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作物学报 ›› 2012, Vol. 38 ›› Issue (12): 2139-2146.doi: 10.3724/SP.J.1006.2012.02139

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

花生蔗糖合酶基因AhSuSy的克隆和干旱胁迫表达分析

何美敬,刘立峰*,穆国俊,侯名语,陈焕英,崔顺立   

  1. 河北农业大学农学院 / 河北省作物种质资源重点实验室, 河北保定 071001
  • 收稿日期:2012-07-15 修回日期:2012-07-15 出版日期:2012-12-12 网络出版日期:2012-10-08
  • 通讯作者: 刘立峰, E-mail: liulifeng@hebau.edu.cn
  • 基金资助:

    本研究由国家现代农业产业技术体系建设专项基金(CARS-14), 河北省应用基础研究计划重点基础研究项目(10960120D)和河北省高等学校科学技术研究重点项目(ZD2010136)资助。

Molecular Cloning of Sucrose Synthase Gene and Expression Analysis under Drought Stress in Peanut (Arachis hypogaea L.)

HE Mei-Jing,LIU Li-Feng*,MU Guo-Jun,HOU Ming-Yu,CHEN Huan-Ying,CUI Shun-Li   

  1. Key Laboratory of Crop Germplasm Resources of Hebei Province / College of Agronomy, Agricultural University of Hebei, Baoding 071001, China
  • Received:2012-07-15 Revised:2012-07-15 Published:2012-12-12 Published online:2012-10-08
  • Contact: 刘立峰, E-mail: liulifeng@hebau.edu.cn

摘要:

蔗糖合酶(sucrose synthase, SuSy)是蔗糖代谢途径中的关键酶, 在植物生长、发育和渗透调节过程中起着重要的作用。本研究利用同源克隆、RACE和TAIL-PCR相结合的方法从花生叶片中分离了蔗糖合酶基因, 命名为AhSuSy (GenBank登录号JF346233)。该基因cDNA序列全长2 790 bp, 包含一个2 421 bp的开放阅读框(ORF), 5′端非编码区57bp, 3¢端非编码区为312 bp。根据编码区预测AhSuSy编码806个氨基酸, 与大豆、拟南芥、玉米等植物中相关蛋白的氨基酸序列同源性达75%以上; 成功构建了该基因的原核表达载体pET32a-AhSuSy, 在IPTG诱导下得到92 kD左右的蛋白, 与理论值一致。半定量RT-PCR分析表明AhSuSy在花生根、茎、叶中均有表达。利用10%PEG模拟干旱处理花生幼苗, 分析胁迫后AhSuSy的转录水平, 同时测定蔗糖合酶活性和蔗糖含量, 发现三者均表现为处理后4~12 h逐渐升高, 相关性分析显示花生中蔗糖含量与蔗糖合酶活性的相关系数达0.993(P=0.007<0.01), 处理后12~24 h逐渐降低。推测该基因响应干旱调控, 在花生抗旱胁迫中可能起着一定的作用。

关键词: 花生, 蔗糖合酶基因, 克隆, 半定量RT-PCR, 表达分析

Abstract:

Sucrose synthase (SuSy), a key enzyme in sucrose metabolism, plays an important role in both plant growth and development and osmotic adjustment. Here, a cDNA sequence of sucrose synthase gene, designated AhSuSy (GenBank accession number: JF346233), was isolated from peanut using homologous cloning, RACE and TAIL-PCR. The full-length of AhSuSy cDNA is 2 790 bp, contained an ORF of 2 421 bp, the 5′UTR and 3′UTR of 57 bp and 312 bp, respectively. A peptide of 806 amino acid residues was deduced according to the ORF, which showed more than 75% homologous to the corresponding proteins in soybean, Arabidopsis and maize. The AhSuSy was ligated into pET-32a(+) and transformed into E. coli competent cell. After induced by IPTG, the fusion protein was obtained and its relative molecular weight was 92 kD, which was consistent with the theoretical value. The tissue specific expression analysis by using semi-quantitative RT-PCR assays indicated that AhSuSy was expressed in root, stem and leaf. After treated with 10%PEG, seedlings of peanut were used to determine transcript level for AhSuSy, sucrose synthase activity and sucrose content. The result showed that all of the three parameters were gradually increased during 4h to 12 h after treatment and the correlation coefficient between sucrose synthase activity and sucrose content was 0.993 (P=0.007<0.01) during this period, then declined from 12h to 24 h after treatment, which indicated that AhSuSy may play an important role in adaptation of plants to drought stress.

 

Key words: Peanut, Sucrose synthase gene, Cloning, Semi-quantitative RT-PCR, Expression analysis

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