棉花3个脂肪酸碳链延长基因的cDNA克隆和表达分析

【目的】克隆棉花脂肪酸合成碳链延长的3个重要基因，分析它们的基因表达及其对逆境胁迫的反应，为棉花高油分育种和抗逆育种提供候选基因。【方法】采用同源克隆的方法，克隆陆地棉脂肪酸合成碳链延长3个重要基因GhKAR、GhHAD和GhENR的cDNA全长；应用生物信息学方法，分析克隆基因编码蛋白的特性；通过RT-PCR，分析目标基因的组织表达特征、时空表达水平和非生物胁迫条件下的表达特性。【结果】GhKAR和GhENR属于NADB Rosemann超基因家族，GhHAD属于hotdog超基因家族；GhKAR、GhHAD和GhENR的cDNA全长分别为1 119、906和1 318 bp，分别编码283、221、394个氨基酸；它们在根、茎、叶及胚珠发育不同时期均有表达。3个基因在高油材料中的基因表达水平都高于低油材料；20DPA（days post anthesis）到30DPA是高低油材料油分积累的主要时期，而30DPA到成熟期是高低油材料油分含量差距产生的关键时期。20DPA低油材料3个基因均有一个表达低值，高油材料基因表达量则持续升高；30DPA时表达量达到最高，其中，对于GhHAD和GhENR的表达量，棉花高油材料为低油材料2倍以上。不同非生物胁迫的诱导表达分析表明，GhKAR、GhHAD和GhENR都不同程度地受低温诱导，GhKAR和GhHAD在ABA诱导时上调表达，但都不受MeJA诱导表达；而GhENR在MeJA诱导2 h时上调表达，之后逐渐下调，受ABA诱导不明显。【结论】获得了GhKAR、GhHAD、GhENR 3个基因cDNA全长，通过基因表达特征分析，3个基因的表达可能对种子油分积累起着重要作用，同时在棉花抗逆方面也起一定的生理作用。

Molecular Cloning and Expression Analysis of Three Chain Extension Genes Related to Fatty Acid Synthesis in Upland Cotton (Gossypium hirsutum L.)

【Objective】Anabolism of fatty acid controls the synthesis of fat and affects responses of plants to abiotic stress. The temporal-spatial expression of related genes directly decides the content of oil in seeds, and affects stress responses of plants associated with fatty acids. The objectives of this study are to clone the important genes related to fatty acid synthesis and to analyze the expression of genes in the process of fatty acid accumulation and the responses of gene expression to stress with the aim to provide candidate genes for high-oil cotton breeding and stress-resistance cotton breeding.【Method】Full length cDNA of GhKAR, GhHAD and GhENR were cloned by means of homologous cloning. Bioinformatics analyses were performed on the obtained genes to describe the characteristics of encoded proteins. The expression model of target genes in tissues, the levels of their temporal-spatial expression and the expression characteristics under abiotic stress were analyzed through RT-PCR.【Result】The cDNA of GhKAR, GhHAD and GhENR were 1 119 bp, 906 bp and 1 318 bp in length, respectively, and encoded 283, 221 and 394 amino acids. GhKAR and GhENR belonged to NADB Rosemann supergene family and GhHAD belonged to hotdog supergene family. The genes expressed variously in different tissues, such as root, stem, leaf and young ovule at different development stages. The expression level of young ovules in high-oil cotton material was slightly higher than that in low-oil cultivars. The oil accumulated majorly from 20DPA to 30DPA in both high-oil and low-oil cotton cultivars, and differed much after 30DPA to maturation. That is, all the 3 genes in high-oil cultivar increased steadily in expression after 20 DPA, and reached the maximum at 30DPA, and expressed at a low value in low-oil cultivars meanwhile. The expression level of GhHAD and GhENR in high-oil cultivars was twice more than that in low-oil ones. The expression level of GhKAR, GhHAD and GhENR induced differently by low temperature. And GhKAR and GhHAD expressed in up-regulation when induced by ABA, and could not be induced by MeJA. The expression level of GhENR was up-regulated at 2h after induction by MeJA, and went down-regulated gradually thereafter, but could not be induced by ABA.【Conclusion】Full length cDNA of GhKAR, GhHAD and GhENR were cloned. Three genes played an important role in oil accumulation in cotton seeds. The characteristics of gene expression inferred these 3 genes involved in responding to physiological stress.