| 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 CaMV 35S 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 differential expressing promoters which have many advantages such as expression in specific tissue, expression at a certain growth stage, high efficiency of gene expression, avoidance of many disadvantages and so on. Although many differential expressing promoters have been successfully cloned in the past years, just a few of them are ideal and have been applied in practice. Maize starch branching enzyme sbeâ…¡b described in this paper is a king of seed differential expressing promoter. Starch branching enzyme that has three isozymes sbe â… , sbe â…¡ and sbe â…¡ b among which sbeâ…¡b has more impact on the content of endosperm amylose is one of the key enzyme in controlling the starch synthesis process. So how to improve the expression level of sbeâ…¡b has become a hotspot in plant genetic engineering. At present researches on the promoter of sbeâ…¡b gene of barley have been found, but there is barely no references about researches on the promoter of sbeâ…¡b gene of maize. This paper aimed to use tissue-specific promoter sbeâ…¡b to replace constitutive promoter CaMV 35S and construct a new plant expression promoter so that the new promoter can start-up and improve specific expression of Maize Starch Branching Enzyme genes in plants, which can increase content of Amylose and improve the quality of maize starch.The genomic DNA of plain maize, yellow waxy corn and white waxy corn were used as templates respectively. According to the known sequence of promoter of sbeâ…¡b gene (GenBank:AF072725), a pair of primers was designed, and partial sequences of promoter of sbeâ…¡b gene were cloned through LA-PCR amplification by using different templates. As the sequence amplified from the template of yellow waxy corn was more specific, we chose it as further study. The sequence of promoter of sbeâ…¡b gene was firstly cloned into pMD18-T Vector and recombinant plasmid. After transformed by Escherichia coli and selected by blue-white-spots method, 17 white spots and 32 blue spots were got.The same length of fragment as aimed fragment was got after extraction of positive clone, PCR amplification and enzymes digestion identification. Then identified bacterium was sent to Dalian Takara Lt Co. to analyze sequence and analyzed by using biological software DNAMAN. The resultindicated that the fragment inserted into the vector whose length was 934bp was the one that we previously designed. But only 14 mutations were found, and the homology was 98.52%. Among the sequence, basic cored sequences TATA-box ( TATAATA) and CAAT-box which can only found in the sequences of promoters of higher plants were found. And 6 copies of GC box as the basic controlled element of eukaryotic promoters were found. Furthermore some specific elements that should exist in seed specific expression also emerged in the sequence such as endosperm expression cis-acting element GCN4 (TGAGTTTC) located at -865bp, factor of seed specific effect element RY-element (CATGCA) located at -113bp, the essential element of endosperm specific promoter EM-motif located at -279bp* sequence AT-rich that has appeared many times and can improve the specific expression. ACGT which has great effect on endosperm expression located at -687bp and so on.The recombinant plasmid and plant expression vector pBI121 were digested byrestriction enzymes BamH I and Hindlll. Plant expression vector pSBE-GUS whichused tissue-specific promoter sbellb to replace constitutive promoter CaMV 35Slocated at the upstream of the GUS gene was constructed by using T4 DNA ligase.After the identification of enzymes digestion, it had been proved that the sequencehad been successfully inserted into the vector. Then plant expression vectorpSBE-GUS was transformed into competent Escherichia coli cells and cultured inorder to increase the quantity of the clones. After that DNA of the plasmid wasextracted, and then the vector was transformed into competent Agrobacteriumtumefaciens cells EHA101 and EHA105 respectively. Agrobacterium tumefacienstransformation was used to transform the promoter of the sbe II b gene into tobaccoand make the untransformed tobacco as negative control. The leave of the transformedtobaccos was co-cultured for 2 days, then delivered to the selection mediumscontaining Kan and Cef. Five transformed tobaccos were got. It was found that whileculturing the Agrobacterium tumefaciens EHA101 and EHA105, the best conditionfor transformation was that the concentration measured by OD600 was 0.5 to 0.6 andkept the wound tobacco leave in the transformed liquid bacterium for 6 minutes.While choosing the untransformed tobacco as negative control,it could be also foundthat the transformation efficiency of EHA105 was better than EHA101. The fivetransformed tobaccos were detected by PCR, and the result was that all of them couldbe amplification the same length of the sequence. Then the positive tobaccos weredetected by southern blot, while untransformed tobacco was chosen as negativecontrol and the digestion of the plasmid with BamH I and Hindlll enzymes aspositive control. The result was that four of the five tobaccos had remarkable bands,which indicated that the aimed gene had successfully integrated into the genome oftobacco.In order to test whether the functions of the promoter > the properties of promoter and its expression level, the tissue chemical analysis of GUS was made in different parts of the tobacco such as seeds > roots ^ stems and leaves transformed by Gene Gun Method and Agrobacterium tumefaciens transformation respectively. The number of blue spots decreased in the order- seeds > leave > stems and roots, and the spots of the stems and roots could barely been seen, which indicated that the promoter ofsbe II b gene was probably expressed specifically in seeds. Then the activity of GUS enzyme was detected by using fluorescence detection, and the same result had been got, which indicated that sbe II b gene could promote GUS gene to express in seeds specifically. This research not only promotes the discovery of the cis-acting element of the Spatio-temporal Expression of sbe lib gene, but also provides base for reconstruction of the plant expression vectors.
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