摘要
花椒中含有的几丁质酶基因ZaCHIT1在植物病程中诱导相关蛋白表达,在植物对抗真菌病害过程中起重要作用。本研究将原核表达载体pMCSG19-ZaCHIT1转化到大肠杆菌BL 21(DE 3)菌株中,之后进行IPTG诱导表达并从诱导温度、时长和IPTG浓度3个方面检测对ZaCHIT 1重组蛋白表达的影响,以获得最优体系,最优体系为诱导温度37℃、IPTG浓度为1 mmol·L^(-1)、诱导时长为14 h。进一步离心后分析各组分发现,ZaCHIT 1重组蛋白多以可溶性蛋白形式存在。利用Ni-NTA亲和层析纯化方法对ZaCHIT 1重组蛋白进行纯化,发现在经过300 mmol·L^(-1)咪唑洗脱液洗脱后的洗脱组分中的ZaCHIT 1重组蛋白表达浓度最大。经Western Blot鉴定发现,在77.51 kDa处有符合ZaCHIT 1重组蛋白大小的特异性条带出现。本研究将为后续研究ZaCHIT1基因在植物中的抗菌作用机制及结构提供依据。
Chitinase ZaCHIT 1 in Zanthoxylum piperitum is a pathogenesis-related protein, which plays an important role in plant resistance to fungal diseases.In this study, the prokaryotic expression vector pMCSG19-ZaCHIT1 was transformed into Escherichia coli strain BL 21(DE 3) and then was induced by IPTG,and the effects of induction temperature, duration and IPTG concentration on the expression of ZaCHIT 1 recombinant protein were detected to obtain the optimal system.The results showed that the optimal system was the induction temperature of 37 ℃,IPTG concentration of 1 mmol·L^(-1) and induction duration of 14 h.After further centrifugation, the ZaCHIT 1 was found to be mostly soluble protein.The ZaCHIT 1 was purified by Ni-NTA affinity chromatography and it was found that the highest concentration of ZaCHIT 1 protein was expressed in the eluted fractions with 300 mmol·L^(-1) imidazole solution.Western blot analysis showed that a specific band corresponding to the size of ZaCHIT 1 recombinant protein appeared at 77.51 kDa.This study would provide a basis for further studies on the antibacterial mechanism and structure of ZaCHIT1 gene in plants.
作者
杨德奕
毛俊文
赵德刚
赵懿琛
YANG Deyi;MAO Junwen;ZHAO Degang;ZHAO Yichen(Guizhou University College of Tea Science/The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education)/Guizhou Key Laboratory of A gricultural Biological Engineering,Guiyang 550025,China;Guizhou University College of Life Sciences/The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education)/Guizhou Key Laboratory of A gricultural Biological Engineering,Guiyang 550025,China;Guizhou Key Laboratory of A gricultural Biological Engineering,Guiyang 550025,China)
出处
《种子》
北大核心
2021年第12期19-25,共7页
Seed
基金
贵州省科技计划项目“无刺花椒快速繁殖技术及产业化示范研究”(黔科合计Z字[2012]4008)
贵州省特色植物种质资源利用与创新人才基地(RCJD 2018-14)
贵州省高层次创新型人才培养项目(黔科合人才[2016]4003号)共同资助。
关键词
花椒
几丁质酶
原核表达
可溶性蛋白
Zanthoxylum piperitum DC
chitinase
prokaryotic expression
soluble protein
