茶树N-甲基转移酶基因启动子克隆及功能分析

咖啡碱是茶叶中重要的功能物质,N-甲基转移酶（NMT）是其生物合成的关键酶。本研究以英红9号茶树新梢为材料,利用hiTAIL-PCR克隆NMT1基因启动子,并采用Plant CARE等在线软件分析其顺式作用元件。根据其元件组成,设计5'递减的启动子与GUS基因一起组建了融合载体转入烟草,利用GUS染色和定量PCR方法,分析了克隆的启动子功能及其对环境因子的响应。结果表明,克隆的NMT1基因启动子长767βbp,含有TATA-box、CAAT-box等真核生物启动子基本元件及多个与植物非生物胁迫相关的响应元件。以5'递减的NMT1启动子替换pBI121中的CaMV35S启动子,构建出与GUS融合的pA、pB、pC、pD载体。在烟草叶片中进行瞬时表达,不同长度的NMT1启动子均具驱动GUS表达功能,且长度越长驱动能力越强。以含全长NMT1启动子载体pA对烟草转基因,转基因烟草各组织均检测到GUS基因的表达且表达量叶>茎>根,叶片中的表达量为根部的3倍。对转基因烟草进行不同程度光照、温度、模拟干旱和脱落酸处理后,除40℃温度条件外,其他处理的叶片中GUS基因表达在处理前后均存在显著变化,表明NMT1启动子功能受外界环境因子的胁迫影响。

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

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.