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长链脂肪酰基Co A合成酶(LACS)在脂肪酸的合成与分解代谢中起着重要作用。本研究以油茶(Camellia oleifera Abel)国家审定品种华硕(Camellia oleifera Huashuo)种仁转录组数据为基础,根据LACS基因Unigene序列设计引物,分离克隆了油茶LACS1基因全长c DNA序列,命名为CoLACS1(Gene Bank登录号:KJ960228),全长2114 bp,开放阅读框2088 bp,编码695个氨基酸;生物信息学分析显示CoLACS1具有3个B1ock,从分子特征可判断CoLACS1属于LACS家族;氨基酸同源比对显示与其他物种的LACS氨基酸序列具有较高的相似性,其中与拟南芥LACS7相似性为78%,与麻风树、大豆、毛果杨等物种LACS6(peroxisomal)相似性可达80%以上;对CoLACS1进行原核表达分析,构建的p ET30a-CoLACS1载体成功转化至BL21(DE3)中经1 mmol/L IPTG诱导表达,菌液检测获得预测的目的蛋白(分子量约为76 k D);分析转录组数据中CoLACS1的Unigene序列RTKM值并对CoLACS1进行实时荧光定量PCR分析,结果表明CoLACS1在油茶华硕种子发育各时期平稳表达,表达丰度变化不大,荧光定量结果变化规律与转录组数据分析一致;同时分析华硕种仁不同时期含油率和脂肪酸成分变化,双变量统计分析发现CoLACS1表达模式与油茶油脂积累规律呈显著相关性。本研究为进一步研究油茶油脂积累与代谢的基因调控提供理论依据。
Long chain fatty acyl Co A synthase (LACS) plays an important role in the synthesis and catabolism of fatty acids. In this study, based on the genome data of Camellia oleifera Huashuo (Camellia oleifera Huashuo), a full-length cDNA sequence of LACS1 gene was isolated and cloned based on the LUC gene Unigene sequence and named as CoLACS1 ( Gene Bank accession number: KJ960228), full-length 2114 bp, open reading frame 2088 bp, encoding 695 amino acids; bioinformatics analysis showed CoLACS1 has three B1ock, molecular features can be judged CoLACS1 belongs to LACS family; amino acid homology comparison Which showed a high similarity with the LACS amino acid sequences of other species. The similarity to LACS7 of Arabidopsis thaliana was 78%, and the similarity with LACS6 (peroxisomal) of Jatropha curcas, soybean, Populus trichocarpa was more than 80% The prokaryotic expression analysis of CoLACS1 was carried out. The p ET30a-CoLACS1 vector was successfully transformed into BL21 (DE3) and induced by 1 mmol / L IPTG. The predicted protein (molecular weight was about 76 kD) The data of Unigene sequence RTKM of CoLACS1 in group data and real-time PCR analysis of CoLACS1 showed that CoLACS1 was expressed steadily in all stages of Camellia sinensis seed development, At the same time, the changes of oil content and fatty acid composition in different kernels of ASUS were analyzed. Bivariate statistical analysis showed that the expression pattern of CoLACS1 was significantly correlated with the accumulation of oleifera oil. This study provides a theoretical basis for further study of gene regulation of oil accumulation and metabolism in Camellia oleifera.