基于DIA LC-MS的高淀粉型与低淀粉型粉葛叶片的定量蛋白质组学差异

Quantitative Proteomics Differences of Leaves in Pueraria thomsonii with the High and Low Starch Content Based on DIA LC⁃MS

[目的]寻求粉葛生长发育的蛋白调控机制。[方法]以高淀粉型赣葛1号(鲜葛根含淀粉20%,干葛根含淀粉35%)和低淀粉型赣葛2号(鲜葛根含淀粉14%,干葛根含淀粉25%)为材料,采用基于全扫描数据非依赖性采集高分辨率色谱-质谱(DIA LC-MS)的定量蛋白质组学技术得到差异蛋白质,并对差异蛋白进行GO富集、KEGG通路和KOG分析。[结果]2个不同粉葛叶片中有178个蛋白(101个上调和77个下调)存在显著差异,主要涉及分子功能,但|log2(FC)|>5.0的差异蛋白仅占11.2%。GO富集分析表明,差异蛋白主要富集在代谢过程、催化活性、水解酶活性、细胞部分、细胞内部分、细胞质部分和细胞内细胞器类别。KEEG富集代谢途径集中在苯丙氨酸生物合成、植物体内的MAPK信号通路、次生代谢物生物合成、丙酮酸代谢和脂肪酸代谢。KOG分析显示,差异蛋白功能主要归为核苷酸转运和代谢、核糖体结构和生物发生、能量产生和转换、信号转导以及翻译后蛋白修饰、周转和伴侣机制。差异程度极大的蛋白依次为上调的类LHCⅡ型1叶绿素a-b结合蛋白、kunitz家族胰蛋白酶和蛋白酶抑制剂前体、凝集素类蛋白和几丁质酶同源物及下调的类溶酶体Pro-X羧肽酶、预测的类dCTP焦磷酸酶1、富含半胱氨酸的受体类蛋白激酶25和色氨酸转氨酶相关蛋白4。[结论]研究结果为粉葛的生长发育、营养成分和活性物质的合成及调控、遗传改良及分子育种等提供数据支撑和技术途径。

[Objective]To seek the protein regulation mechanism for the growth and development of Pueraria thomsonii.[Method]Gange No.1 with high starch content(20%starch in fresh kudzu root,35%starch in dry kudzu root)and Gange No.2 with low starch content(14%starch in fresh kudzu root,25%starch in dry kudzu root)were used as materials,differential proteins were obtained by quantitative proteomics based on full scanning data independent acquisition liquid chromatography⁃mass spectrometry(DIA LC⁃MS),and GO enrichment,KEGG pathway and KOG analysis were performed on the differential proteins.[Result]There were significant differences in 178 proteins(101 up⁃reg⁃ulated and 77 down⁃regulated)in the leaves of two starch kudzu cultivars,most proteins involved molecular functions,but the differential pro⁃teins with|log2(FC)|>5.0 was only 11.2%.GO enrichment analysis revealed that the differential proteins were mainly enriched in the meta⁃bolic process,catalytic activity,hydrolase activity,cell part,intracellular part,cytoplasmic part and intracellular organelle.KEEG pathways were concentrated in phenylalanine biosynthesis,MAPK signaling pathway⁃plants,biosynthesis of secondary metabolites,pyruvate metabolism and fatty acid metabolism.KOG analysis indicated that the differential protein functions were mainly classified as nucleotide transport and me⁃tabolism,ribosomal structure and biogenesis,energy production and conversion,signal transduction,and posttranslational protein modification,turnover,chaperones mechanisms.The proteins with great differences were up⁃regulated chlorophyll a⁃b binding protein of LHC II type 1⁃like,kunitz family trypsin and protease inhibitor precursor,lectin⁃like protein,chitinase homolog and down⁃regulated lysosomal Pro⁃X carboxypepti⁃dase⁃like,predicted dCTP pyrophosphatase 1⁃ike,cysteine⁃rich receptor⁃like protein kinase 25 and tryptophan aminotransferase⁃related protein 4.[Conclusion]These results provide the data support and technical approaches for growth and development,synthesis and regulation of nutri⁃ents and active substances,genetic improvement and molecular breeding of Pueraria thomsonii.