花生膜联蛋白基因启动子的克隆及功能分析

Annexin plays an important role in cell signaling transduction, maintaining the stability of the membrane structure and plant response to stress. The study of Annexin gene promoter provides an important experimental basis for parsing the regulation of gene expression. Our research team has cloned six full-length sequences, named AnnAhs, and functional analysis showed that their expressions had strong stress response characteristics and tissue-specific. In this study, functional analysis on AnnAhs promoters will be conducted so as to explore the mechanisms of AnnAhs genes involved in the peanut adversity response process. By using TAIL-PCR, AnnAhs promoters will be cloned and their full length, 5 'UTR absent and a series of promoter 5' end truncated plant expression vector will be constructed based on the analysis of substructure and its potential cis element in regulatory regions using bioinformatics tools. Functionality and activity of AnnAhs promoters will also be analyzed via determination of transgenic tobacco GUS histochemical staining and fluorescence enzyme activity. Gene function and mechanism will be genetically dissected according to the results of function verification of AnnAhs gene and promoters. Cloning and functional verification of AnnAhs promoters will provide important theoretical basis for the studies of the mechanisms of AnnAhs genes involved in the peanut adversity response and peanut stress resistance and molecular breeding program.

膜联蛋白（Annexin）在细胞信号转导、维持膜结构稳定性以及植物响应逆境中起着重要作用，对其启动子进行功能分析将有助于解析Annexin基因表达调控。基于项目组前期克隆的花生膜联蛋白基因家族（AnnAhs）及其在烟草上的功能鉴定结果，本研究拟从启动子的角度深入研究 AnnAhs在花生抗逆中的作用机制，利用染色体步移（TAIL-PCR）技术克隆AnnAhs家族的启动子序列，运用生物信息学手段对启动子结构及其调控区域中潜在的顺式作用元件进行分析预测，构建启动子全长、缺失启动子植物表达载体，通过转基因烟草GUS的组织化学染色及荧光酶活测定，对启动子活性和功能进行分析，结合启动子调控AnnAhs的作用和AnnAhs在抗逆中的功能，对AnnAhs在花生抗逆中的作用机制和调控机理进行遗传解析。AnnAhs启动子的克隆及功能分析将为膜联蛋白基因介导的抗性机制和花生抗逆分子育种研究提供重要的基础。

干旱是影响我国花生产量和品质提高的关键因素之一。挖掘花生耐性相关基因和研究花生对逆境的分子机制是高效快速得到耐性新品种的切实可行方法。花生膜联蛋白基因在高盐、低温、干旱、重金属等逆境中发挥重要调控作用，推测其启动子对膜联蛋白基因在转录的过程起着较强的调控作用。本项目通过对花生种质资源进行耐盐性、耐旱性、抗果腐病及各种生理指标鉴定，筛选出的耐性极端花生品种为实验材料，克隆膜联蛋白基因启动子序列，针对不同耐性材料中克隆序列并比较进行生物信息学分析，同时，对启动子表达模式功能等进行分析，获得主要研究结果如下：.1、筛选到海花1、花育25、M66和 M101等耐盐材料。开花期下针期耐旱性最强的资源是冀花16，最弱的品种为吉花11；结荚期耐旱性最强的品种为冀花16，耐旱性最弱的为吉花11；全生育期耐旱性最强的品种为冀农花2号，耐旱性最弱的为唐花10。高抗病材料包括Grif14051和PI502111共2份；高感材料包括PI162857，PI270786，PI356004，G5059和G11-15共5份。 .2、膜联蛋白基因转录起始位点上游含有30多个顺式调控元件，即含有真核生物启动子的基本元件TATA-box，CAAT-box和一系列光响应元件如G-box，Box4，SP1，脱落酸响应元件ABRE，水杨酸响应元件TCA-element，厌氧诱导必要的顺式作用元件ARE，干旱响应MYB结合位点MBS，低温响应元件LTR，参与热胁迫响应的顺式元件HSE及防御和胁迫响应元件TC-rich repeats等。.3、以强耐性和耐性敏感品种为材料，克隆不同抗性花生模联蛋白基因启动子序列，对克隆序列进行分析。启动子序列相似度98.34%，有比较高的保守程度。对Annah2和Annah6克隆的启动子序列进行序列比对，结果显示两者的相似性仅有35.08%。.4、通过烟草叶片的瞬时表达研究膜联蛋白基因启动子的活性，表明proAnnah6在烟草中有表达活性，但绿色荧光略低于CaMV35S启动子。但proAnnah2启动子未发现有明显的绿色荧光，说明proAnnah2在烟草中没有表现出启动子的活性，可能的原因是并未克隆到全部的启动子序列，参与转录活性的某些关键顺式作用元件在基因起始位点ATG上游-2000 bp之前。此外，对于proAnnah6连接2000bp启动子后GUS基因的表达最强。