Identification And Functional Analysis Of WY7 Promoter From Erysiphe Quercicol

Promoters are very important for transcriptional regulation and gene expression,and have become invaluable tools for genetic engineering.As a "switch",the promoter is essential for the regulation of genes at the transcriptional level.Inducible promoters can respond to changes in the external environment based on biological stimuli under relevant conditions and reduce energy waste.Erysiphe quercicol is an obligate parasite.In this study,the Promoter Scan software was used to screen out the WY7 promoter from the E.quercicol genome published in the laboratory,and the promoter function was analyzed.The test results are as follows:1.The WY7 promoter((Gen Bank accession number(MN889519))was cloned from the genome of E.quercicol by PCR.Bioinformatics analysis revealed that its sequence contains a cis-acting element involved in defense and stress response,a light-responsive element,a cis-acting element related to meristem expression,and three unknown cis-acting elements.2.The p BI121-WY7 plant expression vector was constructed,and Nicotiana tabacum cv.Xanthi nc was transformed by leaf disc transient transformation method.GUS staining showed that WY7 has a promoter function.Using ATMT method to transform N.tabacum tobacco leaf discs,stably express WY7 promoter,obtain transgenic N.tabacum,and dye it under various induction conditions.The results show that the WY7 promoter can regulate the expression of GUS gene under the induction of low temperature and salt stress,confirming that WY7 is an inducible promoter.3.Using transient transformation method,the WY7 promoter was used in monocot Zea mays Linn,Oryza sativa L.spp.Japonica cv.Nipponbare,dicot plant N.tabacum and Hylocereus undulates Britt for transient transformation,the WY7 promoter can drive GUS gene expression in both monocot and dicot plants.The GUS gene expression level was determined by q RT-PCR.The transient expression level of GUS gene regulated by WY7 promoter is 11.7 times that of 35 S promoter in N.tabacum and 5.13 times that of ACT1 promoter in O.sativa.4.The p BI121-WY7-hpa1 Xoo,p BI121-hpa1 Xoo and p BI121 vectors were constructed and transformed into leaf discs to obtain transgenic N.tabacum.Phenotypic analysis showed that the plant height,weight and chlorophyll content of p BI121-WY7-hpa1 Xoo and p BI121-hpa1 Xoo transgenic N.tabacum were not significantly different,but they were both higher than p BI121 and wild-type N.tabacum,and the difference was significant.The results of soluble protein analysis showed that both p BI121-WY7-hpa1 Xoo and p BI121-hpa1 Xoo transgenic N.tabacum can cause plant allergic reactions,enhance tobacco’s defense response,and significantly improve disease resistance.Moreover,p BI121-WY7-hpa1 Xoo transgenic N.tabacum had a 15.56% reduction in the number of lesions after TMV inoculation of p BI121-hpa1 Xoo transgenic tobacco,indicating that WY7 has a better effect on regulating hpa1 Xoo gene expression in N.tabacum than WY7 and 35 S promoters.5.Using the deletion mutation method,the 5’end of the 2K sequence upstream of the WY7 promoter was deleted and bioinformatics analysis was performed,including 21 cisacting elements involved in abscisic acid,low temperature response,defense and stress response,and salicylic acid response.Four kinds of plant expression vectors were constructed,namely p BI121-WY7Q1,p BI121-WY7Q2,p BI121-WY7Q3,and p BI121-WY7Q4.Using transient transformation method,four fragments were expressed in N.tabacum leaf discs.The WY7Q1,WY7Q2,WY7Q3 and WY7Q4 fragments all have promoter activity,and the highest activity of WY7Q2 is 8.82 times of 35 S.In summary,WY7 is an inducible promoter regulated by low temperature and salt stress.It can drive the expression of reporter genes and functional genes in non-host bodies,and it also has high-efficiency activity in mono-and dicotyledonous plants.It can be used as a plant gene.The potential of engineering application tools.