Cloning Of The CDNA Of ACC Oxidase Gene From Lilies And Its Construction Of Plant Antisense Expression Vector

Lily flower is one of the five world famous cut flowers, and it is widely favored by people for the characters of the big flower, varied flower color, graceful posture, and strong fragrance. The sales of fresh cut flower can be counted in billions around the world. But like other fresh cut flowers, lily will decay rapidly while blooming. How to prolong the longevity of lily flower has become one of the main subjects for studying recently. On the basic research of senescent mechanism in Lily cut flower, through cloning its cDNA of ACC oxidase genes, building anti-sense expression vectors, further transferring them to the cells of Lily, and controlling the senescence of Lily by manipulating the expression of ethylene, the purpose of prolonging the longevity of lily flower can be attained. This research chooses Asia hybrids Pollyanna and Oriental hybrids Sorbonne to be the materials, and extracts the total RNA of the flowers. The cDNA fragments of ACC oxidase genes were obtained by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology. Then inserted the sequence reversely into the plant expression vector with high efficiency, and built the anti-expression vector of ACC oxidase genes in Lily, so that a solid foundation for the research of gene transferring can be made. The results are as follows: 1. Improved CTAB-LiCl method has been built to suit the extraction of the total RNA of the flowers. On the basis of making comparisons as to the effect of extracting total RNA by three methods, which are acid guanidinum thiocyanale, SDS/phenol, and CTAB-LiCl, due to high levels of polysaccharides in Lily tissues, the special stage of getting rid of polysaccharides was added so as to improve the method of CTAB-LiCl in isolating RNA from Sorbonne. The results demonstrate the followings: it can effectively get rid of polysaccharides; in the extracted RNA, the light of 28s rRNA is about twice of that of 18s rRNA;A260/A280 is in the range of 1.8 to 2.0,while A260/23O is 2.0;the production of RNA in Pollyanna and Sorbonne are 36.3μg /g and 10.2μg /g respectively. 2. Obtained the cDNA fragments of ACC oxidase genes in Lily. Chose the petals of Sorbonne and Pollyanna to be materials, extracted and purified the total RNA, then the ds-cDNA was synthesed by reverse transcriptase. The double degenerate primers were designed by the homologous sequence of the ACC oxidase genes. Through PCR, two ACC oxidase genes fragments of Sorbonne and Pollyanna had been obtained. The sequences data had submitted to GeneBank, and the accession number is OHS-1, AY656735, OHS-2, DQ062133, AHP-1, AY656736, AHP-2 and DQ062134 respectively. 3.Building the anti-sense expression vectors of ACC oxidase genes fragment. Through recloning, the enzyme sites of BamH I and Sal I were transferred into the two ends of ACC oxidase genes fragment AHP-2, and the ACO plasmid of Pollyanna was obtained. Cutting the ACO plasmid of Pollyanna and Pwr306 plasmid at the sites of BamH I / Sal I; then after electrophoresis, the small fragment of 500bp and big one were recycled respectively. Both of them were linked and cloned so as to enlarge the reproduction in E.coil DH5 a. And extracted plasmid for next step of research. 4. Cloned the symtomless virus gene fragments in Asian Lily. Anti transcripted the total RNA extracted from the petals of susceptible Asian hybrid 'Pollyanna', and got cDNA. Choosing a section of virus sequence to be the primer, then the virus gene fragment was obtained through PCR. This demonstrates that the kind of Asian Lily were infected by Lily symtomless virus; meanwhile, cloned to the Lily symtomless virus gene fragment of Asian Lily 'Pollyanna'.