向日葵FAD2-1基因克隆与功能分析

Cloning and functional analysis of FAD2-1 gene in sunflower

为解析向日葵Δ12脂肪酸脱氢酶基因的功能,根据前期转录组测序结果,采用RT-PCR方法克隆了向日葵Δ12脂肪酸脱氢酶基因HaFAD2-1的开放阅读框(ORF)序列,其核苷酸序列长度为1137 bp,编码378个氨基酸,生物信息学预测表明HaFAD2-1编码蛋白为碱性且亲水性蛋白,二级结构为α螺旋。系统进化分析发现HaFAD2-1基因与异果菊(Dimorphotheca sinuata DC.)FAD2进化关系最近。qRT-PCR分析表明HaFAD2-1基因是种子中特异高表达的基因,且在低油酸种子中表达量明显高于高油酸种子,相关性分析发现HaFAD2-1表达量与油酸含量呈显著负相关,与亚油酸含量呈显著正相关。亚细胞定位发现HaFAD2-1编码蛋白定位于细胞质中,与野生型对照相比,异源表达转HaFAD2-1拟南芥种子的总不饱和脂肪酸含量显著升高,亚油酸合成下游的大部分脂肪酸含量显著升高,而亚油酸合成上游的大部分脂肪酸含量显著降低,表明HaFAD2-1可能是参与向日葵不饱和脂肪酸合成的重要基因。

To elucidate the function ofΔ12 fatty acid dehydrogenase gene in sunflower,open reading frame(ORF)sequence ofΔ12 fatty acid dehydrogenase gene HaFAD2-1 in sunflower was cloned by RT-PCR,according to the results of transcriptome sequencing in previous research.Results showed that the ORF was 1137 bp,encoding 378 amino acids.Bioinformatics prediction showed that HaFAD2-1 was an alkaline and hydrophilic protein with a secondary structure ofα-helix.Phylogenetic analysis showed that HaFAD2-1 gene was most closely related to FAD2 in Dimorphotheca sinuata DC.qRT-PCR analysis showed that HaFAD2-1 gene was particularly highly expressed in seeds.Its expression level in low oleic acid seeds was significantly higher than that in high oleic acid seeds.Correlation analysis showed that the expression level of HaFAD2-1 was significantly negatively correlated with oleic acid content,and significantly positively correlated with linoleic acid content.HaFAD2-1 coding protein was found to be subcellular localized in cytoplasm.Compared with wild type,the content of total unsaturated fatty acid in transgenic Arabidopsis seeds increased significantly,and content of most fatty acids in the downstream of linoleic acid synthesis increased significantly,while the content of most fatty acids in the upstream of linoleic acid synthesis decreased significantly.These results suggested that HaFAD2-1 was an important gene involved in the synthesis of unsaturated fatty acids in sunflower.