【作者】 李静；

【导师】 李永华；

【作者基本信息】 河南农业大学 ， 园林植物与观赏园艺， 2016， 硕士

【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。更多还原

【Abstract】 △9 Stearoyl-ACP desaturase(SAD)catalyze dehydrogenation between9 and 10 carbon atoms to form the first double bond.SAD participate in the synthesis of cell membrane lipid unsaturated fatty acid.When the content of SAD was changed,it can change the ratio between saturated and unsaturated fatty acid,while it can affect cold resistance of plants.SAD gene expressive patterns under low temperature stress from the plant molecular level was researched in this test,in order to lay the foundation for further cold resistance addition research,cold resistance variety screening and resistance breeding of chrysanthemum,it can be also used for cold resistance seed selection and the ornamental value improvement of chrysanthemum.Chrysanthemum(Chrysanthemum×morifolium Ramat.)variety of cold resistance‘Xingguangcanlan’ was selected as experimental materials.Chrysanthemum’s SAD gene was cloned through improved method of CTAB from Chrysanthemum’s leaf,and its sequence was analyzed.The expression index of chrysanthemum’s SAD was determined using q RT-PCR,and the expressive influence of the gene under different temperatures was analyzed.The main results were as follows:1.Degenerate primers were designed according to homologous sequences of chrysanthemum’s SAD genes.Fragment of chrysanthemum’s SAD genes was cloned from leaf of chrysanthemum ‘Xingguangcanlan’ and the specific primers were designed and a total 1203 bp length of c DNA was obtained through amplification.It can encode the number of 401 amino acids.Its relative molecular weight is 45.57 KD.Registration number is KC529335 in Gene Bank.2.Homologous sequence was compared between chrysanthemum SAD gene and other plants.There is homology of 80.29% between the amino acid encoded by SAD genes fragment of chrysanthemum’s and Xinjiang Snow lotus Herb’s(Saussurea involucrata Kar.Et Kir.Ex Maxim.,ABF66638)of the same family.And there is homology of 79.81% with sunflower(Helianthus annuus,AAB65145.1)of the same family.There is high homology with other plants,such as jatropha(77.62%),paraquat(75.91%),potato(77.86%),Millettia pinnata(77.62%),peanut(77.13%),Excoecaria sebifera(76.89%),cassava(76.89%).It had a higher homology with SAD in otherplants,so the gene was named CmSAD.The protein is soluble and had no transmembrane signal area.There is a transit peptide of chloroplast in N– and a sequence of conservative area of SAD in C– of encoded protein by CmSAD through protein active site analysis.And 3D structure of the protein was predicted.3.The expression quantity of CmSAD gene was decreased in root system with temperature decreasing.When the temperature was 16 ℃,the expression quantity of CmSAD gene is the highest.The expression quantity at 5℃,-4℃ and-8℃ was 0.87,0.11,and 0.03 times than that at 16℃,respectively.With the decrease of temperature,the expression quantity of CmSAD gene in leaves increased first and achieved the highest at 5℃,then it was decreased.The expression quantity at 16℃,-4℃ and-8℃was 1.3,5.6 and 3.5 times than that at 5 ℃.Different temperatures made diffident expression quantity of CmSAD gene.Expression quantity of CmSAD gene in root system was higher than leaves when the temperature were 16℃ and 5℃.Expression quantity of CmSAD gene in leaves was higher than root system when the temperature were 4℃ and-8℃.SAD gene made saturated fatty acid catalyze dehydrogenation to form the first double bond,which changed the content of unsaturated fatty acid.With the decreased of temperature,the expression quantity of CmSAD gene was decreased.Therefore,the expression quantity of CmSAD gene was related to temperature and it also had a close relationship with cold resistance.更多还原