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摘 要 利用5′缺失设计一系列引物，扩增HbFCA启动子质粒，获得5个启动子缺失片段，用这些片段分别替换PBI121载体中的CaMV35S启动子，得到5个HbFCA的5′缺失表达载体，即pA、pB、pC、pD和pE。利用农杆菌介导叶盘法进行烟草遗传转化，对组培瓶内生根较好的烟草叶片GUS染色，结果发现，长度为最小片段的276 bp的pE可以较好地实现HbFCA启动子的功能。对橡胶树体细胞胚瞬时表达检测发现，pE片段也能启动表达，有较好的GUS活性，可以实现HbFCA启动子的功能。

关键词 橡胶树 ；HbFCA ；启动子 ；5′端缺失分析法 ；核心结构域

中图分类号 S794.1 文献标识码 A Doi：10.12008/j.issn.1009-2196.2017.07.013

5' Deletion Analysis of HbFCA Promoter from Hevea brasiliensis

in Transgenic Tobacco

LI Ji DAI Xuemei SUN Fang HUANG Tiandai HUA Yuwei HUANG Huasun

（Rubber Research Institute， CATAS & Ministry of Agriculture Key Laboratory

of Rubber Biology， Danzhou， Hainan 571737）

Abstract Using the 5' end serial deletion analysis method， five HbFCA promoter deletion fragments which had different distances from transcription start site were amplified by PCR， and they were used to replace the CaMV35S promoter of PBI121 carrier to obtain 5 HbFCA 5'deletion expression vectors， i.e. pA， pB， pC， pD and pE. The expression vectors were transformed into tobacco by using agrobacterium-meditated leaf disc method， and the well-rooted transformed tobacco plants in tissue culture vessels were directly detected with GUS staining. The results showed that the pE 267 bp， the shortest among the fragments， had promoter activity. The transient expression analysis of somatic embryo of rubber tree showed that the fragment pE also had better GUS staining， and could function as a HbFCA promoter.

Keywords rubber tree ； HbFCA ； promoter ； 5' deletion analysis method ； functional domain

橡胶树转基因体系已相对完善[1-2]，载体构建时多使用外源组成型启动子（如CaMV35S[3-5]）。但外源性启动子存在着表达效率低下，多呈瞬时表达，稳定表达较少[6-9]，容易引起外源基因的多拷贝整合[10]或转基因沉默[11]，以及影响外源基因的表达水平和表达模式[12]等问题。因此，选择一个橡胶树内源的，高效的组成型表达启动子已成为橡胶树遗传转化系统中的关键环节。

FCA基因的功能具有多样性，除与开花调控外，还与种子休眠、种子千粒重和纤维产量的相关[13]。拟南芥FCA启动子在多个组织中均能启动FCA基因的表达[14]。橡胶树HbFCA启动子具有典型的真核生物核心启动子区域，共有28种顺式调控元件，在橡胶树叶片、茎段、树根和胚状体等组织均能启动报告基因的表达，具有组成型启动子的特点[15]。

启动子缺失分析是研究启动子及其调控元件功能的一种重要、有效的方法。……