Cloning And Functional Identification Of The Oidium Heveae Promoter WY172 And Its Regulatory Analysis On HpaXm

The key problem in plant genetic engineering is ensuring the expression of foreign genes in recipient plant cells.Promoters are important elements in terms of the regulation of gene expression.Using bioinformatics software analysis,a suspected endogenous promoter from Oidium heveae was obtained and named WY172.The promoter function was verified by transient expression,GUS staining experiments,and GUS activity analysis.In addition,the foreign functional gene HpaXm was introduced,and the expression effect of WY172-driven HpaXm gene was investigated via stable expression,micro-hypersensitive response(mHR),tobacco mosaic virus(TMV)inoculation,quantitative real-time PCR,and enzyme activity detection.The types of WY172 promoter were analyzed by inductive element analysis,induction experiments and GUS staining.In order to investigate whether WY172 has the possibility of diversified induction,the 2K sequence upstream of the WY172 promoter was used as the research object,and a gradual deletion mutation was performed.Induction element analysis and promoter activity analysis were performed.The main results are as follows:1.Recombinant expression vectors pBI121-GFP(turnover vector),pBI121-ACT1(positive control),pBI121-WY172 were obtained.WY172 has a promoter function,which can drive the expression of the reporter gene GUS,and can play a promoter role in both monocotyledonous rice and dicotyledonous tobacco.2.The pBI121-HpaXm with 35S(positive control)and PBI121-WY172-HpaXm plant expression vectors were obtained and the corresponding transgenic tobaccos were obtained.The mHR and TMV vaccination experiment proved that WY172 can effectively drive the expression of foreign functional gene HpaXm,stimulate the allergic response of transgenic plants and enhance its disease resistance.At the same time,it was found that TMV may induce the expression of WY172.3.According to bioinformatics analysis,WY172 has an auxin-responsive element,as well as a light-responsive element and two cis-acting elements with unknown functions.Induction experiments and qRT-PCR proved that WY172 may be an IAA-inducible promoter.4.Clone four different length deleted fragments on the 2K sequence upstream of the promoter of the O.heveae WY172.GUS staining and GUS activity results showed that all deletion mutant fragments have promoter activity that regulates GUS gene expression,and the promoter activity is stronger than the 35 S promoter.Among them,WY172Q3 has the highest activity in regulating GUS gene expression.At the same time,it was found that the 2K sequence upstream of the WY172 promoter has abundant cis-acting elements.In conclusion,the endogenous promoter WY172 of the O.heveae was obtained in this study.WY172 was likely to be an IAA-and TMV-inducible promoter with a wide host range and high application potential.