生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 151-161.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0251

• 研究报告 • 上一篇    下一篇

山杏油体蛋白基因PsOLE4克隆及其调控油脂累积功能分析

党瑗(), 李维, 苗向, 修宇, 林善枝()   

  1. 北京林业大学生物科学与技术学院 林木育种国家工程实验室 林木、花卉遗传育种教育部重点实验室 树木花卉育种生物工程国家林业和草原局重点实验室,北京 100083
  • 收稿日期:2022-02-28 出版日期:2022-11-26 发布日期:2022-12-01
  • 作者简介:党瑗,女,硕士研究生,研究方向:植物分子生物学;E-mail:9946125098@qq.com
  • 基金资助:
    国家自然科学基金面上项目(31972592)

Cloning of Oleosin Gene PsOLE4 from Prunus sibirica and Its Regulatory Function Analysis for Oil Accumulation

DANG Yuan(), LI Wei, MIAO Xiang, XIU Yu, LIN Shan-zhi()   

  1. The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration,Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants,Ministry of Education,National Engineering Laboratory for Tree Breeding,Beijing Forestry University,Beijing 100083
  • Received:2022-02-28 Published:2022-11-26 Online:2022-12-01

摘要:

油体蛋白OLE在植物油脂合成累积中具有重要调控作用。依据山杏(Prunus sibirica)不同发育阶段种子的mRNA转录组测序数据,注释获得5个具有完整开放阅读框和典型保守结构域oleosin的油体蛋白OLE家族基因,通过差异转录谱分析及RT-qPCR检测,确定与山杏种子发育及油脂累积密切相关的高表达PsOLE4基因为研究对象,克隆该基因并进行生物信息学分析、组织特异表达检测、亚细胞定位分析、遗传转化拟南芥及其种子油脂含量和脂肪酸组分测定等研究。结果显示,PsOLE4基因全长序列为378 bp,可编码含125个氨基酸且分子量为13 kD的蛋白。该蛋白为疏水性无信号肽的非分泌蛋白,含有18个磷酸化位点、2个跨膜结构域和1个高度保守的脯氨酸结(PX5SP3P)结构域,定位于细胞膜上。PsOLE4基因在山杏种子中的表达量显著高于茎、叶及果实,而且PsOLE4基因表达可有效促进转基因拟南芥种子的脂肪酸含量提高与油脂累积,表明山杏PsOLE4基因具有种子表达特异性,对油脂累积具有重要调控作用。研究结果为后续开展山杏种子PsOLE4功能鉴定及应用奠定基础。

关键词: 油体蛋白, 油脂, 山杏种子, PsOLE4基因克隆, 组织表达特性, 亚细胞定位, 遗传转化, 功能分析

Abstract:

Oleosin(OLE)plays an important role in regulating synthesis and accumulation of plant lipids. Based on our recent mRNA transcriptomics sequencing data for the seeds of Prunus sibirica from different developing stages,a total of 5 OLE family genes with complete open reading frame and typical conserved oleosin domain were screened by functional annotation,and importantly,RT-qPCR and differential transcription profile were used to determine the PsOLE4 with abundant transcript closely related to development and oil accumulation of P. sibirica seeds as research objects. Hence,the PsOLE4 gene was cloned from P. sibirica seeds,and then the analyses of bioinformatics,tissue-specific expression,subcellular localization and ectopic expression in Arabidopsis thaliana as well as oil content and fatty acid composition of transgenic seeds were conducted. The results showed that the full-length cDNA of PsOLE4 contained an open reading frame of 378 bp in length,encoding a protein of 125 amino acid residues with a molecular mass of 13 kD. The PsOLE4 protein,without signal peptide,was a non-secretory and hydrophobic protein containing 18 phosphorylation sites,2 transmembrane domains and a highly conserved proline junction domain(PX5SP3P),which was mainly localized on cell membrane. The expressions of PsOLE4 was much higher in the seeds of P. sibirica than in the stems,leaves and fruits,and PsOLE4 expression efficiently enhanced the contents of oils and its five predominant fatty acids in A. thaliana seeds,revealing that PsOLE4 with seed-expressed specificity may contribute to the oil accumulation of transgenic A. thaliana seeds. In conclusion,our results could provide important foundation for the further studies of functional identification of PsOLE4 and its application for oil yield improvement.

Key words: oleosin, oil, Prunus sibirica seeds, PsOLE4 gene cloning, tissue specific expression, subcellular localization, genetic transformation, function analysis