柑橘溃疡病致病基因pthA的克隆及序列分析

Cloning and sequencing of pthA gene of Xanthomonas axonopodis pv. Citri.

根据pthA基因和溃疡病病菌大质粒序列设计特异性引物,利用long-PCR方法,获得了包括pthA完整编码区及两端部分非编码区在内的约4100bp的DNA片段,再利用net-PCR获得了pthA完整的编码区,经XbaⅠ和SacⅠ双酶切后将其克隆到植物表达载体pBI121中.经双酶切及核酸序列测定鉴定的阳性克隆重组质粒被命名为pBI-pthA.序列分析结果表明,克隆得到的pthA与登录到Genbank上的pthA序列有99.8%的同源性,pthA全长基因编码1163个氨基酸,具有17.5个102bp单元的重复序列,每个单元编码34个氨基酸,重复区后有1个亮氨酸拉链结构(LZ),3个核定位信号(NLS)序列和C末端酸性转录激活(AAD)保守区.

4100bp DNA segments both at pthA complete encoding partition and at the two non-encoding terminus were obtained by long-PCR method, and by designing specific primers based on pthA gene And host range of X. axonopodis pv. Citri.. A pthA complete encoding partition was obtained by way of net-PCR. Then it was cloned into pBI121 plant expression vector after XbaI and SacI double restriction analysis. The recombinant plasmid was named pBI-pthA , identified by restriction digesting and sequencing. The result of sequence analysis showed that pBI-pthA had 99.8% similarity with that ofpthA deposited in Genbank. The pthA has a large open reading frame（ORF） encoding 1 163 amino acids. Sequence of the ORF contained 17.5 of 102 bp tandem repeats with each encoding 34-amino-acids, one leucine zipper（LZ） and an acidic activation to transcriptional domain（AAD） at C-terminus.