Comparative proteomics of two Populus spp.(Section Tacamahaca) allotriploid derived by different types of 2n female gamete and their parents
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摘要:目的研究两种2n雌配子来源的青杨杂种三倍体与亲本的蛋白质组表达差异，从蛋白水平探讨异源三倍体杨树在生长、抗性等方面具有优势的分子基础，为杨树多倍体选育和遗传改良提供科学依据。方法采用同位素标记相对和绝对定量(iTRAQ)技术对青杨三倍体与其亲本进行定量蛋白质组学研究，所提取的杨树蛋白质样品经FASP酶解、iTRAQ试剂标记、高pH-RPLC分离、RPLC-MS分离分析，获取的串联质谱数据通过软件Proteome Discoverer 1.3搜库进行蛋白质鉴定，通过蛋白质相对定量的比较寻找差异表达蛋白，再对差异蛋白质进行GO、KEGG代谢通路分析。结果本研究共鉴定出1 472个蛋白质，差异蛋白202个。FDR和SDR青杨杂种三倍体与母本‘哲引3号杨’、父本‘北京杨’的差异蛋白比率在2.0%~10.1%之间。两种不同2n配子来源三倍体中FDR与亲本差异蛋白比率最高，且两种三倍体与父本的差异蛋白比率均比母本高。通路注释分析显示，差异蛋白显著富集于代谢相关、核糖体组装、光合作用和胁迫响应等通路。结论杂交和加倍后促进了蛋白质的合成以及光合作用的增强，并提高了多倍体的抗逆性和适应性，这些变化促进了杨树异源多倍体营养生长优势的形成。Abstract:ObjectiveAlthough Populus allotriploid has a prominent vegetative growth advantages, the underlying molecular mechanisms have not yet been revealed and elucidated. This work was designed to investigate proteins differentially expressed in the two Populus allotriploid derived by different types of 2n female gamete and their parents. The results will provide the scientific foundation for Populus polyploidy selection and genetic improvement.MethodThe iTRAQ proteomics approach was used in this study. The extracted proteins were digested using FASP method and identified by iTRAQ coupled with LC-MS/MS technology. Raw data were analyzed by Proteome Discoverer 1.3 search engine. Then the pathway analysis was conducted using GO and KEGG.ResultA total of 1 472 proteins were identified and 202 proteins were detected as differentially expressed proteins. The ratio of differentially expressed proteins between FDR and SDR Populus allotriploid and the two parents varied from 2.0% to 10.1%. Compared with the female parent or male parent, the ratio of differentially expressed proteins in FDR Populus allotriploid was higher than in SDR Populus allotriploid. In particular, there was an expression level dominance bias toward the triploid progenitors. Further analysis showed that the differentially expressed proteins were significantly enriched in the pathways such as metabolic related, ribosomes, photosynthesis and response to stress.ConclusionThe results indicated that plyploidization and hybridization could enhance photosynthesis, proteins synthetic and increased the resistance and adaptability of Populus polyploidy. All of these contribute to vegetative growth advantages in allotriploid plants.
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Keywords:
- 2n female gamete /
- Populus allotriploid /
- proteomics /
- iTRAQ
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杨树(Populus)三倍体与二倍体相比具有明显的营养生长优势[1-3]。1936年，Nilsson-Ehle在瑞典发现了一株叶片巨大、生长迅速的巨型三倍体欧洲山杨(Populus tremula)[4]。毛白杨(Populus tomentosa)天然三倍体在材积生长、纤维特性和抗性等方面都优于二倍体对照[5]。Einspahr等在采用四倍体欧洲山杨与二倍体美洲山杨杂交获得了遗传增益显著的人工异源三倍体山杨[6]。通过给毛新杨(Populus tomentosa×Populus bolleana)授以毛白杨天然2n花粉杂交获得的三倍体毛白杨新品种在材积生长和木材材性改良方面表现出的突出优势[7]。青杨(Populus spp.(Section Tacamahaca))异源三倍体群体净光合速率、叶绿素含量和叶面积等生理性状显著高于全同胞二倍体[3]。因此，综合倍性优势和杂交优势的异源多倍体育种是杨树遗传改良的最佳途径[8-9]。
多倍体植物中，许多研究报道了基因组加倍和杂合性对基因表达的影响。Ng等以拟南芥(Arabidopsis thaliana)异源四倍体为材料发现了拟南芥四倍体中胁迫响应蛋白表达增加[10]；相对于二倍体水稻，三倍体水稻除了叶片面积偏大、叶绿素含量增加且有13个的叶绿体蛋白表达量显著增加[11]。运用蛋白质组学定量研究技术(iTRAQ)研究表明，菊科植物多倍体9%~10%的蛋白相对于二倍体亲本存在差异[12]。此外，Suo等[13]发现三倍体杨树总甲基化水平低于亲本；杨树miRNA表达无剂量效应可能在多倍体营养生长优势的分子机制中起重要作用[14]；以青杨异源三倍体及其全同胞二倍体的顶芽为材料，Cheng等[15]分析了多倍体的基因表达特点，显示青杨杂种三倍体在蛋白质代谢过程、碳固定、胁迫响应以及热激蛋白等基因表达模式与二倍体不同。这些研究在一定程度上解释了多倍体营养生长优势的分子基础，受林木杂种多倍体创制技术的限制，基于蛋白水平对林木多倍体营养生长优势的遗传调控的研究还需进一步深入。
本研究以两种不同2n雌配子来源的‘哲引3号杨’(Populus pseudo-simonii × Populus nigra var. italica)ב北京杨’(Populus × beijingensis)杂种三倍体青杨[16]，及其母本‘哲引3号杨’和父本‘北京杨’为材料，采用同位素标记相对定量和绝对定量(isobaric tags for relative and absolute quantification，iTRAQ)技术，研究FDR、SDR青杨杂种三倍体与亲本蛋白质组差异，从蛋白质组学角度揭示杨树多倍体营养生长优势的分子基础，为进一步科学开展杨树多倍体育种奠定理论依据。
1. 材料与方法
1.1 试验材料
本试验以‘哲引3号杨’为母本、‘北京杨’为父本，通过高温处理人工诱导雌配子染色体加倍(第一次减数分裂抑制产生FDR型2n雌配子，第二次减数分裂抑制产生SDR型2n雌配子)获得两种不同途径的青杨异源三倍体群体(first-division restitution，FDR；second-division restitution，SDR)。利用SSR标记区分来源于FDR型和SDR型2n配子的FDR群体和SDR群体[17-19]。于2015年8月，每个群体选择5个基因型，取完全舒展的成熟叶片放入液氮中速冻，然后储存于-80 ℃超低温冰箱保存。
1.2 蛋白提取
将每个群体选取的5个基因型样品叶片等量混合，称取1 g叶片样品采用三氯乙酸-丙酮法[20]独立提取每个群体的叶片总蛋白。叶片样品于液氮中研磨成粉末并悬浮于10倍体积预冷的-20 ℃丙酮溶液(含10%三氯乙酸和0.07%β-巯基乙醇)中。充分混匀后，于-20 ℃静置2 h；4 ℃、12 000 r/min离心30 min，弃上清液。重复用预冷的-20 ℃丙酮溶液(含0.07%β-巯基乙醇)悬浮沉淀并离心多次直至沉淀为白色，后将沉淀真空干燥。称取0.1 g沉淀于2 mL裂解液(8 mol/L尿素、4% CHAPS、1 mmol/L PMSF、2 mmol/L EDTA、10 mmol/L DTT)中裂解4 h，4 ℃、12 000 r/min离心30 min，取上清液，上清液即为叶片总蛋白溶液。采用Bradford法测定提取的蛋白质溶液的浓度，分装后，-80 ℃保存备用[21]。
1.3 FASP酶解
取200 μg蛋白溶液置于离心管中，加入4 μL还原剂(Reducing Reagent)，60 ℃反应1 h后加入2 μL半胱氨酸封闭剂(Cysteine-Blocking Reagent)，室温10 min。将还原烷基化后的蛋白溶液转入10 kD超滤离心管中，12 000 r/min离心20 min，弃去管底溶液，加入1 mol/L TEAB 100 μL，12 000 r/min离心20 min，弃去管底溶液，重复3次。按质量比1:50加入胰蛋白酶及1 mol/L TEAB，使最终体积为50 μL，过夜酶解。次日，离心收集酶解消化后的肽段溶液，并干燥保存。
1.4 iTRAQ标记
将6个酶解后的肽段样品各取100 μg，分别用113、114标记FDR三倍体子代，117标记母本(P1)，118标记父本(P2)，119、121标记SDR三倍体子代(具体操作按照试剂盒说明书进行)后，等量混合。
1.5 蛋白鉴定和生物信息学分析
采用Proteome Discoverer软件(版本1.3，Thermo Fisher Scientific，MA，USA)对肽段MS/MS的数据在NCBI(http://www.ncbi.nlm.nih.gov/protein?term=txid3694[Organism])毛果杨蛋白数据库进行检索。选择置信度在95%以上的结果进行报告。蛋白至少含有一个唯一肽段，并且阳性结果错误率(FDR)≤1%才被认为鉴定有效。对于蛋白定量，只有蛋白的定量信息至少存在于2次生物学重复中才作进一步分析。以2次生物学重复的平均值作为最终蛋白表达的倍率。对其进行t检验，P≤0.05且蛋白质平均比率≥1.5或≤0.67分别为上调和下调的差异蛋白。采用GO数据库(http://www.geneo-ntology.org/)注释工具对差异蛋白进行蛋白功能聚类GO (gene ontology)分析，采用KEGG(kyoto encyclopedia of genes and genomes)通路数据库(http://www.genome.ad.jp/kegg/)对差异蛋白涉及的代谢通路进行分析。
2. 结果与分析
2.1 青杨杂种三倍体与亲本的差异蛋白数据统计
质谱原始数据经通过Proteome Discoverer1.3软件对毛果杨蛋白质组数据库进行检索，报告置信度在95%以上的蛋白质共1 472个，差异蛋白数202个。与母本‘哲引3号杨’(P1)相比，FDR三倍体植株差异蛋白43个，上调25个，下调18个；SDR三倍体差异蛋白29个，上调15个，下调14个。与父本‘北京杨’(P2)相比，FDR三倍体植株差异蛋白148个，上调60个，下调89个；SDR三倍体植株差异蛋白76个，上调27，下调49个，见表 1。
表 1 两种青杨杂种三倍体子代与二倍体亲本蛋白质组差异Table 1. Proteome differences between two Populus allotriploid and the diploid parents组别
Group差异蛋白数
Number of DEPs占总蛋白数的百分率
Percentage in total proteins/%上调个数
Up-regulated number下调个数
Down-regulated numberFDR vs P1 43 2.9 25 18 FDR vs P2 148 10.1 60 89 SDR vs P1 29 2.0 15 14 SDR vs P2 76 3.1 27 49 注：P1代表母本，P2代表父本。下同。Notes: P1, female parent; P2, male parent. The same below. FDR、SDR三倍体植株是由第1次减数分裂抑制产生FDR型2n雌配子和第2次减数分裂抑制产生SDR型2n雌配子的方式授粉后杂交产生的全同胞家系。从差异蛋白数量上看，FDR、SDR三倍体植株与母本‘哲引3号杨’差异蛋白数分别为43和29，而FDR、SDR三倍体植株与父本‘北京杨’的差异蛋白数分别为148和76。如图 1所示，两种2n雌配子来源的三倍体子代与父本的蛋白表达差异大于母本。
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图 1 两种青杨杂种三倍体子代与亲本差异蛋白维恩图分析Figure 1. Venn diagrams of proteins differentially expressed in two Populus allotriploid and diploid parents2.2 青杨杂种三倍体与亲本的差异蛋白GO功能聚类
为了深入分析不同2n配子来源的三倍体杨树与‘哲引3号杨’、‘北京杨’差异表达蛋白的生物功能，每个比较组合的差异蛋白进行GO功能聚类分析。GO注释主要是对蛋白质的生物过程(biological process)、分子功能(molecular function)、细胞位置(cellular component)进行分析。其中，FDR型2n雌配子来源的三倍体与母本‘哲引3号杨’差异蛋白主要参与的生物过程有15个，其中占比例较高的前5个是光呼吸作用、氧化还原过程、脂质代谢过程、镉离子响应、糖酵解；分子功能所占比例较高的是氧化还原酶活性、水解酶活性、羧酸酯水解酶活性、过氧化物酶活性、辅酶结合；细胞位置所占比例较高的前5个是叶绿体类囊体、呼吸链、细胞壁、叶绿体包膜、细胞质，如图 2A所示。FDR型2n雌配子来源的三倍体与父本‘北京杨’差异蛋白参与的生物过程有15个，所占比例较高的前5个是翻译、核糖体合成、RNA甲基化、香豆素生物合成过程、镉离子响应；分子功能所占比例较高的前5个是核糖体的结构成分、rRNA结合、RNA结合、细胞骨架的结构成分、甲硫氨酸腺苷转移酶活性；细胞位置所占比例较高的前5个是核糖核蛋白复合物、核糖体、细胞质、胞质大核糖体亚基、胞质核糖体，如图 2B所示。
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图 2 FDR青杨杂种三倍体与二倍体亲本差异蛋白GO功能分类PO.光呼吸作用；OR.氧化还原过程；LC.脂质代谢过程；RC.镉离子响应；G.糖酵解；HP.过氧化氢分解代谢过程；DR.对细菌的防御反应；ET.电子传递链；NR.催化活性的负调控；MC.单酰甘油分解代谢过程；DP.二酰甘油代谢过程；PN.嘌呤核苷酸转运；RG.呼吸气体交换；GC.甘氨酸分解代谢过程；CP.叶绿素生物合成过程；OA.氧化还原酶活性；HA.水解酶活性；CE.羧酸酯水解酶活性；PER.过氧化物酶活性；CO.辅酶结合；CI.铜离子结合；AO.替代氧化酶活性；AL.酰基甘油脂肪酶活性；AT.ATP：ADP逆向转运蛋白活性；FR.果糖二磷酸醛缩酶活性；NA.NADH脱氢酶(泛醌)活性；DO.δ4-3-氧代甾族5β-还原酶活性；PEP.肽酶活性；ER.烯酮还原酶活性；EI.内肽酶抑制剂活性；CT.叶绿体类囊体；RE.呼吸链；CW.细胞壁；CH.叶绿体包膜；CY.细胞溶质；EX.细胞外区域；AP.质外体；CM.叶绿体类囊体膜；MI.线粒体内膜；RCC.呼吸链复合体Ⅰ；MRC.线粒体呼吸链复合体Ⅰ；CHL.叶绿体；ME.线粒体被膜；MM.线粒体膜；TH.类囊体；TR.翻译；RB.核糖体生物合成；RM.RNA甲基化；CBP.香豆素生物合成过程；RS.盐胁迫响应；PMP.苯丙素类代谢过程；GO.高尔基组织；CMA.细胞修饰氨基酸生物合成过程；SB.S-腺苷甲硫氨酸生物合成过程；WT.水分运输；RH.根毛伸长；PBP.苯丙素类生物合成过程；RHL.激素水平调节过程；SC.核糖体的组成结构；RR. rRNA结合；RN. RNA结合；SCC.细胞骨架的结构成分；MA.甲硫氨酸腺苷转移酶活性；SMA.结构分子活性；PAP.苯丙氨酸解氨酶活性；ALA.氨解酶活性；HY.水解酶活性、作用于酯键；GT.谷胱甘肽转移酶活性；RIB.核糖体结合；3D.3-脱氧-7磷酸庚酮酸合酶活性；RIC.核糖核蛋白复合体；RI.核糖体；CL.胞质大核糖体亚基；CR.细胞质核糖体；IN.细胞内；P.胞间连丝；CS.细胞质小核糖体亚基；NU.核仁；GOA.高尔基体；CYT.细胞质；LR.大亚基。下同。Figure 2. GO analysis of proteins differentially expressed in FDR Populus allotriploid and the diploid parentsPO, photorespiration; OR, oxidation-reduction process; LC, lipid catabolic process; RC, response to cadmium ion; G, glycolysis; HP, hydrogen peroxide catabolic process; DR, defense response to bacterium; ET, electron transport chain; NR, negative regulation of catalytic activity; MC, monoacylglycerol catabolic process; DP, diacylglycerol catabolic process; PN, purine nucleotide transport; RG, repiratory gaseous exchange; GC, glycine catabolic process; CP, chlorophyll biosynthesis process; OA, oxidoreductase activity; HA, hydrolase activity acting on ester bonds; CE, carboxylic ester hydrolase activity; PER, peroxidase activity; CO, coenzyme binding; CI, copper ion binding; AO, alternative oxidase activity; AL, acylglycerol lipase activity; AT, ATP:ADP antiporter activity; FR, fructose-bisphosphate aldolase activity; NA, NADH dehydrogenase (ubiquinone) activity; DO, delta4-3-oxosteroid 5beta-reductase activity; PEP, peptidase activity; ER, enone reductase activity; EI, endopeptidase inhibitor activity; CT, chloroplast thylakoid; RE, respiratory chain; CW, cell wall; CH, chloroplast envelope; CY, cytosol; EX, extracellular region; AP, apoplast; CM, chloroplast thylakoid membrane; MI, mitochondrial inner membrane; RCC, respiratory chain complex Ⅰ; MRC, mitochondrial respiratory chain complex Ⅰ; CHL, chloroplast; ME, mitochondrial envelope; MM, mitochondrial membrane; TH, thylakoid; TR, translation; RB, ribosome biogenesis; RM, RNA methylation; CBP, coumarin biosynthetic process; RS, response to salt stress; PMP, phenylpropanoid metabolic process; GO, Golgi organization; CMA, cellular modified amino acid biosynthetic process; SB, S-adenosylmethionine biosynthetic process; WT, water transport; RH, root hair elongation; PBP, phenylpropanoid biosynthetic process; RHL, regulation of hormone levels; SC, structural constituent of ribosome; RR, rRNA binding; RN, RNA binding; SCC, structural constituent of cytoskeleton; MA, methionine adenosyltransferase activity; SMA, structural molecule activity; PAP, phenylalanine ammonia-lyase activity; ALA, ammonia-lyase activity; HY, hydrolase activity, acting on ester bonds; GT, glutathione transferase activity; RIB, ribosome binding; 3D, 3-droxy-7-phosphoheptulona; RIC, ribonucleoprotein complex; RI, ribosome; CL, cytosolic large ribosomal subunit; CR, cytosolic ribosome; IN, intracellular; P, plasmodesma; CS, cytosolic small ribosomal subunit; NU, nucleolus; GOA, Golgi apparatus; CYT, cytoplasm; LR, large ribosomal subunit. The same below.SDR型2n雌配子来源的三倍体与母本‘哲引3号杨’差异蛋白主要参与的生物过程有15个，所占比例较高的前5个是光呼吸作用、香豆素生物合成、蛋白水解、氧化还原过程、冷胁迫响应；分子功能所占比例较高的前5个是肽酶活性、铜离子结合、丝氨酸型肽酶活性、氧化还原酶活性、丝氨酸型内肽酶活性；细胞位置所占比例较高的前5个是细胞壁、细胞质、呼吸链、胞间连丝、叶绿体包膜，如图 3A所示。SDR型2n雌配子来源的三倍体与父本‘北京杨’差异蛋白主要参与的生物过程有15个，所占比例较高的前5个是翻译、RNA甲基化、镉离子响应、对盐胁迫的反应、糖酵解；分子功能所占比例较高的前5个是核糖体结构成分、rRNA结合、铜离子结合、细胞骨架结构成分、甲硫氨酸腺苷转移酶活性；细胞位置所占比例较高的前5个是核糖核蛋白复合物、核糖体、胞质大核糖体亚基、胞质核糖体、细胞质，如图 3B所示。
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图 3 SDR青杨杂种三倍体与二倍体亲本差异蛋白GO功能分类PRO.蛋白水解；REC.冷响应；LM.脂质代谢过程；PF.类黄酮生物合成过程的正调控；LO.脂质氧化；RW.对伤害的反应；CRB.叶绿体二磷酸核酮糖羧化酶复杂的生物发生；SPA.丝氨酸型肽酶活性；SE.丝氨酸型内肽酶活性；LLA.亚油酸酯13S-脂氧合酶活性；OAA.氧化还原酶活性，作用于具有掺入分子氧的单个供体；UDP.UDP-葡糖基转移酶活性；RCA.核酮糖二磷酸羧化酶活性；LAL.长链脂肪酸-CoA连接酶活性；AEA.醛糖1-差向异构酶活性；CHR.叶绿体二磷酸核酮糖羧化酶复合物；PCW.植物型细胞壁；CMP.碳水化合物代谢过程；LB.木质素生物合成过程；RO.氧化应激反应；OM.单碳代谢过程；GR.生长；AGA. 4-α-葡聚糖转移酶活性；DA.二磷酸果糖-6-磷酸1-磷酸转移酶活性；GH.甘氨酸羟甲基转移酶活性；DL.UDP-葡萄糖醛酸脱羧酶活性；CHS.叶绿体基质。下同。Figure 3. GO analysis of proteins differentially expressed in SDR Populus allotriploid and diploid parentsPRO, proteolysis; REC, response to cold; LM, lipid metabolic process; PF, positive regulation of flavonoid biosynthetic process; LO, lipid oxidation; RW, response to wounding; CRB, chloroplast ribulose bisphosphate carboxylase complex biogenesis; SPA, serine-type peptidase activity; SE, serine-type endopeptidase activity; LLA, linoleate 13S-lipoxygenase activity; OAA, oxidoreductase activity, acting on single donors with incorporation of molecular oxygen; UDP, UDP-glucosyltransferase activity; RCA, ribulose-bisphosphate carboxylase activity; LAL, long-chain fatty acid-CoA ligase activity; AEA, aldose 1-epimerase activity; CHR, chloroplast ribulose bisphosphate carboxylase complex; PCW, plant-type cell wall; CMP, carbohydrate metabolic process; LB, lignin biosynthetic process; RO, response to oxidative stress; OM, one-carbon metabolic process; GR, growth; AGA, 4-alpha-glucanotransferase activity; DA, diphosphate-fructose-6-phosphate 1-phosphotransferase activity; GH, glycine hydroxymethyltransferase activity; DL, UDP-glucuronate decarboxylase activity; CHS, chloroplast stroma. The same below.2.3 青杨杂种三倍体与亲本的差异蛋白KEGG通路分析
将全部202个差异蛋白进行KEGG代谢通路分析，表明差异蛋白显著富集的代谢通路(P≤0.05)一共32个，其中富集程度最高的前10个代谢通路为苯丙氨酸代谢、乙醛酸和二羧酸代谢、核糖体、精氨酸和脯氨酸代谢、黄酮类生物合成、果糖和甘露糖代谢、氮代谢、光合作用，如图 4所示。
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图 4 FDR和SDR青杨杂种三倍体与二倍体亲本差异蛋白KEGG通路分析PB.苯丙素生物合成；PM.苯丙氨酸代谢；GD.乙醛酸和二羧酸代谢；APM.精氨酸和脯氨酸代谢；NM.氮代谢；FB.类黄酮生物合成；LA.亚油酸代谢；PC.卟啉和叶绿素代谢；AA.丙氨酸天冬氨酸和谷氨酸代谢；BS.次生代谢物的生物合成；MP.代谢途径；ALM.α-亚麻酸酸代谢；GS.甘氨酸、丝氨酸和苏氨酸代谢；CC.柠檬酸循环(TCA循环)；GM.谷胱甘肽代谢；NN.烟酸酯代谢；PR.蛋白酶体；OP.氧化磷酸化；GB.硫代葡萄糖苷生物合成；GG.糖酵解/糖异生；FF.黄酮和黄酮醇生物合成；ZB.玉米素生物合成；FA.脂肪酸延伸；PYM.丙酮酸代谢。SM.硒代化合物代谢；FM.果糖和甘露糖代谢；PH.吞噬体；PA.光合作用-天线蛋白；BM.丁酸代谢；PPM.丙酸酯代谢；VL.缬氨酸亮氨酸和异亮氨酸降解；AS.氨基糖和核苷酸糖代谢；CM.半胱氨酸和蛋氨酸代谢；GB.鞘糖脂生物合成-globo系列；SP.剪接体；PS.聚酮化合物糖单元生物合成；FAD.脂肪酸降解；C5.C5-支链二元酸代谢；PT.苯丙氨酸酪氨酸和色氨酸生物合成；GL.糖鞘脂生物合成；PHO.光合作用；SD.二苯乙烯类、二芳基庚烷类和姜醇生物合成；PE.过氧化物酶体；AAM.抗坏血酸和醛酸代谢；GLD.糖胺聚糖降解；TM.色氨酸代谢；PPE.内质网中的蛋白质加工；GLM.甘油脂代谢；CA.氰氨基酸代谢；OC.叶酸-碳库；GA.半乳糖代谢；CB.鞘糖脂生物合成-ganglio；MB.单萜生物合成；PP.磷酸戊糖途径。*表示显著差异(P<0.05)。Figure 4. KEGG pathway analysis of proteins differentially expressed in two Populus allotriploid and diploid parentsPB, phenylpropanoid biosynthesis; PM, phenylalanine metabolism; GD, glyoxylate and dicarboxylate metabolism; APM, arginine and proline metabolism; NM, nitrogen metabolism; FB, flavonoid biosynthesis; LA, linoleic acid metabolism; PC, porphyrin and chlorophyll metabolism; AA, alanine aspartate and glutamate metabolism; BS, biosynthesis of secondary metabolites; MP, metabolic pathways; ALM, alpha-linolenic acid metabolism; GS, glycine, serine and threonine metabolism; CC, citrate cycle (TCA cycle); GM, glutathione metabolism; NN, nicotinate and nicotinamide metabolism; PR, proteasome; OP, oxidative phosphorylation; GB, glucosinolate biosynthesis; GG, glycolysis/gluconeogenesis; FF, flavone and flavonol biosynthesis; ZB, zeatin biosynthesis; FA, fatty acid elongation; PYM, pyruvate metabolism; SM, selenocompound metabolism; FM, fructose and mannose metabolism; PH, phagosome; PA, photosynthesis-antenna proteins; BM, butanoate metabolism; PPM, propanoate metabolism; VL, valine, leucine and isoleucine degradation; AS, amino sugar and nucleotide sugar metabolism; CM, cysteine and methionine metabolism; GB, glycosphingolipid biosynthesis-globo series; SP, spliceosome; PS, polyketide sugar unit biosynthesis; FAD, fatty acid degradation; C5, C5-branched dibasic acid metabolism; PT, phenylalanine, tyrosine and tryptophan biosynthesis; GL, glycosphingolipid biosynthesis; PHO, photosynthesis; SD, stilbenoid, diarylheptanoid and gingerol biosynthesis; PE, peroxisome; AAM, ascorbate and aldarate metabolism; GLD, glycosaminoglycan degradation; TM, tryptophan metabolism; PPE, protein processing in endoplasmic reticulum; GLM, glycerolipid metabolism; CA, cyanoamino acid metabolism; OC, one carbon pool by folate; GA, galactose metabolism; CB, clycosphingolipid biosynthesis-ganglio; MB, monoterpenoid biosynthesis; PP, pentose phosphate pathway. * represents significant difference at P<0.05 level.3. 讨论
由于蛋白质组是基因表达的产物，直接执行细胞的生物功能并体现细胞的表型[22]，差异蛋白与生理变化和性状变异的关联更为密切[23-24]。因此，蛋白质组学的研究有助于揭示杨树多倍体营养生长优势形成的分子机制。本研究以两个不同2n雌配子来源的杂种三倍体青杨及其亲本为材料，分别比较两种基因型三倍体子代与亲本‘哲引3号杨’、母本‘北京杨’的蛋白质组学差异，讨论三倍体营养生长优势的蛋白质基础。
本实验中，青杨三倍体的FDR和SDR型2n配子来源不同，其杂合度不同，FDR、SDR型2n配子通过不同染色体传递的杂合性平均值分别为74.80%、39.58%[25]。与母本‘哲引3号杨’相比，FDR和SDR型青杨杂种三倍体植株的差异蛋白比率分别为2.9%和2.0%；与父本‘北京杨’相比，FDR和SDR型青杨杂种三倍体差异蛋白比率分别为10.1%和3.1%。显然，FDR型青杨杂种三倍体与亲本的差异表达蛋白数多于SDR型青杨杂种三倍体与亲本的表达差异蛋白数与FDR型2n配子通过不同染色体传递亲本杂合性较高有关。
此前，Wang等以青杨杂种三倍体和其同亲本二倍体为材料，比较了不同倍性的全同胞家系子代的蛋白质组表达差异，结果显示全同胞三倍体中核糖体蛋白表达上调如60S核糖体蛋白L3-1、L8-3、L24-1(60S ribosomal protein L8-3、L3-1、L8-3、L24-1)、40S核糖体蛋白S11-3(40S ribosomal protein S11-3)等以及光合作用通路相关蛋白表达上调如光合系统Ⅱ蛋白H(photosystem Ⅱ protein H)，从而促进了三倍体的营养生长[26]。而本研究中青杨杂种三倍体子代与其亲本的比较中202个差异蛋白主要富集于代谢相关通路、核糖体组装通路、光合作用等。
代谢相关蛋白包括碳水化合物、次生产物、核苷酸和脂类等相关蛋白[27]，其中淀粉合成酶(starch synthase，SSS)、蔗糖合成酶家族蛋白(sucrose synthase family protein)在FDR三倍体中的表达量分别是父本‘北京杨’的1.9倍和2倍，同时淀粉合成酶和蔗糖合成酶家族蛋白在SDR三倍体的表达量是父本的1.9倍和1.7倍。淀粉合成酶和蔗糖合成酶是控制淀粉和蔗糖合成代谢的关键酶[28]，这些酶的高表达可能促进了三倍体中淀粉和蔗糖的合成。核糖体是细胞蛋白质合成的主要场所，其中FDR三倍体中30S核糖体蛋白(30S ribosomal protein 3-1)的表达量是父本的1.7倍，叶绿体核糖体蛋白S19(30S ribosomal protein S19 (chloroplast))在SDR三倍体中的表达量是母本的3.4倍, 核糖体通路的蛋白表达增加可能促进蛋白质的合成[29]。关于光合作用通路相关蛋白对营养生长的影响，一般认为光合作用相关蛋白表达上调与三倍体叶绿素含量高、光合作用强有关[11]。本研究中青杨三倍体核糖二磷酸羧化酶(RuBisCO)、光合系统Ⅱ L蛋白(PSII L protein)、光合系统Ⅱ细胞色素b559蛋白(PSII cytochrome b559)等富集于光合作用通路的蛋白表达增加，可能促进了三倍体的光合作用并固定更多的碳水化合物，从而形成了杨树三倍体相对于二倍体亲本的营养生长优势。
此外，我们的研究还发现了29个防御相关差异蛋白，如热激蛋白、超氧化物歧化酶、谷胱甘肽转移酶等，在拟南芥和油菜中有类似发现，拟南芥经过加倍后抗胁迫相关蛋白表达增加[11]，油菜六倍体中响应胁迫生物过程的蛋白与亲本差异显著[30]。多倍体中的抗胁迫相关蛋白高表达与多倍体的抗性强有关[31]，表明杨树三倍体与亲本相比具有更强的抗逆性。
4. 结论
利用高温处理杨树雌花芽的方法获得的两种三倍体子代与母本‘哲引3号杨’、父本‘北京杨’蛋白表达差异显著。由于不同2n配子来源的三倍体杂合性不同，其中FDR型2n配子来源的三倍体与亲本的蛋白表差异程度高于SDR型2n配子来源的三倍体。差异蛋白主要参与代谢相关、核糖体组装、光合作用、胁迫响应等通路，显示三倍体在能量代谢、蛋白合成、光合作用、抗逆性等方面与亲本有不同的表达模式，杂交和加倍后促进蛋白质的合成、光合作用的增强，提高了多倍体的抗逆性和适应性，这些变化促进了杨树异源多倍体营养生长优势的形成。
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图 1 两种青杨杂种三倍体子代与亲本差异蛋白维恩图分析
Figure 1. Venn diagrams of proteins differentially expressed in two Populus allotriploid and diploid parents
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图 2 FDR青杨杂种三倍体与二倍体亲本差异蛋白GO功能分类
PO.光呼吸作用；OR.氧化还原过程；LC.脂质代谢过程；RC.镉离子响应；G.糖酵解；HP.过氧化氢分解代谢过程；DR.对细菌的防御反应；ET.电子传递链；NR.催化活性的负调控；MC.单酰甘油分解代谢过程；DP.二酰甘油代谢过程；PN.嘌呤核苷酸转运；RG.呼吸气体交换；GC.甘氨酸分解代谢过程；CP.叶绿素生物合成过程；OA.氧化还原酶活性；HA.水解酶活性；CE.羧酸酯水解酶活性；PER.过氧化物酶活性；CO.辅酶结合；CI.铜离子结合；AO.替代氧化酶活性；AL.酰基甘油脂肪酶活性；AT.ATP：ADP逆向转运蛋白活性；FR.果糖二磷酸醛缩酶活性；NA.NADH脱氢酶(泛醌)活性；DO.δ4-3-氧代甾族5β-还原酶活性；PEP.肽酶活性；ER.烯酮还原酶活性；EI.内肽酶抑制剂活性；CT.叶绿体类囊体；RE.呼吸链；CW.细胞壁；CH.叶绿体包膜；CY.细胞溶质；EX.细胞外区域；AP.质外体；CM.叶绿体类囊体膜；MI.线粒体内膜；RCC.呼吸链复合体Ⅰ；MRC.线粒体呼吸链复合体Ⅰ；CHL.叶绿体；ME.线粒体被膜；MM.线粒体膜；TH.类囊体；TR.翻译；RB.核糖体生物合成；RM.RNA甲基化；CBP.香豆素生物合成过程；RS.盐胁迫响应；PMP.苯丙素类代谢过程；GO.高尔基组织；CMA.细胞修饰氨基酸生物合成过程；SB.S-腺苷甲硫氨酸生物合成过程；WT.水分运输；RH.根毛伸长；PBP.苯丙素类生物合成过程；RHL.激素水平调节过程；SC.核糖体的组成结构；RR. rRNA结合；RN. RNA结合；SCC.细胞骨架的结构成分；MA.甲硫氨酸腺苷转移酶活性；SMA.结构分子活性；PAP.苯丙氨酸解氨酶活性；ALA.氨解酶活性；HY.水解酶活性、作用于酯键；GT.谷胱甘肽转移酶活性；RIB.核糖体结合；3D.3-脱氧-7磷酸庚酮酸合酶活性；RIC.核糖核蛋白复合体；RI.核糖体；CL.胞质大核糖体亚基；CR.细胞质核糖体；IN.细胞内；P.胞间连丝；CS.细胞质小核糖体亚基；NU.核仁；GOA.高尔基体；CYT.细胞质；LR.大亚基。下同。
Figure 2. GO analysis of proteins differentially expressed in FDR Populus allotriploid and the diploid parents
PO, photorespiration; OR, oxidation-reduction process; LC, lipid catabolic process; RC, response to cadmium ion; G, glycolysis; HP, hydrogen peroxide catabolic process; DR, defense response to bacterium; ET, electron transport chain; NR, negative regulation of catalytic activity; MC, monoacylglycerol catabolic process; DP, diacylglycerol catabolic process; PN, purine nucleotide transport; RG, repiratory gaseous exchange; GC, glycine catabolic process; CP, chlorophyll biosynthesis process; OA, oxidoreductase activity; HA, hydrolase activity acting on ester bonds; CE, carboxylic ester hydrolase activity; PER, peroxidase activity; CO, coenzyme binding; CI, copper ion binding; AO, alternative oxidase activity; AL, acylglycerol lipase activity; AT, ATP:ADP antiporter activity; FR, fructose-bisphosphate aldolase activity; NA, NADH dehydrogenase (ubiquinone) activity; DO, delta4-3-oxosteroid 5beta-reductase activity; PEP, peptidase activity; ER, enone reductase activity; EI, endopeptidase inhibitor activity; CT, chloroplast thylakoid; RE, respiratory chain; CW, cell wall; CH, chloroplast envelope; CY, cytosol; EX, extracellular region; AP, apoplast; CM, chloroplast thylakoid membrane; MI, mitochondrial inner membrane; RCC, respiratory chain complex Ⅰ; MRC, mitochondrial respiratory chain complex Ⅰ; CHL, chloroplast; ME, mitochondrial envelope; MM, mitochondrial membrane; TH, thylakoid; TR, translation; RB, ribosome biogenesis; RM, RNA methylation; CBP, coumarin biosynthetic process; RS, response to salt stress; PMP, phenylpropanoid metabolic process; GO, Golgi organization; CMA, cellular modified amino acid biosynthetic process; SB, S-adenosylmethionine biosynthetic process; WT, water transport; RH, root hair elongation; PBP, phenylpropanoid biosynthetic process; RHL, regulation of hormone levels; SC, structural constituent of ribosome; RR, rRNA binding; RN, RNA binding; SCC, structural constituent of cytoskeleton; MA, methionine adenosyltransferase activity; SMA, structural molecule activity; PAP, phenylalanine ammonia-lyase activity; ALA, ammonia-lyase activity; HY, hydrolase activity, acting on ester bonds; GT, glutathione transferase activity; RIB, ribosome binding; 3D, 3-droxy-7-phosphoheptulona; RIC, ribonucleoprotein complex; RI, ribosome; CL, cytosolic large ribosomal subunit; CR, cytosolic ribosome; IN, intracellular; P, plasmodesma; CS, cytosolic small ribosomal subunit; NU, nucleolus; GOA, Golgi apparatus; CYT, cytoplasm; LR, large ribosomal subunit. The same below.
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图 3 SDR青杨杂种三倍体与二倍体亲本差异蛋白GO功能分类
PRO.蛋白水解；REC.冷响应；LM.脂质代谢过程；PF.类黄酮生物合成过程的正调控；LO.脂质氧化；RW.对伤害的反应；CRB.叶绿体二磷酸核酮糖羧化酶复杂的生物发生；SPA.丝氨酸型肽酶活性；SE.丝氨酸型内肽酶活性；LLA.亚油酸酯13S-脂氧合酶活性；OAA.氧化还原酶活性，作用于具有掺入分子氧的单个供体；UDP.UDP-葡糖基转移酶活性；RCA.核酮糖二磷酸羧化酶活性；LAL.长链脂肪酸-CoA连接酶活性；AEA.醛糖1-差向异构酶活性；CHR.叶绿体二磷酸核酮糖羧化酶复合物；PCW.植物型细胞壁；CMP.碳水化合物代谢过程；LB.木质素生物合成过程；RO.氧化应激反应；OM.单碳代谢过程；GR.生长；AGA. 4-α-葡聚糖转移酶活性；DA.二磷酸果糖-6-磷酸1-磷酸转移酶活性；GH.甘氨酸羟甲基转移酶活性；DL.UDP-葡萄糖醛酸脱羧酶活性；CHS.叶绿体基质。下同。
Figure 3. GO analysis of proteins differentially expressed in SDR Populus allotriploid and diploid parents
PRO, proteolysis; REC, response to cold; LM, lipid metabolic process; PF, positive regulation of flavonoid biosynthetic process; LO, lipid oxidation; RW, response to wounding; CRB, chloroplast ribulose bisphosphate carboxylase complex biogenesis; SPA, serine-type peptidase activity; SE, serine-type endopeptidase activity; LLA, linoleate 13S-lipoxygenase activity; OAA, oxidoreductase activity, acting on single donors with incorporation of molecular oxygen; UDP, UDP-glucosyltransferase activity; RCA, ribulose-bisphosphate carboxylase activity; LAL, long-chain fatty acid-CoA ligase activity; AEA, aldose 1-epimerase activity; CHR, chloroplast ribulose bisphosphate carboxylase complex; PCW, plant-type cell wall; CMP, carbohydrate metabolic process; LB, lignin biosynthetic process; RO, response to oxidative stress; OM, one-carbon metabolic process; GR, growth; AGA, 4-alpha-glucanotransferase activity; DA, diphosphate-fructose-6-phosphate 1-phosphotransferase activity; GH, glycine hydroxymethyltransferase activity; DL, UDP-glucuronate decarboxylase activity; CHS, chloroplast stroma. The same below.
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图 4 FDR和SDR青杨杂种三倍体与二倍体亲本差异蛋白KEGG通路分析
PB.苯丙素生物合成；PM.苯丙氨酸代谢；GD.乙醛酸和二羧酸代谢；APM.精氨酸和脯氨酸代谢；NM.氮代谢；FB.类黄酮生物合成；LA.亚油酸代谢；PC.卟啉和叶绿素代谢；AA.丙氨酸天冬氨酸和谷氨酸代谢；BS.次生代谢物的生物合成；MP.代谢途径；ALM.α-亚麻酸酸代谢；GS.甘氨酸、丝氨酸和苏氨酸代谢；CC.柠檬酸循环(TCA循环)；GM.谷胱甘肽代谢；NN.烟酸酯代谢；PR.蛋白酶体；OP.氧化磷酸化；GB.硫代葡萄糖苷生物合成；GG.糖酵解/糖异生；FF.黄酮和黄酮醇生物合成；ZB.玉米素生物合成；FA.脂肪酸延伸；PYM.丙酮酸代谢。SM.硒代化合物代谢；FM.果糖和甘露糖代谢；PH.吞噬体；PA.光合作用-天线蛋白；BM.丁酸代谢；PPM.丙酸酯代谢；VL.缬氨酸亮氨酸和异亮氨酸降解；AS.氨基糖和核苷酸糖代谢；CM.半胱氨酸和蛋氨酸代谢；GB.鞘糖脂生物合成-globo系列；SP.剪接体；PS.聚酮化合物糖单元生物合成；FAD.脂肪酸降解；C5.C5-支链二元酸代谢；PT.苯丙氨酸酪氨酸和色氨酸生物合成；GL.糖鞘脂生物合成；PHO.光合作用；SD.二苯乙烯类、二芳基庚烷类和姜醇生物合成；PE.过氧化物酶体；AAM.抗坏血酸和醛酸代谢；GLD.糖胺聚糖降解；TM.色氨酸代谢；PPE.内质网中的蛋白质加工；GLM.甘油脂代谢；CA.氰氨基酸代谢；OC.叶酸-碳库；GA.半乳糖代谢；CB.鞘糖脂生物合成-ganglio；MB.单萜生物合成；PP.磷酸戊糖途径。*表示显著差异(P<0.05)。
Figure 4. KEGG pathway analysis of proteins differentially expressed in two Populus allotriploid and diploid parents
PB, phenylpropanoid biosynthesis; PM, phenylalanine metabolism; GD, glyoxylate and dicarboxylate metabolism; APM, arginine and proline metabolism; NM, nitrogen metabolism; FB, flavonoid biosynthesis; LA, linoleic acid metabolism; PC, porphyrin and chlorophyll metabolism; AA, alanine aspartate and glutamate metabolism; BS, biosynthesis of secondary metabolites; MP, metabolic pathways; ALM, alpha-linolenic acid metabolism; GS, glycine, serine and threonine metabolism; CC, citrate cycle (TCA cycle); GM, glutathione metabolism; NN, nicotinate and nicotinamide metabolism; PR, proteasome; OP, oxidative phosphorylation; GB, glucosinolate biosynthesis; GG, glycolysis/gluconeogenesis; FF, flavone and flavonol biosynthesis; ZB, zeatin biosynthesis; FA, fatty acid elongation; PYM, pyruvate metabolism; SM, selenocompound metabolism; FM, fructose and mannose metabolism; PH, phagosome; PA, photosynthesis-antenna proteins; BM, butanoate metabolism; PPM, propanoate metabolism; VL, valine, leucine and isoleucine degradation; AS, amino sugar and nucleotide sugar metabolism; CM, cysteine and methionine metabolism; GB, glycosphingolipid biosynthesis-globo series; SP, spliceosome; PS, polyketide sugar unit biosynthesis; FAD, fatty acid degradation; C5, C5-branched dibasic acid metabolism; PT, phenylalanine, tyrosine and tryptophan biosynthesis; GL, glycosphingolipid biosynthesis; PHO, photosynthesis; SD, stilbenoid, diarylheptanoid and gingerol biosynthesis; PE, peroxisome; AAM, ascorbate and aldarate metabolism; GLD, glycosaminoglycan degradation; TM, tryptophan metabolism; PPE, protein processing in endoplasmic reticulum; GLM, glycerolipid metabolism; CA, cyanoamino acid metabolism; OC, one carbon pool by folate; GA, galactose metabolism; CB, clycosphingolipid biosynthesis-ganglio; MB, monoterpenoid biosynthesis; PP, pentose phosphate pathway. * represents significant difference at P<0.05 level.
表 1 两种青杨杂种三倍体子代与二倍体亲本蛋白质组差异
Table 1 Proteome differences between two Populus allotriploid and the diploid parents
组别
Group差异蛋白数
Number of DEPs占总蛋白数的百分率
Percentage in total proteins/%上调个数
Up-regulated number下调个数
Down-regulated numberFDR vs P1 43 2.9 25 18 FDR vs P2 148 10.1 60 89 SDR vs P1 29 2.0 15 14 SDR vs P2 76 3.1 27 49 注：P1代表母本，P2代表父本。下同。Notes: P1, female parent; P2, male parent. The same below. 
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