Cloning And Functional Analysis Of HMGR Gene In Taraxacum Kok-saghyz Rodin

Object Natural rubber is a biopolymer with exceptional qualities that can’t be completely replaced using synthetic alternative. Natural rubber biosynthesized via the isoprenoid pathway by domestic plant sources, such as Rubber tree, Eucommia ulmoides, Partheninu argentatum(guayule), Taraxacum kok-saghyz Rodin(Russian variety) etc. Rubber-producing TKS plant has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. TKS plant synthesises high-quality natural rubber(NR) in there roots, and they are the more economically sustainable with improved yields and so were apromising aternativ global source of this raw material. The objective of this study is to modify isoprenoid production in TKS through overexpression of the HMGR gene. Because of this, it was of great practice significance to increase the rubber concent in TKS through overexpressing the enzyme genes in rubber bio-synthesis.Methods Based on the construction of high frequency regeneration and genetic transformation system of TKS. Tk HMGR gene and Hb HMGR1 promoter are resperctively cloned from TKS and Rubber Tree, and the study analyses the HMGR. The prokaryotic expression vector for this gene was constructed and then successfully induced to express by IPTG in BL21. Cell fusion expression vector was construted, conwersing GV3101 and infecting the onion cell, and gene expression is observed in the confocal microscope. The HMGR and Hb HMGR promoter were transformed into TKS and expression location were cattyed out. It was showed that the gene had integrated into the genome of TKS, and was analysised in the transgenetic TKS.Results(1) Tk HMGR gene and Hb HMGR1 promoter are resperctively cloned from TKS and Rubber Tree, the HMGR gene contains 1749 bp basic group, codes 582 AAs, andv HMG-Co A reductase belongs to the family of HMG-Co A reductase member.(2) A transformation plasmid PET30a-HMGR was constructed with a vector of the HMGR gene. Results of SDS-PAGE show that the specific fusion protein was successfully induced to express by IPTG, and the molecular weight is 62.6591 KDa, the expression of 6h is the best.(3) Cell fusion expression vector was construted, and according to the result of test, the fusion protein of HMGR-GFP mainly locate in the cell membrane and nuclear membrane.(4) Optimized protocol of TKS was developed. The best explant for tissue culture is petioles, the optimum induction medium of Adventitious bud elongation growth is 1mg/L 6-BA and 0.1mg/L NAA, and the most suitable mudium for adventitions rooting is 0.1mg/L NAA.(5) Overexpression vectors of p CAMBIA-2300-35S-HMGR and p CAMBIA-1303-Hb HMGR1-HMGR were constructed, and transgenetic TKS was transformed via leaf disc transformation. We obtained the genetic modified TKS after the PCR, and analysised via Real-Time PCR, and the tesult showed that the gene had integrated into genome of TKS and were transcribed on RNA level.(6) The terpen and rubber were extracted from genetic TKS, and was analyzed and measured. Results show that the overexpression of HMGR gene doesn’t improve the rubber content, but the premise material of rubber had increased significantly, which laid a foundation for follow-up study of increasing rubber production.