棉花GhSAD2基因的克隆与功能验证

发布时间：2018-01-25 05:27

  本文关键词： 陆地棉 基因克隆 遗传转化 脂肪酸 抗寒性　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：本研究以Gh SAD2为目的基因,采用电子拼接结合RT-PCR的方法克隆得到陆地棉GhSAD2基因的c DNA全长并对其进行相关的生物信息学分析;通过qRT-PCR研究了Gh SAD2基因在棉花不同组织中的表达特点,并探究目的基因在棉子发育过程中和低温胁迫下的表达模式;本研究构建了Gh SAD2基因的过表达载体,并通过农杆菌介导遗传转化到烟草和新陆早33号,初步分析目的基因的功能;此外,以转GhSAD2干涉载体的棉花T3代和受体材料2074B为研究材料,分析其幼苗抗寒能力及脂肪酸成分变化,主要研究结果如下:1.克隆了陆地棉GhSAD2基因,并对该基因进行了生物信息学分析。Gh SAD2在NCBI注册的登录号为KX197920,其cDNA全长1188 bp,编码396个氨基酸,含有2个组氨酸簇是一个典型的脂肪酸去饱和酶家族蛋白。GhSAD2基因编码的分子量大小为45.3 kD,理论等电点为5.88,平均亲水系数(GRAVY)为-0.464,为亲水性蛋白。系统进化分析表明,该基因与可可树的同源基因进化关系非常接近。2.构建了目的基因的过表达载体,并转化了烟草和棉花。将植物过表达载体pBI121-GhSAD2通过根瘤农杆菌介导的叶盘法和愈伤组织转化法将其分别转入‘山西烟草’和‘新陆早33号’,共得到9株GhSAD2基因过表达的转基因烟草植株,其中烟草的转化率为75%;得到了棉花阳性愈伤组织,并进行了GUS检测。3.通过荧光定量PCR技术,研究了目的基因的组织表达特征。结果表明,GhSAD2基因在棉花叶片中的表达量高于茎和根;Gh SAD2基因在棉花种子发育过程中的表达量呈现出先上升后逐步下降的变化趋势,在花后25 d的种子中表达量最高,在成熟种子中的表达相对比较微弱;Gh SAD2基因能够响应低温胁迫,在4℃和15℃胁迫6 h时其表达量最大,胁迫12h后,表达量基本保持稳定状态,是对照组的1.3-2.3倍。4℃低温胁迫48h后,GhSAD2过表达的转基因烟草的电导率显著低于野生型烟草,由此可以推测Gh SAD2基因可能在棉花冷胁迫调控中起一定的作用。4.测定了转GhSAD2干涉载体的棉花T3代株系的农艺性状及棉子中脂肪酸的含量。结果表明:转基因棉花与受体2074B相比,衣分增长了7%-8%,衣指含量增长了0.8-1.6g,籽指含量减少了1.3-2.4g。其转基因棉子中的总脂肪酸含量、棕榈酸含量及亚油酸含量显著降低,总蛋白含量、硬脂酸含量及油酸含量显著升高。5.通过荧光定量PCR技术,研究了目的基因在转基因植株种子发育过程中的表达特性,结果显示,除花后15d其它时期均低于受体2074B,表达模式和受体植株相同,呈现出先上升后下降的变化趋势,在花后30d的种子中表达量最高。6.测定了转基因植株叶片的电导率、丙二醛含量及脯氨酸含量。结果表明,RNAi的转基因植株相比CK,其抗寒能力减弱,耐热能力增强,推测棉花幼苗的抗寒能力可能会与GhSAD2基因在棉花幼苗中的表达量相关。
[Abstract]:In this study, Gh SAD2 was used as the target gene. The full length of c DNA of GhSAD2 gene of Upland cotton was cloned by electronic splicing combined with RT-PCR and its bioinformatics analysis was carried out. The expression characteristics of Gh SAD2 gene in different tissues of cotton were studied by qRT-PCR, and the expression pattern of Gh SAD2 gene during cotton seed development and under low temperature stress was explored. In this study, the overexpression vector of Gh SAD2 gene was constructed and transformed into tobacco and Xinluzao 33 by Agrobacterium tumefaciens to analyze the function of the target gene. In addition, the T 3 generation and receptor material 2074B of cotton transformed with GhSAD2 interference vector were used as the research materials to analyze the changes of cold resistance and fatty acid composition of cotton seedlings. The main results are as follows: 1. The GhSAD2 gene of Upland cotton was cloned and analyzed by bioinformatics. Gh SAD2 was registered as KX197920 in NCBI. Its cDNA length is 1188bpand encodes 396 amino acids. Two histidine clusters are a typical fatty acid desaturase family protein. GhSAD2 gene encodes a molecular weight of 45.3 KD and a theoretical isoelectric point of 5.88. The average hydrophilic coefficient (GRAVY) is -0.464, which is a hydrophilic protein. Phylogenetic analysis shows that GRAVY is a hydrophilic protein. The evolutionary relationship between this gene and the coca homologous gene is very close to .2. the overexpression vector of the target gene was constructed. The plant overexpression vector pBI121-GhSAD2 was transformed into 'Shanxi tobacco' and 'Xinluzao 3' by Agrobacterium tumefaciens mediated leaf disk and callus transformation, respectively. Three. Nine transgenic tobacco plants with overexpression of GhSAD2 gene were obtained, and the conversion rate of tobacco was 75%. The positive callus of cotton was obtained and detected by GUS. The expression characteristics of the target gene were studied by fluorescence quantitative PCR. The expression of GhSAD2 gene in cotton leaves was higher than that in stems and roots. The expression of Gh SAD2 gene increased firstly and then decreased gradually during the development of cotton seeds, and the highest expression was found in the seeds 25 days after anthesis. The expression in mature seeds was relatively weak. The expression of Gh SAD2 gene was the highest at 4 鈩，

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