菊花去饱和酶基因CmSAD的克隆与表达分析
发布时间：2018-10-22 17:30
【摘要】：△9硬脂酰-ACP去饱和酶(Stearoyl-ACP desaturase,简称:SAD)催化脱氢形成第一个双键,其参与细胞膜脂不饱和脂肪酸的合成,其含量的变化能够改变饱和脂肪酸与不饱和脂肪酸之间的比例,从而影响植物的抗寒性。本试验从植物分子水平上研究SAD基因在低温胁迫下的表达模式,为深入研究菊花抗寒机制、筛选抗寒品种以及进一步进行抗性育种奠定基础,也可用于抗寒品种的选育,并提高菊花的观赏价值。以秋菊抗寒品种‘星光灿烂’为试材,通过改良的CTAB法从菊花叶片中克隆出菊花SAD基因的中间片段,并对其序列进行分析。利用实时荧光定量的方法对菊花SAD的表达量进行测定,分析此基因在不同温度下的表达情况。主要研究结果如下:1.根据菊花SAD基因的同源序列设计简并引物,从菊花‘星光灿烂’叶片中克隆出菊花SAD基因的中间片段,并设计特异引物扩增得到c DNA全长共有1203bp,编码401个氨基酸,相对分子量为45.57 KD,在Gene Bank注册号为KC529335。2.对菊花SAD基因进行同源序列比较,菊花SAD基因片段编码的氨基酸与同科的新疆雪莲(Saussurea involucrata Kar.et Kir.ex Maxim.,ABF66638)SAD基因的同源性为80.29%与向日葵(Helianthus annuus,AAB65145.1)SAD基因的同源性为79.81%。与其它物种SAD基因也具有较高的同源性,如麻疯树(77.62%),百脉根(75.91%),马铃薯(77.86%),水黄皮(77.62%),花生(77.13%),乌桕(76.89%),木薯(76.89%)。与其他植物的SAD基因有较高的同源性,因此命名为CmSAD。该蛋白是一个可溶性蛋白,没有跨膜信号区,N-端含有一段叶绿体转运肽,蛋白活性位点分析发现在CmSAD编码的蛋白C-端有1个SAD的保守区域序列,并对该蛋白3D结构进行预测。3.根系中CmSAD基因的表达量随着温度的降低而降低,16℃时表达量最高,5℃、-4℃和-8℃分别是16℃的0.87、0.11和0.03倍。叶片中CmSAD表达量随着温度的降低先升高再降低,5℃时最高,分别是16、-4、-8℃条件下的1.3、5.6、3.5倍。随着温度的下降,CmSAD基因的表达量呈下降趋势。在16℃和5℃时,CmSAD基因在根系中的表达量高于叶片;-4℃和-8℃时,叶片中CmSAD的表达量高于根系。SAD基因使饱和脂肪酸脱氢形成第一个氢键,使不饱和脂肪酸的含量发生改变。CmSAD基因的表达量随着温度的降低而降低,因此,CmSAD基因的表达量与温度有关,同时与抗寒性也有着密切的关系。
[Abstract]:9 stearyl-ACP desaturase (Stearoyl-ACP desaturase, for short) catalyzes dehydrogenation to form the first double bond, which is involved in the synthesis of unsaturated fatty acids of cell membrane. The change of the content of stearyl-ACP desaturase (Stearoyl-ACP desaturase,) can change the ratio of saturated fatty acids to unsaturated fatty acids. Thus affecting the cold resistance of plants. In this study, the expression pattern of SAD gene in chrysanthemum under low temperature stress was studied at the molecular level, which laid a foundation for further study on chrysanthemum cold resistance mechanism, selection of cold resistant varieties and further resistance breeding, and could also be used in breeding cold resistant varieties. And improve the ornamental value of chrysanthemum. The intermediate fragment of chrysanthemum SAD gene was cloned from chrysanthemum leaves by improved CTAB method and its sequence was analyzed with chrysanthemum cold resistant variety 'Xingguangguanghuang'. The expression of SAD in chrysanthemum was determined by real-time fluorescence quantitative method, and the expression of this gene at different temperature was analyzed. The main results are as follows: 1. According to the homologous sequence of chrysanthemum SAD gene, degenerate primers were designed and the intermediate fragment of SAD gene was cloned from chrysanthemum 'starlight brilliant' leaves. The total length of c DNA was 1203bp, encoding 401 amino acids. Relative molecular weight 45.57 KD, at Gene Bank registration number KC529335.2. The homology of SAD gene in chrysanthemum was 80.29% and 79.81% respectively in chrysanthemum SAD gene and Helianthus annuus,AAB65145.1 SAD gene. The amino acid sequence of chrysanthemum SAD gene was 80.29% compared with that of SAD gene of Saussurea Xinjiang Saussurea in the same family. There were also high homology with other species, such as Jatropha curcas (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebiferum (76.89%), cassava (76.89%), Chinese tallow (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebifera (76.89%) and cassava (76.89%). The SAD gene has high homology with other plants, so it is named CmSAD.. The protein is a soluble protein with no transmembrane signal region, and the N-terminal contains a chloroplast transport peptide. The protein activity site analysis shows that there is a conserved region of SAD at the C-terminal of the protein encoded by CmSAD, and the 3D structure of the protein is predicted. The expression of CmSAD gene in roots decreased with the decrease of temperature. The expression of CmSAD gene was the highest at 16 鈩，
本文编号：2287830
[Abstract]:9 stearyl-ACP desaturase (Stearoyl-ACP desaturase, for short) catalyzes dehydrogenation to form the first double bond, which is involved in the synthesis of unsaturated fatty acids of cell membrane. The change of the content of stearyl-ACP desaturase (Stearoyl-ACP desaturase,) can change the ratio of saturated fatty acids to unsaturated fatty acids. Thus affecting the cold resistance of plants. In this study, the expression pattern of SAD gene in chrysanthemum under low temperature stress was studied at the molecular level, which laid a foundation for further study on chrysanthemum cold resistance mechanism, selection of cold resistant varieties and further resistance breeding, and could also be used in breeding cold resistant varieties. And improve the ornamental value of chrysanthemum. The intermediate fragment of chrysanthemum SAD gene was cloned from chrysanthemum leaves by improved CTAB method and its sequence was analyzed with chrysanthemum cold resistant variety 'Xingguangguanghuang'. The expression of SAD in chrysanthemum was determined by real-time fluorescence quantitative method, and the expression of this gene at different temperature was analyzed. The main results are as follows: 1. According to the homologous sequence of chrysanthemum SAD gene, degenerate primers were designed and the intermediate fragment of SAD gene was cloned from chrysanthemum 'starlight brilliant' leaves. The total length of c DNA was 1203bp, encoding 401 amino acids. Relative molecular weight 45.57 KD, at Gene Bank registration number KC529335.2. The homology of SAD gene in chrysanthemum was 80.29% and 79.81% respectively in chrysanthemum SAD gene and Helianthus annuus,AAB65145.1 SAD gene. The amino acid sequence of chrysanthemum SAD gene was 80.29% compared with that of SAD gene of Saussurea Xinjiang Saussurea in the same family. There were also high homology with other species, such as Jatropha curcas (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebiferum (76.89%), cassava (76.89%), Chinese tallow (77.62%), Potato (77.86%), Peanut (77.13%), Sapium sebifera (76.89%) and cassava (76.89%). The SAD gene has high homology with other plants, so it is named CmSAD.. The protein is a soluble protein with no transmembrane signal region, and the N-terminal contains a chloroplast transport peptide. The protein activity site analysis shows that there is a conserved region of SAD at the C-terminal of the protein encoded by CmSAD, and the 3D structure of the protein is predicted. The expression of CmSAD gene in roots decreased with the decrease of temperature. The expression of CmSAD gene was the highest at 16 鈩，
本文编号：2287830
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