油葵种子特异启动子Ha ds10 G1的克隆及功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2017.02.011

摘要/Abstract

摘要： 从油葵中克隆得到LEA蛋白基因家族Ha ds10 G1基因的启动子序列,并对其进行功能分析。利用PCR 技术从油葵品种“矮大头“基因组DNA中分离Ha ds10 G1基因上游的调控序列,将其与GUS 基因融合,构建种子特异性表达载体pBI121-PHa ds10,通过根癌农杆菌介导法转化烟草（Nicotiana tabacum）NC89,对再生植株进行PCR、RT-PCR和GUS组织化学分析,以检测GUS基因在转基因烟草中的表达情况。结果表明,油葵Ha ds10 G1基因启动子长度为 1 417 bp,与已报道的向日葵Ha ds10 G1基因启动子序列同源性为89.42%。作用元件分析发现该区域除了具有启动子核心调控序列外,还含有多个与组织特异性、激素、逆境等表达相关的顺式作用元件,如RY重复元件、ABRE元件、TC-rich元件等。转基因植株的PCR结果显示,成功地获得了转基因阳性植株;GUS活性检测表明,该启动子序列仅能够驱动GUS基因在烟草种子表达,而在根、茎、叶等组织中均未检测到GUS基因表达。因此,油葵LEA蛋白基因家族Ha ds10 G1基因上游1 417 bp片段具有种子特异性启动子功能。研究结果为油葵等油料作物的油脂遗传改良提供组织特异性启动子。

关键词: 油葵, 种子特异性启动子, GUS染色, 烟草

Abstract: The objective of this work is to clone and identify the seed-specific promoter of Ha ds10 G1 in a LEA（late embryogenesis abundant）protein gene family from oil sunflower. The upstream regulatory region of Ha ds10 G1 gene was isolated from the genomic DNA of sunflower “Aidatou” by PCR method. The cloned region was fused to the GUS reporter gene,and plant expression vector pBI121-PHa ds10 was constructed,which was introduced into Nictiana tabacum NC89 by medicating Agrobacterium tumefaciens. The expression of GUS gene in the transgenic tobacco plants was detected by PCR,RT-PCR and GUS. As results,the gene promoter of Ha ds10 G1 was 1 417 bp,and it had homology with the reported sequence at 89.42%. Cis-acting elements search in the segment revealed that in addition to kernel regulatory sequences of promoter,there was the presence of a number of motifs related to tissue specificity,hormone,and adverse environment,such as RY-repeat elements,ABRE motif and TC-rich element,etc. PCR results showed that the transgenic plants were obtained successfully. GUS activity assays indicated that this promoter drove the expression of GUS gene only in tobacco seed embryo,and not in the other tissues such as roots,stems and leaves. Conclusively,the cloned 1 417 bp upstream regulatory region of Ha ds10 G1 gene from the LEA protein gene family of oil sunflower is seed-specific promoter. The results may provide the tissue-specific promoter for genetic improvement of oilseed crops such as oil sunflower.

Key words: oil sunflower, seed-specific promoter, GUS staining, tobacco

孙黎, 周茜萍, 齐梓云, 王梦瑶, 陈福龙. 油葵种子特异启动子Ha ds10 G1的克隆及功能鉴定[J]. 生物技术通报, 2017, 33(2): 72-79.

SUN Li, ZHOU Xi-ping, QI Zi-yun, WANG Meng-yao, CHEN Fu-long. Cloning and Identification of Seed-specific Promoter Ha ds10 G1 from Oil Sunflower（Helianthus annuus）[J]. Biotechnology Bulletin, 2017, 33(2): 72-79.

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