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在转录组测序的基础上,采用逆转录聚合酶链式反应(RT-PCR)技术,从三七主根中分离到三七病程相关蛋白10(pathogensis related protein 10,PR10)基因,命名为PnPR10-2,测序结果表明该序列长465 bp,编码154个氨基酸。氨基酸序列同源性及系统发育树分析发现,PnPR10-2与人参的PR10-2蛋白同源性最高,具有典型的病程相关蛋白Bet v I保守结构域。构建了重组载体pET32a(+)-PnPR10-2,在宿主菌Escherichia coli BL21中诱导表达融合蛋白,优化诱导条件,PnPR10-2融合蛋白在E.coli BL21中以可溶性和包涵体蛋白2种形式大量表达。纯化上清中的重组蛋白,运用纸片法分析纯化重组蛋白的体外抑菌活性,其对三七根腐病病菌腐皮镰刀菌Fusarium solani和坏损柱孢菌Cylindrocarpon destructans具有一定的抑制作用,猜测该基因可能参与了三七抗根腐病的防御反应。
Based on the transcriptome sequencing, the PR10 gene of Panax notoginseng was isolated from Panax notoginseng by reverse transcriptase-polymerase chain reaction (RT-PCR) and named PnPR10- The sequencing results showed that the sequence was 465 bp in length and encoded 154 amino acids. Amino acid sequence homology and phylogenetic tree analysis showed that PnPR10-2 shared the highest homology with PR10-2 protein of ginseng and had a typical Bet v I conserved domain of disease-related protein. The recombinant vector pET32a (+) - PnPR10-2 was constructed and expressed in Escherichia coli BL21. The conditions of induction were optimized. PnPR10-2 fusion protein was expressed in two kinds of soluble and inclusion protein in E.coli BL21 expression. The recombinant protein was purified and the antibacterial activity of purified recombinant protein was analyzed by disk method. The antibacterial activity of purified recombinant protein was inhibited to Fusarium solani and Cylindrocarpon destructans, It is speculated that this gene may be involved in the defensive reaction of Panax notoginseng.