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作物学报 ›› 2016, Vol. 42 ›› Issue (03): 389-398.doi: 10.3724/SP.J.1006.2016.00389

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大赖草6-SFT基因的克隆及其转基因烟草抗旱和抗寒性分析

贺晓岚1,王建伟2,李文旭3,陈真真1,赵继新1,武军1,王中华1,陈新宏1,*   

  1. 1 西北农林科技大学农学院 / 陕西省植物遗传工程育种重点实验室, 陕西杨凌 712100;2 凯里学院环境与生命科学学院, 贵州凯里 556011;3 河南省农业科学院小麦研究所, 河南郑州 450002
  • 收稿日期:2015-07-22 修回日期:2015-11-20 出版日期:2016-03-12 网络出版日期:2015-12-18
  • 通讯作者: 陈新宏, E-mail: cxh2089@126.com; Tel: 029-87082854
  • 基金资助:

    本研究由国家自然科学基金(31571650)项目和西北农林科技大学唐仲英育种基金资助。

Cloning of 6-SFT Gene from Leymus racemosus and Analysis of Tolerance to Drought and Cold Stresses in Transgenic Tobacco

HE Xiao-Lan1,WANG Jian-Wei2,LI Wen-Xu3,CHEN Zhen-Zhen1,ZHAO Ji-Xin1,WU Jun1,WANG Zhong-Hua1,CHEN Xin-Hong1,*   

  1. 1 Shaanxi Provincial Key Laboratory of Plant Genetic Engineering Breeding / College of Agronomy, Northwest A&F University, Yangling 712100, China; 2 College of Environment and Life Science, Kaili University, Kaili 556011, China; 3 Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
  • Received:2015-07-22 Revised:2015-11-20 Published:2016-03-12 Published online:2015-12-18
  • Contact: 陈新宏, E-mail: cxh2089@126.com; Tel: 029-87082854
  • Supported by:

    This study was supported by the National Natural Science Foundation of China (31571650) and the Tang Zhong-Ying Breeding Funding Project of the Northwest A&F University.

摘要:

果聚糖合成酶(6-SFT)在植物抵御逆境胁迫中起重要作用利用RACE结合RT-PCR技术从大赖草(Leymus racemosus中克隆到6-SFT基因,其完整开放阅读框为1863 bp,被命名为Lr-6-SFT (GenBank登录号KT387273),编码620个氨基酸,其推导氨基酸序列含有保守的果糖基转移酶结构域。氨基酸序列比对及进化树分析表明,大赖草6-SFT与华山新麦草、普通小麦、西尔斯山羊草和大麦6-SFT具有高度相似性。采用基因重组技术构建p1300-35SN-Lr-6-SFT表达载体,利用农杆菌介导法将该载体转入烟草品种W38中。对经过抗性筛选、PCR和RT-PCR验证的转基因植株进行抗旱和抗寒性鉴定,发现转基因植株与对照相比,抗旱和抗寒性明显增强;在逆境胁迫条件下,转基因植株的果聚糖、可溶性糖、脯氨酸含量都显著高于对照,而丙二醛的含量显著低于对照。本研究表明,Lr-6-SFT基因是典型的GH32家族成员,, 2n= 4x = 28, NsNsXmXm)其表达能够提高烟草对干旱和寒冷胁迫的抗性。

关键词: 6-SFT基因, 干旱, 寒冷, 大赖草, 生理指标, 转基因烟草

Abstract:

The fructan biosynthesis enzyme (6-SFT) plays an important role in plant responses to abiotic stresses. In this study, a full-length cDNA encoding sucrose:fructan-6-fructosyltransferase, designated as Lr-6-SFT (GenBank accession No. KT387273), was cloned from Leymus racemosus (2n = 4x = 28, NsNsXmXm) using reverse transcriptase PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques. The full-length open reading frame comprises 1863 bp and encodes 620 amino acids. The predicted protein structure of the gene containes a conserved fructosyltransferase domain. Multiple sequence alignment and phylogenetic analysis showed that the Lr-6-SFT protein shared high similarity with 6-SFT proteins from Psathyrostachys huashanica, Triticum aestivum, Aegilops searsii, and Hordeum vulgare subsp. vulgare. The Lr-6-SFT gene was transferred into tobacco (Nicotiana tabacum L. cv. W38) via Agrobacterium-mediated transformation. The screened plants were tested by PCR and RT-PCR, and the transgenic tobacco plants exhibited much higher tolerance to drought and cold compared with the non-transgenic plants. Under drought and cold stresses, the Lr-6-SFT expressions were associated with the increased accumulation of stored carbohydrate and proline and the decreased malondialdehyde storage. These results suggest that Lr-6-SFT is a typical member of the glycoside hydrolase 32 (GH32) family and may be linked to enhanced tolerance to drought and cold stresses.

Key words: 6-SFT gene, Drought, Freezing, Leymus racemosus, Physiological indices, Transgenic tobacco

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