麻疯树油质蛋白JcOle14.3基因克隆及序列分析

Cloning and sequence analysis of oleosin gene JcOle14.3 in Jatrophacurcas

作为最丰富的油体结合蛋白,油质蛋白在油体的发生到分解消失过程中起着重要的生物学功能。本实验应用生物信息学和分子生物学技术分离克隆了麻疯树Jatrophacurcas全长593 bp的油质蛋白JcOle14.3基因序列,无内含子结构。JcOle14.3基因转录的m RNA包含了完整的开放阅读框,推测编码由137个氨基酸残基组成、相对分子质量为14.3 k D的稳定、两性的油质蛋白JcOle14.3。JcOle14.3理论等电点为9.65,正电荷残基(Arg+Lys)总数为10个,负电荷残基(Asp+Glu)总数为6个;不稳定系数为25.90,属于稳定性蛋白;其二级结构的主要构件为α-螺旋、随机卷曲和延伸链。根据高级结构预测结果推测JcOle14.3含有N-末端两亲性结构域、中间疏水性结构域以及C-末端两亲性结构域。C-末端两亲性结构域包含1个α-螺旋结构域,中间疏水性结构域包含由3个α-螺旋构成的跨膜结构域,并具有oleosin蛋白特征性的高度保守的脯氨酸结模体,推测在oleosin蛋白与单层磷脂层和油体锚定结合上起着重要的作用。该研究为后期JcOle14.3的异源表达和功能研究奠定了基础。

Oleosins as the most abundant oil body-associated proteins have been suggested to play an important biological function in the stability ofoil bodies, and in their synthesis and metabolism. In this study, the gene JcOle14.3 containing no intron and encoding 14.3 k D oleosin was isolated by PCR combined with bioinformatics analysis and molecular biological technique from Jatrophacurcas L.. The full-length m RNAcomprised 593 nucleotides consisting of an open reading frame of 414 nucleotides encoded a putative JcOle14.3 comprising 137 amino acid residues with molecular weight of 14.3 k D. The isoelectric point of 9.65.and the total number of positively charged residues（Arg＋Lys） was 10, and total number of negatively charged residues（Asp＋Glu） was 6. JcOle14.3 with unstable coefficient 25.90 belongs to the stable protein. The main parts of predicted secondary structures of JcOle14.3 were α-helices, random coilsand extended strand. The JcOle14.3 gene product consists of a highly hydrophobic central domain flanked byrelatively polar N- and C-terminal domains. The results indicated thatthe C-terminal region forms an amphipathicα-helix. The JcOle14.3 central domain forms three α-helices and α1 and α2 were separated by the proline-knot forming a 180 turn. The oleosin central domain is highly conserved between all oleosinssequenced to date. This workalso suggested that the highly conservedproline-knotplayimportantrolesfor oil body targeting. The study results established the foundation for the heterologous expression and functional analysis of JcOle14.3.