玉米蛋白磷酸酶2C基因ZmPP2C26两个可变剪接体的功能分析  

Functions of two alternative spliced variants of protein phosphatase type 2C gene ZmPP2C26 of maize

作　　者：张盼娃 张夏玮 于好强 盘林秀 付凤玲[1] 李晚忱[1] ZHANG Panwa;ZHANG Xiawei;YU Haoqiang;PAN Linxiu;FU Fengling;LI Wanchen(Maize Research Institute,Sichuan Agricultural University,Chengdu,Sichuan 611130,China)

机构地区：[1]四川农业大学玉米研究所,四川成都611130

出　　处：《西北农林科技大学学报（自然科学版）》2018年第7期23-31,72,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家转基因生物新品种培育重大专项(2016ZX08003-004);国家自然科学基金项目(31071433)

摘　　要：【目的】探讨蛋白磷酸酶2C在植物生长发育和逆境抗性中的作用。【方法】利用逆转录PCR扩增玉米蛋白磷酸酶2C基因ZmPP2C26的两个可变剪接体,用生物信息学工具预测其推导蛋白的特性后,转化野生型拟南芥,并进行功能验证和亚细胞定位。【结果】在较短剪接体中切除的213nt第一内含子,在较长剪接体中被保留。第一个内含子的剪接位点为5′-CC..CG-3′,与植物中通常的组成型剪接位点5′-GT..AG-3′不同。保留内含子及其侧翼序列的G/C含量较高,相当于其他内含子及其侧翼序列的2倍。两个剪接体在野生型拟南芥中的异源表达均可增加其对干旱胁迫的敏感性。两个剪接体推导蛋白的预测三维结构均与PP2C相似,较长剪接体保留内含子编码的71个冗余氨基酸序列保持随机螺旋状态,预测为叶绿体定位信号肽。绿色荧光蛋白标记法定位表明,较长剪接体定位于细胞核、细胞膜和细胞器,而较短剪接体定位于细胞核和细胞膜。【结论】ZmPP2C26基因转录后加工过程中发生内含子保留型可变剪接。第一个内含子的保留没有改变其编码蛋白的PP2C功能,但有可能使其作用由细胞核和细胞膜延伸至叶绿体。【Objective】This study investigated the role of protein phosphatase type 2C（PP2C）in plant development and environmental response.【Method】Two alternative spliced variants of the ZmPP2C26 gene were amplified by reverse transcription PCR,the properties of their putative proteins were bioinformatically predicted,and they were transformed into wild type Arabidopsis for functional validation and subcellular localization.【Result】The first intron of 213 nt removed in the shorter spliced variant was retained in the longer variant.Its splice site was 5′-CC..CG-3′,which was different from the constitutive splicing site（5′-GT..AG-3′）of plants.The G/C content of the retained intron and its flanking sequences was two times of that of the other two introns and their flanking sequences.The heterologous expression of the two variants increased the sensitivity of wild type Arabidopsis to drought stress.The 3-dimensional structures of the two putative proteins were predicted to be similar to the PP2C domain,while the redundant amino acid sequence encoded by the retained intron of the long variant remained as random coil,which was predic-ted to be a signal peptide targeting to the chloroplast.The longer spliced variant was localized in the nucleus,cytoplasmic membrane and organelles,whereas the shorter spliced variant was localized in the nucleus and cytoplasmic membrane by the method of green fluorescent protein labeling.【Conclusion】The alternative splicing of intron retention occured in the post-transcriptional processing of the ZmPP2C26 gene.The retention of the first intron did not change its encoded protein functioning as a PP2 C,but probably expanded its effect from nucleus and cytoplasmic membrane to the chloroplast.

关 键 词：可变剪接 干旱 异源表达 玉米 蛋白磷酸酶2C 

分 类 号：Q78[生物学—分子生物学]