水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离

Construction of a Genomic Library of Xanthomonas oryzae pv.oryzae Strain GX1329 and Isolation of Clones Containing the Genes Encoding TAL Effectors

由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50%以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3/pthA家族基因的情况,本研究应用AluⅠ部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHⅠ酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27.7kb,最大为58.5kb,平均大小为39.9kb,文库克隆容量约为2.8×103Mb,该文库中包含基因组中任一个基因的概率为99.4%。利用来自水稻白叶枯病菌菲律宾菌株PXO86的无毒基因avrXa10的第252位～第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3/pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3/pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3/pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3/pthA家族基因的功能奠定了基础。

Bacterial leaf blight （BLB）, caused by Gram-negative bacterium Xanthomonas oryzae pv. oryzae （Xoo）, is a serious threat to yield losses in the main regions of rice growth including Asia, North America and Africa. BLB could lead to a crop loss of up to 50%. It is known that the virulence of Xoo mainly relies on type BI secre- tion system （T3SS） and its secreted effectors. To know the numbers of genes encoding avrBs3/pthA family mem- bers in Guangxi Xoo strain GX1329, a genomic DNA library containing 736 clones was successfully constructed by partially digesting the genomic DNA with A lu I . Restriction enzyme BamH I digestion analysis of plasmids from 15 randomly chosen library clones showed that the cloned DNA in the genomic library was highly random. The size of the smallest eloned DNA in one clone was 27.7kb, the size of the biggest cloned DNA in one clone was 58.5 kb, and the average size of cloned DNA in one clone was 39.9 kb. The cloning capacity of the library is about 2.8x103 Mb with high randomness, and the probability of any one gene contained in the library was about 99.4%. Thirty-seven positive clones were screened out from the GX1329 genomic library by colony in situ hybridization using the 252^th to 486^th bp sequence of avrXalO from Xoo strain PXO86 as probe. Southern hybridization analysis of the 17 clones showed that they contain at least 13 different avrBs3/pthA genes. The results also showed that the avrBs3/pthA family genes occurred in individual or clusters in the genome of strain GX1329. This work defined the number of avrBs3/pthA family genes in the genome of GX1329, which may provide a solid basis for further studying the function of the genes.