三七病程相关蛋白PR102基因克隆表达及功能初步分析お.docVIP

三七病程相关蛋白PR102基因的克隆、表达及功能初步分析お 　　[摘要]在转录组测序的基础上，采用逆转录聚合酶链式反应（RTPCR）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PnPR102，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，PnPR102与人参的PR102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（+）PnPR102，在宿主菌Escherichia coli BL21中诱导表达融合蛋白，优化诱导条件，PnPR102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。 　　[关键词]三七； 根腐病； 病程相关蛋白； 原核表达 　　Molecular cloning， bacterial expression analysis and functional 　　characterization of pathogenesisrelated PR102 gene in Panax notoginseng 　　YANG Dan1， BAO Yi1， CHEN Limei1， CUI Xiuming1，2， LI Kunzhi1* 　　（1Faculty of Life Science and Technology， Kunming University of Science and Technology， Kunming 650500， China； 　　2 Key Laboratory of sustainable utilization of Panax notoginseng Resources of Yunnan Province， Kunming 650500， China） 　　[Abstract]Base on the transcriptome analysis and RTPCR techniques，a pathogenesisrelated protein 10 gene was isolated from Panax notoginseng root and named as PnPR102 Bioinformatics and phylogenetic trees analysis revealed that open reading frame （ORF） of PnPR102 was 465 bp in length，encoding 154 amino acids，containing one typical conserved domain of pathogenesis related protein Bet v I family， and showed high similarity with that from P ginseng The recombinant expressed plasmid pET32a（+）PnPR102 was expressed in Escherichia coli BL21 The expression conditions were optimized and it could be expressed well in soluble and inclusion body protein Purified PnPR102 recombinant protein from the supernatant of cells was used to analysis the pathogen resistance activity by paper method The purified recombinant protein could inhibit typical root rot disease pathogen （Fusarium solani and Cylindrocarpon destructans）growth evidently， we conjecture that PnPR102 may participated in defense response of P notoginseng resistance to root rot disease pathogen