燕麦GPAT基因家族鉴定及AsGPAT9-1调控油脂合成的分子机制

Identification of oat GPAT gene family and molecular mechanism of AsGPAT9-1 regulating lipid synthesis

摘要:

甘油-3-磷酸酰基转移酶(glycerol-3-phosphate acyltransferase, GPAT)是催化三酰甘油(triacylglycerol, TAG)生物合成的关键限速酶，在油料作物油脂合成过程中发挥重要作用。燕麦(Avena satiba L.)胚乳中的脂肪含量在粮食作物中居于首位，作为唯一在胚乳中大量积累油脂的禾本科作物，其GPAT基因的功能尚不清楚。本研究从燕麦基因组中鉴定出58个编码AsGPATs的基因，不均匀地分布在20条染色体上，根据其系统发育关系可分为3个不同的类群，同一亚家族的AsGPAT成员具有相似的基因结构和4个保守的酰基转移酶结构域；转录组测序分析表明AsGPAT9-1基因在燕麦不同发育时期的种子中表达量最高，且其编码蛋白定位于内质网；酵母表达系统分析表明，AsGPAT9-1具有很强的GPAT酶活性，对TAG组装至关重要，并且外源脂肪酸(fatty acids, FA)饲喂试验证实其对C18:1具有底物偏好性；过表达AsGPAT9-1显著提高了转基因烟草叶片及种子的总油脂含量，此外，转基因烟草叶片淀粉和可溶性糖含量降低，蛋白含量无明显变化，种子中淀粉、可溶性糖及蛋白含量均显著降低。研究结果表明AsGPAT9-1可作为基因代谢工程中调控油料作物脂肪酸含量的理想靶点，为燕麦种子油脂合成代谢的深入研究奠定了理论基础。

Abstract:

Glycerol-3-phosphate acyltransferases (GPATs) are key rate-limiting enzymes that catalyze the biosynthesis of triglycerides (TAG) during the lipid synthesis of oilseed crops. Oat (Avena satiba L.) has the highest fat content among cereal crops, and it is the only gramineous crop that accumulates a large amount of oil in the endosperm. However, the functions of the GPAT genes in oat remain unclear. In this study, 58 genes encoding AsGPATs were identified from the oat genome, and they presented uneven distribution across 20 chromosomes. According to phylogenetic relationships, they were assigned into three different subfamilies. AsGPAT members within the same subfamily exhibited highly conserved gene structures and four conserved acyltransferase domains. The transcriptome sequencing results showed that AsGPAT9-1 had the highest expression level in oat seeds at different developmental stages, and its encoded protein was localized to the endoplasmic reticulum. Functional analysis using a yeast expression system demonstrated that AsGPAT9-1 had strong GPAT activity, which was crucial for TAG assembly. The experiments with exogenous addition of fatty acids (FAs) confirmed that AsGPAT9-1 had a substrate preference for C18:1. Overexpression of AsGPAT9-1 significantly increased the total FA content in the leaves and seeds of transgenic tobacco. Additionally, transgenic tobacco leaves showed reduced starch and soluble sugar content and no significant change in protein level. Moreover, the starch, soluble sugar, and protein content in the seeds of transgenic tobacco all significantly reduced. The results indicated that AsGPAT9-1 served as an ideal target for regulating the FA content of oil crops in genetic metabolic engineering, laying a theoretical foundation for in-depth research on the lipid synthesis and metabolism of oat seeds.