Cloning、Function Identification And Application Exploration Of Endogenous Strong Promoter WY193 Of Oidium Heveae B.A.Steinmann

The promoter is an important cis-acting element for regulating the gene expression and is the focus of molecular biology.Filamentous fungi are important industrial enzyme producing bacteria.Its endogenous gene expression is very high,and there may be strong endogenous promoters,which has great potential for development.But so far,fewer promoters have been developed for filamentous fungi.Oidium heveae B.A.Steinmann is a special parasitic filamentous fungus.The genome of the pathogen was first reported on the basis of our previous research work and several suspected promoters were found.Therefore,the purpose of this study is to develop endogenous promoter in Oidium heveae B.A.Steinmann,to provide new materials for plant genetic engineering,to understand the genetic composition and evolution of the powdery mildew of the rubber tree from the molecular level.Based on genomic data of Oidium heveae B.A.Steinmann HO-73 strain genomic data,a suspected promoter was obtained by the online software Promotor Scan,named WY193.The expression of the GUS gene was measured by qRT-PCR.The expression of the GUS gene driven by WY193 was significantly higher than that of the positive control CaMV35 S promoter,which is about 8.34 times of 35 S.WY193 can be used to drive GUS gene to express in dicotyledonous plants like tobacco and dragon fruit,and monocotyledonous plants such as coconuts.qRT-PCR was used to determine the expression of GUS gene regulated by WY193 in the dicotyledonous dragon fruit,which is about 4.46 times of positive control 35S(35S)promoter.qRT-PCR was used to determine the expression of GUS gene regulated by WY193 in the monocotyledonous coconuts,which is about 3.70 times of positive control Ubiquitin(Ubi)promoter.WY193-hpaXm transgenic tobacco plants were obtained by ATMT method.To inoculate TMV for T1 transgenic plants,the results showed that the TMV resistance of WY193-hpaXm transgenic tobacco plants was significantly improved compared with that of wild-type tobacco and positive control 35S-hpaXm transgenic tobacco.Compared with wild-type tobacco,the average number of necrotic spots in the T1 generation of WY195-hpaXm transgenic plants decreased by 66.44%,while that in the T1 generation of WY195-hpaXm transgenic plants decreased by 52.48% compared with the positive control.Compared with wild-type tobacco,WY193-hpaXm transgenic tobacco had an average necrotic spots reduction of 64.40%,and compared with the positive control35S-hpaXm transgenic tobacco,the averagenecrotic spots reduction was 41.32%.qRT-PCR results showed that the expression of hpaXm gene in WY193-hpaXm transgenic tobacco without TMV was 2.87 times higher than that of 35S-hpaXm without TMV.Whereas WY193-hpaXm transgenic tobacco inoculated with TMV showed 1.65 times more expression of the driven hpaXm gene than WY193-hpaXm without TMV.Preliminarily identified the WY193 promoter as the TMV-induced promoter.To further confirm whether WY193 belongs to inducible promoter,using online bioinformatics software PlantCARE to analyze the composition of cis-acting elements of WY193,and to conduct the corresponding induction expression experiment of T1 transgenic tobacco seedlings by stable expression,using salt and other inducible factors.Preliminarily identified TMV and salt as one of the inducers of the WY193 promoter,WY193 may be the promoter of the pathogen-induced or salt-induced type.Successful prediction,screening and identification of WY193 promoters provide a new tool and option for plant transgenic engineering.