Studies On Differential Proteomics During Fruit Ripening In Litchi(Litchi Chinensis Sonn.)

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.