Study On Cloning And Function Aanlysis Of StNCED1 Gene In Potato

As the rate-limiting enzyme of ABA biosynthesis, 9-cis-epoxycaro-tenoid dioxygenase(NCED) could regulate and control ABA biosynthesis. Neoxanthin cracked into zeaxanthin aldehyde by catalytic reaction of NCED which is a first C15 in-termediate and play a significant role in ABA biosynthesis. In this study, potato cultivar “Longshu 3” was used as dornor, special primers for StNCED1 gene were designed according to the sequences of StNCED1 gene registered in GenBank. StNCED1 gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR). The over-expression vector pBIC-StNCED1 and interference expression vector pCBP121-amiRNA were condtructed and transformed them into potato cultivar “Longshu 3” by Agrobacterium tumefaciens-mediated method. Quantitative real-time polymerase chain reaction(Qrt-PCR) was used for determinating the relative transcription level of StNCED1 gene in microtuber of the transgenic plants. High performance liquid chromatography(HPLC) was used to measure ABA content of transgenic microtuber. The result of the study could provide foundation for further research of function of StNCED1 gene. The research results are as following:1. The full-length cDNA of StNCED1 gene was amplified by RT-PCR, which was 1952 bp and CDS was 1851 bp. The homologous identity analysis showed that homology of potato StNCED1 gene was 95% with tomato slNCED1 gene. The amino acid sequence analysis of NCED showed that it contained 603 amino acid residues, the weight of total molecular was 67.132 KD, and PI was 6.16. The highest homology of protein was 98% with tomato.2. The over-expression vector pBIC-StNCED1 was constructed by ligating StNCED1 gene with expression vector pBIC. The mature sequence of amiRNA was designed using WMD3 software platform. The precursor sequence of amiRNA was obtained using multiplex PCR method. The interference expression vector pCBP121-amiRNA was contructed by ligating the precursor sequence of ami RNA with expression vector pCBP121.3. The putative transformed plants of potato cultivar “Longshu 3” were obtained by Agrobacterium tumefaciens-mediated method trasformd with pBIC-StNCED1 and pCBP121-amiRNA. Five transgenic plants were obtained from pBIC-StNCED1 transformation and 6 transgenic plants were obtained from pCBP121-amiRNA transformation after kanamycin screening and PCR analysis.4. The microtubers were obtained from the transgenic plants and further used to determine the relative expression of StNCED1 gene and ABA content. The result showed that the relative expression amount of StNCED1 gene in the transgenic plants 1-5 increased 2-8 times, and ABA content increased 1.10-1.76 times compared with the control plants. The relative expression amount of StNCED1 gene in the transgenic plants 1-6 transformed with pCBP121-ami RNA decreased 53%-85%, and ABA content decreased 34%-51% compared with the control.In conclusion, NCED synthesis could be regulated by controlling the expression of StNCED1 gene, and further regulating ABAcontent. The result demonstrated that StNCED1 gene played a significant role in ABA biosynthesis, and provided foundation for further elucidate the function of StNCED1 gene.