Analysis Of The Role Of RY Element In AtBGLU19 Promoter

With the development and utilization of molecular biology in genetic engineering,genetic engineering has become an important tool for obtaining transgenic plants,which has profound significance for the development of contemporary agriculture.In transgenic technology,the function of foreign genes and related regulatory mechanisms should be clarified,as well as their expression sites and expression levels in plants.In addition,because the expression of foreign genes in plants needs to be regulated by binding transcription factors,it is important to identify the promoter of the foreign gene and the function of the regulatory elements on the promoter.Our laboratory have shown that the promoter of Arabidopsis ?-glucosidase 19(AtBGLU19)gene can drive the specific expression of foreign gene in seeds,which proves that it is a seed specific promoter.At the same time,the full-length sequence of the AtBGLU19 promoter was analyzed,and many cis-acting elements related to its expression were identified.Among the cis-acting elements related to seed-specific expression,the RY element is closely related to seed-specific expression.Green fluorescent protein(GFP)is a kind of bioluminescent protein,which can replace GUS gene as a molecular marker of transgenic plants.Therefore,in this study,we used the GFP gene as a reporter gene and designed the mutant sequence of the three RY elements on the AtBGLU19 promoter sequence,and then amplified the RY element mutant promoter fragment by PCR amplification of the mutant primers,and then ligated into a plant expression vector.The above-mentioned expression vector was transformed into Arabidopsis thaliana using the Agrobacterium-mediated dip flower method,and transgenic Arabidopsis thaliana was continuously screened to obtain transgenic homozygous lines.Thereafter,the GFP fluorescence was detected and the RY element on the AtBGLU19 promoter was functionally identified by semi-quantitative RT-PCR.The main experimental results of this paper are as follows:(1)Sequence analysis of AtBGLU19 promoter and each RY element.PlantCARE software was used to analyze the full-length AtBGLU19 promoter to obtain each RY element site.There are three RY elements in RY1,RY2,and RY3.(2)Construction of At BGLU19 promoter expression vector with RY elements mutated at different sites.Site-directed mutation of the RY element by PCR,followed by electrophoresis detection of its amplified product.The electrophoretic band was digested as expected,and the digested band was recovered and ligated to the plantexpression vector.Finally,the sequencing results were compared,indicating that the plant expression vectors of the three mutant fragments were successfully constructed.According to the different positions of the RY element on the AtBGLU19 promoter,The resulting RY element expression vectors at different sites were named pMUTRY1-GFP,pMUTRY2-GFP and pMUTRY3-GFP in sequence.(3)Three plant expression vectors were introduced into Agrobacterium tumefaciens GV1301,and then transgenic Arabidopsis plants were obtained by Agrobacterium-mediated dipping method.T3 generation transgenic Arabidopsis homozygous strains were obtained through hygromycin resistance screening and isolation ratio screening.(4)The T3 generation transgenic Arabidopsis plants were seeded for qualitative analysis of GFP fluorescence activity.The results showed that the mutation of the RY element at different sites caused the AtBGLU19 promoter to drive down-regulated GFP gene expression in seeds,and the degree of down regulation.The same indicates that the RY element plays an important role in the transcriptional activity of the AtBGLU19 promoter in seeds.The fluorescence detected by the seeds of pMUTRY1-GFP,pMUTRY2-GFP,and pMUTRY3-GFP was reduced to different degrees compared with the fluorescence intensity detected by the seeds of the wild-type promoter.Comparing these three mutant promoters,the pMUTRY1-GFP transgenic lines had the strongest fluorescence intensity,followed by pMUTRY2-GFP,and the weakest was pMUTRY3-GFP.The fluorescence intensity indicates that the RY element at the pMUTRY3-GFP site has the strongest positive regulatory effect on the AtBGLU19 promoter-driven GFP gene expression in seeds,followed by pMUTRY2-GFP and pMUTRY1-GFP.(5)The semi-quantitative RT-PCR method was used to quantitatively analyze the seeds of the T3 generation transgenic Arabidopsis plants.The results of electrophoresis showed that compared to the wild-type promoter,the GFP expression of the pMUTRY1-GFP transgenic lines in the three mutant promoters The highest amount was followed by pMUTRY2-GFP,and the lowest GFP expression was pMUTRY3-GFP.Then by combining the detection of GFP activity and the results of semi-quantitative RT-PCR,the following conclusions can be drawn: In the At BGLU19 promoter,the main positive regulatory function is the RY3 element,followed by RY2 and RY1.