Studies On Maintenance Of Orchid Transgenic Cultures And Cloning And Expression Analysis Of ACO In Oncidium

The research of genetic transformation of antisense ACS gene had been carried out in our laboratory in Dendrobium and Oncidium. In this experiment, the PLBs of the transgenic antisense ACS gene were used as the materials for the research of maintenance orchid transgenic cultures, optimization of plant regeneration system and molecular detection of Oncidium of the transgenic antisense ACS gene; gene cloning and relative expression analysis of ACO in Oncidium.The main results were as follows:1Maintenance of the orchid transgenic cultures and optimization of plant regeneration systemThe Oncidium and Dendrobium PLBs of transgenic antisense ACS gene were used as materials for the research of optimization of plant regeneration system including PLBs proliferation, differentiation of protocorms, growth and transplanting of plantlets. The effects of different additives (apple juice, banana juice, activated carbon) and concentrations on the proliferation and differentiation were compared. The results showed that1/2MS media supplemented with0.5mg/L NAA and50g/L or100g/L of apple juice were the best media for proliferation, and the proliferation coefficient were13.88or13.21, respectively; the best medium for the PLBs differentiation was1/2MS medium supplemented with0.5mg/L IBA,0.5mg/L NAA and1g/L active carbon, with the differentiation rate of63.75%. Comparing the effects of factors of Lg (34) for proliferation in Oncidium and Dendrobium PLBs, the best medium for Oncidium PLBs differentiation was1/2MS medium supplemented with0.4mg/L IBA,0.6mg/L NAA and1.5g/L active carbon, with the score of9.27, according to comprehensive evaluation method; the best medium for Dendrobium PLBs differentiation was1/2MS medium supplemented with1.0mg/L6-BA,0.4mg/L IBA,0.6mg/L NAA and1.5g/L active carbon, with the score of9.13. Comparing the effects of factors of L9(34) for plantlets growth in Oncidium. The results showed that1/2MS medium supplemented with0.6mg/L IBA,1.5mg/L NAA,150g/L of apple juice was the best medium for protocorms in Oncidium, with the score of9.67. Comparing the effects of the three basal media (1/2MS,1/4MS,1/8MS) on plantlets growth in Dendrobium of the transgenic antisense ACS gene,1/4MS (as the basal culture medium) could be better for the growth of Dendrobium plantlets. Comparing the effects of the different matrix on growth in Oncidium of the transgenic antisense ACS gene, the mix of palm and aquatic plants was more suitable for it.2Molecular detection of Oncidium of the transgenic antisense ACS'geneThe materials of Oncidium of the transgenic antisense ACS gene were selected, including transplant seedlings, adult seedlings, young seedlings, PLBs and the materials of the non-modified Oncidium, including transplant seedlings, mature seedlings, young seedlings, PLBs, in total eight different stages of the materials. Using PCR detection, the purpose gene strip appeared in the materials of Oncidium of the transgenic antisense ACS gene, but the purpose gene strip didn't appear in the materials of the non-modified Oncidium.At the same time using the18S rRNA for reference gene, with SYBR Green Ⅱ fluorescent dyes method of real-time PCR detected the report gene gus in different stages of materials. The results showed that gus gene had expressed in the materials of Oncidium of the transgenic antisense ACS gene, the non-modified Oncidium couldn't detect gus expression. It proofed that antisense ACS gene has been integrated into the DNA of Oncidium. Relative expression of gus gene decreased from transplant seedlings, adult seedlings, young seedlings to PLBs.3Gene cloning and relative expression analysis of ACO in OncidiumCombined homology cloning with RACE, the PLBs were used as materials to clone the ACO gene in Oncidium'little cherry'. The full-length cDNA sequence of ACOwas1349bp, and5'UTR was152bp, while3'UTR was128bp. The3'end also contained20bp poly (A). The sequence of ACO encoded323amino acids, with ATG as start codon and TGA as stop codon. Accession number of ACO in GenBank was JN994719; the relative molecular weight of protein ACO was36543.8Da; the isoelectric point (pi) was5.96; the molecular formula was C1628H2554N442O482S16; the total number of atoms was5122; the negatively charged residues (Asp+Glu) was45and the positively charged residues (Arg+Lys) was40; the ACO protein was a hydrophilic protein, which didn't contain signal peptide and transmembrane domains. The maximum credibility that its sub-cellular localization was in the cytoplasm was0.650. The ACO protein had the amino acid sequence of synthesis of the existing morphine N double oxygen enzyme super family (as haem dioxygenase in morphine synthesis N-terminal) and Fe2+add oxygen dependence on the enzyme super family (2OG-Fe (II) oxygenase superfamily) conservative structure domain; The structure and function domain had a Fe (2+)2-oxoglutarate dioxygenase amino acid base sequence in the158-258position. Secondary structure prediction showed that the ACO protein was with45%α-helices,21%β-strands,19%β-corner and16%coil. Three-dimensional structure prediction showed that it contained a large amount of protein alpha helix, as the same as the secondary structure prediction result. When establishing the evolutionary tree in Neighbor-Joining system found that Oncidium and other Orchidaceae plants were in the same branch, including Cymbidium hybrid cultivar, Dendrobium hybrid cultivar, Cattleya bicolor, Papilionanthe hookeriana x Papilionanthe teres, Cattleya intermedia, x Sophrolaeliocattleya'Love Castle', etc.Using the18SrRNA for the reference gene, with SYBR Green Ⅱ fluorescent dyes method of real-time PCR, relative expressions were analysed at different stages in Oncidium. Relative expressions of ACO gene in eight samples presented in the following three rules. Firstly, no matter what stage-developmental materials, the relative expressions of Oncidium of the transgenic antisense ACSgene were higher than those of the non-modified Oncidium. The relative expression of PLBs was the lowest among all transgenic samples, with the relative expression of0.37, but it was also higher than those of all the non-modified samples. Secondly, the relative expression of Oncidium of the transgenic antisense ACS gene, in turn, transplant seedlings>adult seedlings> young seedlings>PLBs. The relative expressions of ACO gene were different at the different stages. The relative expression of seedling stage was higher than that at the bulb stage. Finally, the order of the relative expressions of the non-modified Oncidium was:young seedlings>transplant seedlings>PLBs>adult plants. The relative expression of young seedlings was the highest, and the adult seedlings was the lowest.