Molecular Cloning And Characterization Of Several Genes Involved In Longan Cotyledon Embryo Development

Longan (Dimocarpus Longan Lour), which belongs to Sapindaceae Lour, is one of the most important and characteristic tropical and subtropical woody fruit trees in those provinces located in South of China. Due to the complex genetic background of Longan zygotic embryos and long juvenile period, the research on genes related to embryonic development is hindered. In the present study, with the cotyledon embryos as plant materials, the cDNA-AFLP technology system was established and then used to investigate the differentially expressed genes concerning embryonic development of Longan, which aimed to lay the foundation for the research on Longan embryonic development. Secondly, the RACE system was established and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in Longan was successfully isolated which could serve as a reference gene in the subsequent study. Moreover, with the RACE system established here, a series of genes relevant to Longan embryonic development were cloned and the expression profile of these genes during embryonic development was also demonstrated.1. Differentially expressed analysis in Longan cotyledon embryo by cDNA-AFLPThe'honghezi'Longan (Dimdcarpus longan Lour.) cotyledon embryo, of which the flowers had faded and flied for more than thirty-five days (early cotyledon embryos) or fifty days (late cotyledon embryos) , was utilized as plant materials to isolate the RNA. Then, the corresponding RNA was used for double-stranded cDNA synthesis, enzyme digest, anchors ligation, pre-amplification and selective amplification. The result of polyacrylamide gel electrophoresis showed that, with strong signal and equal distribution, the amplification bands of cDNA-AFLP were clear and easy to discriminate. Thus, the cDNA-AFLP system suitable for the differential expression analysis was established successfully. Then, 41 differentially expressed fragments were recovered, cloned and sequenced. The corresponding sequences of 20 differentially expressed fragments were found to highly homologous with some cloned genes with known function, the function mainly involved in the abaxial polarity of lateral organ, glycolysis, energy metabolism, ion transport, cell wall elongation, cell cycle regulation, RNA transcription, RNA translation and regulation, protein phosphorylation regulation and protein degradation, signal transduction and so on; while the sequences of the remaining genes had low homologous with function-known genes or even had no homologous with any function-known genes. Finally, seven genes were selected for RACE amplification, and the expression of these genes was also detected with RT-PCR.2. Full-length cDNA sequence cloning and sequence analysis of GAPDH gene in Longan cotyledon embryo According to the features of Super SMARTTM PCR cDNA Synthesis Kit, the primers anchored at both ends were designed, and at the same time in accordance with the sequence of EST which shows highly homologous to cloned GAPDH gene, nested primers were also designed. Then, the 3 'UTR and 5' region of GAPDH gene in Longan were successfully amplified. The amplification indicated that the method is suitable for RACE amplification of gene in Longan cotyledon embryo.The result of sequence analysis showed that the full-length cDNA sequence of GAPDH gene in Longan was 1395 bp long, including a 1008 bp long open reading frame (ORF) encoding 336 amino acids. The nucleotide and amino acid sequence of this cloned GAPDH gene were found to be highly homologous with GAPDH genes in other plant species. Through RT-PCR analysis, the expression of this GAPDH gene cloned here was constitutively expressed and could be used as a reference gene.3. Cloning and sequence analysis of abaxial polarity gene YABBY2 in Longan cotyledon embryoUsing the method of nested RACE amplification, the abaxial polarity YABBY2 genes which shared high homology with Arabidopsis abaxial polarity gene YABBY2 were obtained. In total, there were three full-length cDNA sequences YAB2-1, YAB2-2 and YAB2-3 and three partial fragments YAB2-4, YAB2-5 and YAB2-6.Bioinformatics analysis indicated that these genes were highly homologous with each other in the corresponding ORF regions and had different kinds of 3 'UTR and 5' non-coding region. In the 5 'end of YAB2-1, YAB2-2 and YAB2-3, the putative non-coding region of the IRES was found.Semi-quantitative RT-PCR analysis showed that YAB2-3 was significantly expressed in the early cotyledon embryo and only a small amount of expression value existed in the late cotyledon embryo; the expression of both YAB2-1 and YAB2-2 in both early and late cotyledon embryos were obviously; however, YAB2-4, with only trace amounts of expression, had a relatively more amounts of expression in later embryos.4. Cloning and sequence analysis of FVE, Remorin, CCR4-NOT genes in Longan cotyledon embryoThe nested RACE method was used and the full-length cDNA sequences of FVE, Remorin and CCR4-NOT genes were successfully obtained, plus the 5 'sequence of sulfur transfer protein gene and the 3' sequences of chitinase gene and dehydration-responsive protein-related gene. The expression of FVE genes was significant and especially high in the early phase; Remorin gene was expressed with a large amount in the early embryo and the expression of this gene in late embryonic expression was only faint; CCR4-NOT gene was highly expressed in both early late embryos and the expression in early embryo was relatively larger; Sulfur transfer protein gene, which was assumed a gene with tiny expression, was expressed at a relatively higher amount in the early embryo; The expression of chitinase gene was also tiny and the expression of this gene in late embryo was larger than that in early embryo; dehydration-responsive protein-related gene was found to be expressed significantly in the early embryo, but its expression in late embryo was only trace.