Cloning And Functional Analysis Of Arabidopsis β - Glucosidase 19 Gene Promoter

When improving the crop resistance, the yield and the quality, we should move the exogenous genes into the particular crop, and the exogenous genes need to be expressed under the regulation of the gene promoters. Due to the limited number of plant promoters specially used for genetical improvement, the research focus on a promoter of the Arabidopsis β-Glucosidase 19(Beta-glucosidase 19, BGLU19) gene to obtain a suitable gene promoter, which can be used for the crop genetic improvement and the plant genetic engineering.In this study, the sequence structure of the β-Glucosidase gene promoter was analyzed, and the elements were predicted. The specific primers were designed to clone the AtBGLU19 gene promoter, and the plant expression vector was constructed. Then the construct was introduced into Agrobacterium tumefaciens strain EHA105, and from there to A. thaliana using the floral dip procedure. Transgene homozygotes, selected in the T3 generation, were used for the analysis of GUS expression. At last, the activities of the transient and stable gene expression directed by the AtBGLU19 promoter were analyzed by GUS histochemical staining. The results showed that the AtBGLU19 promoter directed high expression of the GUS reporter gene in the seed and could be expressed in the heterologous plant. In a word, we provided a suitable plant promoter for crop genetic improvement through this study. The main results that we obtained are as follows:1. We screened high specificity expressed genes in the seed by using the bioinformatics methods, and analyzed the expression data of the AtBGLU19 genes by using AtGenExpress Visualization Tool and Genevisible Expression Data. We discovered that AtBGLU19 gene was highly expressed in seeds. Therefore, the AtBGLU19 promoter may be a seed specific promoter.2. Analysis of the AtBGLU19 promoter by using manual search, PLACE and PlantCARE online softwares indicated that this promoter contains many cis-acting elements, which were associated with tissue specific expression and responsed to abiotic stresses. In this study, only three seed-specific regulatory elements were analyzed, which were the GCN4 element(TGAGTCA), AACA element(AACAAAA) and ACGT element(GTACGT). We also found the other cis-acting elements, which were responsed to low-temperature, ABA or drought stresses, such as four ABA response elements(ABRE element: TACGTG, GCCACGTACA, CGTACGTGCA and GACACGTGGC), one as-1element(TGACG) and one ACGTCA element which were related to cold stress, and a MBS element(TAACTG) which was in response to drought stress.3. The specific primers were designed to clone the AtBGLU19 promoter sequence by PCR amplification. The resulting PCR fragments were digested and inserted into the plant expression vector to generate the plasmid pBGLU19-GUS. The results of restriction enzyme digestion and DNA sequence analysis showed that the AtBGLU19 gene promoter was cloned and the pBGLU19-GUS plant expression vector was constructed successfully.4. We transformed the pBGLU19-GUS plant expression vector into the Arabidopsis thaliana, and obtained the T3 homozygous seeds by screening in antibiotics. The seeds, leaves, flowers and siliques of the T3 homozygous plants were examined by GUS staining experiments. The GUS staining analysis showed that the GUS gene was highly expressed in the seeds. So the AtBGLU19 gene promoter was a seed specific expression promoter.5. In this study, Agrobacterium suspensions transformed the pBGLU19-GUS plant expression vector were infiltrated into the leaves of 5-week-old N. benthamiana plants, and transient expression was monitored 48 h after infiltration. GUS expression analysis was carried out by staining samples with X-Gluc. GUS histochemical taining results showed that the tobacco leaves which injected into pBGLU19-GUS plasmid were stained blue by X-Gluc solution, and the wild type tobacco leaves were not. Therefore, our results showed that the AtBGLU19 gene promoter could direct GUS transient expressions in the heterologous tobacco system.6. The pBGLU19-GUS plant expression vector was also used to obtain transgenic tobacco plants by Agrobacterium-mediated leaf disc transformation method. After selection on hygromycin containing medium, several plants were regenerated.