摘要
杂种优势是一种普遍存在的生物学现象，指两个遗传性不同的亲本杂交产生的杂交种在生长势、生活力、抗逆性以及产量、品质等方面优于其双亲的现象。杂种优势的利用获得了巨大的经济和社会效益。但对杂种优势机理的探索远远落后于对它的实践应用。
近年来对杂种优势分子基础的研究成为一个热点，在mRNA转录水平发现了许多杂种基因表达偏离亲本平均水平的非加性表达(nonadditive expression)现象，但在蛋白质水平的研究却很少。本研究采用荧光差异凝胶电泳，双向电泳和MALDI-TOF-MS技术，对强优势杂交水稻——汕优63及其亲本的胚、苗期第3叶以及根系的蛋白质组进行比较研究，探寻它们在蛋白质组水平上的基因表达特征，研究结果如下：
(1)对强优势杂交水稻——汕优63及其亲本的成熟胚蛋白质组进行分析。从不育系珍汕97A、保持系珍汕97B、恢复系明恢63及杂种一代汕优63的胚中分别分离到965．7±19．2，964．7±16．7，961±18．7，982．7±19个蛋白质点。发现同核异质的珍汕97A与珍汕97B的胚蛋白质组电泳图谱基本相同，珍汕97A有19个稳定可重复的蛋白质点不同于恢复系明恢63，明恢63有17个稳定可重复的蛋白质点不同于珍汕97A，这些亲本间的差异蛋白质都在F_1汕优63中表达，表现为共显性或显性效应，未发现杂种特异表达的蛋白质，这些蛋白质主要呈加性表达，少数为非加性表达。
经MALDI-TOF-MS／MS鉴定了其中20个差异蛋白，9个获得有显著性意义的鉴定结果。其中2种与代谢相关，分别为推定的磷酸甘油酸变位酶和二氢硫辛酸脱氢酶；1种可能与防御相关的几丁质酶；另有1种蛋白质含晚期胚胎丰富蛋白序列，其余为推定的球蛋白。
(2)对汕优63组合3叶1心期地上部分性状进行了观察分析，发现汕优63在苗期的株高、叶长和叶宽表现为不同程度的中亲优势，分别为13．14％、14．79％和5．95％，均未超亲。但叶面积表现为显著的超高亲优势，达15．72％。分析认为叶面积的超亲优势与叶长和叶宽两个子性状间的乘数效应有关。
对汕优63及其双亲苗期第3叶蛋白质组进行DIGE定量比较分析，分离到1667个以上的蛋白质点。发现有23个蛋白质点在三个品系间存在显著差异(p＜0．01)。其中16个双亲间的差异表达蛋白质在汕优63的丰度接近于双亲均值，占全部69．6％；2个蛋白质点的丰度对双亲均值偏离超过20％，其余5个蛋白质点偏离幅度在10-20％之间，表现为不同程度的非加性表达效应。
经MALDI-TOF-MS分析，有22个差异蛋白得到成功鉴定，包括9对存在差异的等位基因。其中2种与呼吸代谢相关，分别为推定的磷酸甘油酸变位酶以及二氢硫辛酸脱氢酶；1种蛋白质与基因表达调控有关，为RPS5包含蛋白；1种铁氧还蛋白-NADP还原酶是光合作用的重要酶类；1种推定的β-羟基类固醇脱氢酶/异构酶，可能与类固醇代谢有关；另3种蛋白质可能与保护防御相关，分别是肽-甲硫氨酸亚砜还原酶、推定的肽-甲硫氨酸亚砜还原酶和GSH依赖脱氢抗坏血酸还原酶。其中铁氧还蛋白-NADP还原酶的正向非加性表达，可能与F1光饱和点较高等优良光合性状有关。
(3)对汕优63及其亲本3叶1心期根系蛋白质组进行比较分析，通过双向电泳和考马斯亮蓝染色，发现23个可重复的差异蛋白质点。双亲间所有的差异点均在F_1汕优63中出现，有4个蛋白质表现为明显的非加性表达效应，未发现杂种特异表达的蛋白质，差异点19在杂种中的表达受到明显抑制。
对23个蛋白质点进行MALDI-TOF-MS／MS鉴定，均得到有显著性的结果．有7种蛋白质与物质能量代谢相关，分别为推定的磷酸甘油酸变位酶，烯醇化酶，甲基丙二酸半醛脱氢酶以及脱氢抗坏血酸还原酶，胞浆苹果酸脱氢酶以及一种推定的β-1，3-葡聚糖酶；有2种酶与信号转导有关，分别是腺苷甲硫氨酸合成酶和N-氨甲酰腐胺酰胺酶；腺苷甲硫氨酸合成酶及槲皮素3-o-甲基转移酶1与次生物质代谢相关。还发现可能与蛋白质相互作用有关的kelch重复片段包含蛋白，推测其可能与上位性效应有关。
(4)探讨了杂种优势理论无法充分解释育种实践上的杂种优势的现象及原因。认为局部优势并不一定导致整体的杂种优势，已有的理论可以解释局部优势，但无法解释做为整体的杂种优势。对局部优势与整体优势的关系做初步分析，提出局部性状优势主要通过乘数关系组织为整体杂种优势的观点，认为乘数关系和乘数效应是杂种优势形成的重要基础。对这一观点做初步的展开论述，并得出一些与杂交水稻高产育种工作相关的推论。
本研究首次利用蛋白质组学的方法对杂交水稻不同器官的蛋白质组进行比较分析，得到杂交水稻在蛋白质组水平上的基因表达特点和有关数据，鉴定了一些有价值的蛋白质，并发现一些双亲间的新等位基因，包括一些功能尚未完全明确的水稻基因。此外还分析了局部优势与整体优势的关系，提出局部性状优势主要通过乘数关系组织为整体杂种优势，乘数关系和乘数效应是杂种优势形成的重要基础的观点。这些工作为了解强优势杂交水稻杂种优势在蛋白质组水平上的基因表达基础积累了一些必要资料，是前人在转录水平上的研究的必要互补，为更好地阐明杂种优势的机制提供依据。
Heterosis is a fundamental biological phenomenon,refers to the superiorperformance of hybrid in terms of increased yield,quality,growth vigor,speed ofdevelopment and resistance to climatic rigours relative to both parent lines.Theapplication of heterosis to agriculture has produced enormous social and economicbenefit,but the mechanism underlying heterosis remains elusive.Recently,manyresearches focus on the molecular basis of heterosis,several studies analyzed heterosisassociated gene expression pattern at the mRNA translation level,and nonadditive geneexpression was found in hybrids of different plants.But few studies were carried out at theprotein lever.To study gene expression pattern of hybrid rice at protein level,theproteome of embryo,leaf and root from an elite hybrid rice Shanyou63 and its parents wasanalyzed with 2-DE or DIGE,MALDI-TOF-MS and MALDI-TOF-MS/MS.
Embryo proteins in mature seeds of elite hybrid rice shanyou 63 and its 3-lines(sterile line,maintainer line and restorer line) were separated with 2-DE.The number ofdetected protein spots was 965.7±19.2 (SD,n=3),964.7±16.7,961±18.7 and 982.7±19(n=6),from Zhenshan97A (sterile line,female parent),Zhenshan97B (maintainer line),Minghui63 (restorer line,male parent) and Shanyou63 respectively.No repeatabledifference was detected between Zhenshan97A and its isogene line Zhenshan97B.However,there were many differentially expressed proteins between Zhenshan97A andMinghui63,showing 19 reaptably differential protein spots detected only in Zhenshan97A,and 17 ones only in Minghui63.All of these differential proteins were expressed in hybrid.Furthermore,there were 21 protein spots with different abundance in parents.It isremarkable that the abundance of all those differentially expressed proteins in the hybridwere different from that of corresponding proteins expressed in its parents,and main ofthem were close to the mid-parent values,although some of them deviated relativelymid-parent predictions,exhibiting somewhat nonadditive expression.20 differentiallyexpressed proteins were analyzed through MALDI-TOF-MS/MS and database searching.Of the 9 identified proteins,dihydrolipoyl dehydrogenase and putative phosphoglyceratemutase involved in respiration metabolism,chitinase correlated to disease and defend, and others were putative globulin and putative LEA domain-containing protein.
Morphological traits of aerial part in Shanyou63 and its parents was surveyed,different midparent heterosis(MPH) was found in plant height,leaf length and leaf wide atseedling stage,only leaf area showed a significant best parent heterosis(BPH) of 15.72 %.It suggested that multiplicative effect is a important factor in the formation of BPH of leafarea.
Differentially expressed leaf proteins among the three lines were examined usingDIGE.Of the＞1677 protein spots reproducibly detected on each gel,23 protein spotswere found different accumulation among the three lines (ANOVA-1 analyzed,p＜0.01).Inthe hybrid,16 proteins were accumulated in a additive fashion in the hybrid compared tothe average of their parental lines,while the standard volume of 2 proteins showed anonadditive expression＞20%,while the other 5 protein spots showed a nonadditive withina range of 10-20%.
22 of the differentially expressed proteins were identified by MALDI-TOF-MS anddatabase searching,included 2 respiration metabolism proteins,dihydrolipoyldehydrogenase and putative phosphoglycerate mutase;a ferredoxin--NADP reductaserevolving in photosynthetic electron transport chain;a Putative 3-beta hydroxysteroiddehydrogenase/isomerase may be an enzyme in steroid metabolism;a RPS5 containingprotein may revolve in gene expression and regulation;and 3 anti-oxidatant enzymes:GSH-dependent dehydroascorbate reductase 1,peptide methionine sulfoxide reductaseand a putative peptide methionine sulfoxide reductase.
Differentially expressed root proteins among the three lines were examined with 2-DEand CBB staining。23 protein spots were found different accumulation among the threelines (Student's t-test,p＜0.05),4 of them showed a nonadditive expression＞20%,noprotein was found specially expressed in hybrid,but spot 19 showed a obvious decreasein F1.
Using MALDI-TOF-MS/MS and database searching,all of the differentially expressedroot proteins were identified.8 of them was manifested involving in matter and energymetabolism,while 2 were related to signal transduction,and a kelch repeat containingprotein might involve in protein-protein interaction,possible related to epistasis.
The puzzle of why dominance or over-dominance hypothesis cannot explain theheterosis mechanism properly was discussed.It is considered that global heterosis,suchas heterosis of yield,dose not necessarily be the result of local heterosis which could beexplained fully by these hypothesis.It is proposed that,among the local traits,there is amultiplicative relationship by which the heterosis of local traits was organized to formglobal heterosis.It is hold that multiplicative relationship and multiplicative effect wereimportant basis of global heterosis,and then some conclusion concerning breedingpractice were deduced.
引文
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