全 文 ：作物学报 ACTA AGRONOMICA SINICA 2013, 39(5): 845854 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

本研究由国家杰出青年科学基金(30925023)和国家自然科学基金重点项目(31230054)资助。
* 通讯作者(Corresponding author): 倪中福, E-mail: nzhf2002@yahoo.com.cn
第一作者联系方式: E-mail: gbj9999@163.com
Received(收稿日期): 2012-09-05; Accepted(接受日期): 2012-12-16; Published online(网络出版日期): 2013-02-22.
URL: http://www.cnki.net/kcms/detail/11.1809.S.20130222.1110.002.html
DOI: 10.3724/SP.J.1006.2013.00845
玉米杂交种与亲本雌穗花器官形成期蛋白差异表达谱分析
郭宝健 宋方威 冯万军 隋志鹏 孙其信 倪中福*
农业生物技术国家重点实验室 / 中国农业大学杂种优势研究与利用教育部重点实验室 / 作物遗传改良北京市重点实验室 / 国家植
物基因研究中心(北京), 北京 100193
摘 要: 为探讨玉米穗粒数杂种优势形成的分子机制, 以玉米强优势杂交种豫玉 22及其亲本综 3和 87-1的花器官形
成期雌穗为材料, 采用高通量的 2-DE 和 MALDI TOF MS 技术, 建立蛋白差异表达谱, 并对差异蛋白进行了质谱鉴
定。结果显示, 在检测到的 1 290个蛋白点中, 有 114个(8.84%)在杂交种与亲本之间的表达差异达到显著水平, 其中
表现单亲沉默、偏高亲、中亲表达、偏低亲、杂种上调、杂种下调、亲本特异和杂种特异表达模式的蛋白点分别为
27、25、15、13、11、11、10 和 2 个。另外, 成功鉴定出其中的 104 个差异表达蛋白点, 涉及代谢、信号转导、能
量、转录、蛋白质合成、蛋白运输与储藏、细胞生长与分裂、细胞结构、抗病防御、次生代谢、转座子及功能未知
和假定蛋白等 12个功能类别。玉米杂交种与亲本蛋白在丰度上的明显差异及涉及多个功能类别, 可能与玉米穗粒数
杂种优势的形成有关。
关键词: 玉米; 雌穗; 杂种优势; 蛋白差异表达谱
Differentially Expressed Protein Profiling during Ear Floral Development be-
tween Maize Hybrid and Its Parents
GUO Bao-Jian, SONG Fang-Wei, FENG Wan-Jun, SUI Zhi-Peng, SUN Qi-Xin, and NI Zhong-Fu*
State Key Laboratory for Agrobiotechnology / Key Laboratory of Crop Heterosis and Utilization, Ministry of Education / Beijing Key Laboratory of
Crop Genetic Improvement, China Agricultural University / National Plant Gene Research Centre (Beijing), Beijing 100193, China
Abstract: To gain an insight into the molecular basis of heterosis related to kernel number per ear in maize, we established a dif-
ferentially expressed protein profiling between highly heterotic hybrid Yuyu 22 and its parental lines Zong 3, 87-1 during ear flo-
ral development by using a combined methods of 2-DE and MALDI TOF MS. A total of 1290 protein spots were detected, among
which 114 were found to be differentially expressed with a significant difference at P<0.05. The number of protein spots belongs
to the models of expression in hybrid and uniparent but not in another parent (UPF1), hybrid is equal to the highly expressed par-
ent (HDH), hybrid is equal to the mid-parent (MPE), hybrid is equal to the lowly expressed parent (LDH), up-regulated in hybrid
(URH), down-regulated in hybrid (DRH), dominant expression of uniparental proteins but not in hybrids (UPnF1), hybrid-specific
expressed protein spots (F1nBP) was 27, 25, 15, 13, 11, 11, 10, and 2, respectively. In addition, 104 proteins were identified by
using MALDI TOF MS, which are involved in 12 functional categories, including metabolism, signal transduction, energy, gene
transcription, protein synthesis, protein transport, and storage, cell growth, cell division, cell structure, disease and defense, sec-
ondary metabolism, transposons, unknown and putative proteins. Taken together, expression differences between hybrid and its
parents at protein abundances and multiple functions of in hybrid may contribute to the heterosis related to kernel number per ear.
Keywords: Maize; Ear; Heterosis; Differentially expressed protein profiling
杂种优势(heterosis)是指遗传基础不同的亲本
杂交产生杂种 F1代, 在生长势、抗逆性、繁殖力、
适应性、产量和品质等方面优于亲本的现象[1], 但其
形成机制仍是一个百年生物学难题。研究表明, 表
型变异的分子基础在于基因表达的变化。从基因组
组成上看, 杂交种的全部基因组来自 2个亲本, 并没
有新的基因出现 , 但其性状并非亲本的简单组合 ,
这可能与来自亲本的基因在杂种一代的表达方式改
附表 玉米杂交种与亲本雌穗花器官形成期差异表达蛋白的质谱鉴定结果
Appendix Identified differentially expressed proteins between hybrid and its parents in ear floral organ formation phase
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
Metabolism
90 UPF1 Squalene/phytoene synthase gi|212722786 Z. mays 71 26
92 DRH UbiE/COQ5 methyltransferase family protein gi|293335467 Z. mays 52 19
96 HDH Dolichyl-phosphatebeta-D-mannosyltransferase gi|226530361 Z. mays 56 15
172 MPE Glutathione S-transferase GSTU6 gi|226510544 Z. mays 44 9
188 UPF1 Protein prenyltransferase gi|115482066 O. sativa 37 43
194 MPE Phytoene synthase gi|226532946 Z. mays 73 26
292 HDH Citrate lyase beta subunit gi|255605888 R. communis 40 12
495 HDH Pyridine nucleotide-disulphide oxidoreductase gi|308081758 Z. mays 22 13
817 URH Glutamate-1-semialdehyde 2,1-aminomutase gi|224134068 P. trichocarpa 48 25
925 HDH Acireductone dioxygenase gi|222628645 O. sativa 19 5
972 LDH Oxidoreductase,zinc-binding dehydrogenase family protein gi|308081586 Z. mays 50 39
979 HDH Dihydrolipoamide dehydrogenase 1 gi|224099079 P. trichocarpa 28 12
1067 DRH Patatin T5 gi|226492557 Z. mays 26 7
1076 HDH Flavonoid 3-monooxygenase gi|226494123 Z. mays 72 12
1092 UPF1 Trehalose synthase-like protein gi|224028441 Z. mays 21 15
1096 UPF1 Putative trehalose-6-phosphate synthase gi|24308617 O. sativa 24 14
1098 HDH GTP cyclohydrolase II (RibA) gi|303280293 M. pusilla 15 5
1143 LDH Oxidoreductase gi|195624808 Z. mays 28 8
Energy
129 URH Phosphoglycerate mutase gi|293336560 Z. mays 45 15
169 MPE Carbonic anhydrase gi|226495621 Z. mays 37 10
415 LDH V-type ATP synthase subunit I gi|222641344 O. sativa 38 19
467 DRH Mitochondrial lipoamide dehydrogenase gi|134142802 P. tremuloides 26 11
536 HDH Sulfotransfer_2 gi|159481436 C. reinhardtii 19 8
575 LDH Putative sex determination protein tasselseed 2 gi|22296337 O. sativa 41 9
587 URH UGP(UDP-glucose pyrophosphorylase) gi|15228498 A. thaliana 50 18
1041 DRH Glycosyltransferase gi|223945287 Z. mays 46 7
1100 LDH ATP synthase D chain, mitochondrial gi|226507194 Z. mays 64 12
Cell growth/division
343 MPH DNA polymerase I gi|195654367 Z. mays 28 7
586 DRH Cyclin-related gi|42569848 A. thaliana 43 46
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
950 LDH Ribosome biogenesis protein NEP1 gi|226495343 Z. mays 48 10
976 LDH ATEXO70C1 (exocyst subunit EXO70 family protein C1) gi|212275810 Z. mays 50 14
1025 HDH Intraflagellar transport protein 172 gi|159467108 C. reinhardtii 15 19
1081 DRH Programmed cell death protein 5 gi|226500664 Z. mays 52 8
Transcription
40 URH GRAS family transcription factor gi|223949083 Z. mays 48 37
79 URH Helicase gi|302822204 S. moellendorffii 32 20
137 LDH Putative splicing factor gi|115454963 O. sativa 30 13
175 MPE mTERF family protein gi|195605386 Z. mays 46 16
201 MPE DEAD-like helicases superfamily gi|302820920 S. moellendorffii 29 19
281 MPE DEAD-like helicases superfamily gi|302820920 S. moellendorffii 22 11
312 HDH KU80; double-stranded DNA binding/protein binding gi|18402015 A. thaliana 35 20
1037 DRH DEAD-box helicases gi|302820920 S. moellendorffii 19 14
Protein synthesis
1006 HDH Mitochondrial ribosomal protein L5 gi|30351950 Z. mays 55 10
1012 UPF1 Eukaryotic translation initiation factor 3 subunit 2 gi|226533034 Z. mays 100 12
Protein transport and storage
77 HDH Chaperonin CPN60-2, mitochondrial precursor gi|162460375 Z. mays 35 18
227 MPE RING-finger domain protein gi|224088100 P. trichocarpa 36 11
446 UPF1 RING-finger domain protein gi|224088100 P. trichocarpa 38 12
507 MPE E3 ubiquitin-protein ligase UPL6OS UPL6_ARATH A. thaliana 32 35
783 HDH Armadillo/beta-catenin repeat family protein gi|297819214 A. lyrata 16 8
1074 HDH D1 protease-like protein precursor gi|19774135 T. aestivum 32 12
1083 DRH Armadillo/beta-catenin-like repeats gi|212275842 Z. mays 31 15
Cell structure
33 UPF1 Ankyrin-like protein gi|47497783 O. sativa 71 27
248 UPF1 Tubulin beta-6 chain gi|162459800 Z. mays 28 15
615 LDH Annexin gi|224057357 P. trichocarpa 34 12
781 LDH MIT domain protein gi|15223173 A. thaliana 56 7
793 UPF1 Putative ankyrin-like protein gi|48716529 O. sativa 41 14
966 UPF1 Ankyrin repeats protein gi|224115944 P. trichocarpa 43 21
Signal transduction
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
34 URH CDC2+/CDC28-related protein kinase R2 gi|195640128 Z. mays 28 7
69 UPF1 Leucine-rich repeat-containing protein, putative gi|255578414 R. communis 32 41
136 UPF1 Lectin-like receptor kinase 7 gi|226508034 Z. mays 40 6
162 LDH Glycogen synthase kinase-3 MsK-3 gi|226533162 Z. mays 30 11
217 UPF1 Mitochondrial carrier protein gi|297724283 O. sativa 72 9
279 HDH Serine/threonine-protein phosphatase 2A activator 2 gi|195640094 Z. mays 52 13
349 UPF1 Putative phototropic response protein gi|115472647 O. sativa 23 8
453 UPF1 Pentatricopeptide repeat-containing protein gi|75268015 A. thaliana 40 6
552 HDH Pentatricopeptide repeat-containing protein gi|297797201 A. lyrata 19 13
676 URH Putative BLE2 protein gi|23617104 O. sativa 21 18
678 MPE Calmodulin gi|293334895 Z. mays 39 6
927 HDH Pentatricopeptide repeat-containing protein gi|297797201 A. lyrata 20 12
1011 HDH WD-40 repeat family protein gi|308080163 Z. mays 57 9
1069 UPF1 WD-40 repeat family protein gi|218192583 O. sativa 31 10
Disease and defence
31 URH Remorin protein gi|293336051 Z. mays 24 9
112 UPF1 TIR-NBS-LRR resistance protein gi|111146892 P. vulgaris 17 17
147 UPF1 Heat shock 70 kDa protein 4 gi|226498820 Z. mays 15 8
198 DRH Powdery mildew resistance protein PM3A gi|62912003 T. aestivum 46 62
233 UPF1 Disease resistance protein RPP13 gi|15231449 A. thaliana 43 32
497 MPE Putative PEP-CTERM system TPR-repeat lipoprotein gi|224029551 Z. mays 24 21
626 F1nBP NBS-LRR type resistance protein gi|2792230 O. sativa 59 8
663 UPF1 Late embryogenesis abundant protein gi|242044772 S. bicolor 52 14
767 HDH Disease resistance protein (NBS-LRR class), putative gi|15231862 A. thaliana 33 29
815 F1nBP NBS-LRR protein gi|21616910 O. sativa 45 10
988 HDH TUP1-like enhancer gi|293336752 Z. mays 40 12
1004 MPE Bacterial trigger factor protein (TF) gi|225434088 V. vinifera 59 15
1066 DRH BED finger-nbs-lrr resistance protein gi|224145670 P. trichocarpa 20 10
Secondary metabolism
48 LDH Cytochrome P450 CYP74A19 gi|195613496 Z. mays 52 22
553 MPE Cytochrome P450 CYP76M15 gi|195646472 Z. mays 37 25
Unknown and putative protein
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
93 HDH Expressed protein gi|108707573 O. sativa 26 15
193 UPF1 Predicted protein gi|303288630 M. pusilla 35 18
294 URH Unknown protein gi|18413400 A. thaliana 38 39
378 URH Predicted protein gi|303282581 M. pusilla 42 9
379 HDH Hypothetical protein gi|40786588 O. sativa 68 9
384 MPE Hypothetical protein OsJ_26893 gi|222640350 O. sativa 42 9
480 HDH Hypothetical protein gi|115477703 O. sativa 19 7
540 UPF1 Predicted protein gi|297817898 A. thaliana 44 12
711 URH DUF538 domain protein gi|226491968 Z. mays 61 12
911 UPF1 Hypothetical protein gi|218196215 O. sativa 38 16
964 LDH Hypothetical protein OsJ_26893 gi|222640350 O. sativa 43 10
1043 UPF1 Conserved hypothetical protein gi|255570033 R. communis 76 5
1065 DRH Unknown protein gi|186489637 A. thaliana 93 8
1108 UPF1 Hypothetical protein ARALYDRAFT_356151 gi|297805920 A. lyrata 21 11
Transposons
154 UPF1 Transposase gi|212275225 Z. mays 20 7
383 MPE Retrotransposon protein, putative, Ty3-gypsy subclass gi|77553948 O. sativa 23 20
408 HDH Retrotransposon protein, putative gi|108864382 O. sativa 43 33
689 HDH Transposase_24 gi|226492958 Z. mays 28 18
1159 UPF1 Retrotransposon protein, putative, Ty3-gypsy sub-class gi|62733790 O. sativa 20 32


846 作 物 学 报 第 39卷

变有关[2-8]。
玉米是我国播种面积最大的粮食作物, 同时也
是杂种优势研究与利用的模式植物。研究表明, 在
产量构成因素中, 玉米杂交种在穗粒数上的优势最
为明显[9-10]。最近, Stupar等[11]以 B73和 Mo17及其
杂交种不成熟幼穗为材料, 采用基因芯片技术研究
发现, 20%左右的基因在杂交种中为非加性表达, 并
且还检测到许多基因在亲本和杂交种中特异表达。
采用同样的研究策略, Li 等[12]和 Pea 等[13]以不同杂
交组合为材料, 分别分析小穗分化期和小花分化期
的雌穗转录组差异表达谱, 筛选出了一些差异表达
基因, 但是在蛋白水平上的研究迄今尚未见报道。
强优势杂交种豫玉 22 曾经是黄淮海玉米区第
一大推广种, 在农业生产中发挥了重要作用。为了
探讨该杂交组合产量杂种优势形成的遗传学基础 ,
Tang等[9]采用永久 F2 (IF2)设计方法研究, 检测到 13
个 QTL, 包括 3 个产量 QTL、7 个穗长 QTL、1 个
穗行数 QTL 及 2 个百粒重 QTL, 且所有 QTL 均表
现超显性作用。本研究中, 我们采用双向电泳技术,
结合质谱鉴定方法, 建立了杂交种与亲本雌穗花器
官形成期的蛋白差异表达谱, 并对差异蛋白进行了
质谱鉴定, 以期为解析玉米穗粒数杂种优势形成的
分子机制提供蛋白水平的数据和资料。
1 材料与方法
1.1 供试材料及取材方法
选用花器官形成期的豫玉22及其亲本综 3、87-1
的雌穗。用锋利的刀片从植株叶腋处剥取带苞叶的
玉米雌穗, 放在平整的桌面上小心地剥取雌穗(长度
2.5 cm), 把苞叶去除干净。将幼穗取出, 迅速包好放
入液氮中, 于80℃保存。
1.2 蛋白的提取和定量
采用 SDS裂解 buffer法提取杂蛋白样品。参照
Bradford[14]的方法测定蛋白质含量。
1.3 双向电泳
参照 Song等[15]方法。
1.4 双向电泳胶图处理
用 Labscan (GE Healthcare, USA)进行图像扫描,
分辨率为 600 dpi, 并用 Imagemaster 2D Platinum
Software Version 5.0 (GE Healthcare, USA)进行图像
分析。首先是蛋白点的自动检测 , 将参数 smooth,
minimum area和 saliency分别设置为: 2、15、8, 随
后进行手动编辑(点的增减、切割、合并和轮廓修正);
在软件自动均一化处理后, 将所有胶与参比胶自动
匹配, 并手动修改。应用分析程序, 分别根据蛋白点
在线性胶条中的迁移率和与蛋白分子量 Marker(购
自中国科学院上海生命科学研究院生物化学与细胞
生物学研究所)的相对位置 , 确定其等电点和分子
量。
1.5 胶内酶切
参照 Song等[15]方法。
1.6 质谱鉴定和肽指纹质谱匹配
使用 AUTOFLEX II TOF-TOF(Bruker Daltonics,
Germany)进行肽指纹图谱及二级质谱测定。
分析峰图用 Mascot distiller(Matrix Science)软
件。将 PMFs肽段列表输入Matrix Science (http://www.
Matrixscience.com/)搜索引擎在 NCBInr 库或 MSDB
库中进行匹配 : 分类学种类选择 Viridiplantae
(Green Plants); 每次匹配允许 1个酶切位点的丢失;
所有肽段质量峰都为单同位素峰并质子化; PMF 肽
段质量精确度为 0.2 Dalton; 分别选择甲硫氨酸氧化
和半胱氨酸脲基甲基化作为可变和固定修饰。被认
定是阳性结果的条件是: (1)MOWSE 分值高于或等
于 MASCOT所给出的 5%显著性水平的分值; (2)至
少有 4 个不同肽段与已知蛋白的肽段匹配; (3)匹配
肽段的覆盖率高于 10%。对于有阳性结果的蛋白,
根据其已有的氨基酸序列在 KEGG (http://www.
genome.jp/kaas-bin/kaas_main)或 UniProt 数据库
(http://beta.uniprot.org/)中进行在线功能分类。对于
在这 2 个数据库中均不能有效分类的蛋白质, 利用
其结构域或查找文献进行人工分类。对于以上两种
方法仍不能确定其功能的蛋白质, 则认为是功能未
知蛋白。
2 结果与分析
2.1 杂交种与亲本之间的蛋白差异表达谱
采用高通量蛋白质双向电泳技术, 建立了玉米
未成熟雌穗花器官形成期的蛋白差异表达谱(图 1)。
选用的 pH值分离范围为 4~7, 蛋白质分子量范围为
7~175 kD, 每个基因型至少设立 3个生物学重复, 并
且在每个基因型中 95%的蛋白质点能够重复出现。
对 3个重复胶图蛋白点均一化后, 通过 t测验鉴定杂
交种与双亲间的差异表达蛋白点。
在雌穗花器官形成时期有 114 个蛋白点的表达
差异达到显著水平(P<0.05), 占该时期检测到的蛋
白点总数(1290个)的 8.84%, 说明杂交种与亲本在蛋
第 5期 郭宝健等: 玉米杂交种与亲本雌穗花器官形成期蛋白差异表达谱分析 847


白质水平上发生了明显的表达差异。
杂交种与亲本之间的蛋白差异表达模式表现为
质和量的差异。质的差异模式可归纳为 3种类型: (A)
亲本特异表达, 只在 1个亲本表达, 另 1个亲本和杂
交种均不表达; (B)杂种特异表达, 基因仅在杂种 F1
中表达; (C)单亲沉默, 只在 1个亲本和杂交种表达,
另 1 个亲本不表达。量的差异模式可顺序归纳为以
下 5 种类型: (D)杂种上调表达, 在杂交种和 2 个亲
本都表达, 但杂交种的表达量高于亲本; (E)杂种下
调表达, 在杂交种和 2个亲本都表达, 但杂交种的表
达量低于亲本; (F)杂种偏高亲表达, 在杂交种和亲
本都表达, 且杂交种表达量与高亲表达量差异不显
著 ; (G)杂种偏低亲表达 , 在杂交种和亲本都表达 ,
且杂交种表达量与低亲表达量差异不显著; (H)中亲
表达, 杂交种中的表达量介于双亲表达量之间, 且
与双亲的表达量差异显著(图 2)。分析发现, 本实验
中, 蛋白差异表现质差异的(39 个)明显少于量差异
的(75个)。其中单亲沉默模式(27个)和偏高亲模式(25
个)的较多, 其次为中亲表达(15个)、偏低亲表达(13
个)、杂种上调表达(11 个)、杂种下调表达(11 个)及
亲本特异表达模式 (10 个 ), 而杂种特异的最少 (2
个)(表 1)。

图 1 玉米杂交种与亲本雌穗花器官形成期蛋白表达谱
Fig. 1 Protein expression profiling of hybrid and its parental lines during ear floral organ formation


图 2 杂交种与亲本之间的蛋白差异表达模式
Fig. 2 Differential expression patterns of protein spots be-
tween hybrids and its parents
A: 单亲沉默; B: 亲本特异表达; C: 杂种特异表达; D: 中亲表
达; E: 杂种偏高亲表达; F: 杂种偏低亲表达; G: 杂种上调表达;
H: 杂种下调表达。
A: expression in hybrid and uniparent but not in another parent;
B: dominant expression of uniparental proteins but not in hybrids;
C: hybrid-specific expressed protein spots; D: hybrid is equal to the
mid-parent; E: hybrid equal to the highly expressed parent; F: hy-
brid equal to the lowly expressed parent; G: up-regulated in hybrid;
H: down-regulated in hybrid.

2.2 差异表达蛋白点质谱鉴定
从 2-DE 胶挖取全部差异表达蛋白点进行质谱鉴
定, 将已鉴定的 104 个蛋白点归为 12 个功能类别(见
附表), 其中代谢类最多(18 个), 其次为信号转导类
(14 个)、功能未知及假定蛋白类(14 个)、抗病防御
表 1 杂交种与亲本雌穗花器官形成期的蛋白差异表达模式
及其数目
Table 1 Pattern and number of differentially expressed
protein spots between hybrid and its parents during ear floral
organ formation
项目
Item
差异表达模式
Differentially
expressed pattern
蛋白质点总数
Total No. of
spots
亲本特异 UPnF1 10
杂种特异 F1nBP 2
单亲沉默 UPF1 27
质的表达差异
Qualitative
difference
小计 Sum 39
杂种上调 URH 11
杂种下调 DRH 11
偏高亲 HDH 25
偏低亲 LDH 13
中亲表达 MPE 15
量的表达差异
Quantitative
difference
小计 Sum 75
总计 Total 114
UPnF1: expression in dominant expression of uniparental
proteins but not in hybrids, F1nBP: expression in hybrid-specific
expressed protein spots, UPF1: expression in hybrid and uniparent
but not in another parent, URH: expression in up-regulated in hy-
brid, DRH: expression in down-regulated in hybrid, HDH: expres-
sion in hybrid is equal to the highly expressed parent, LDH: ex-
pression in hybrid is equal to the lowly expressed parent, MPE:
expression in hybrid is equal to the mid-parent.
848 作 物 学 报 第 39卷

类(13 个)、能量类(9 个)、转录类(8 个)、蛋白运输
与储藏类(7 个)、细胞生长与分裂类(6 个)、转座子
类(5个)、细胞结构类(6个)、蛋白质合成类(2个)及
次生代谢类(2个)。
3 讨论
关于玉米产量杂种优势形成的遗传学基础, 目
前已经有许多研究报道[9,16-18]。早在 1992年, Stuber
等[16]首次利用 QTL 的方法, 采用 “Design III”设计
对 B73×Mo17 组合进行了研究, 分析认为超显性是
玉米产量杂种优势的遗传学基础, 这与此后 Lu 等[17]
以 LH200×LH216 组合为材料获得的研究结果相类
似。后来, Frascaroli 等[18]以 B73×H99 组合为材料,
采用“TTC”的设计方法, 检测到 17 个穗粒数相关的
QTL, 其中表现为超显性、显性、部分显性和加性
效应的位点数分别为 10、5、1和 1个。最近, Tang
等[9]以豫玉 22 组合为基本材料, 利用“IF2”群体, 共
检测到 13 个 QTL, 包括 3 个产量 QTL、7 个穗长
QTL、1 个穗行数 QTL及 2 个百粒重 QTL, 且所有
QTL 均表现超显性作用方式。另外, 他们还检测到
一些上位性互作位点, 也可能起重要作用。有研究
表明, 1个超显性位点有可能分解为 2个相斥相连锁
的显性位点, 造成假超显性现象。例如, 以玉米自交
系 B73 和 Mo17 杂交组合为基础材料检测到的 1 个
超显性产量主效 QTL, 经过精细作图分析证明由 2
个较小的表现为显性效应的 QTL组成[16]。因此, 显
性和超显性等不同类型的遗传互作可能对玉米杂种
优势形成都有贡献, 并不是互作排斥的。本研究建
立了强优势杂交种豫玉 22 及其亲本综 3 和 87-1 雌
穗花器官形成期的蛋白差异表达谱, 检测到 114 个
差异表达蛋白点, 包括单亲沉默、偏高亲、中亲表
达、偏低亲、杂种上调、杂种下调、亲本特异和杂
种特异等多种差异表达模式, 其中单亲沉默、杂种
偏高亲表达和杂种偏低亲等差异蛋白点可被解释为
显性效应(65个), 表现亲本特异表达、杂种特异表
达、杂种上调表达和杂种下调表达的差异蛋白点可
被解释为超显性效应(34个)。据此, 我们认为显性和
超显性效应可能均与玉米穗粒数杂种优势的形成有
重要关系, 这也与遗传上的研究结果相吻合。
在蛋白差异表达谱分析的基础上 , 我们利用
MALDI TOF MS质谱技术鉴定了 104个在杂交种与
亲本之间差异表达的蛋白点, 涉及代谢、信号转导、
抗病防御、能量、转录、蛋白运输与储藏、细胞生
长与分裂、转座子、细胞结构、蛋白质合成、次生
代谢及功能未知和假定蛋白 12个功能类别。尽管这
些差异表达蛋白与玉米穗粒杂种优势形成的关系还
不清楚, 但是其中部分基因涉及的代谢途径在玉米
雌穗发育过程中起重要作用, 例如, 6-磷酸海藻糖磷
酸酶基因的突变导致玉米雌穗形态变化[19]。本研究
检测到两个海藻糖代谢基因在杂交种与亲本之间差
异表达, 即海藻糖合酶和 6-磷酸海藻糖合酶, 且均
为单亲表达型。因此, 这些基因的差异表达可能导
致杂交种中海藻糖代谢途径的改变, 进而影响玉米
杂种优势的形成。
在杂种优势机制研究上, 已经定位了大量的重
要性状杂种优势 QTL, 并明确亲本基因在杂交种中
发生了明显的表达改变, 需要进一步回答的问题是
这些基因是如何互作并导致优势产生的, 即杂种优
势形成的遗传网络 [4,20], 其中表观遗传调控可能在
其中起重要作用。例如, 基因组水平上的研究结果
显示, 水稻、拟南芥和玉米等植物杂交种与亲本在
DNA甲基化、染色质结构修饰和/或小分子 RNA等表
观遗传调控修饰状态上也发生了明显的改变[21-26]。拟
南芥的生物量杂种优势与生物钟调控基因 CCA 和
LHY 启动子区域的组蛋白修饰状态改变有密切关
系 [23]。最近研究发现 , 拟南芥表观遗传调控基因
HEN1 突变后导致苗期生物量杂种优势明显降低 ,
进一步说明表观遗传调控对杂种优势形成有重要的
贡献[26]。本研究检测到许多表现为非加性表达的蛋
白点, 但差异表达的分子调控机制是一个有待探讨
的问题。比如, 编码蛋白的基因在转录水平是否差
异表达？表观遗传调控是否参与玉米穗粒数杂种优
势的形成？我们相信对这些基因及其编码蛋白功能
以及调控机制的深入研究, 将有助于进一步阐明玉
米穗粒数杂种优势形成的分子生物学基础。
转座子和反转录转座子普遍存在于植物界, 在
植物基因和基因组进化过程中起重要作用, 可引起
染色体重排和基因的移码突变等[27]。这些转座元件
可作为调控序列, 改变其他基因的表达方式。以小
麦杂交种与亲本间叶片为材料的研究发现, 编码转
座酶和反转录转座子基因也发生了差异表达[28-29]。
本研究也发现, 5 个差异蛋白点(点 154、点 383、
点 408、点 689和点 1159)与转座酶或反转录转座子
具有较高同源性, 这可能是在杂合状态下, 转座酶
或者反转录转座子的活性受到激发, 从而调控其他
基因在杂交种中发生表达变化。
第 5期 郭宝健等: 玉米杂交种与亲本雌穗花器官形成期蛋白差异表达谱分析 849


4 结论
建立了强优势杂交种豫玉 22 及其亲本综 3 和
87-1 花器官形成期的雌穗蛋白质差异表达谱, 检测
到 114个差异表达蛋白点, 包括单亲沉默、偏高亲、
中亲表达、偏低亲、杂种上调、杂种下调、亲本特
异和杂种特异表达模式等多种差异表达模式 ;鉴定
出 104 个差异表达蛋白点, 归为 12 个功能类别, 以
代谢类最多(18个), 特别是与玉米幼穗发育密切相
关的海藻糖代谢基因存在明显的表达差异, 可能在
穗粒数杂种优势形成过程中起重要作用。
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附表 玉米杂交种与亲本雌穗花器官形成期差异表达蛋白的质谱鉴定结果
Appendix Identified differentially expressed proteins between hybrid and its parents in ear floral organ formation phase
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
Metabolism
90 UPF1 Squalene/phytoene synthase gi|212722786 Z. mays 71 26
92 DRH UbiE/COQ5 methyltransferase family protein gi|293335467 Z. mays 52 19
96 HDH Dolichyl-phosphatebeta-D-mannosyltransferase gi|226530361 Z. mays 56 15
172 MPE Glutathione S-transferase GSTU6 gi|226510544 Z. mays 44 9
188 UPF1 Protein prenyltransferase gi|115482066 O. sativa 37 43
194 MPE Phytoene synthase gi|226532946 Z. mays 73 26
292 HDH Citrate lyase beta subunit gi|255605888 R. communis 40 12
495 HDH Pyridine nucleotide-disulphide oxidoreductase gi|308081758 Z. mays 22 13
817 URH Glutamate-1-semialdehyde 2,1-aminomutase gi|224134068 P. trichocarpa 48 25
925 HDH Acireductone dioxygenase gi|222628645 O. sativa 19 5
972 LDH Oxidoreductase,zinc-binding dehydrogenase family protein gi|308081586 Z. mays 50 39
979 HDH Dihydrolipoamide dehydrogenase 1 gi|224099079 P. trichocarpa 28 12
1067 DRH Patatin T5 gi|226492557 Z. mays 26 7
1076 HDH Flavonoid 3-monooxygenase gi|226494123 Z. mays 72 12
1092 UPF1 Trehalose synthase-like protein gi|224028441 Z. mays 21 15
1096 UPF1 Putative trehalose-6-phosphate synthase gi|24308617 O. sativa 24 14
1098 HDH GTP cyclohydrolase II (RibA) gi|303280293 M. pusilla 15 5
1143 LDH Oxidoreductase gi|195624808 Z. mays 28 8
Energy
129 URH Phosphoglycerate mutase gi|293336560 Z. mays 45 15
169 MPE Carbonic anhydrase gi|226495621 Z. mays 37 10
415 LDH V-type ATP synthase subunit I gi|222641344 O. sativa 38 19
467 DRH Mitochondrial lipoamide dehydrogenase gi|134142802 P. tremuloides 26 11
536 HDH Sulfotransfer_2 gi|159481436 C. reinhardtii 19 8
575 LDH Putative sex determination protein tasselseed 2 gi|22296337 O. sativa 41 9
587 URH UGP(UDP-glucose pyrophosphorylase) gi|15228498 A. thaliana 50 18
1041 DRH Glycosyltransferase gi|223945287 Z. mays 46 7
1100 LDH ATP synthase D chain, mitochondrial gi|226507194 Z. mays 64 12
Cell growth/division
343 MPH DNA polymerase I gi|195654367 Z. mays 28 7
586 DRH Cyclin-related gi|42569848 A. thaliana 43 46
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
950 LDH Ribosome biogenesis protein NEP1 gi|226495343 Z. mays 48 10
976 LDH ATEXO70C1 (exocyst subunit EXO70 family protein C1) gi|212275810 Z. mays 50 14
1025 HDH Intraflagellar transport protein 172 gi|159467108 C. reinhardtii 15 19
1081 DRH Programmed cell death protein 5 gi|226500664 Z. mays 52 8
Transcription
40 URH GRAS family transcription factor gi|223949083 Z. mays 48 37
79 URH Helicase gi|302822204 S. moellendorffii 32 20
137 LDH Putative splicing factor gi|115454963 O. sativa 30 13
175 MPE mTERF family protein gi|195605386 Z. mays 46 16
201 MPE DEAD-like helicases superfamily gi|302820920 S. moellendorffii 29 19
281 MPE DEAD-like helicases superfamily gi|302820920 S. moellendorffii 22 11
312 HDH KU80; double-stranded DNA binding/protein binding gi|18402015 A. thaliana 35 20
1037 DRH DEAD-box helicases gi|302820920 S. moellendorffii 19 14
Protein synthesis
1006 HDH Mitochondrial ribosomal protein L5 gi|30351950 Z. mays 55 10
1012 UPF1 Eukaryotic translation initiation factor 3 subunit 2 gi|226533034 Z. mays 100 12
Protein transport and storage
77 HDH Chaperonin CPN60-2, mitochondrial precursor gi|162460375 Z. mays 35 18
227 MPE RING-finger domain protein gi|224088100 P. trichocarpa 36 11
446 UPF1 RING-finger domain protein gi|224088100 P. trichocarpa 38 12
507 MPE E3 ubiquitin-protein ligase UPL6OS UPL6_ARATH A. thaliana 32 35
783 HDH Armadillo/beta-catenin repeat family protein gi|297819214 A. lyrata 16 8
1074 HDH D1 protease-like protein precursor gi|19774135 T. aestivum 32 12
1083 DRH Armadillo/beta-catenin-like repeats gi|212275842 Z. mays 31 15
Cell structure
33 UPF1 Ankyrin-like protein gi|47497783 O. sativa 71 27
248 UPF1 Tubulin beta-6 chain gi|162459800 Z. mays 28 15
615 LDH Annexin gi|224057357 P. trichocarpa 34 12
781 LDH MIT domain protein gi|15223173 A. thaliana 56 7
793 UPF1 Putative ankyrin-like protein gi|48716529 O. sativa 41 14
966 UPF1 Ankyrin repeats protein gi|224115944 P. trichocarpa 43 21
Signal transduction
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
34 URH CDC2+/CDC28-related protein kinase R2 gi|195640128 Z. mays 28 7
69 UPF1 Leucine-rich repeat-containing protein, putative gi|255578414 R. communis 32 41
136 UPF1 Lectin-like receptor kinase 7 gi|226508034 Z. mays 40 6
162 LDH Glycogen synthase kinase-3 MsK-3 gi|226533162 Z. mays 30 11
217 UPF1 Mitochondrial carrier protein gi|297724283 O. sativa 72 9
279 HDH Serine/threonine-protein phosphatase 2A activator 2 gi|195640094 Z. mays 52 13
349 UPF1 Putative phototropic response protein gi|115472647 O. sativa 23 8
453 UPF1 Pentatricopeptide repeat-containing protein gi|75268015 A. thaliana 40 6
552 HDH Pentatricopeptide repeat-containing protein gi|297797201 A. lyrata 19 13
676 URH Putative BLE2 protein gi|23617104 O. sativa 21 18
678 MPE Calmodulin gi|293334895 Z. mays 39 6
927 HDH Pentatricopeptide repeat-containing protein gi|297797201 A. lyrata 20 12
1011 HDH WD-40 repeat family protein gi|308080163 Z. mays 57 9
1069 UPF1 WD-40 repeat family protein gi|218192583 O. sativa 31 10
Disease and defence
31 URH Remorin protein gi|293336051 Z. mays 24 9
112 UPF1 TIR-NBS-LRR resistance protein gi|111146892 P. vulgaris 17 17
147 UPF1 Heat shock 70 kDa protein 4 gi|226498820 Z. mays 15 8
198 DRH Powdery mildew resistance protein PM3A gi|62912003 T. aestivum 46 62
233 UPF1 Disease resistance protein RPP13 gi|15231449 A. thaliana 43 32
497 MPE Putative PEP-CTERM system TPR-repeat lipoprotein gi|224029551 Z. mays 24 21
626 F1nBP NBS-LRR type resistance protein gi|2792230 O. sativa 59 8
663 UPF1 Late embryogenesis abundant protein gi|242044772 S. bicolor 52 14
767 HDH Disease resistance protein (NBS-LRR class), putative gi|15231862 A. thaliana 33 29
815 F1nBP NBS-LRR protein gi|21616910 O. sativa 45 10
988 HDH TUP1-like enhancer gi|293336752 Z. mays 40 12
1004 MPE Bacterial trigger factor protein (TF) gi|225434088 V. vinifera 59 15
1066 DRH BED finger-nbs-lrr resistance protein gi|224145670 P. trichocarpa 20 10
Secondary metabolism
48 LDH Cytochrome P450 CYP74A19 gi|195613496 Z. mays 52 22
553 MPE Cytochrome P450 CYP76M15 gi|195646472 Z. mays 37 25
Unknown and putative protein
(续附表)
蛋白点编号
Spot No.
表达模式
Expression pattern
蛋白功能
Protein function
序列号
Accession No.
物种
Species
肽段覆盖率
Sequence coverage (%)
肽段匹配数
Matched peptide No.
93 HDH Expressed protein gi|108707573 O. sativa 26 15
193 UPF1 Predicted protein gi|303288630 M. pusilla 35 18
294 URH Unknown protein gi|18413400 A. thaliana 38 39
378 URH Predicted protein gi|303282581 M. pusilla 42 9
379 HDH Hypothetical protein gi|40786588 O. sativa 68 9
384 MPE Hypothetical protein OsJ_26893 gi|222640350 O. sativa 42 9
480 HDH Hypothetical protein gi|115477703 O. sativa 19 7
540 UPF1 Predicted protein gi|297817898 A. thaliana 44 12
711 URH DUF538 domain protein gi|226491968 Z. mays 61 12
911 UPF1 Hypothetical protein gi|218196215 O. sativa 38 16
964 LDH Hypothetical protein OsJ_26893 gi|222640350 O. sativa 43 10
1043 UPF1 Conserved hypothetical protein gi|255570033 R. communis 76 5
1065 DRH Unknown protein gi|186489637 A. thaliana 93 8
1108 UPF1 Hypothetical protein ARALYDRAFT_356151 gi|297805920 A. lyrata 21 11
Transposons
154 UPF1 Transposase gi|212275225 Z. mays 20 7
383 MPE Retrotransposon protein, putative, Ty3-gypsy subclass gi|77553948 O. sativa 23 20
408 HDH Retrotransposon protein, putative gi|108864382 O. sativa 43 33
689 HDH Transposase_24 gi|226492958 Z. mays 28 18
1159 UPF1 Retrotransposon protein, putative, Ty3-gypsy sub-class gi|62733790 O. sativa 20 32