Cloning And Transformation Of IFS, CHS Genes Of Trifolium Pratense Minshan

ObjectivesIn the present work, in order to obtain the new trifolium pretense lines of high-yielding isoflavone,we cloned IFS and CHS gene, transformed them into trifolium pretense callus.And by genetic engineering means into the minshan trifolium pretense, obtain high expression of IFS trifolium pretense callus,which induced by tissue engineering for IFS gene expression of transgenic plants. And construct the CHS-LA-IFS gene cluster.MethodsTrifolium pretense minshan was used as materials, cotyledon, hypocotyl, Root and petioleas explants.The IFS and CHS gene were cloned from Minshan trifolium pretense callus using RT-PCR. Isoflavone content of the trifolium pretense and the trifolium pretense callus were determined by HPLC. Sequence analysis indicated that IFS and CHS gene has a high homology with the same gene of other plants. After comparition and evolution analysis, the gene was ligated into expression vector pRI101-AN-IFS and pRI101-AN-CHS. And introduced IFS gene into agrobacterium tumefaciens strain LBA4404, The IFS overexpressing callus was acquired through introducing LBA4404harboured pRI101-AN-IFS vector into trifolium pretense callus.Results1. The callus could be obtained at the temperature of25±2℃with MS medium, which contains sucrose30g/L,2,4-D2mg/L,6-BA0.5mg/L and powered agar8g/L (pH=5.8). HPLC determination of isoflavone content in the callus was far higher than that of the west, that in cotyledon callus induction of callus in the highest levels of isoflavones.2. The IFS and CHS gene were cloned.The was constructed plant binary expression of pRI101-AN-CHS and pRI101-AN-IFS. By the freezing and thawing method respective-ly two carrier into agrobacterium LBA4404, successful transformation.3. CHS-LA-IFS series gene cluster was acquired by overlapping PCR cloned from2A and LP4before nine peptide amino acid sequence of connection link of CHS-LA-IFS series IFS gene cluster. Build PMD18-T-CHS-LA-IFS carrier.4. Transformation of agrobacterium LBA4404into trifolium pretense.After comparition and evolution analysis, the gene was ligated into expression vector pRI101-AN-IFS and introduced into agrobacterium tumefaciens strain LBA4404, The IFS overexpressing callus was acquired through introducing LBA4404harboured IFS-pRI101-AN-IFS vector into trifolium pretense callus. HPLC measured the content of transgenic callus isoflavone higher than untransgenic callus.ConclusionsEstablished the minshan trifolium pretense callus induction system, as the next callus isoflavones as a raw material for industrial production provide a theoretical basis.Cloned minshan trifolium pretense CHS, IFS gene and CHS-LA-IFS gene cluster, so as to increase the red clover isoflavones content and lay the foundation for genetical engineered.Success will be the IFS gene into the minshan trifolium pretense callus, obtain the IFS gene expression of transgenic callus, and induce engineering seedlings at the same time. To cultivate high yield new strains and isoflavones in red clover isoflavones bioengineering research lay the foundation.