小种中具有最广的抗谱。最后经检测发现9311-Pid3基因在我国籼稻育种中已经得到广泛利用,而Pid3及其同源基因在粳稻育种中还有很大的利用空间。9311-Pid3与野生稻40-1-Pid3对稻瘟病菌具有不同的抗谱,它们在预测的蛋白序列上的差异只出现在LRR区域,预示着LRR区域在这两个基因间对稻瘟病菌小种特异识别方面起作用。经过对Pid3同源基因间LRR区域的互换重组,发现野生稻38-1-PID3与地谷PID3间对稻瘟病菌小种特异识别区域是CC-NBS;38-1-PID3与A4-PID3间以及A4-PID3与地谷PID3间主要由CC-NBS区域起小种特异识别作用,但LRR区域也会产生一定影响。在对地谷中Pid3基因与野生稻15-3-Pid3基因进行LRR区域互换重组研究时,发现重组基因DG15出现了植株矮化表型,经抗性鉴定表明其仍具有抗稻瘟病功能；在对Pid3基因MHD保守模体点突变体研究中也发现有植株矮化表型出现,但这种突变体却并不抗病,与这两种突变类似的其他突变体以前在烟草叶片中作瞬时表达研究时常常都归为自发HR(Hypersensitive Response)反应,本研究结果表明它们的分子机制可能是不相同的。由于绝大多数抗稻瘟病基因都属于NBS-LRR类型,本研究利用这一结构特点对抗稻瘟病基因的人工进化做了初步探索。将Pid3基因与同属于CC-NBS-LRR类型的广谱抗稻瘟病基因Pi9以及抗稻瘟病QTL Pbl做了不同结构域间的互换重组,在这些重组基因中,发现Pid3Pi9、Pid3aPi9、Pid3aPb1、Pid3aaPb1仍然具有抗稻瘟病功能,尽管这些重组基因的抗性没有重组前的基因强,但这些结果说明不同抗稻瘟病基因间人工重组进化是可行的。同时为了探索大规模人工进化抗稻瘟病基因的途径,本研究利用multisite gateway技术成功构建了Pid3基因的3片段重组基因,尽管此基因目前未检测到具有抗稻瘟病功能,但我们相信此重组技术将会为大规模人工进化抗稻瘟病基因提供技术支持。最后本研究将得到的Pid3同源基因进行了杂交聚合,还利用组织特异性表达启动子PD540(-544)将Pid3基因进行了水稻绿色部位特异表达,使其在水稻胚和胚乳中不表达,进行了一些抗稻瘟病基因转基因生物安全方面的研究探索。更多还原

【Abstract】 Rice blast, caused by the filamentous ascomycete Magnaporthe oryzae (M. oryzae), is one of the major diseases that drastically damage rice production. The use of plant varieties with resistance (R) genes is, and will continue to be, the most cost-effective and environmentally-friendly way to control this disease. Thus far, more than70blast R genes have been described at the genetic level,24of which have been cloned and characterized. Most of these cloned blast R genes be long to the nucleotide binding-site leucine-rich repeat (NBS-LRR) family, and some have been found to be alleles. We previously cloned Pid3from the indica variety Digu by performing a genome-wide comparison of paired NBS-LRR genes and their pseudogene alleles between two rice genome-sequenced varieties,9311(indica) and Nipponbare (japonica), Pid3alleles in most japonica varieties were identified as pseudogenes due to the presence of a nonsense mutation, but this pseudogene mutation did not occur in the indica varieties that were tested, or in common wild rice species.In this study,41orthologs of Pid3in common wild rice and in cultivated rice were identified using an allele mining approach, and11of which were transformed into the rice blast susceptible variety TP309. After resistance evaluation for transgenic plants,5Pid3orthologs were found to be functional rice blast resistance genes. The ortholog in indica variety9311had the same blast resistance spectrum as Pid3, while the remaining4orthologs had different resistance spectrum between each other, and the ortholog in common wild rice A4had the broadest resistance spectrum as to these blast strains we used. Additional, we found the Pid3ortholog in9311had been broadly used as resistance resources in our Country’s indica rice breeding program, and these Pid3orthologs could be important blast resistance resources for japonica rice breeding in the future.The predicted40-1-PID3and9311-PID3proteins differed solely in the LRR region, thus, specificity differences between orthologs could be determined by the LRR region. Functional analysis in transgenic plants of recombinant orthologs constructed in vitro provided further information:DG38and A438recombinants, encoding the LRR of38-1-Pid3, conferred Pid3and A4-Pid3resistance specificity, respectively;38DG recombinant, encoding the LRR of Pid3, conferred38-1-Pid3resistance specificity;38A4recombinant, encoding the LRR of A4-Pid3, conferred different resistance specificity from38-1-Pid3and A4-Pid3, thus, specificity differences between orthologs could be determined by the CC-NBS domain too. DG15recombinant, encoding the LRR of15-3-Pid3, exhibited dwarf, abnormal morphology phenotype and conferred blast resistance, whereas the point mutate Pid3(D508V), which had a point mutation in the MHD motif, exhibited moderately dwarf, abnormal morphology phenotype and didn’t confer blast resistance, thus we believed that the molecular mechanism between the two mutates was different, although both were considered the same HR phenotypes in other transient expression researches in tobacco leaves.Considering that most of the cloned blast R genes belong to NBS-LRR type, we constructed some recombinants in vitro between Pid3and other NBS-LRR type rice blast R genes to explore artificial evolution of rice blast R genes. Pid3Pi9and Pid3aPi9recombinants encoding the LRR region and ARC-LRR region of broad blast R gene Pi9, respectively, also conferred blast resistance; The analogue Pid3aPbl and Pid3aaPbl recombinants encoding the LRR region and ARC-LRR region of blast resistance QTL Pb1, respectively, had the blast resistance ability too. These results demonstrated the possibility of artificial evolution of rice blast R genes in the future. Meanwhile, in order to explore large-scale artificial evolution of rice blast R genes, we constructed three-fragment recombinants using the multi-site gateway technology. Although the recombinant did not confer blast resistance, we believed this approach would do favor for artificial evolution of rice blast R genes in the future.Finally, we pyramided Pid3orthologs by crossing transgenic plants with each ortholog, and constrained Pid3expression only in rice green tissues not in embryo and endosperm by using tissue specific promoter PD540(-544) for transgenic bio-safety research.更多还原