菊花去饱和酶CmSAD基因的克隆与表达分析

以秋菊(Chrysanthemum morifolium Ramat.)抗寒品种‘星光灿烂’为材料,利用RT-PCR和RACE方法从叶片中克隆△9硬脂酰-ACP去饱和酶(Stearoyl-ACP desaturase,SAD)基因,命名为CmSAD,Gen Bank登录号为KC529335。该基因cDNA全长1 203 bp,编码401个氨基酸,相对分子质量为45.57 k D,等电点为6.48,编码的氨基酸序列与新疆雪莲同源性最高(80.29%)。通过对该蛋白进行生物信息学分析得出其没有跨膜结构,主要存在于叶绿体基质中,N端含有一段包含60个氨基酸的叶绿体转运肽。通过实时荧光定量PCR研究低温胁迫下叶片和根系中CmSAD基因的表达量变化,该基因在叶片和根系中均有表达。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;叶片中CmSAD表达量随着温度的降低先升高再降低,5℃时最高。随着温度的降低,菊花叶片和根系中CmSAD基因的表达情况表现出一定的差异。CmSAD基因的表达变化与温度有关,为低温胁迫下菊花脂肪酸代谢的分子机理研究提供了基础。

Cloning and express analysis of fatty acid desaturase gene CmSAD in chrysanthemum

Chrysanthemum (Chrysanthemum morifolium Ramat.) variety of cold resistance ‘ Xingguangcanlan’ was selected as experimental materials.△9 Stearoyl-ACP desaturase (SAD) gene were separated and cloned from the leaf by RT-PCR and RACE,named CmSAD,accession number is KC529335 in GenBank.The full length cDNA of CmSAD is 1203 bp,encoding a protein of 401 amino acids.Its molecular mass is 45.57 kD,pI is 6.48,and the homology of coding sequence of amino acids is the highest with Saussurea involucrata(80.29％).This protein has no transmembrane structure,and mainly exists in the chloroplast stroma,and a chloroplast transit peptide at N-terminal,which contains 60 amino acids.Quantitative PCR research by real-time fluorescent was conducted on quantity alteration in leaves and root under low temperature stress CmSAD gene expression.The expression quantity of CmSAD gene decreased in root system with temperature decreasing.When the temperature was 16 ℃,the expression quantity of CmSAD gene was the highest.With the decrease of temperature,the expression quantity of CmSAD gene in leaves increased first and achieved the highest at 5 ℃,then decreased.Different temperatures made diffident expression quantity of CmSAD gene.With the decrease of temperature,the expression quantity of CmSAD gene decreased.Therefore,the expression quantity of CmSAD gene was related to temperature and it also had a close relationship with cold resistance.