陆地棉GhSAD2基因克隆与表达特征研究

Molecular Cloning and Expression Analysis of Stearoyl-ACP Desaturase Gene(GhSAD2)in Upland Cotton(Gossypium hirsutum L.)

Δ9硬脂酰ACP脱氢酶基因(GhSAD2)是脂肪酸合成代谢过程中关键的去饱和酶基因,为明确该基因在棉花脂肪酸合成代谢中的功能,该研究克隆了陆地棉GhSAD2基因,并对该基因的序列特征、进化关系及表达特性进行分析。序列分析显示,GhSAD2基因(GenBank登录号为KX197920)cDNA全长1 188bp,编码396个氨基酸,具有脂肪酸去饱和酶家族2个高度保守的组氨酸簇EENRHG和DEKRH,分别位于氨基酸的185和271位。系统进化分析显示,GhSAD2基因与可可树的同源基因进化关系非常接近。qPCR分析显示,GhSAD2基因在叶中的表达量高于茎和根,且在花后25d的种子中表达量达到最高值。低温胁迫诱导结果表明,GhSAD2基因在不同程度低温处理下均有上调表达,6h表达量最大,之后逐渐下调。研究表明,GhSAD2基因可能对棉子油不饱和脂肪酸的合成具有重要作用,同时在棉花抗寒方面也起一定的生理作用。

Δ9stearoyl ACP desaturase gene（GhSAD2）is a fatty acid synthesis and metabolism in the process of key desaturase gene.To clarify the gene function in biosynthesis of cotton fatty acids,we cloned the GhSAD2 gene from upland cotton,and analyzed the gene sequence features,evolutionary relationship and expression characteristics.In this study,a full-length cDNA of stearoyl-ACP desaturase gene was cloned from upland cotton ‘Xinluzao 33＇leaves successfully by using electronic stitching and RT-PCR,which named GhSAD2 and access number was KX197920 in GenBank.The gene nucleotide and amino acid sequences were analyzed by using NCBI and DNAMAN and a phylogenetic tree was constructed by using Clustalw 1.83 and MAGE 5.0software,which would help analyze the gene evolutionary relationships a-mong different species.Result showed that the GhSAD2 gene open reading frame had 1188 bp,encoding a polypeptide of 396 amino acids.Sequence analysis revealed that this gene GhSAD2 had two highly conserved histidine clusters of ferritin-like superfamily：EENRHG and DEKRH were located at amino acid 185 and 271.Phylogenetic analysis showed that GhSAD2 homologous gene evolutionary relationship with the cacao tree is very close,which is consistent with the status of the species in the classical taxonomy.qPCR showed that GhSAD2 in G.hirsutumhad a much higher expression in leaves than that in stems and roots.GhSAD2 express highest at 25days（s25）after flowering seed.GhSAD2 in leaves expression under cold stress analysis showed that the gene were upregulated under varying low temperature treatments.It has the highest expression at 6hours treatment,and then gradually reduced.Studies have shown that GhSAD2 gene may play an important role at cotton seed oil unsaturated fatty acid synthesis.At the same time,it has a certain physiological role in the cold resistance of cotton.