| Mfhb-1is a kind of HD-Zip transcription factor genes which was isolated during the early stages of theinduction of somatic embryogenesis in alfalfa by subtractive clonging technology. Currently, sense andantisense technology were used to investigate the biological function of the mfhb-1gene during somaticembryogenesis in alfalfa. The transformed plant derived from47/15were generated and they are F4(containing mfhb-1coding sequence), D3(containing mfhb-1coding sequence in antisense orientation), C5(containing full length mfhb-1sequence, including CDS and uORF). Previous research results suggest thatoverexpression of the mfhb-1gene should enhance the induction and formation of somatic embryos inalfalfa. In this study, somatic embryogenesis was inducted in4kinds of alfalfa above-mentioned. Twodimensional electrophoresis was conducted to analysis the differential expression proteins during the earlystage of the induction of somatic embryogenesis in alfalfa. Then relevant differential expression proteinswere indentified and analyzed by mass spectrometry technology. The main results and conclusions are asfollows.1. The method “TCA-acetone precipitation” was used to extract the total protein of alfalfa embryos.Two dimensional electrophoresis results showed that the protein samples which extracted by TCA-acetoneprecipitation and purified can meet the needs of the research.2.2-D Quant kit method was used as determination of protein concentration. The method is a kind ofmicro protein quantitative method, which is more simple and accurate than others. In this study, thecorrelation of the standard curve obtained is high and reliable (R2=0.9951).3. In pre-experiment,7cm IPG strips of pH3-10was used for isoelectric focusing. Based on thepre-experiment results,18cm linear IPG strips of pH4-7were selected for the first dimensionalelectrophoresis. To separate the tolal proteins, the selected separating gel concentration for the seconddimensional electrophoresis was at13%.4. Under the standard we setted, compared to control47/15,39differentially expressed proteins wereidentified in D3. They were:17proteins up-regulated and5proteins down-regulated in D3,12proteinsexisted only in D3but5proteins existed only in47/15.45differentially expressed proteins were identifiedin F4. They were:20proteins up-regulated and7proteins down-regulated in F4,9proteins existed only in F4and9proteins existed only in47/15.37differentially expressed proteins were identified in C5. Theywere:7proteins up-regulated and9proteins down-regulated in C5,11proteins existed only in C5but10proteins existed only in47/15.5.27for3times up-regulated differentially expressed proteins between F4and47/15were selected tocarry out MALDI-TOF/MS and13positive results were obtained. Only5proteins matched with thedatabase,1protein was unknown,7proteins were hypothetical proteins.6. The alfalfa database analysis results are as follows. Compared with control47/15,4up-regulatedprotein spots were identified. Spot21was identified as electron-transfer flavoprotein-ubiquinoneoxidoreductase. Spot276was identified as phosphoribosyl transferase. Spot530was identified as BTB andMATH domain-containing protein. Spot626was identified as actin. Spot50was identified as ribulose-1,5-bisphosphate carboxylase small subunit which was down-regulated protein spot. Spot682was unknownprotein. The others were all hypothetical proteins.7. According to the protein information acquired preliminary, it is speculated that the mechanisms ofthe gene mfhb-1regulating the development of embryos as follows. On the one hand, gene mfhb-1mayinduce the expression of products such as enzymes associated with energy metabolism and the synthesis ofthe cytoskeletal protein. On the other hand, it may regulate the expression of the transcription factors whichregulate the enbryogenesis related genes.The overexpression of the electron-transferflavoprotein-ubiquinone oxidoreductase promotes the synthesis of ATP, which ensures the adequate energysupply for somatic embryogenesis. The overexpression of actin promotes the reconstruction of the cell walland cell membrane. Also, the overexpression of phosphoribosyl transferase accelerates the synthesis ofneucleotide and gene synthesis and mRNA transcription speed up. So the division and differentiation ofcell speed up, the number of embryos increase. Ribulose-1,5-bisphosphate carboxylase plays a negativerole in somatic embryogenesis. It makes the organics in cell consumed, then lead to the shortage of the rawmaterial for cell growth and differentiation, the number of embryos increase. Other proteins such as BTBand MATH domain-containing protein may be as a kind of regulatory factor involving in the regulation ofsomatic embryogenesis. The fuction of some hypothetical proteins and unknown proteins should be furtherstudied.In a word, several differential proteins were indentified preminarily during the somatic embryogenesis in alfalfa. The possible molecular regulatory mechanisms of mfhb-1gene expression were analyzed. Thisstudy provides the basic data for the biological fuctional investigation of the gene mfhb-1in alfalfa andreference information for further separating and identifying proteins related to somatic embryogenesis inalfalfa. The study may enrich and developemt the study achievements on the molecular genetic mechanismand regulatory network of somatic embryogenesis. |
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