香青菜亚麻酸含量性状相关miRNA的鉴定

Identification of miRNA Associated with Linolenic Acid Content Traits in Brassica campestris ssp.chinensis

本研究旨在发掘香青菜(Brassica campestris ssp.chinensis L.Makino,NHCC)中影响亚麻酸含量的相关miRNA及其对应靶基因,探究它们的调控机制。以香青菜亚麻酸含量差异大的两个亚种‘绣花筋’和‘黑叶香青菜’的成熟叶片为材料,采用小RNA高通量测序来鉴定两个材料中差异表达的miRNA,预测其靶基因并进行基因功能注释。试验共鉴定得到miRNA 236个,包括159个已知miRNA,77个新的miRNA。筛选出差异表达的miRNA 63个,其中,以‘黑叶香青菜’为对照,上调的有28个,下调的有35个。差异表达的miRNA对应的靶基因中共6041个得到功能注释,其涉及的主要通路多个与脂肪酸代谢相关,如α-亚麻酸代谢、脂肪酸合成、不饱和脂肪酸的生物合成、脂肪酸降解等。差异表达miRNA的靶基因主要是一些亚麻酸合成或代谢重点的关键酶,如脂氧合酶(lipoxygenase,LOX)、3-酮酰基-CoA硫酶2(3-ketoacyl-CoA thiolase,KCS)等。香青菜通过对这些miRNA在不同品种中的差异表达负调控LOX等脂肪酸代谢关键基因和KCS等亚麻酸合成相关基因的表达,一方面减少亚麻酸的代谢损失,另一方面增加亚麻酸的合成量,从而引起亚麻酸含量的增高。

The purpose of this study is to explore the miRNA and its target genes that affect linolenic acid content in Xiangqingcai(Brassica campestris ssp.chinensis L.Makino,NHCC),and to clarify its molecular function in the regulation of this character.In this research,using the mature leaves of ’Xiuhuajin’ and ’Heiyexiangqingcai’ which have largest difference in linolenic acid content as materials,the differentially expressed miRNA was identified by high-throughput microRNA sequencing,and the target gene function was predicted and further function annotated.In total 236 miRNAs were detected,of which 159 were known miRNA and 77 were novel ones.In these miRNAs,28 were up-regulated and 35 were down regulated.A total of 6 041 target genes corresponding to the differentially expressed miRNA were annotated,including many fatty acid metabolism pathways,such as α-linolenic acid metabolism,fatty acid synthesis,unsaturated fatty acid biosynthesis and fatty acid degradation,etc.The target genes of differential expressed miRNA mainly are some key enzymes of linolenic acid synthesis or metabolism,such as lipoxygenase(LOX),3-ketoacyl-CoA thiolase(KCS),which play an important role in the synthesis and transformation of linolenic acid.Through negatively regulating their expression and other key genes of fatty acid metabolism and the related genes of linolenic acid synthesis such as KCS through the differential expression of these miRNAs in different varieties,on the one hand reduces the metabolism loss of linolenic acid,on the other hand increases the synthesis amount of linolenic acid,thus resulting in the increase of linolenic acid content.