木薯MePPD3基因的克隆及功能分析
Cloning and physicochemical characterization of MePPD3gene from cassava(Manihot esculenta)
贾素行 1朱寿松 1符仁稳 1李春霞 1陈银华1
作者信息
- 1. 海南大学热带作物学院,海口 570228
- 折叠
摘要
为了探究PsbP蛋白是否在木薯(Manihot esculenta)抗病中发挥功能,以木薯'华南 8号'总RNA为模板,通过 RT-PCR扩增 MePPD3基因(Phytozome数据库编号:Manes.05G127800).序列分析结果显示,MePPD3基因全长 807 bp,编码 268个氨基酸,在氨基酸序列第 106～266位存在PsbP结构域,推测其为psbP蛋白.蛋白多序列比对、进化树和保守结构域分析表明,木薯 MePPD3蛋白与巴西橡胶(Hevea brasiliensis)中PsbP家族蛋白的同源性最高,同源率高达 99%.亚细胞定位显示,MePPD3蛋白定位在叶绿体.qRT-PCR结果显示,MePPD3基因在木薯不同组织中的表达量具有较大差异,在叶片特别是成熟叶片中表达量最高;此外,该基因表达量受到菜豆黄单胞菌木薯萎蔫致病变种(Xanthomonas phaseoli pv.manihotis,Xpm)诱导后显著上升.利用VIGS技术沉默MePPD3基因,qRT-PCR结果表明,MePPD3基因沉默成功,其表达量显著下降;Xpm侵染植株后,沉默植株pCsCMV-MePPD3叶片的病斑面积显著小于野生型植株,故推测MePPD3可能负调控木薯对细菌性枯萎病的抗病性.
Abstract
The subcellular localization and expression analysis of Psbp protein MePPD3(Manihot esculenta PsbP domain-containing protein 3,Phytozome database number:Manes.05G127800)were performed to explore whether MePPD3 is involved in disease resistance of cassava.MePPD3 gene was amplified by RT-PCR.Sequence analysis showed that MePPD3 gene was 807bp in length,encoding 268 amino acids,with PsbP domain located at position 106-266 aa.Bioinformatics analysis of MePPD3 protein was conducted by NetPhos 3.1 Server,SignalP 5.0 Server,TMHMM Servervr,PSIPRED and PHYRE2 online,respectively.The results indicated that MePPD3 protein contained 32 phosphorylation sites,5 glycosylation sites,and 1 transmembrane domain.The secondary structure of the protein was composed of 21.3%Helix(Helix),26.5%fold(strand)and 52.2%random curl(loop).The multiple sequence alignment,phylogenetic tree analysis and conserved domain analysis indicated that PPD3 protein had a high genetic relationship among different plants.Subcellular localization showed that MePPD3 protein was localized in chloroplast.QRT-PCR results revealed that the expression level of MePPD3 gene in different cassava tissues was significantly different,and was the highest in mature leaves.In addition,the expression of this gene was induced by Xanthomonas phaseoli pv.manihotis(Xpm),indicating MePPD3 is involved in the resistance of cassava to Xpm.After MePPD3 gene was silenced by VIGS technique,the leaf lesion area of silent plants was significantly smaller than that of the control plants,which implies that MePPD3negatively regulates cassava resistance to bacterial fusarium wilt caused by Xpm.
关键词
木薯/MePPD3/生物信息学分析/Xpm/CHN11Key words
cassava/MePPD3/bioinformatics analysis/Xpm CHN11引用本文复制引用
基金项目
国家自然科学基金地区科学基金(31860485)
出版年
2024
NETL
NSTL
维普
万方数据