甘蓝型油菜乙酰转移酶基因的克隆及功能验证

doi:10.7668/hbnxb.20191383

摘要/Abstract

摘要： 为探究乙酰转移酶基因对甘蓝型油菜脂肪酸代谢的影响，以低亚麻酸油菜20~35 d自交种cDNA为模板，克隆了乙酰转移酶基因（Acetyltransferase gene，AT）的2个拷贝（114和148），并进行生物信息学分析以及构建了过表达载体和干扰载体，并转化拟南芥，经潮霉素筛选T₀种子，PCR鉴定抗性苗后，收获T₁种子，用气相色谱法检测T₁种子脂肪酸组成。结果显示，114编码的蛋白偏中性而148编码的蛋白偏碱性，114与148编码的蛋白均为稳定蛋白，亲水性差，属于脂溶性蛋白，且定位在细胞质，无跨膜结构； AT基因的2个拷贝114与148对饱和脂肪酸的转化有影响，114基因与148基因在脂肪酸代谢途径中，抑制软脂酸向硬脂酸转化。过表达114与148基因均会导致软脂酸和亚油酸含量的增加，硬脂酸含量的减少；过表达114基因，会使花生烯酸含量下降，干扰148基因的表达会导致芥酸含量的增加，结果表明，这2个拷贝对改良甘蓝型油菜脂肪酸成分作用明显。

关键词: 甘蓝型油菜, 乙酰转移酶基因, 基因克隆, 过表达, RNA干扰

Abstract: To explore the effect of acetyltransferase gene on the fatty acid composition of Brassica napus. By using the cDNA of 20-35 d inbred of rapeseed with the lower linolenic acid content, 2 copies(114 and 148) of acetyltransferase gene(AT) were cloned and conducted a bioinformatics analysis.The overexpression vector and interference vector were constructed and transformed into Arabidopsis thaliana. The T₀ seeds were screened by hydomycin and the resistant seedlings were identified by PCR. The T₁ seeds were obtained and the fatty acid composition of T₁ seeds were determined by gas chromatography. The results showed that the protein encoded by 114 was neutral while the protein encoded by 148 was alkaline. The proteins encoded by 114 and 148 were both stable proteins with poor hydrophilicity. They were lipid soluble proteins and located in the cytoplasm without transmembrane structure. The two copies of AT gene, 114 and 148, had an effect on the transformation of saturated fatty acids. In the metabolic pathway of fatty acids, 114 and 148 genes inhibit the transformation from palmitic acid to stearic acid. Overexpression of both 114 and 148 genes leads to an increase in palmitic acid and linoleic acid content, and a decrease in stearic acid content. And it was found that overexpressed 114 genes decreased arachidonic acid content, interfering with the expression of the 148 gene increased in erucic acid content. The results of this study showed that these two copies had a significant effect on improving the fatty acid composition of Brassica napus.

Key words: Brassica napus, Acetyltransferase gene, Gene cloning, Overexpression, RNAi

中图分类号:

S635.03
Q78

程潜, 官梅, 张振乾, 常涛, 谭敏, 官春云. 甘蓝型油菜乙酰转移酶基因的克隆及功能验证[J]. 华北农学报, 2021, 36(1): 36-43. doi: 10.7668/hbnxb.20191383.

CHENG Qian, GUAN Mei, ZHANG Zhenqian, CHANG Tao, TAN Min, GUAN Chunyun. Cloning and Functional Verification of Acetyltransferase Gene in Brassica napus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(1): 36-43. doi: 10.7668/hbnxb.20191383.

http://www.hbnxb.net/CN/Y2021/V36/I1/36

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