Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172

doi:10.13560/j.cnki.biotech.bull.1985.2019-0728

Abstract

Abstract: The aim of this study is to clone 4 different-length deletion fragments of Oidium heveae promoter WY172 and its upstream 2K sequence in order to analyze the expression activity of each fragment of the promoter. Based on the prior research in our laboratory,the 2K sequence upstream of WY172 was used as the research object to conduct the gradual deletion mutation,and 4 fragments in different lengths which might have promoter activity were obtained. In combination with WY172,the pBI121 vector was used as the backbone,and the CaMV35S promoter before the GUS gene was replaced,then the recombinant expression vector was constructed respectively,and the Agrobacterium was transformed by ATMT method. The enzyme activities of the WY172 promoter and fragments of different lengths were analyzed by GUS staining and enzyme activity assay. Five recombinant plant expression vectors including pBI121-WY172,pBI121-WY172Q,pBI121-WY172Q1,pBI121-WY172Q2 and pBI121-WY172Q3 were constructed. GUS gene was expressed in all of the recombinant plant expression vectors,and its expression（blue occurred）was stronger than the positive control CaMV35S promoter. Moreover,the transient expression of pBI121-WY172Q3 recombinant vector was the most intense. The results of GUS enzyme activity assay showed that all deletion mutant fragments presented promoter activity regulating gene expression,and the activation activity was stronger than that of CaMV35S promoter. Among them,the GUS enzyme activity regulated by WY172Q3 was the highest. Therefore,we infer that WY172 and its four 2K-length deletion fragments on the upstream 2K sequence have promoter activity,and WY172Q3 promoter fragment has the strongest expression activity.

Key words: endogenous promoter WY172 of Oidium heveae, GUS staining, deletion mutation, GUS activity

YIN Jin-yao, WANG Yi, XU Liang-xiang, ZHU Li, WANG Chen, LIU Wen-bo, MIAO Wei-guo. Cloning and Expression Analysis of Different-length Fragments of Oidium heveae（HO-73）Promoter WY172[J]. Biotechnology Bulletin, 2020, 36(1): 29-36.

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