摘要
水稻白叶枯病(Xanthomonas oryzae pv.oryzae,Xoo)作为水稻中三大病害之一,是威胁我国水稻生产的最主要细菌病害。由于致病小种发生变异,已有抗病品种丧失了抗性,从野生稻中发掘、利用抗白叶枯病新基因资源成为抗病育种的有效途径。利用不对称体细胞杂交技术,从疣粒野生稻(Oryza meyeriana L.)获得了抗白叶枯病基因资源。利用蛋白组学方法从该抗病新种质SH5和SH76中筛选出了一个生长素调节蛋白,是一类由生长素调节基因家族编码的蛋白,并命名为XooIARP(Xanthomonas oryzae pv. oryzae-induced auxin regulated protein)。本论文以感病品种8411、抗病新种质SH5和SH76为研究材料,试图用转基因技术研究XooIARP基因的功能；同时分析了热激转录因子(heat shock factor,HSF)在抗白叶枯病过程中的表达。研究结果如下：
1.根据其它抗病材料的基因芯片表达谱结果筛选出7个在接种前后差异表达的OsHsfs.通过半定量RT-PCR分析8411、SH5和SH76水稻叶组织在接种白叶枯菌小种P6(PX099)后12h、24h、48h、72h的表达情况。相比于感病亲本8411,抗病新种质SH5和SH76中OshsfB2c2基因表达上调,其他基因则无显著变化。
2.序列分析表明,XooIARP编码蛋白含2个重复的谷胱甘肽-S-转移酶的N末端结构域,推测该蛋白有可能是谷胱甘肽-S-转移酶。本研究将XooIARP cDNA克隆到表达载体pSKB.4(含T7启动子)中,构建重组原核表达质粒pSKB.4-XooIARP,并转化至E.coli菌株BL21(DE3)中,经IPTG诱导后,SDS-PAGE胶获得预测重组融合蛋白条带。
3.构建了XooIARP基因的超表达和RNAi载体,通过农杆菌介导法转化水稻,将超表达载体转入白叶枯病感病品种日本晴和8411；而RNAi载体转入抗病新种质SH5。并获得转基因植株,PCR鉴定证明目的基因已经整合到基因组中。
Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae(Xoo) was one of the three destructive diseases of rice(Oryzae sativa L.), causing the major loss of rice production in China. With existing resistance overcomed by new Xoo race, it is necessary to get new genes resistant to BB from wild rice. SH5 and SH76 contain novel BB resistance gene that was introduced into a cultivated japonica rice variety 8411, via somatic hybridization using the wild Oryzae meyeriana as the donor. By using the proteomic analyses, an auxin-regulated protein from the new hybrids was found, which was named XooIARP(Xanthomonas oryzae pv. oryzae.-induced auxin regulated protein).In this paper the function of XooIARP was studied by using transgenic method. Meanwhile, the expression patterns of seven heat shock factors(OsHSFs) were analysed in 8411, SH5 and SH76. Our results are summarized as follows.
1. Seven differentially expressed OsHSFs were chosen from the microarray results, and their expression patterns were analyzed in leaves inoculating Xoo for 12h,24h,48h and 72h by reverse transcriptase polymerase chain reaction (RT-PCR) In contrast with 8411, only OshsfB2C2 was up-regulated in two new germplasms.
2. Sequence analysis indicated that XooIARP contained two repeats of the N-terminal domain of glutathione S-transferase (GST) and may function as glutathione S-transferase. The prokaryotic expression vector pSKB.4 plus T7 promoter was constructed. After IPTG inducing, the fusion protein band with predicted molecular mass appeared on SDS-PAGE.
3. XooIARP was first reported to be involved in the plant-pathogen interaction. In order to understand its role in resistance to BB, the over-expression and RNAi vectors of XooIARP gene under the regulation of cauliflower mosaic virus 35s promoter were constructed. Then the rice calluses were infected by Agrobacterium tumefaciens EHA105 with vectors, and hygromycin resistant seedings were obtained. XooIARP was integrated into the genome of the plants by PCR identification.
引文
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