水稻条斑病细菌hrp调节基因hrpG_(Xooc)和hrpXooc的克隆和序列分析

Cloning and sequencing hrp regulatory genes, hrpG_(Xooc) and hrpXooc,from Xanthomonas oryzae pv. oryzicola

Xanthomonasoryzaepv.oryzicola基因文库的hrp基因克隆pUHRS138携 39.3 kb大小的hrp基因片段。经系列亚克隆和对hrp 突变体的功能互补验证 ,4 .5 kbBamHI KpnI为最小功能片段 ,该片段可使X .o .pv .oryzicola的hrp 突变体恢复在烟草上激发产生HR和在水稻上具致病性。序列测定和分析显示 ,4 .5 kbhrp片段中含hrpXooc和hrpGXooc基因。单独的hrpGXooc和hrpXooc不能功能互补hrp 突变体。hrpXooc与其它黄单胞菌中已克隆的hrpX的同一性达 83%以上 ,推测的蛋白质水平上的差别主要在32、14 1、16 4、175、2 13、2 4 7和 35 7位点上。HrpX序列中α 螺旋 转 α 螺旋结构在黄单胞菌中高度保守。hrpGXooc与水稻白叶枯病菌的hrpGXoo同一性达 96 % ,与X .campestrispv.vesicatoria的hrpGXcv同一性达 87% ,与Ralstoniasolanacearum的hrpGRs同源性较低 ,4种HrpG蛋白质水平上的差别主要集中在 2 2、2 9、115和 2 5 2位点上。HrpGXooc和HrpGXcv同列比较显示 ,3～ 9和 2 16～ 2 2 0区域的氨基酸序列有所不同 ,可能反映了HrpG蛋白在感知环境信号和调节hrp基因表达方面的差别。

An hrp gene clone, pUHRS138, obtained from a genomic library of Xanthomonas oryzae pv. o ryzicola (Xooc) by using biparental conjugation protocol, contained a 39.3 kb foreign DNA fragment. Series of subclones were achieved from the 39.3 kb hrp fragment in pUHRS138 and hrp phenotype complementation by the subclones showed that a 4.5 kb Bam HI Kpn I DNA fragment could complement hypersensitive response (HR) elicitation on tobacco to all hrp mutants, but only restored the pathogenicity to an hrp mutants, M1005, on rice. The nucleotide sequencing revealed that hrpG Xooc and hrpXooc genes were located in the 4.5 kb DNA fragment. Neither of these two hrp regulatory genes could restore HR or pathogenicity to hrp mutants of Xooc . Sequence comparison of hrpXooc with other hrpX genes of xanthomonads in GenBank showed at least 83% identity. The differences in amino acids between HrpXooc and the other HrpX proteins were found at the sites 32, 141, 164, 175, 213, 247 and 357. Interestingly, no amino acid substitution was found in the region coding for the alpha helix turn alpha helix structure which was conserved in HrpXooc and the other HrpX proteins. The comparison of the four putative HrpG proteins indicated that the HrpG in xanthomonads had a low similarity with the HrpG from Ralstonia solanacearum . The comparison of HrpG Xooc of Xooc with HrpG Xoo of Xanthomonas oryzae pv. oryzae revealed four amino acid alternatives at the sites 22, 29, 115, and 252. The alignments of HrpG Xooc with HrpG Xoo and HrpG Xcv showed that the amino acid residues at two sites 3-9 and 216-220 were different. These suggest that HrpG in xanthomonads may sense environmental signals and regulate hrp genes differently in different modes.