三个耐冻性不同的马铃薯野生种中FAD2基因的克隆及表达分析

Molecular Cloning and Expression Analysis of FAD2 Gene from Three Wild Potato Species with Different Levels of Freezing Tolerance

以3个耐冻性和冷驯化能力不同的马铃薯野生种Solanum commersonii、S.acaule和S.cardiophyllum为材料,利用RT-PCR技术克隆出不饱和脂肪酸合成途径中的关键酶基因ω-6脱氢酶基因(FAD2),分别命名为Cmm-FAD2(GenBank登录号为KF214782)、Aca-FAD2(KF214781)和Cph-FAD2(KF214783)。序列分析表明,该基因在3个马铃薯野生种中核苷酸长度都为1326 bp,编码441个氨基酸残基。3个野生种FAD2基因核苷酸序列和蛋白序列比对结果表明,在18个位点的差异核苷酸中有8个位点的差异导致对应的氨基酸残基变化,其中第11和第44氨基酸残基处,耐冻且有冷驯化能力的S.commersonii和S.acaule有相同的氨基酸,而冷冻敏感S.cardiophyllum的氨基酸却不同。二级结构预测结果表明S.commersonii和S.acaule的FAD2蛋白与S.cardiophyllum FAD2蛋白的α-螺旋、延伸链和随机卷曲存在明显差异。蛋白多序列比对和进化树分析表明,3个马铃薯野生种的FAD2基因与番茄的亲缘关系最近,其次是与马齿笕。qPCR分析表明,12 d冷驯化使FAD2基因在3个马铃薯野生种中都上调表达,S.commersonii和S.acaule的FAD2基因表达水平均高于S.cardiophyllum,且差异显著。

Using reverse transcription polymerase chain reaction（RT-PCR）,three full-length cDNAs of ω-6 desturases（FAD2） were obtained from three wild potato species（Solanum commersonii,S.acaule,and S.cardiophyllum） with different levels of freezing tolerance and cold acclimation capacity.These genes were designated Cmm-FAD2（GenBank accession No.KF214782）,Aca-FAD2（KF214781）,and Cph-FAD2（KF214783）,respectively.They are all 1326 bp in length,coding 441 amino acids.The alignment result indicated that eight of 18 different nucleotides result in the corresponding variation of coding proteins in the three species.At the amino acid positions of No.11 and No.44,amino acid residues of S.commersonii and S.acaule are different from that of S.cardiophyllum.The results of secondary structure prediction showed obvious differences in alpha-helix,extend chain,and random crimp of S.commersonii and S.acaule as compared to those of S.cardiophyllum.According to the alignment result of multiple protein sequences and the phylogenetic tree,the coding products of FAD2 genes from the three wild potato species are highly homologous to those from tomato and purslane.The results of qPCR showed that FAD2 expression was up-regulated in the three potato wild species after 12-day cold acclimation;however,the relative expression level of FAD2 in S.commersonii and S.acaule were significantly higher than that in S.cardiophyllum.