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棉花学报 ›› 2016, Vol. 28 ›› Issue (6): 527-537.doi: 10.11963/issn.1002-7807.201606002

• 研究与进展 • 上一篇    下一篇

棉花脂肪酸合成酶基因GhKARGhENR表达载体构建及其功能初探

刘丽1,2(),王玉美3,赵彦朋1,王丹1,赵鹏1,刘正杰1,华金平1,*()   

  1. 1. 中国农业大学棉花遗传育种与基因组研究实验室,北京 100193
    2. 新疆农垦科学院棉花研究所/农业部西北内陆区棉花生物学与遗传育种重点实验室,新疆 石河子832000
    3. 湖北省农业科学院经济作物研究所,湖北 武汉 430064
  • 收稿日期:2016-02-17 出版日期:2016-11-15 发布日期:2016-11-15
  • 通讯作者: 华金平 E-mail:cottonliuli@sina.com;jinping_hua@cau.edu.cn
  • 作者简介:刘丽(1979―),女,博士,副研究员,cottonliuli@sina.com
  • 基金资助:
    国家自然科学基金——棉花脂肪酸关键基因的表达特征研究及功能分析(31360349);国家科技重大专项(2014ZX08005002-004);新疆农垦科学院科技引导计划(YYD2010-5)

Construction of Expression Vectors and a Preliminarily Functional Analysis of Fatty Acid Synthetase Genes of GhKAR and GhENR in Upland Cotton

Liu Li1,2(), Wang Yumei3, Zhao Yanpeng1, Wang Dan1, Zhao Peng1, Liu Zhengjie1, Hua Jinping1,*()   

  1. 1. Laboratory of Cotton Genetics, Genomics and Breeding, China Agricultural University, Beijing 100193, China
    2. Cotton Institute, Xinjiang Academy Agricultural and Reclamation Science / Northwest Inland Region Key Laboratory of Cotton Biology and Genetic Breeding (Xinjiang), Ministry of Agriculture, China, Shihezi, Xinjiang 832000, China
    3. Institute of Cash Crops, Hubei Academy of Agricultural Sciences, Wuhan, Hubei 430064, China
  • Received:2016-02-17 Online:2016-11-15 Published:2016-11-15
  • Contact: Jinping Hua E-mail:cottonliuli@sina.com;jinping_hua@cau.edu.cn

摘要: 酮脂酰ACP还原酶(β-ketoacyl-(ACP) reductase,KAR)和烯脂酰ACP还原酶(β-enoyl-(ACP) reductase,ENR)是脂肪酸合成过程中2个关键的还原酶,主要负责脂肪酸碳链的延伸。为明确棉花同源基因的功能,克隆了陆地棉GhKARGhENR基因,并构建表达载体;应用花粉管通道法转化棉花,经过PCR检测和Southern杂交,筛选出阳性单拷贝植株,分析其后代株系中目的基因对油分含量以及抗寒性的影响。通过对转基因T4株系含油量的测定,发现转基因株系种子的含油量比野生型对照提高10.2%~14.1%,与对照差异极显著。通过气相色谱法分析转基因株系种子的脂肪酸成分,发现不饱和脂肪酸含量比野生型对照增加4.04%~6.02%。测定4 ℃低温胁迫下各个转基因株系不同时间生理指标的变化,结果表明:转基因株系的相对电导率和脯氨酸含量积累明显高于对照,而丙二醛含量低于对照,在不同胁迫时间与对照表现出显著或极显著差异,过表达的转基因株系的抗寒性有所提高。

关键词: 陆地棉; 脂肪酸合成; 含油量; 抗寒性

Abstract:

Cotton is an important economic crop, and fatty acids account for 25%-40% of cottonseeds. A positive correlation exists between the unsaturated fatty acid content and cold tolerance. GhKAR and GhENR are two reductase genes that are responsible for carbon-chain extension, which is related to fatty acid synthesis in cotton. Here, we built two vectors that over-expressed GhKAR and GhENR independently, and then separately transferred the vectors into upland cotton by means of the pollen tube channel. The positive transformants containing single vector copies were obtained after being identified by PCR and Southern hybridization. The objectives of this research were to explore the gene functions and to provide candidate genes for high-oil and abiotic stress-resistance breeding in cotton. The oil contents in transgenic T4 lines were 10.2%-14.1% higher than that of the control (wild type). The fatty acidcompositions of transgenic lines were analyzed by gas chromatography and revealed that the unsaturated fatty acidcontent increased by 4.04%-6.02% compared with the control. Additionally, the cold tolerance of the transgenic lines was investigated under low-temperature (4 ℃) stress. The changes in the physiological indexes were evaluated. The relative electric conductivity and proline contents of the transgenic lines were remarkably or extremely higher than those of the control, but the malonaldehyde contents were remarkablylower in all periods of stress treatment. The results indicated that the transgenic lines could potentially be used in improving cotton's cold tolerance.

Key words: upland cotton; fatty acid synthesis; oil content; cold resistance