陆地棉Ghuhrf1基因及其启动子的克隆与功能分析  

Cloning and Functional Analysis of Ghuhrf1 Gene and Promoter from Gossypium hirsutum

作　　者：杨江涛[1,2] 王旭静 哈斯阿古拉[2] 王志兴[1] YANG Jiangtao;WANG Xujing;HASI Agula;WANG Zhixing(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;College of Life Sciences,Inner Mongolia University,Huhhot 010021,China)

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]内蒙古大学生命科学学院,呼和浩特010021

出　　处：《中国农业科技导报》2020年第6期17-25,共9页Journal of Agricultural Science and Technology

基　　金：国家转基因生物新品种培育重大专项(2016zx08005-003)。

摘　　要：在分子育种过程中,优良基因和组织特异表达启动子的缺乏是制约棉花纤维品质改良的重要因素之一。分析转录组数据库中差异基因表达谱发现,Ghuhrf1为开花当天胚珠优势表达基因,推测该基因可能参与或调控棉纤维的合成。根据其EST序列设计引物,通过RACE技术获得Ghuhrf1基因的全长cDNA序列。荧光定量PCR分析表明,Ghuhrf1在开花当天的胚珠中显著表达。通过同源序列分析获得Ghuhrf1的上游启动子Ghuhrf1-Pro。Ghuhrf1-Pro全长为1 417 bp,含有GC-box、CAAT-box、CAT-box、CCGTCC-box和ACGT-motif等顺式作用元件和组织特异性调控元件。根据组织特异性调控元件的分布位置构建了4个不同程度的缺失体转化烟草和拟南芥进行分析。GUS组织化学染色结果表明,全长启动子Pro和缺失体Pro1(-1 180^+226 bp)、Pro2(-867^+226 bp)、Pro3(-726^+226 bp)都能特异地驱动Gus基因在转基因拟南芥的叶片边缘尖部和莲座叶基部等分化生长旺盛部位表达,说明与分生组织表达相关的CAT-box元件和CCGTCC-box元件在启动子种子特异表达中起到重要的作用。同时,通过启动子缺失分析也表明,Ghuhrf1基因可能不是直接参与棉纤维的合成,而是间接的参与棉纤维原始细胞分化起始的调控。该结果为棉花纤维品质基因Ghuhrf1的功能研究提供了参考,并为棉花分子育种工程提供了新的调控元件。The absence of excellent gene and promoter is one of the main factors to restrict the development of genetic engineering in cotton fiber improvement. By analyzing the differential expression profiles in transcriptome databases, it was found that Ghuhrf1 was the dominant expressed at 0 DPA(days post anthesis) flowering in ovules, so it was speculated to be involved in the synthesis of cotton fibers. The full-length cDNA of Ghuhrf1 gene was cloned by RACE techniques according EST sequence. Real-time PCR analysis indicated that Ghuhrf1 was significantly expressed at the initial stage of fiber cell differentiation. 1 417 bp sequence of Ghuhrf1 promoter was cloned according to the homologous sequence. There were lots of cis-acting factors and the tissue specific regulation elements, such as GC-box, CAAT-box, CAT-box, CCGTCC-box and ACGT-motif. Based on the distribution of the regulatory elements, four truncations of Ghuhrf1-Pro were obtained and transformed into tobacco and Arabidopsis thaliana through Agrobacterium. The results of GUS histochemical staining indicated that the full-length promoter Ghuhrf1-Pro and the truncations Pro1(-1 180^+226 bp), Pro2(-867^+226 bp), and Pro3(-726^+226 bp) could specifically drive the gus gene to express in the differentiated and vigorous growth sites of the leaf tip and petiole base of the transgenic Arabidopsis thaliana, which indicated the meristem expression-related CAT-box and CCGTCC-box elements played important roles in Ghuhrf1 promoter. At the same time, the promoter deletion analysis further indicated that the Ghuhrf1 gene might not be directly involved in the synthesis of cotton fiber, but indirectly involved in the regulation of the initiation of cotton fiber primitive cell differentiation. Above results provided reference for understanding Ghuhrf1 and regulatory elements for genetic engineering of cotton fiber quality improvement.

关 键 词：Ghuhrf1基因 启动子 缺失分析 克隆 顺式作用元件 

分 类 号：S562[农业科学—作物学] Q78[生物学—分子生物学]