山药块茎发育过程中淀粉积累及差异蛋白分析

Analysis of starch accumulation and differentially expressed proteins during the development of Chinese yam tuber

【目的】明确山药Dioscorea spp.块茎发育过程中淀粉积累规律,为高产优质山药栽培及育种提供理论依据。【方法】以‘GH16’为材料,测定山药块茎发育过程中淀粉积累变化,并通过石蜡切片观测块茎组织、细胞及淀粉粒变化,采用蛋白质双向电泳技术寻找与块茎物质积累相关的关键蛋白。【结果】在块茎形成初期(4—5月)未见淀粉积累;块茎膨大初期(6月)淀粉粒均匀分布于薄壁组织细胞中,随后块茎细胞、淀粉粒随着山药块茎的发育逐渐增大,淀粉迅速积累;至膨大中后期(9月后),淀粉粒大量积累于靠近维管束周围的薄壁组织细胞中;到膨大后期(11月)块茎细胞达到最大值,细胞纵径和横径分别为121.685和89.572μm,淀粉积累达到高峰,淀粉粒长径最长达27.608μm (12月),短径最长达16.450μm (11月);3个发育时期共有179个蛋白表达发生了显著变化。选取其中差异较大的52个蛋白点进行质谱鉴定,其中31个差异表达的蛋白被鉴定,这些差异蛋白主要是与糖代谢和淀粉合成相关的酶,如6–磷酸葡萄糖酸脱氢酶、果糖–1,6–二磷酸醛缩酶、ATP合成酶α亚基、UTP–葡萄糖–1–磷酸尿苷酰转移酶、磷酸甘油酸激酶1等,它们在膨大初期后表达上调。山药块茎特异蛋白Dioscorin在膨大后期大量积累。【结论】山药块茎的膨大是细胞分裂与细胞膨大及淀粉积累共同作用的结果;块茎发育过程中的主要差异蛋白在糖代谢及淀粉合成过程中起关键作用。

【Objective】To understand the rules of starch accumulation during development of Chinese yam(Dioscorea spp.)tuber,and provide theoretical references for breeding and cultivation of Chinese yam with high yield and quality.【Method】Using‘GH16’as material,starch accumulation during development process of Chinese yam tuber was measured.The changes of tuber tissues,cells and starch grains were studied through paraffin sectioning.The key proteins related to tuber development were discovered by two-dimensional electrophoresis.【Result】There was no starch accumulation in early stage of tuber formation(April to May).In the early expansion stage(June),starch grains distributed uniformly in parenchyma cells.With the development of yam tuber,tuber cells and starch grains enlarged gradually,and starch accumulated rapidly.In the mid and late expansion stages(after September),substantial starch grains distributed near parenchyma cells around vascular bundle.In the late expansion stage,tuber cell size reached the maximum in November with cell longitudinal diameter and transverse diameter reaching 121.685 and 89.572μm respectively,and starch accumulation reached the peak with longitudinal diameter of starch grain being up to 27.608μm(December),and transverse diameter being up to 16.450μm(November).There were 179 differentially expressed proteins to be found.Among them,Fifty-two proteins with relatively larger differences in expression were analyzed by mass spectra,and 31 differentially expressed proteins were identified.Most of them were enzymes related to sugar metabolism and starch synthesis,such as glucose 6-phosphate acid dehydrogenase,fructosebisphosphatealdolase cytoplasmic isozyme-like,ATP synthetase alpha subunit,UTP-glucose-1-phosphate uridylyltransferase and phosphoglycerate kinase 1,and these were up-regulated after the early stage of tuber formation.Chinese yam tuber specific protein,Dioscorin,was accumulated in the late stage of tuber expansion.【Conclusion】The expansion of Chinese yam tuber is the results of cell division,cell amplification and starch accumulation.The main differentially expressed proteins during development of Chinese yam tuber play the key roles of sugar metabolism and starch synthesis.