Isolation Of The ACC Oxidase Gene LlACO1 And Establishment Of Genenic Transformation Acceptor System In Lilium Longiflorum Thunb.

Lily (Lilium) is one of the most important ornamental bulb flowers in the world. Its cut flowers are of great commercial value, but the flowers were senescencing rapidly as other cut flowers when they were blooming. It is important to prolong the longevity of lily flowers and presently many scientists are focusing on this subject. It has been earlier confirmed that the senescence of lily flower was relative to ethylene. ACC oxidase was the last key enzyme in ethylene's biosynthetic pathway and it is of great significance to clone Lily's ACC oxidase (ACO) gene and to control its expression.In this study, a series of expriments were carried out, including ACO gene cloning, bioinformatic analysis and expression characteristics analysis, establishment of genetic transformation acceptor system, construction of plant antisense expression vector, and Agrobacterium mediated-transformation. The results are as follows:1. The full-length cDNA sequence of a 1- aminocyclopropane -1-carboxylate oxidase gene LlACO1 (GenBank Accession No. EU249333)was isolated from Lilium longiflorum Thunb for the first time. LlACO was 1067 bp long including 26 bp of 5'UTR, and 91 bp of 3'UTR and an ORF of 954 bp, encoding a 36.18 kDa protein with 317 amino acid residues. It was highly homologous to ACO of tulip with amino acid sequence homology of 86%. Multiple alignment analysis showed that LlACO1 contained many conserved domains, and there were few differences among ACOs from different kinds of plants. LlACO1 had the homologous functional domain with oxgenase family. Expression profiles of LlACO1 were studied by semi-quantitative one-step RT-PCR.2. The genomic gene of LlACO1 was isolated from L. longiflorum Thunb. for the first time. Genomic LlACO1 had four extrons and three introns, which located in nt 132-312, nt 618-969 and nt 1342-1671. All the introns had GT at 5'end and AG at 3'end.3. The transformation acceptor system for Lily was investigated. Using tender tissue from L. Longiflorum Thunb flowers as explants, the optimum callus-inducing explant, callus-inducing culture medium, proliferating culture medium, differentiating culture medium and rooting culture medium were screened out. The optimum concentrations of kanamycin and hygromycin for selecting cultures were also screened.4. The anti-sense expression vector for L1ACO1 gene antiL1ACO1 was constructed, the anti-sense fragment of L1ACO1. The antiLlACO1 was cloned into plant binary expression vector pCAMBIA1304, without removing the report gene gus. The expression vector was transformed into Escherichia coli DH5αfor propagation. Then the plasmid was extracted and transformed into Agrobaterium tumefactions EHA105 which can be used for lily transformation.5. Agrobacterium-mediated transformation was used to transform L. Longiflorum Thunb. The transient expression of foreign gene in calli was studied through GUS staining. Some hyg resistant calli and regenerated plants were obtained through hygromycin selection.