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Cloning And Expression Of CDC48 And GPX Genes In The Process Of Somatic Embryogenesis In Dimocarpus Longan Lour.

Posted on:2010-07-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y T ChenFull Text:PDF
GTID:1103360275485026Subject:Pomology
Abstract/Summary:PDF Full Text Request
In this experiment, the embryogenic calli (EC) of longan (Dimocarpus longan Lour. cv. Honghezi) were used as the materials to study the following eight aspects:①cDNA and DNA cloning of longan EC CDC48 gene (cell division cycle 48 gene);②prokaryotic expression of CDC48 gene;③analysis of bioinformatics of longan EC CDC48 gene;④c DNA and DNA cloning of longan EC GPX (glutathione peroxidase gene);⑤p rokaryotic expression of GPX gene;⑥a nalysis of bioinformatics of longan EC GPX;⑦u sing both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes in the embryogenic cultures at different developmental stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR;⑧analysis the changes of longan GPX activities at different stages during longan somatic embryogenesis and under stress treatment. The main results showed as follows:1. Obtaining full length of cDNA and DNA of CDC48 gene from longan ECUsing longan EC as the material, the full length of cDNA (2620 bp ) of CDC48 from longan EC was obtained by homology cloning combined with RACE technology, which contained 17bp 5'UTR,187bp 3'UTR, and 3'-end involved 13 poly(A)tails. The results showed the sequence of CDC48 gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 2415 bp open reading frame, encoded 805 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLCDC48 and registered in GenBank. The registered No. was EU606206. The DNA of CDC48 gene which had no intron by sequence analysis was also cloned from longan EC, which was also registered in GenBank, and the registered No. was FJ590953.2. Prokaryotic expression of longan EC CDC48 geneAccording to sequence analysis of full-length cDNA of longan EC CDC48 gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and the molecular weight was about 89 kD by SDS-PAGE analysis. The protein molecular weight of the induced expression was near with that of the theoretical derivation from longan EC CDC48.3. Analysis of bioinformatics of longan EC CDC48 geneLongan EC CDC48 nucleotide sequence and corresponding amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein CDC48 was 89564.8 Da; the theoretical isoelectric point ( pI ) was 4.92; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the nucleus; three regions were most likely to form coiled-coil containing with 40.87%α-spiral, 15.28% extending chain and 43.85% irregular curl; and the phosphorylation sites were 39. The longan CDC48 gene had two typical ATPase modules and specific N-terminal by analysis of conservative structure domain and function domain. Through prediction and analysis, it was speculated that the function of this gene was relevant with cell division. By analyzing phylogenetic trees of its amino acid sequence, it was concluded that the evolution of CDC48 reflected the evolution of plants to some degree. Furthermore, the three-dimensional structure of CDC48 enzyme molecules was predicted and analyzed, etc..4. Obtaining the full length of cDNA and DNA of GPX gene from longan ECUsing longan EC as the material, the full length of longan EC GPX gene 947 bp was obtained by homology cloning combined with RACE technology, which contained 195bp 5'UTR, 245bp 3'UTR with a typical add-tail signal AATAA and a poly (A) tail. The results showed that the sequence of GPX gene from longan EC was highly homologous with that of other plants reported in GenBank. Sequence analysis revealed that splicing of the cDNA contained a 504 bp open reading frame, encoded 168 amino acids, and ATG was the initiation codon and TAG was the stop codon. The gene was named as DLGPX, and registered in GenBank. The registered No. was EU364813. The DNA of GPX gene was cloned from longan EC, which had 1736 bp nucleotide sequence with ATG initiation codon and TAA stop codon. The gene was registered in GenBank and the No. was EU680970. By the analyses of the DNAMAN 6.0 software, the GPX gene consisted of 5 exons and 4 introns, and the splice sites of all introns were accorded with eukaryotic GT-AG rule. 5. Prokaryotic expression of longan EC GPX gene According to sequence analysis of full-length cDNA of longan EC GPX gene, a pair of specific primers added in restriction enzymes were designed in the possible ORF region to amplify ORF, and then it was inserted into the expression vector pET-28 a, and finally the well Constructed expression vector was taken into the E. coli expression host BL21 (DE3) for fusion expression. After induction, there was a new protein emerging from the host bacteria and molecular weight about 23 kD by SDS-PAGE analysis. In this study, SacI and XhoI were used as the enzyme restriction sites, and 38 amino acids translated additionally in vector were also expressed. The theoretical molecular weight of gene product encoded by longan GPX gene was 18.54 kD, added with the theoretical derivation molecular weight of translating additionally amino acids 4.08 KD(38 amino acids theoretical molecular weight is 39.45 KD), and the total was 22.62KD, which was accorded with the results of this experiment. 6. Analysis of bioinformatics of longan EC GPX gene Longan EC GPX nucleotide sequence and amino acid sequences were analyzed by using bioinformatics softwares. The results showed that the molecular weight of protein GPX gene was 89564.8 Da; the theoretical isoelectric point (pI ) was 7.18; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, and was mainly located in the cytoplasm; a region was most likely to form coiled-coil containing with 27.98%α-spiral, 20.249% extending chain and 51.79% irregular curl, and the phosphorylation sites were13. The amino acid sequence of longan EC GPX gene contained two characteristics sequences of PHGPX. It was speculated that the cloned gene was possibly the cDNA sequence of PHGPX which was protective to membrane injury in GPXs family. Furthermore, the three- dimensional structure of GPX enzyme molecules was predicted and analyzed, etc..7. Analysis of the transcriptional expression levels of longan CDC48 and GPX genes by fluorescence quantitative PCRUsing both UBQ and EF-1a genes in longan as the reference genes, the transcriptional expression levels of CDC48 and GPX genes at different stages during longan somatic embryogenesis were analyzed by fluorescence quantitative PCR. The results showed that longan CDC48 expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of embryogenic callus(EC), and the lowest was at the stage of globular embryo; longan GPX also expressed to some extents in the process of somatic embryogenesis; the expression level was the highest at the stage of tight-spherical embryogenic structure, next was at the stage of cotyledon embryo, and the lowest was at the stage of embryogenic callus (EC).8. Changes of GPX enzymatic activity in the process of longan somatic embryogenesisIn the process of longan somatic embryogenesis, GPX activity also expressed to some extents. The expression level was the highest at the stage of the tight-spherical embryogeic structure, next was at the stage of cotyledon embryos, and the lowest was at the stage of EC. The results were consistent with the results of gene relative quantitative expression in the process of longan somatic embryogenesis. Using longan EC lines LC2 as materials, the changing rules of GPX enzyme activity of longan EC under NaCl, light and temperature stress treatments were also studied in this experiment. The study indicated that GPX in cells played a role of response to external stimuli in face of adversity stress, and the expression of activity was regular. It was speculated that the changes of GPX activity and the resistance of embryogenic cells were of positive correlation, which was one of the main indicators of plant defensing ROS injury in adversity stress.In conclusion, in this experiment two closely related genes in the process of somatic embryogenesis had been cloned. The expressions of the two genes were analyzed; and the structure and function were systematically predicted and analyzed; and the mechanism of somatic embryogenesis of longan was further understood. By this study, some new information could be provided for building the gene network of longan somatic embryogenesis; valuable evidences could be provided for exploring the mechanism of cell developmental regulation and removal of reactive oxygen in the process of longan somatic embryogenesis; and the gene resources could also be provided for genetic improvement of embryonic developmental regulation in longan and other plants, etc..
Keywords/Search Tags:longan, somatic embryogenesis, CDC48, GPX, gene cloning, prokaryotic expression, real-time quantitative PCR
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