摘要
本研究以荔枝品种‘乌叶’(Litchi chinensis Sonn.cv.‘Wuye’)和‘下番枝’(Litchi chinensis Sonn.cv.‘Xiafanzhi’)果实为试验材料,利用双向电泳和质谱技术对荔枝果实不同成熟时期进行了差异蛋白质组学研究。获得了与荔枝果实成熟衰老相关的差异蛋白,并对部分差异蛋白进行cDNA克隆及相对定量表达分析。主要研究结果如下:
1.建立了适合荔枝果皮和假种皮中总蛋白质提取的方法和双向电泳体系。以荔枝果皮和假种皮为材料,比较了TCA-丙酮、丙酮和酚3种提取方法在总蛋白产量、单向SDS-PAGE和双向电泳等方面的区别,并对荔枝果皮和假种皮的双向电泳体系进行探索。结果表明,酚抽提法最佳,提取的总蛋白得率最高;所获蛋白在单向SDS-PAGE中形成条带数目最多,最清晰;经双向电泳分离用银染显色,2D图谱分辨率较好,蛋白点清晰,主要分布在pH4-8,15.0-85.0 kD,可有效解析荔枝果皮和假种皮的蛋白质组。
2.采用18 cm pH4-7和pH3-10 IPG胶条相结合的方法对‘乌叶’果皮不同成熟期进行蛋白质分离,随着果皮的成熟蛋白点数总体表现出先上升后下降的变化。其中,以阶段Ⅲ检测到的蛋白点数目最多,分别为753和937个,这个时期刚好处在荔枝果皮的转色期;以阶段Ⅵ完全转红期的蛋白点数最少,分别为651和604个。从pH4-7胶条分离的蛋白中选取79个和pH3-10胶条分离的蛋白中选取28个,总107个差异蛋白进行MALDI-TOF/TOF质谱分析,获得了107个完整的肽指纹图谱,61个差异蛋白获得成功鉴定,成功率约为57.0%。通过生物信息学分析,这些鉴定成功的已知蛋白参与了糖和能量代谢(29.51%)、蛋白质合成和代谢以及稳定(11.48%)、基因表达调控(11.48%)、抗氧化(9.84%)、细胞代谢和周期调控(4.92%)、氨基酸代谢(4.92%)、转录与翻译(4.92%)、信号转导(1.64%)等生命活动功能,功能未知的蛋白为21.31%。
3.采用18 cm pH4-7和pH3-10 IPG胶条相结合的方法对‘下番枝’果皮不同转色期进行蛋白质分离,随着果皮转色的进程蛋白点数表现出上升趋势。其中,以果皮转红阶段检测到的蛋白点数目最多,分别为750和1106个,在果皮为青色期的蛋白点最少,分别为643和1041个。从pH4-7胶条分离的蛋白中选取55个和pH3-10胶条分离的蛋白中选取10个,总65个差异蛋白进行MALDI-TOF/TOF质谱分析,获得了65个完整的肽指纹图谱,43个差异蛋白获得成功鉴定,成功率约为66.15%。通过生物信息学分析,这些鉴定成功的蛋白参与了糖和能量代谢(20.93%)、蛋白质合成和代谢以及稳定(16.28%)、基因表达调控(13.95%)、信号转导(6.98%)、氨基酸代谢(6.98%)、转录与翻译(6.98%)、次生代谢(4.65%)、抗氧化(4.65%)、细胞结构相关(2.33%)、激素相关(2.33%)等生命活动功能,功能未知的蛋白为13.95%。
4.采用18 cm pH4-7胶条对‘乌叶’假种皮不同成熟期进行蛋白质分离,随着果皮转色的进程蛋白点数表现出先上升再下降的变化趋势。其中,以阶段Ⅲ检测到的蛋白点数目最多1374个,这个时期刚好处在荔枝果实的转色期;以阶段Ⅵ果实完全转红期的蛋白点数最少914个;其它时期检测到的蛋白点为:阶段Ⅰ1144个、阶段Ⅱ1330个、阶段Ⅳ1096个、阶段Ⅴ1012个。这与乌叶果皮转色过程中蛋白质点数的总体变化趋势相似,在阶段Ⅲ能检测到的蛋白质最多,在阶段Ⅵ完全转红期的蛋白质最少。选取64个差异蛋白进行MALDI-TOF/TOF质谱分析,获得了64个完整的肽指纹图谱,43个差异蛋白获得成功鉴定,成功率约为67.19%。通过生物信息学分析,这些鉴定成功的蛋白参与了糖和能量代谢(27.91%)、蛋白质合成和代谢以及稳定(11.63%)、氨基酸代谢(6.98%)、细胞结构和周期调控(6.98%)、基因表达调控(4.65%)、转录与翻译(9.3%)、信号转导(4.65%)、抗氧化(4.65%)、次生代谢(4.65%)等生命活动功能,功能未知的蛋白为18.6%。
5.荔枝果实成熟过程中部分相关蛋白的基因克隆及其在果皮中的表达。
①克隆了荔枝果皮Actin1基因的cDNA序列,该序列全长1418 bp,其中开放阅读框(ORF)共有1134 bp,编码377个氨基酸。Actin1在荔枝果皮不同成熟期的蛋白水平和转录水平的表达相对稳定,变化不大。因此,在研究荧光定量表达分析中,所克隆的Actin1基因可作为其它目的基因的内参基因。
②克隆了荔枝果皮UFGT基因的cDNA序列,该序列全长1577 bp,其中开放阅读框(ORF)共有1362 bp,编码453个氨基酸。荔枝果皮UFGT基因在转录水平随着果皮着色的加深呈不断上升变化,与其在果皮成熟过程中的蛋白水平表达趋势一致。
③克隆了荔枝果皮OEE1基因的cDNA序列,该序列全长1217 bp,其中开放阅读框(ORF)共有1002 bp,编码333个氨基酸。荔枝果皮OEE1基因在转录水平随着果皮着色的加深呈不断下降变化,与其在果皮成熟过程中的蛋白水平表达趋势一致。
④克隆了荔枝果皮rbcs基因的cDNA序列,该序列全长868 bp,其中开放阅读框(ORF)共有567 bp,编码188个氨基酸。荔枝果皮rbcs基因在转录水平随着果皮着色的加深呈不断下降变化,与其在果皮成熟过程中的蛋白水平表达趋势一致。
⑤克隆了荔枝果皮Aconitase基因的cDNA序列,该片段序列长2945 bp,编码883个氨基酸。荔枝果皮Aconitase基因在转录水平随着果皮着色的加深呈不断上升变化,与其在果皮成熟过程中的蛋白水平表达趋势一致。
⑥克隆了荔枝果皮ASR基因的cDNA序列,该片段序列长515 bp,编码99个氨基酸。荔枝果皮ASR基因在转录水平随着果皮着色的加深呈先上升后下降的变化,在阶段Ⅱ的表达量最高,与其在果皮成熟过程中的蛋白水平表达趋势一致。
⑦克隆了荔枝果皮TCTP基因的cDNA序列,该序列全长848bp,其中开放阅读框(ORF)共有507 bp,编码168个氨基酸。荔枝果皮TCTP基因在转录水平随着果皮着色的加深呈先上升后下降再上升的变化,与其在果皮成熟过程中的蛋白水平表达趋势不太一致。
⑧克隆了荔枝果皮14-3-3基因的cDNA序列,该序列全长1084 bp,其中开放阅读框(ORF)共有792 bp,编码261个氨基酸。荔枝果皮14-3-3基因在转录水平随着果皮着色的加深呈先上升后下降再上升的变化,与其在果皮成熟过程中的蛋白水平表达趋势不太一致。
⑨克隆了荔枝果皮VSP基因的开放阅读框(ORF),该序列长678 bp,编码225个氨基酸。荔枝果皮VSP基因在转录水平随着果皮着色的加深呈不断下降变化,与其在果皮成熟过程中的蛋白水平表达趋势一致。
In this experiment,the pericarp and aril of litchi(Litchi chinensis Sonn.) were utilized as materials,and the techniques of two dimensional electrophoresis(2-DE),Mass Spectrometry(MS)and real-time fluorescence quantified PCR(qRT-PCR)were performed to study the mechanism of fruit ripening in litchi.The main results were as follow:
1.In order to develop an efficient protein extraction method which was suitable for litchi pericarp and aril proteome analysis, three protein extraction methods based on trichloroacetic acid(TCA)- acetone,and phenol respectively,were compared in total protein yield and the protein resolution in one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and two dimentional gel. It was found that the phenol extraction method was the best one resulting in the highest total protein yield,the most protein bands and the best resolution in one dimentional SDS-PAGE;about 890 protein spots were detected by 2-DE which could obtain clear map and better separation effect followed by silver staining.Most of isoelectric points were found between pH4-8,and most of relative molecular weight of protein was between 15.0 to 85.0 kD.
2.The proteins of‘wuye’pericarp at different stages during fruit ripening were separated by two dimensional electrophoresis with immobilized pH4-7and pH3-10 gradients. The results indicated that the general numbers of protein increased then decreased during fruit ripening,the most protein numbers could be detected at stageⅢabout 753 and 937 spots,the lowest protein numbers appeared at stageⅥabout 651 and 604 spots.Then 107 spots of differential protein during pericarp ripening were selected and identified through mass spectrometry(MS) analysis,61(57.0%)of them were identified,and they were classified to nine functional classes,which including 18 proteins involved in energy metabolism(29.51%),7 proteins involved in gene regulation(11.48%),7 proteins involved in protein synthesis and metabolism(11.48%),6 proteins involved in stress response and defense(9.84%),3 proteins involved in amino acid metabolism(4.92%),3 proteins involved in cell structural(4.92%),3 proteins involved in transcription factor(4.92%),1 proteins involved in signal transduction(1.64%)and 13 unknown functional proteins(21.31%).
3.The proteins of‘xiafanzhi’pericarp at different stages during color-changing period were separated by two dimensional electrophoresis with immobilized pH4-7and pH3-10 gradients. The results indicated that the numbers of protein increased during color-changing period,the most protein numbers could be detected at red period about 750 and 1106 spots seperately,the lowest protein numbers appeared at green period about 643 and 1041 spots.Then 65 spots of differential protein during pericarp ripening were selected and identified through mass spectrometry(MS) analysis. 43(66.15%)of them were identified,and they were classified to eleven functional classes,which including 9 proteins involved in energy metabolism(20.93%),7 proteins involved in protein synthesis and metabolism(16.28%),6 proteins involved in gene regulation(13.95%),3 proteins involved in amino acid metabolism(6.98%),3 proteins involved in transcription facto(r6.98%),3 proteins involved in signal transduction(6.98%),2 proteins involved in stress response and defens(e4.65%),2 proteins involved in secondary metabolism(4.65%),1 proteins involved in cell structural(2.33%),1 proteins involved in hormonal phase(2.33%),and 6 unknown fuctional proteins(13.95%).
4.The proteins of‘wuye’aril at different stages during fruit ripening were separated by two dimensional electrophoresis with immobilized pH4-7 gradients . The results indicated that the general numbers of protein increased then decreased during fruit ripening.The most protein numbers could be detected at stageⅢabout 1374 spots.The lowest protein numbers appeared at stageⅥabout 914 spots.Then 64 spots of differential protein during aril ripening were selected and identified through mass spectrometry(MS) analysis,43(67.19%)of them were identified,and they were classified to 10 functional classes,which including 12 proteins involved in energy metabolism(27.91%),5 proteins involved in protein synthesis and metabolism(11.63%),4 proteins involved in transcription factor(9.3%),3 proteins involved in amino acid metabolism(6.98%),3 proteins involved in cell structuraln and division cycle(6.98%),2 proteins involved in stress response and defense(4.65%),2 proteins involved in gene regulation(4.65%),2 proteins involved in secondary metabolism(4.65%),2 proteins involved in signal transduction(4.65%),and 8 unknown fuctional proteins(18.6%).
5.Cloning of several ripening associated protein genes were expressed at transcript level during different ripening stages in pericarp.
①Actin1 gene was cloned and its cDNA full-length was 1418 bp,and its ORF was 1418 bp which encoded 377 amino acids. Actin1 gene expressed stablely at transcript and protein levels in pericarp.Thus the Actin1 gene was used as reference gene which was used to detect other target genes expression at transcript level during fruit ripening.
②The full-length cDNA sequence of UFGT gene was cloned with 1577 bp.It contained a 1362 nucleotides open reading frame which encoded protein of 453 amino acid residues.Transcription level of UFGT gene was increasing with the deepening of pericarp color,which was also consistent with the change at protein level.
③The OEE1 gene was cloned with full-length 1217 bp and its ORF was 1002 bp which encoded 333 amino acids.The relative expression of OEE1 gene was decreasing during fruit ripening at transcription level and reached the lowest level at stageⅥ,which was also consistent with the trend at protein level.
④Rbcs gene was cloned and its cDNA full-length was 868 bp,and its ORF was 567 bp which encoded 188 amino acids. The relative expression of rbcs gene was decreasing duing fruit ripening at transcription level and reached the lowest level at stageⅥ,which was also consistent with the change at protein level.
⑤The partial sequence of Aconitase gene isolated from pericarp by RACE was 2945 bp,encoding 883 amino acid residues. Quantitative real-time RT-PCR analysis indicated that Aconitase mRNA accumulated during pericarp ripening and reached the highest level at periodⅥ,the trend of transcription level was similar with the change of protein level.
⑥The fragment cDNA of ASR gene contains 515 bp length was cloned and encoding 99 amino acids. The relative expression of ASR gene was first increased and then decreased changes during fruit ripening at transcription level ,and reached the highest level at stageⅡ,which was also consistent with the change at protein level.
⑦The full-length cDNA clone of TCTP gene contained 848 with an open reading frame of 507 nucleotides and encoded 168 amino acids. The relative expression of TCTP gene was first increased from stageⅠtoⅡ,then decreased to stageⅣ,further addition to stageⅥduing fruit ripening at transcription level.The change of transcription level was not consistent with the tendency at protein level.
⑧The 14-3-3 gene was cloned and its cDNA full-length was 1084 bp.Its ORF was 792 bp which encoded 261 amino acids. The relative expression of 14-3-3 gene was first increased from stageⅠtoⅢ,then decreased to stageⅤ,further addition to stageⅥduing fruit ripening at transcription level.The change of transcription level was not consistent with the tendency at protein level.
⑨The ORF sequence of VSP gene was cloned with 678 bp encoding 225 amino acids. The relative expression of VSP gene was reducing duing fruit ripening at transcription level and reached the lowest level at stageⅥ,which was also consistent with the change at protein level.
引文
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