茶树N-甲基转移酶基因启动子克隆及功能分析

doi:10.13305/j.cnki.jts.2018.06.003

Abstract

Abstract: Caffeine is an important bioactive compound in tea plant, and N-methyltransferase (NMT) is the key enzyme for its biosynthesis. In this research, the promoter of NMT1 was isolated from Camellia sinesis Yinghong 9 by hiTAIL-PCR, and its cis-elements were analyzed by Plant CARE and other online software. According to cis-elements composition, serial 5' shorten promoter were re-amplified from NMT1 promoter and cloned into pBI121 vector fusion with GUS gene respectively. The constructed vectors then transformed into tobaccos. The function of cloned promoter and its response to environmental factors were analyzed by GUS staining and quantitative PCR. The results showed, the isolated NMT1 gene promoter was 767βbp, containing basic cis-acting elements such as TATA-box, CAAT-box and some other response elements involved in the plant abiotic stress. Using recombinant technique, the CaMV35S promoter in pBI121 was replaced by the 5' shorten NMT1 promoters, resulted in 4 new vectors fusion with GUS and marked as pA, pB, pC and pD. The transient expression with the four constructed vectors in tobacco leaves revealed that NMT1 promoters with different lengths all could regulate the expression of GUS, and the activity of GUS enhanced along with promoter length increased. The pA vector which owned full cloned NMT1 promoter was selected for transgenosis and successfully gained transgenic tobacco. Expression of GUS could be detected in different tissues of transgenic tobacco, The expression level followed leaf > stem > root and the expression level in leaves reached 3 times higher than that in roots. When transgenic tobacco treated with light, temperature, simulated drought and abscisic acid, there were significant changes in the expression of GUS in leaves, except at the temperature of 40℃. The results showed that the function of NMT1 promoter was influenced by the environmental stresses.

Key words: tea plant, N-methyltransferase, promoter, hiTAIL-PCR, GUS gene

CLC Number:

S571.1
Q52

LIU Ping, REN Qiujing, KANG Xin, ZHANG Yuanyuan, LIN Xiaorong, LI Bin, GAO Xiong, CHEN Zhongzheng. Isolation and Functional Analysis of Promoter for N-methyltransferase Gene Associated with Caffeine Biosynthesis in Tea Plants (Camellia sinensis)[J]. Journal of Tea Science, 2018, 38(6): 569-579.

References 31

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Isolation and Functional Analysis of Promoter for N-methyltransferase Gene Associated with Caffeine Biosynthesis in Tea Plants (Camellia sinensis)

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