利用酵母双杂交筛选豌豆叶绿体镁离子螯合酶D亚基相互作用蛋白

Screening for Proteins Interacting with Mg-chelatase D Subunit in Pea Chloroplast Using Yeast Two-hybrid System

植物叶绿体镁离子螯合酶是四吡咯化合物生物合成途径中合成叶绿素分支(镁分支)的第一个酶,它催化镁离子螯合到原卟啉IX中,形成镁原卟啉IX.镁离子螯合酶是1个由3个亚基H、D和I组成的多亚基酶,3个亚基均由细胞核编码,进入叶绿体发挥功能.该酶不仅控制着叶绿素的合成,其各个亚基还具有很多其它的功能:H亚基既是ABA受体,又参与叶绿体到细胞核的反向信号传导;D亚基也与叶绿体到细胞核的反向信号传导有关.本文利用酵母双杂交技术,将编码豌豆镁离子螯合酶D亚基的cDNA片段构建到诱饵载体pGBKT7中,分别用共转化的方法筛选豌豆叶片细胞核编码的均一化cDNA文库和用Mating的方法筛选豌豆叶片叶绿体编码的均一化cDNA文库,共得到121个候选克隆,其中有60个克隆共编码21个叶绿体蛋白质,19个来自于核基因编码,2个来自于叶绿体基因编码.这些候选蛋白参与叶绿素合成、卡尔文循环、叶绿体蛋白质翻译和叶绿体基因转录等多个代谢过程.酵母点对点和GST-pull down对其中的4个蛋白做了进一步的验证.这些结果将为D亚基的功能研究提供进一步的线索.

Plant chloroplast Mg-chelatase,the first enzyme at the branch of chlorophyll biosynthesis pathway,catalyzes the incorporation of Mg2＋ into protoporphyrin IX.Its three subunits,H,D and I,are products of nuclear coding genes,then imported into and function at the chloroplast.These subunits are not only involved in chlorophyll biosynthesis along with the enzyme,also exert other functions.Subunit H was found as an ABA receptor,and both H and D subunits were involved in the retrograde signaling from chloroplast to nucleus.Using a yeast two-hybrid system,we constructed a pGBKT7 bait vector containing pea Mg-chelatase D subunit cDNA and screened both a normalized nuclear-encoded and chloroplast-encoded cDNA library of pea leaves by co-transformation and mating method,respectively.In the total of 121 putative positive clones obtained,60 clones encode 21 chloroplast proteins,of which 19 were nuclear genes and 2 were chloroplast genes.The identified proteins were involved in different biological functions,such as chlorophyll biosynthesis,Calvin cycle,and regulation of protein translation and transcription in chloroplast,etc.By two hybrid re-cotransformation and GST pull-down assay,4 proteins were validated.Our results provided valuable clues for the functional studies of chloroplast Mg-chelatase D subunit.