‘鸭梨’及其自交亲和性突变体‘金坠梨’花粉蛋白质组比较分析  

Comparative proteome analysis of pollens from ’Yali’ pear and its self-compatible mutant,’Jinzhui’ pear

作　　者：沈辉 殷亚蕊 武军凯 王海静 黄佳 左波 张立彬[2] 于凤鸣[1] SHEN Hui;YIN Yarui;WU Junkai;WANG Haijing;HUANG Jia;ZUO Bo;ZHANG Libin;YU Fengming(College of Life Science and Technology,Hehei Normal University of Science&Technology,Qinhuangdao 066004,Hebei,China;College of Horticultural Science and Technology,Hehei Normal University of Science&Technology,Qinhuangdao 066004,Hebei,China)

机构地区：[1]河北科技师范学院农学与生命科技学院,河北秦皇岛066004 [2]河北科技师范学院园艺科技学院,河北秦皇岛066004

出　　处：《果树学报》2019年第9期1101-1111,共11页Journal of Fruit Science

基　　金：国家自然科学基金(31401853)

摘　　要：【目的】分析‘鸭梨’和‘金坠梨’花粉蛋白差异,探讨‘金坠梨’花粉侧自交亲和突变的机制。【方法】以‘鸭梨’及其自交亲和性芽变‘金坠梨’花粉为材料,利用双向电泳技术(two-dimensional electrophoresis,2-DE)对二者花粉总蛋白进行分离,并对差异蛋白进行功能和质谱分析,通过qRT-PCR进行相关差异基因的表达验证。【结果】分离得到23个差异蛋白,其中14个蛋白在‘金坠梨’中上调表达,9个蛋白在‘金坠梨’下调表达;对其中差异显著的10个蛋白点质谱检测结果表明,1个为半胱氨酸甲基转移酶(cysteine S-methyltransferase,4 803),1个为存储蛋白(Storage protein,5 413),4个为蛋白酶类:果糖激酶(fructokinase,5 316)、顺乌头酸水合酶(aconitate hydratase,5 811)、烯酰-ACP-还原酶(Enoyl-ACP reductase,7 303)和JPR ORF1蛋白(JPR ORF1 protein,7 506),4个为HSP家族蛋白:HSP70伴侣26(HSP70-type chaperone 26,8 633)、HSP 90.5(8 822)和2个ER结合蛋白(ER-binding protein,4 209和8 805)。这些蛋白涉及到遗传信息处理、碳水化合物代谢、能量代谢、酶和氨基酸代谢等生理过程;qRT-PCR对8个基因进行验证,发现部分基因在蛋白水平与基因表达水平有明显差异。这些差异的蛋白在不同的信号途径中参与了‘金坠梨’花粉侧自交亲和突变。【结论】明确了‘鸭梨’和‘金坠梨’花粉侧自交亲和性突变中差异表达蛋白,为进一步研究梨亚科自交不亲和性的调控机理提供理论依据。【Objective】The differentially expressed proteins of pollens from‘Yali’pear(self-incompatibility)and‘Jinzhui’pear(self-compatible mutant of‘Yali’)were analyzed,and the mechanism of selfcompatible mutation in‘Jinzhui’pear was discussed.【Methods】The pollens of‘Yali’pear and selfcompatible pollen-part mutant‘Jinzhui’pear were used as the materials,and the pollen proteins were separated.Thereafter,the proteins were tested by dyeing technology and BIO-RAD GS-900 Calibrated Densitometer scanning.PDQuest 8.0.1 software was used to analyze the gel images,and the differential protein spots were obtained.The differentially expressed proteins were finally identified through enzymatic hydrolysis of differentially expressed proteins and LC-MS/MS analysis in the UniProt database.The raw file of mass spectrometry data was processed by the Mascot software,and meanwhile the quality of data was controlled.The results of identified proteins were obtained.Using the online website NCBI and Gene Ontology Database(GO),the identified proteins were compared and analyzed according to cell components,biological processes and molecular functions.Protein sequences were submitted to Kyoto Encyclopedia of Gene and Genomes(KEGG)and bi-directional best hit(BBH)method was used to compare similarities to find the most similar proteins and determine the KO(KEGGORTHOLOG)classification of retrieval proteins.According to the gene sequences of differential proteins,gene-specific primers were designed by NCBI on-line primer design tool Primer-BLAST,and the transcription level of related genes was verified by qRT-PCR.【Results】The results of two-dimensional electrophoresis(2-DE)of total pollen proteins showed that the protein spots were clearly visible and the separation quality was high.It was found that the protein spots were mainly distributed in the region of pH 3-10,the relative molecular weights were about 10-200 kDa,and about 550 protein spots could be detected.Using PDQuest 8.0.1 software,23 differential proteins

关 键 词：梨 花粉 自交不亲和 双向电泳 蛋白质组学 

分 类 号：S661.2[农业科学—果树学]