Cloning And Functional Analysis Of GGPPS Gene In Taraxacum Kok-saghyz

Taraxacum kok-saghy, compositae mouse-ear perennial type perennial root herb, and Hevea brasiliensis, guayule called three largest rubber-producing plant all over the world. The nature rubber on structure and performance from T.kok-saghy root and Hevea brasiliensis are fairly. From T.kok-saghyz root, natural rubber is not only extracted, as a important strategic material, but also the clean energy ethanol and health products inulin can be harvested. It is accepted that T.kok-saghyz is one of the promising second natural rubber to commercial exploitation. Through genetic engineering methods, to improve rubber content in T.kok-saghy, will have a vital significance on resolving the shortage of rubber resources. But T.kok-saghyz, as a kind of herb adhesive production plants, has low rubber content which is difficult to collect and high production costs, which limits its application scope.Therefore, Tk GGPPS expression was altered in T.kok-saghy by over-expression methond, to change its original expression level, speed up the synthetic rubber, this improve the rubber production. On the other hand, Tk GGPPS expression was altered using RNA interference methods, to research the role of GGPPS gene in rubber biosynthesis, which will provide certain theoretical guidance and technical support for further study to synthetic mechanism of natural rubber. The main research content were as follows:(1)A new c DNA sequence encoding GGPPS(designated as Tk GGPPS) from T.kok-saghyz was cloned by RT-PCR and RACE techniques and assayed by bioinformatics. The c DNA sequence of Tk GGPPS was of 1140 bp encoding 379 amino acids; Sequence analysis showed that Tk GGPPS was one of the members of short chain prenyltransferases super family; Amino acid sequence homology alignment and phylogenetic analysis showed that the similarition of Tk GGPPS and Ha GGPPS from Helianthus annuus were more than 74.67%.(2)Tk GGPPS gengs expression in different groups and different development period in T.kok-saghyz was detected by the q RT-PCR technology. The results showed that Tk GGPPS genes expressed in 6th-week T.kok-saghyz root and mature seeds significantly. In addition, Tk GGPPS induced by methyl jasmonic acid(Me JA), and millimoles concentration is 3 mmol·L-1, relative expression of Tk GGPPS reached the maximum.(3)Tk GGPPS over-expressing vector and Tk GGPPS-RNAi vector were constructed, respectively.leaf and petiole of T.kok-saghyz were converted by agrobacterium mediated transformation method successfully to obtain genetically modified T.kok-saghy. Through PCR test, the positive transgenic plants of Tk GGPPS over-expressing lines was five, and Tk GGPPS-RNAi lines was four.(4)Tk GGPPS over-expressing lines had slightly higher levels of rubber in their roots and of photosynthetic pigment in their leafs, relative to the control, Tk GGPPS-RNAi lines showed significant decreases in root rubber content and leaf photosynthetic pigment content.(5)Relative transcript level of Tk GGPPS was detected by Real-time quantitative between Tk GGPPS over-expressing lines and Tk GGPPS-RNAi lines. It turned out that the expression level of Tk G-GPPS over-expressing lines was up-regulated, and the expression level of Tk GGPPS-RNAi lines was down-regulated.(6)The TkGGPPS-GFP fusion expression vector was constructed. Based on Agrobacterium mediated transformation method, the onion epidermal cells was converted. The result showed that Tk GGPPS was located in the cell wall.(7)The promoter sequence of geranylgeranyl pyrophosphate synthase(GGPPS) gene from T.kok-saghyz was obtained by thermal asymmetric interlaced PCR(TAIL-PCR). It was designated as Pro Tk GGPPS and the full length of the fragment is 1131 bp. The sequence which was analyzed by Plant Care software, showed that the promoter sequence contained the basic cis-acting element: TATA box and CAAT box. In addition, the promoter sequence contained multiple Cis-acting elements involved in light-regulated responsiveness(ACE, Box I, ATC-motif and so on) and a number of stress-related cis-acting elements(HSE, ARE, LTRE and so on).(8)The p CAMBIA1304-Pro Tk GGPPS-GFP plant expression vector was constructed. Based on Agrobacterium mediated transformation method, the onion epidermal cells was converted. The result showed that the promoter could drive the downstream reporter GFP gene expression.Here, The c DNA sequence of Tk GGPPS gene and its promoter region were cloned from T.kok-saghyz. Functional studies showed that GGPPS gene expresses in different groups and different development period, and associated with the biosynthesis of rubber.