Cloning And Functional Analysis Of The Promoter Of Storage Protein Gene In Soybean

Nowadays the main problems existed in gene engineering are around problems such as the expression level of exogenous genes,the expressing position of exogenous gene and so on. The research on promoters as one of the most important elements in gene expression controlling has become the key work while solving problems in gene engineering. There are three kinds of promoters that are constitutive promoter, tissue-specific promoter and inducible promoter. So far CaMV35S a kind of constitutive promoter is widely used in plant expression vectors. Its prominence characteristics is that it can result in the expression in every position and at every stage of plant growth, which to certain extent can be regarded as a kind of waste of energy and can also lead to changes in plant shape and influence plant growth. So that people have paid more attention to the research and application in specific expressing promoters. Specific promoters direct exogenous gene orientation express in presumptive time and space(tissue or organ).Not only can cause the expression product of target gene accumulate in a certain space and increase the district expression quantity, but also make the expression of exogenous gene in the control of human. Ensure the exogenous gene effective express and reduce harmful effects to plant. It is considered that to use specific promoter is a very important method to get high express target gene ,and it is also the first choic to culture efficient and safe transgenic plants.11s glycinin is the main storage protein in the seed of soybeans, encoded by multigene family, So far seven soybean glycinin genes have been cloned and sequenced already, Including from Gyl to Gy7. The storage protein in the seed of soybeans have the function with the seed-specific time sequence expression. Ripe middle and later periods instantaneity formate and store on seed protein among the body in a large amount in seed. All sorts of the function that specific time sequence expression these mainly depend on seed storage protein genetic the upper reaches adjust and control arrays. Therefore the promoter of the storage protein become the first-selected material which has become specific expression of the higher plant gene of studying and adjusted and controlled. The upstream regulatory region of the storage protein was separated from Glycine max variety "Ji Nong T7018" genomic DNA, according to published glycinin 11S G7 subunit (Gy7) gene(GenBank AF319776) sequence, a pair of primers was desgeined, in order to amplify the array before Gy7 gene initiation codon (ATG). Then cloned into pMD18-T Vector, transforms E.coli competence cells and selected by blue-white-spots method. Chosed the white spot and extracted plasmid by alkaline lysis, by PCR and restriction enzymes digested identification. Then identified strain was sent to Dalian TaKaRa biotech Co.,Ltd to sequencing. The result indicates the length of the clone sequence is 963bp and shares a similarity of 45.24 % with the published Glycine max glycinin subunit G7 (Gy7) gene promoter.but have more similar to quantity and distance of promoter elements, they all contained typical TATA-box, CAAT-box and necessary regulatory motifs of seed-specific expression.Take this promoter constructed into plant expression vector pBI121 to substituted for the CaMV35s promoter, linked with GUS(β-glucuronidase) reporter gene, thus a new type recombinanted plant expressign vector pGy-GUS obtained. The plasmid pGy-GUS pBI121 was transformed into Agrobacterium tumefaciens LBA4404 competence cells by LN freeze thawing method. Then transferred to tobacco variety NC89 by Agrobacterium-mediated transformation. After cultivated in the selection mediums containing Kan and Cef, six transformed tobaccos(carrying pGy-GUS construct ) were got. The six transformed tobaccos were detected by PCR,the plasmid pGy-GUS as positive control ,non-transformed tobacco plant as negative control, and the result showed that all of them could be amplification the same length of the sequence with positive control. Then the positive tobaccos were detected by southern blot.The result showed that four of the six tobaccos had remarkable bands, which indicated that cloned promoter had integrated into the genome of tobacco, successfully.In order to verificate whether that cloned promoter has the function of specificity expression in seeds, We have analysed the diffent tissues(including root, stem segment ,leaf and seeds ) of transgenic tobacco plants by Histochemical staining of GUS activity. The result showed: There were non-coloration in root, stem segment and leaf of transgenic tobacco plants carrying the pGy-GUS construct, but have the number of blue spot in seeds, manifest seed specificity, markedly. So we confirmed that GUS gene controlled by this promoter gave specific-expression in seeds. The completion of this thesis not only offers theoretical evidence to the study of expressed regulatory mechanism for soybean storage protein gene, but also produce useful protein or other metabolites in differential part or developed step at plants, thus we can timed, fixed-point, quantitive stereo-precise regulatory the expression of exogenous genes.