Cloning And Reliminary Analysis Of FPS And CPT1 Gene In Taraxacum Kok-saghyz Rodin

Taraxacum kok-saghyz Rodin is asteraceae perennial herbaceous plants, the roots of perennial plants contain up to 20% rubber,which structure and function are similar to Hevea brasiliensis rubber. TKS is a rubber production plant which has good development prospects, and also can be used to study the mechanism of rubber production glue as a model plant. Planting TKS, not only obtains the important strategic materials of rubber, but also harvests clean energy of alcohol and health products of inulin, It is accepted that TKS is one of the promising second natural rubber to commercial exploitation. Nowadays TKS has high production costs and low Rubber content, limit its application fields.Because of this, it is of great practical significance to lower down the cost of TKS rubber and promote its wide employment by increasing the rubber content in TKS through overexpressing the enzyme genes in rubber bio-synthesis.This thesis focuses on increasing rubber content through overexpressing TkFPS and TkCPT1 genes in rubber metabolic pathways. The results of the study are as follows:(1) Cloning, sequence analysis of the farnesyl pyrophosphate synthase gene and the cis-prenyltransferase gene from Taraxacumkok-saghyz Rodin, and the prokaryotic expression vector for these gene was constructed. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG..(2) Analysis of the FPS and CPT1 gene expression in different tissues and roots at different times in TKS. The results showed that TkFPS and TkCPT1 genes are expressed in the roots, petioles, leaves,pedicels, flowers, seeds, the highest of the Tk FPS expression in leaves, lowest in flower; The highest expression of TkCPT1 gene was in seeds, the lowest was peduncle. Meanwhile, it is the highest expression level of TkFPS and TkCPT1 gene when rubber grass grows to 6 months.(3) Transgenic plants were obtained through Agrobacterium tumefaciens-mediated transformation.PCR detections demonstrated the transgenic statuses of 6 independent plants transformed with FPS, 9with CPT1.(4) Real-time PCR showed that the expression levels of FPS and CPT1 were up-regulated in transgenic plants analyzed.(5) Extracted rubber through modified alkali boiling to detect the rubber content in transgenic plants,the results showed that, transgenic FPS gene plant growth slow,which have high rubber content but low total rubber content in the root; rubber content in the transgenic CPT1 gene plant were higher than wild type.In conclusion, FPS and CPT1 gene play an important role in rubber synthesis, it could improve rubber content through overexpressing Tk FPS and TkCPT1 genes in rubber metabolic pathways.