RNAi Vector Construction of FAH12、FAD2and FAE1Gene and the Transformation of Castor(Ricinus Communis L.)
|School||Hunan Agricultural University|
|Keywords||Ricinus communis High oleic acid RNAi vector Regeneration system Genetic transformation|
Castor seeds contain50%-60%castor oil which is an ideal lubrication oil widely used in machinery, aerospace, plastics and other industrial field. However, as a new raw material for biodiesel, castor oil requires high oleic acid content. Therefore, it is an effective way to further castor as a raw material for biodiesel applications by blocking the down stream of oleic acid biosynthetic to accumulate oleic acid through RNAi technology. In this study, fragments of three key genes involving the synthesis of ricinoleic acid（FAH12）, linoleic acid（FAD2） and long chain fatty acids （FAEl）were cloned to construct a seed-specific expression vector that can interfere these three genes at the same time. Then, the triple-gene RNAi vector was transformed into castor. The main results were as the follows:1. Primers were designed according to FAH12gene （XM002528081）, FAD2gene （XM002530658） and FAE1gene （XM002531584） in GeneBank. Three conservative sequences were cloned, one was162bp from FAH12gene, one was250bp from FAD2gene and the last was332bp from FAE1gene. Then, this three fragments were ligated together to form a longer complex fragment named Fahde.2. A seed-specific expression vector that can interfere three genes at the same time in castor have been constructed for the first time:the complex fragment Fahde was inserted to pFGC1008plasmid with a seed specific expression Albumin promoter in forward and reverse direction respectively, to construct a RNAi vector which was used to interfere the three gene at the same time.3. A regeneration system for Ricinus communis was established:the hypocotyl of Zi NO.5and Xiubi NO.2mature seeds were used as explants. Fasciculate buds forming adventitious buds were induced on optimized MS medium with TDZ. An average of4.0normal buds was produced on each explant. The plantlets developed from vigorous buds by elongating and rooting survived. The amounts of adventitious buds as well as the capacity of root differentiation produced in the two selected castor species showed few differences, which reveals that there exists no significant differences between the two selected castor genotypes though the establishment of in vitro regeneration system.4. The preliminary construction of genetic transformation system for Ricinus communis:embryo axes pre-cultured for5days under dark conditions were excised. Then they were immersed in bacterial suspension （OD600=0.2-0.5） for10min. Subsequently, the explants were co-cultivated for3days. Following co-cultivation, the explants were transferred to the resistant screening medium containing20mg/L Hyg. The whole process takes about75days. Resistant seedlings were proved to be positive transgenic plants by PCR.5. Results of genetic transformation:we obtained64resistant seedlings in total and one transgenic plant, the rate is5.12%and0.08%respectively.