【作者】 李广旭；

【导师】 何晨阳；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2009， 博士

【摘要】 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)引起的水稻白叶枯病是世界水稻生产上最严重的细菌病害之一,水稻—Xoo互作系统是植物—病原物互作研究的一个重要模式系统。然而,至今对于其互作的分子过程及水稻抗/感病性调控的分子机理所知甚少。在前期研究工作中,本实验室通过对水稻品种日本晴(Oryza sativa L. cv. Nipponbare)与Xoo菌株(JXO I)亲和互作后的表达谱分析,发现水稻转录因子基因OsBTF3受Xoo侵染而显著地被诱导上调表达。推测OsBTF3可能是水稻对Xoo侵染的抗/感病性表达中重要的调控因子之一。鉴此,本研究对OsBTF3基因的结构、定位、表达和功能进行了系统研究,试图揭示其在水稻—Xoo分子互作、抗逆性以及生长发育中的作用和生物学功能。利用生物信息学方法,对OsBTF3基因结构及其同源性进行了分析。发现该基因位于第3号染色体上,具有5个外显子和4个内含子,成熟mRNA全长有528核苷酸;可编码具有175个氨基酸的蛋白质(分子量=19.2KDa、等电点=5.26)。OsBTF3在N端22–26区域有RRKKK核定位信号(NLS),在34–92区域具有NAC结构域,而在C端171–174区域存在内质网滞留信号(KKES)。在水稻基因组中存在2个OsBTF3同源基因(10号染色体上的Os10g0483000和3号染色体上的Os03g085100),其与OsBTF3具有极为相似的基因结构;不同物种BTF3同系物氨基酸序列以及NAC结构域长度、位置和序列具有物种特异性和保守性,其意味着BTF3在生物的生命活动中可能具有重要的、基础的功能。利用GFP融合蛋白技术对OsBTF3进行了亚细胞定位分析,发现其定位于细胞核内和细胞膜区域,表明其具有核蛋白和胞质蛋白的特性。利用RT-Q-PCR方法对OsBTF3基因表达进行测定,发现其在水稻根、茎、叶、叶鞘和小穗组织中均可组成性表达;OsBTF3具有病原菌、信号分子以及逆境诱导应答特性。Xoo菌株JXO I、水稻细菌性条斑病菌(Xooc)菌株RS63和稻瘟菌(Mg)菌株YN1均能诱导OsBTF3的表达增强,但表达反应速度和强度有差异;脱落酸(ABA)诱导活性最强,茉莉酸甲酯(MeJA)和乙烯(ETH)次之,水杨酸(SA)诱导作用不显著;干旱和高盐胁迫抑制其表达;伤口和光不诱导其表达。利用基因过量表达和RNAi沉默方法,进行了OsBTF3基因的生物学功能分析。通过农杆菌介导法,获得了159个过量表达和RNAi转基因水稻株系。对苗期、分蘖期和孕穗期的T1代转基因水稻株系对Xoo菌株(JXO I和PXO99A)的抗病性进行测定,发现所有测试的过量表达和RNAi株系均比对照株更加感病;PXO99A接种后,几种代表性防卫基因(PAL,AOS、LOX、PR1、PR4和PBZ1)在野生型、过量表达和RNAi株系中的表达发生了不同程度的变化;此外,过量表达株系OE37、OE39、OE17和RNAi株系RI30、RI32、RI19比野生型对Xooc菌株(RS63)更加感病。对T2代转基因水稻株系的抗逆性进行测定,发现过量表达株系对高盐(200mM NaCl)和低温(4℃)胁迫抗性显著增强,但抗旱性下降,RNAi株系表型正好相反。表明OsBTF3在水稻对生物和非生物逆境胁迫的反应中起着重要的作用。对T1代转基因水稻株系的生长发育进行分析,发现与野生型对照相比,过量表达株系植株高大、生长旺盛,而RNAi株系严重矮化、生长较弱;尽管所有转基因植株的节数未发生变化,但节长度发生了明显改变,造成株高的显著差异;过量表达株系的节中薄壁细胞分裂加速,叶片气孔变小;过量表达株系的光合作用显著增强,穗长、穗粒数和千粒重显著增加,而RNAi株系则明显降低。上述结果表明OsBTF3在水稻生长发育中具有重要的生物学功能。总之,本研究基本明确了水稻基因OsBTF3的结构特征和表达特性,阐述了其在水稻对Xoo和Xooc抗/感病性中的作用,揭示了其在非生物胁迫抗性以及生长发育中的生物学功能。因此,本研究通过对OsBTF3这一新基因的功能发掘,为其将来可能应用于转基因水稻的研究提供了重要的科学依据和信息。更多还原

【Abstract】 Bacterial blight of rice caused by Xanthomonas oryzae pv. Oryzae (Xoo) is a serious worldwide bacterial disease. The interaction between rice and Xoo is a model system of the study of plant-pathogen interaction. But untill now, little is known about the interaction process and molecular mechanism of resistance/susceptibility. In previous research, OsBTF3, a rice transcriptional factor gene, was found to be up-regulated during the interaction between rice(Oryza sativa L. cv. Nipponbare)and Xoo strain JXOI. We speculated that OsBTF3 may be a crucial regulating facter for rice resistance/susceptibility to Xoo infection. So the gene structure, localization, expression and function were studied to elucidate the biological function of OsBTF3 in rice-Xoo interaction, tolerance to abiotic stress and development of rice.Bioinformatic was applied to analysed the gene structure and sequence characters of OsBTF3. OsBTF3 was located in Chromosome 3 with 5 exons and 4 introns, the full length mRNA is 528bp which can encode a protein of 175 aa with predicted molecular weight 19.2 KD and PI 5.26. There exist a RRKKK nuclear localization signal in the 22-26 residues of N-terminal , a NAC domain in 34-92 residues and an ER retention signal KKES in the 171-174 residues of C-terminal. Os03g085100 and Os10g0483000 identified as two homologous genes of OsBTF3 in rice genome have a high similar gene structure with OsBTF3. BTF3 is found to be species-specific and conserved on the base of sequence alignment of BTF3 homologues from diferent species and charaters of length, position and sequence of their NAC domain, which implies that BTF3 may play important and fundamental roles in biology organism.Subcellular location analysis of GFP-fusion protein showed that OsBTF3 located in nucleus and cell membrane with a property of nucleus and cytoplasm protein. OsBTF3 was constitutive expressed in root, stem, leaf, sheath and panicle of rice revealed by real-time quantitative PCR. OsBTF3 have the charaters of response to pathogen, signal molecule and abiotic stress. Expression of OsBTF3 can be induced by Xoo strain JXO I、Xooc strain RS63 and Mg strain YN1 with different speed and strength. OsBTF3 was strongly induced by ABA, followed by MeJA and ETH but with no obvious response to SA. OsBTF3 can be suppressed by drought and salt stress. It can not be induced by light and wound.The biological function of OsBTF3 had been analyzed through over-expression and RNAi method. 159 over-expression and RNAi transgenic lines of OsBTF3 were obtained using agrobacterium-mediated rice transformation. Resistance detection of T1 transgenic rice lines had been conducted by inoculation with Xoo strain JXOI and PXO99A in seedling, tillering and booting stage of rice, the results showed that all over-expression lines and RNAi lines were more sensitive to Xoo than wild type. After inoculation by PXO99A, several representative defence-related genes (PAL, AOS, LOX, PR1, PR4 and PBZ1) showed various degree of expression level. Furthermore, over-expression lines OE37、OE39、OE17 and RNAi lines RI30、RI32、RI19 were also more sensitive to Xooc strain RS63. Abiotic stress response detection of T2 transgenic lines showed that the tolerance of over-expression lines to salt and cold is highly improved but the tolerance to drought is depressed, while the result of RNAi lines is reverse. This showed that OsBTF3 has a close relationship with the response of rice to biotic and abiotic stress.The effectes of OsBTF3 on growth and development were analysed in T1 transgenic rice lines. Compared to wild type rice, the over-expression lines of OsBTF3 grow higher and stronger while the RNAi lines are shorter and weaker; Though the stem nodes number of all transgenic lines maintain unchanged, changes of the length of stem nodes led to significant difference on height of over-expression and RNAi rice plants; SEM results revealed that, for over-wxpression lines, fission of parenchymatous cells in stem node were accelerated, while leaf stomas were smaller than normal; Photosynthesis analysis showed that the photosynthesis ability of over-expression lines were obviously enhanced while the RNAi lines were depressed; Survey on several biological charaters including spike length, seeds number per spike and thousand seeds weight showed significantly increased tendency in overexpression lines and obvious decrease tendency in RNAi lines; Our results implies that OsBTF3 may play important and fundamental roles in growth and development of rice.In conclusion, we have made clear the gene structure and expression characters of OsBTF3, and have elucidated the effect of OsBTF3 on rice resistance/susceptibility to Xoo and Xooc and illuminated its biological function in tolerance to abiotic stress and development of rice. Therefore, the finding of biological function of OsBTF3 will afford scientific foundation and information for its potential applying on the study of transgenic rice.更多还原