Research On Expression Regulation And Management Of Maize Starch Branch Enzyme Genes

The purpose of this research is to increase the content of direct-chain starch in maize. With genetic engineering method, the SBE gene encoding a starch branch enzyme and SBE promoter were cloned, a highly efficient antisense expression vector and siRNA expression system were constructed. Then the gene constructs were introduced into maize inbred lines mediated by Agrobacterium mediation and pollen tube passage method in order to restrain the expression of starch branch enzyme and regulate the biological composing process of starch. The main results are as fellow :1.Using the genomic DNA of maize variety "Jin Nuo 1" as template, the SBE promoter was isolated by PCR and was cloned into pMD18-T Vector. The size of this promoter is 934bp. Sequencing analysis of this SBE promoter through DNASIS soft ware showed that the cloned fragment contained all the essential motifs which only expressed in seeds, such as TATA motif, CAAT motif, TACACAT motif, CATGCATG motif, GTAAAAGT motif, GCN4 motif and ACGT motif. Only 14 nucleotides showed changes comparing to the reported sequence and homologous rate is 98.50%. To construct the SBE promoter-GUS chimera gene, the SBE promoter was then cloned into expression vector pBI121 to replace the 35S promoter, and inserted upstream of the GUS gene encoding region. We then introduced pSBE-gus into tobacco by Agrobacterium mediation. Analysis of gus activity showed that the gus only expressed in seeds. This confirmed that the cloned promoter is a seed specific promoter.2. By using ordinary corn of inbred line 7922 as experimental material, the transformation conditions of Agrobacterium mediation were optimized. After 10 days of pollination, Infantembryos were nanspiamed onto the MB improved medium and most embryo callus inducedshowed bright, firm and light yellow . The rate of callus induction was 82.5% and that of embryo callus induction was 39.16%. After two weeks cultivation, the callus were transplanted into regulating and managing medium. The concentration of 2,4-D was 2mg/L. The changing rate of callus was 45.716%. The time of Agrobacterium contamination was 25 minutes, the concentration of Agrobacterium was 0.6 OD600 and the time of cocultivation was 3 days. The rate of transformation was 3.3%.3. Using PCR technology, the 1698bp gene encoding a maize branch enzyme was amplified and cloned into pMD18-Tvector. The array analysis of DNASIS soft showed that the sequence of gene was the same with the genes of those publicized before. We then insertedthe DNA fragments of gene downstream of the 35S promoter in reverse orientation, and cloned into pCAMBIA 1301 to generate recombinant plasmid pCJS2b,Then it were introduced into maize inbred lines by Agrobacterium mediation. To identify the transgenic plants, using geromic DNA of the regenerated plants' leaves as template, PCR amplification was performed with 35S promoter primers. The 1800bp PCR product was obtained respectively from the transgenic plants. While this product disappeared from those control plants that have not been transplanted. Then, Southern blot analysis was performed to further identify the number of copies that of gene integrated into the geromic DNA of the transgenic plant, And the result indicates that hybrid signal occurred in both the transgenic plant lines with one or two copies, respectively. But there was no hybrid signals shown in plants that hadn't been transplanted. We can infer from that the introduced genes can be descended in the following generations. And the separating rate fits to Model's genetic law. The total RNA was isolated from transgenic maize seeds and subject to Northern blot analysis using SBE cDNA as probe labelled with DIG, and then we found that the expression of SBE mRNA significant decreased..4.The second fragments 935bp of maize starch branch enzyme was also obtained by PCR using gene-specific-primers, then the anti-sense and sense gene fragments were inserted upstream of 35S promoter and cloned into pBI121 to construct RNAi expression vector pCJSBE2b. We introduced it into maize inbred lines by pollen pipe passage method and obtained TOgeneration transgenic seeds. When the seeds sprouted, using the genomic DNA from single plant's leaves as template, and PCR amplification was performed with 35S promoter primers. The 2500bp PCR product was obtained respectively from three transgenicpiaiiu>. vv iiuc ima jjiuuuci uisappcdicu iiuin muse coliuui piaius Uicu nave nui utcntransplanted. Then, Southern blot analysis was performed to further identify the number of copies that of gene integrated into the geromic DNA of the transgenic plant, And the result indicates that hybrid signal occurred in all the three plants. Combining the locating conditions of expression substance Hindlll enzyme cutting positions, we can infer that the inserted copy numbers of introduced genes are 1, 1 and 2 respectively. No hybrid signals occured in plants that had not been transplanted. And that indicates that introduced genes have been intergrated into the genomic DNA of transgenic maize. The PCR results of the TI generation transgenic plant indicates that SBE gene descended in the following generation. And the separating rate fits to Model's genetic law. The total RNA was isolated from transgenic maize seeds andsubject to Northern blot analysis using SBE cDNA as probe labelled with DIG, and we found that the expression of SBE mRNA sharply decreased. This result indicates that the RNAi technology inhibited SBE gene expression and lead to the decrease of origin starch branch enzyme mRNA.5. From the generation transgenic seeds, the activity of Starch branch enzyme was assayed using the dividing ray method. The result indicates that the activity of branch enzyme of transgenic maize was 4.895U; 7.3945U; 4.8015U; 9.611U and 10.524U, which decreased by 85%; 78%; 85%; 71%; 68% When compared to the 33.285U of control. And the activity indicates that the insert of introduced has efficiently restrained the expression of starch branch enzyme.6. The content of direct chain starch and branch chain starch were also assayed from TO generation transgenic maize using double wavelength method to test. The result of transgenic starch content indicates that the total starch content are 680mg, 690mg, 673mg and 687mg(net content),respectively. There is basically no sharp change compared to the control 693mg/g (net content). The content of direct chain starch has been raised obviously (that of the origin one is 23%) and reached respectively 50%; 47%; 51%; 43%; 41%. That indicates the use of anti sense and RNAi technology has effectively regulated the combination process of corn starch, and raised the content of direct chain starch.