| Anthurium andraeanum is the Araceae Anthurium high-grade indoor ornamental potted plants, belonging to the spadix plants. A. andraeanum is deeply loved by many people due to its beautiful shape and bright colour. It is the best choice for home decoration and gift for relatives and friends. It takes up a great percentage of flower market especially in the Lunar New Year Flower Market. Therefore, its planting area has been rapidly increasing in our province. Moreover, with the improvement of people’s living standard and the change of comsumption concept, the demand for high-grade potted flower is becoming greater and greater and it has huge potential in the market. However, as A. andraeanum is native to the tropical area and sensitive to temperature, it requires the growth temperature above15℃and cold damage could easily take place below12℃. Low temperature is one of the most important factors which constrain the growth of A. andraeanum. The production of Anthurium in our province is facing the problems of high energy cost, which reduces the market competitiveness and affects the sustainable and healthy development of the industry of A. andraeanum potted flower. The most effective way to solve this problem is to use the breeding method to improve the cold resistance of A. andraeanum plants. In this study, using A. andraeanum ’alabama’ as material, three full-length genes of alternative oxidase, catalase and ascorbate peroxidase were cloned by PCR combined with RACE technology and analyzed by bioinformatic tools. The real-time PCR technique was used to analyze the gene expression in different organs of A. andraeanum and under different low temperature stresses. The plant expression vector of ascorbate peroxidase gene from A. andraeanum was constructed and APX gene was transformed into tobacco plants in order to indentify its function. Finally, the fatty acid desaturase gene was transferred to A. andraeanum and the resistant plants were obtained. The results are as follows:1. The cloning and sequence analysis of a full length alternative oxidase gene (AnAOX) from A. andraeanumAn alternative oxidase gene was cloned from A. andraeanum leaves. The full-length AOX cDNA sequence was1170bp, which contained an open reading frame of1038bp. This gene was named as AnAOX and the genbank accession number is JX163297. The sequence encoded a protein of345amino acid residues with a calculated molecular weight of38.0kD consisting of an isoelectric point of8.51. The amino acid sequence of AnAOX gene code was analyzed using DNAMAN5.2.2software. The results showed that this gene has the whole3and5 terminal and it is the full length sequence of alternative oxidase gene of A. andraeanum.2. The cloning and sequence analysis of a full length hydrogen peroxide oxidase gene (AnCAT) of A. andraeanumA hydrogen peroxide gene was cloned from A. andraeanum ’alabama’ leaves. The full-length CAT cDNA sequence was1704bp, which contained an open reading frame of1476bp. This gene was named as AnCAT and the genbank accession number is JX163298. The sequence encoded a protein of492amino acid residues with a calculated molecular weight of57.1kD consisting of an isoelectric point of6.89. The homology between the CAT of tentative amino acid sequence of A. andraeanum and that of Zantedeschia aethiopica, Elaeis guineensis, Musa acuminata and Oryza sativa Indica are91%,87%,87%and81%, respectively.3. The cloning and sequence analysis of a full length ascorbate peroxidase gene(AnAPX) of A. andraeanumAn ascorbate peroxidase gene was cloned from A. andraeanum’alabama’leaves. The full-length APX cDNA sequence was888bp, which contained an open reading frame of750bp. This gene was named as AnAPX and the genbank accession number is JQ8385071. The sequence encoded a protein of250amino acid residues with a calculated molecular weight of27.4kD consisting of an isoelectric point of5.4. According to the prediction of physicochemical property of AnAPX protein by ProtParam online software, it suggested that the tentative APX protein molecular formula was C1232H1904N330O363S7. The molecular weight was about27.4kD with an isoelectric point of5.4, half-life period of about30h and the unstable parameter of32.55, belonging to stable protein. The homology between the AnAPX of tentative amino acid sequence and those of Zantedeschia aethiopica, Vitis pseudoreticulata, Gossypium hirsutum, Elaeis guineensis and Zea mays were93%,87%,87%and86%, respectively.4. The gene expression analysis of AnAOX, AnCAT and AnAPX under cold stressUsing the quantitative real time PCR, the gene expression levels of the three genes in the A. andraeanum leaves were determined after different time of low temperature stress (6℃), we observed that the expression of AnAOX, AnCAT and AnAPX genes were all up-regulated. The AnAOX gene upregulation of the expression was not obvious after12h of low temperature treatment, and became obvious after24h low temperature treatment. The expression level of24h is obviously higher than that of12h. After more than24h low temperature treatment, the expression level slightly went down and there was no obvious difference during the period of36～48h. The AnCAT gene expression upregulation was obvious after12h low temperature treatment and its expression level began to drop after more than12h. The expression level closed to or even slightly below to the control when low temperature treatment reached48h. The AnAPX gene expression upregulation was obvious after12h low temperature treatment. Along with the time of the low temperature treatment, its expression rose gradually and reached to the peak when treated48h with low temperature. It is speculated that the change of the expression level of AnAPX gene was most closely related to the low temperature and its time course, followed by AnAOX, and finally AnCAT gene.5. The building of plant expression vector of AnAPX.Design the peculiar primer on the basis of AnAPX gone full length sequence, taking the A. andraeanum’alabama’as test material, we got the whole open reading frame of AnAPX gene. The obtained section was connected with the pMD18-T vector and measured, and the results suggested that the inserted section was correct. After double enzyme digestion cut, the clone section has been inserted into the plant expression vector pCAMBIA2300and the expression vector of AnAPX gene has been built. Having built the expression vector of AnAPX successfully, we used electroporation to transform recombinant plasmid into the bacterial strain of agrobacterium tumefaciens, which established the basis of the follow-up function identification of AnAPX.6. The gene transformation of A. andraeanumUsing meloidogynosis sonication-assisted Agrobacterium-mediated transformation, taking callus of A. andraeanum as infection material, we converted the green fluorescent protein (GFP) gene and fatty acid desaturase (FAD3) gene into A. andraeanum. By screening with G418and kanamycin, we have gotten the transgenic plants of A. andreanum. Further identification of the transgenic plants showed that the target gene FAD3had been integrated into the genome of partial transgenic plants. Therefore, we have successfully obtained the transgenic plants of A. andreanum with FAD3gene. |
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