Analysis of starch accumulation and differentially expressed proteins during the development of Chinese yam tuber
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摘要:目的
明确山药Dioscoreaspp.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89.572 μm，淀粉积累达到高峰，淀粉粒长径最长达27.608 μm (12月)，短径最长达16.450 μm (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。
Abstract:ObjectiveTo understand the rules of starch accumulation during development of Chinese yam (Dioscoreaspp.) tuber, and provide theoretical references for breeding and cultivation of Chinese yam with high yield and quality.
MethodUsing ‘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.
ResultThere 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 89.572 μm respectively, and starch accumulation reached the peak with longitudinal diameter of starch grain being up to 27.608 μm (December), and transverse diameter being up to 16.450 μm (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, fructose-bisphosphatealdolase 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.
ConclusionThe 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.
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Keywords:
- Dioscoreaspp. /
- tuber /
- growth and development /
- starch accumulation /
- microstructure /
- proteomics
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图 1 山药块茎发育过程中的形态变化
各图中数值上凡是具有一个相同小写字母者，表示不同月份间差异不显著(P>0.05，LSD法)
Figure 1. Morphological changes during development of Chinese yam tuber
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图 2 山药块茎发育过程中的营养组分含量变化
各图中数值上凡是具有一个相同小写字母者，表示不同月份间差异不显著(P>0.05，LSD法)
Figure 2. Changes of nutrient constituents during development of Chinese yam tuber
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图 3 山药块茎发育的细胞学观察
A、B和C分别为4、6和12月块茎皮层及其附近组织横切面结构，D、E、F、G、H和I分别为4、5、6、9、11和12月块茎薄壁组织横切面结构；Ep：表皮，Co：皮层，Vc：维管束，Xy：木质部，Pc：薄壁细胞，S：淀粉粒；标尺=100 μm
Figure 3. The cytology observation during development of Chinese yam tuber
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图 4 山药块茎发育过程中薄壁细胞与淀粉粒大小变化
各图中数值上凡是具有一个相同小写字母者，表示不同月份间差异不显著(P>0.05，LSD法)
Figure 4. Changes of parenchyma cell and starch grain sizes during development of Chinese yam tuber
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图 5 山药块茎发育过程中的蛋白质双向电泳图
红色数字为已鉴定的差异蛋白的序号
Figure 5. Two-dimensional electrophoresis of proteins during development of Chinese yam tuber
表 1 等电聚焦参数
Table 1 Isoelectric focusing (IEF) parameters
步骤 电压/V 升压模式 时间/h S1 50 线性 12 S2 500 线性 1 S3 1 000 线性 1 S4 1 000~10 000 快速 1 S5 10 000 快速 11 S6 500 快速 任意时长 
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表 2 部分差异蛋白质信息列表
Table 2 Information of partial differentially expressed proteins
蛋白
序号Mascot数据库
登陆号蛋白名称 得分 Mr 等电点 不同月份相对表达量比值1) 5/6 5/11 6/11 126 tr|W7NY18 几丁质酶 75 26 204 4.89 2.73 50.22 18.41 211 sp|P80052 酸性内切壳多糖酶 106 28 859 4.80 – 0.28 0.49 6207 gi|33312115 核酮糖–1,5–二磷酸羧化酶/加氧酶大亚基 151 32 695 5.90 2.15 9.04 4.20 8427 gi|731391631 阳离子过氧化物酶 61 33 147 4.43 3.38 6.24 – 8119 gi|586751268 肽基脯氨酸顺反异构酶 80 26 347 9.26 1.55 3.44 2.20 1411 gi|46917481 块茎储藏蛋白 318 35 982 5.69 0.16 – 7.93 7522 gi|719974028 果糖–1,6–二磷酸醛缩酶 193 40 958 6.04 0.26 0.06 0.23 7618 gi|603221 6–磷酸葡萄糖酸脱氢酶 110 67 906 8.40 0.43 10.11 23.61 8707 gi|20160920 ATP合成酶α亚基 66 61 537 8.49 – – 196.00 9111 gi|1079652 胚胎发育晚期丰富蛋白 145 17 567 9.49 0.12 – 13.29 5724 gi|449456208 UTP–葡萄糖–1–磷酸尿苷酰转移酶 96 52 062 8.08 0.39 0.41 – 3005 gi|672122878 40S核糖体蛋白质 106 18 124 5.82 0.10 – 19.18 3522 gi|102139814 磷酸甘油酸激酶 198 42 269 9.58 0.30 – 2.80 4209 gi|12229919 蛋白酶α亚基 63 27 506 5.75 0.18 – 2.63 4526 gi|732559013 磷酸甘油酸激酶1 89 53 026 5.65 0.12 – 5.24 5311 tr|C7E3T8 Dioscorin蛋白 283 31 815 6.10 – 0.10 0.11 1）“–”表示该蛋白在2个时期的表达量差异不显著（P>0.05，t检验）
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