Cloning And The Expression Of PCD Related Genes Of Somatic Embryogenesis In Longan

In this experiment paper, the embryogenic calli (EC) of longan (Dimocarpus longan Lour. cv. Honghezi) were used as the materials to study the following three aspects:①Cloning longan PCD related genes CP,COX Vb and Caspase during somatic embryogenesis;②analysis of bioinformatics of longan CP gene, COX Vb gene and Caspase gene;③the expression profiles of CP gene, COX Vb gene and Caspase gene in the embryogenic cultures at different developmental stages during longan somatic embryogenesis; The main results showed as follows: 1 Obtaining full length of cDNA of Longan PCD genes CP gene, COX Vb gene and Caspase gene during somatic embryogenesisUsing longan EC as the material, the full length of cDNA of CP gene,COX Vb gene and Caspase gene was obtained by homology cloning combined with RACE technology. The results showed that:①CP cDNA (823bp) contained 163bp 5'UTR, 258bp 3'UTRand 3'-end involved 20 bp poly(A)tail. The results showed that the sequence of CP 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 411 bp open reading frame, encoded 136 amino acids, and ATG was the initiation codon and TAA was the stop codon. The gene was named as DLCP and registered in GenBank. The registered No. is JF733788.②The full length of cDNA ( 627bp ) of COX Vb gene from longan EC was obtained which contained 140bp 5'UTR, 66bp 3'UTR, and 3'-end involved 15bp poly(A)tail. Sequence analysis revealed that splicing of the cDNA contained a 420 bp open reading frame, encoded 139 amino acids, and TAG was the stop codon. The gene was named as DLCOXVb and registered in GenBank. The registered No. was JF733786.③The full length of cDNA (868bp ) of Caspase gene from longan EC was obtained which contained 73bp 5'UTR, 272bp 3'UTRand without poly(A)tail. According to DNAMAN6.0 software speculation splicing of the cDNA contained a 519 bp open reading frame, encoded 173 amino acids, and TGA was the stop codon. The gene was named as DLCPS-1 and registered in GenBank. The registered No. was JF JF733787.2 Analysis of bioinformatics of longan CP gene, COX Vb gene and Caspase gene during somatic embryogenesisLongan EC CP gene, COX Vb gene and Caspase gene nucleotide sequence and corresponding amino acid sequences were analyzed by using bioinformatics softwares.The results showed that:①CP protein formula is C623H948N174O200S17; the molecular weight of protein CP was 14620.4 Da; the theoretical isoelectric point ( pI ) was 6.76; it was a kind of hydrophilic cytoplasmic protein with transmembrane domain and without signal peptide, 4.41% extending chain and 95.59% irregular curl; Through prediction and analysis, it was speculated that the function of this gene was relevant to cell death. Furthermore, the three-dimensional structure of CP enzyme molecules was predicted and analyzed, etc.②The molecular weight of protein COXVb was 15229.6Da; the theoretical isoelectric point ( pI ) was 7.02; it was a kind of hydrophilic cytoplasmic protein without transmembrane domain and with signal peptide, with 29.29%α-spiral 12.14% extending chain and 58.57% irregular curl; with the possible 12 amino acid sites. Through prediction and analysis, it was speculated that the function of this gene was relevant with PCD Control. Furthermore, the three-dimensional structure of COX Vb enzyme molecules was predicted and analyzed, etc.③The molecular weight of protein Caspase was 19502.2Da; the theoretical isoelectric point ( pI ) was 9.32; It was a kind of hydrophilic cytoplasmic protein with transmembrane domain and without signal peptide, the secondary structure includes 17.06%α-spiral 12.35% extending chain and 70.59% irregular curl; and the phosphorylation sites were 6.Through prediction and analysis, it was speculated that the function of this gene was relevant to cell death.3. Analysis of the transcriptional expression profiles of longan CP gene,COX Vb gene and Caspase gene by fluorescence quantitative PCRBased on the obtain of the different stages of synchronized cultures somatic embryogenesis in Longan, those different stages meterial total RNA were extracted respectively, and reverse transcription PCR were done according to the requirements of quantitative PCR. Based on available CP, COXVb and Caspase cDNA full-length sequence, longan CP, COXVb and Caspase quantitative gene special primers were designed.Using both UBQ and EF-1a genes as the reference genes, the transcriptional profile of these three genes at different stages were analyzed by fluorescence quantitative PCR during longan somatic embryogenesis, the results shows as follows:①The CP expression level was the higher at the stage of embryogenic callus II embryogenic structure.The changes of CP expression was low, from embryonic compaction of spherical structure to globular embryonic stage and increased sharply at stage of heart-shaped embryo. It indicated that the embryonic development metabolic process began at the early embryogenesis phase. Followed by the gradual decline and the lowest expression was at the stage of mature cotyledon embryo; The expression of CP related cell differentiation, The relationship of expression of longan CP and the cell differentiation degree was proportional rate.②During longan somatic embryogenesis, COXVb gene expression from the embryogenic callus to the globular embryo stage was more constant. The COXVb gene expression from globular embryogenic structure to heart-shaped embryogenic structure was the most dramatic changes, expression of heart-shaped embryogenic structure increased rapidly, may herald the advent of the new developmental state. Later declined, and almost the same level of the previous expression. In the mature cotyledon embryo stage, expression of COXVb was highest. COXVb expression and cell metabolism is proportional. COXVb was maintaining the integrity of mitochondrial function. COXVb provided the energy for active metabolic activity, which played an important role in promoting the normal development of somatic embryos.③At the early embryogenic development of longan, caspase expression level was higher, and from embryogenic callus II to the stage not completely tight structure was higher.expression gradually increased. However, expression of Caspase from not completely spherical embryogenic structure to the stage of spherical structure was reduced.At the Heart-shaped embryogenic callus stage, Caspase gene expression increased, and reaching the peak of expression, which may be associated with programmed cell death.Followed by gradual decline. The expression level was the lowest at the stage of the mature cotyledon. Longan Caspase played a role in the process of programmed cell death.In conclusion, in this experiment three closely related genes in the process of somatic embryogenesis had been cloned. The expressions of the three genes were analyzed; and the structure and function were systematically predicted and analyzed; and the mechanism of PCD during somatic embryogenesis of longan was further understood. New ideas were provided in the molecular level on the regulation of embryo development and other aspects of genetic improvement in longan and other plants.