黄独微型块茎低温离体保存的蛋白质组学分析

Proteomic Analysis of Dioscorea bulbifera L. Microtubers Conserved in vitro at Low Temperature

本研究利用TMT标记＋LC-MS/MS技术来探明黄独低温离体保存微型块茎的差异蛋白。研究表明,所有肽段的mass error进行统计,大多数mass error 〈0.02 Da,且其分布均在0附近,MS数据比较精确,符合检测要求。大多数肽段的长度分布在8～16之间,符合tryptic酶切肽段的理论值,表明样本的前处理也符合要求。差异蛋白为106个,其中上调差异蛋白61个,下调差异蛋白45个。排名前10位差异蛋白分别为伸长因子3、6-磷酸葡萄糖酸脱氢酶、磷酸甘油酸激酶、蔗糖合成酶、光系统Ⅱ的贮藏蛋白质、未知蛋白、分子伴侣DNAK、α-淀粉酶、S-腺苷甲硫氨酸合成酶和淀粉磷酸化酶,其中伸长因子3、6-磷酸葡萄糖酸脱氢酶、磷酸甘油酸激酶、蔗糖合成酶、分子伴侣DNAK和S-腺苷甲硫氨酸合成酶为在4℃低温离体保存中上调的蛋白,而未知蛋白、α-淀粉酶、淀粉磷酸化酶为在4℃低温离体保存中下调的蛋白。经过wego分析后,所有检测到的蛋白主要聚集于代谢过程、细胞过程细胞、绑定、催化等GO terms。通过富集,晚期内体膜、钙离子结合、谷胱甘肽转移酶活性、蛋白定位到液泡、高尔基液泡运输、液泡运输、氨基末端肽结合空泡分选和披网格蛋白小泡膜等GO term富集显著。富集的KEGG pathway主要有乙醛酸盐代谢、光合作用、甲烷代谢、碳水化合物的消化和吸收、半胱氨酸和蛋氨酸代谢、淀粉和蔗糖代谢和碳代谢。黄独低温离体保存微型块茎差异蛋白的初步发现为进一步了解其低温离体保存的分子机制奠定了基础,也为低温离体保存黄独微型块茎的破除休眠以及其后续萌发提供了理论依据。

In order to verify differential proteins ofDioscorea bulbifera L. microtubers conserved in vitro at 4℃, LC-MS/MS technique labeled with TMT was applied. The results showed that the mass error of all peptides was statistically analyzed, the distribution of mass error was near 0, and most of them were less than 0.02 Da, which indicated that the accuracy of MS data met the requirements. The length distribution of most peptides was between 8-16, which was consistent with the theoretical value of the tryptic peptide segment and indicated that the sample pretreatment was also in line with the requirements. There were 106 differentially expressed proteins, of which the difference protein was up-regulated by 61 and down-regulated by 45. The top 10 differential proteins were elongati onfactor 3, 6-phosphogluconate dehydrogenase, phosphoglycerate kinase, sucrose synthase, photosystem If protein storage, unknown protein, molecular chaperone DNAK, alpha amylase, S-adenosylmethionine synthetase and starch phosphate synthase. In Dioscorea bulbifera L. microtubers conserved in vitro at 4℃, elongation factor 3, 6-phosphogluconate dehydrogenase, phosphoglycerate kinase, sucrose synthase, molecular chaperone DNAK and S-adenosine methionine synthetase was up-regulated, and unknown protein, alpha amylase and starch phosphorylase was down-regulated. After wego analysis, all the proteins were mainly concentrated in the metabolic process, cell process cells, binding, catalysis and other GO terms. After enrichment, late endosome membrane, calcium ion binding, glutathione transferase activity, protein targeting to vacuole, Golgi to vacuole transport, vacuolar transport, amino-terminal vacuolar sorting propeptide binding and clathrin-coated vesicle membrane and so on concentrated significantly. KEGG pathway is the main enrichment of glyoxylate and dicarboxylate metabolism, photosynthesis, methane metabolism, carbohydrate digestion and absorption, cysteine and methionine metabolism, starch and sucrose metabolism and carbon metabolism. The preliminary finding of differential proteins ofD. bulbifera L. microtubers conserved in vitro at low temperature laid the foundation for further understanding the molecular mechanism of conservation in vitro at low temperature and provides a theoretical basis for low temperature conservation breaking dormancy and its subsequent germination D. bulbifera L. microtubers.