Cloning And Identification Of ACC Oxidase Genes In Several Vegetables

Ethylene, a phytohormone, plays an extensive role in plant growth and development, such as seed germination, floral differentiation, sex determination and fruit setting as well as in various stresses: wounding, freezing, drought, plant disease. Ethylene biosynthesis in plants has been discovered. ACC oxidase is the key enzyme in the course. The antisense transgenic plants have been acquired in tomato. The research in cloning, expression of ACO and ACS gene is important to learn the role of ethylene and the mechanism of manipulation.The objective of this research was to clone the ACO genes of tomato, cucumber and cauliflower, and analyze the gene structures and expression patterns. Then, plant expression vectors with sense and antisense orientations and RNA interfering have been constructed and the manipulation of endogenous gene expression was done with the Agrobacterium-mediated transformation.1. The gDNA and cDNA of tomato ACO gene (LeACO11) was cloned using PCR. The full length of LeACO11 gDNA is 1697 bp with four exons and three introns. The length of LeACO11 cDNA is 1018 bp, including the coding sequence 951 bp and poly--A. It encoded 316 amino acids. The alignment results of the gDNA and cDNA showed that AG was always on the splicing site.2. 2000 bp upstream of LeACO11 gene was obtained using the method of vector-mediated PCR. Sequence analysis showed that it contains the regulatory elements: TATA box (-29 ~ -22), CAAT box (-193 ~ -189), wound and drought response elements. -1724 ~ -1300 and -1037 ~ -592 were repeated. 100 transcription factor binding sites exist in the upstream.3. Northern blotting of different tissues was conducted. The expression of LeACO11 is week in both leaves and the mature green fruits, however it expressed strongly in breaker and red ripen fruits.4. Five plant expression vectors were constructed, which consist of the sense, antisense, RNAi of LeACO11 cDNA sequence and two fragments of upstream sequences. Transgenic plants were acquired using the Agrobacterium-mediated transformation. Expression analysis showed the inhibition of the endogenous gene.5. The promoter regulatory elements were chereterized with histochemical analysis of GUS activity in various tissues. The results showed that the two fragments of the promoter were able to direct fruit-specific gene expressions. The expression driven by 2000 bp fragment of regulatory element increased with the maturation of fruits. The fragment of 1200 bp could direct uidA gene expression in fruits with the induction of drought and wound. The regulatory region for fruit-specificity was probably located in the 1200 bp of 5'-flanking sequence and some positive regulatory elements or enhancers may exist in the region from 1200 to 2000 bp.6. The cucumber ACO gene of CsACO4 was cloned using the method of homologous sequence with the GenBank accession number: AY450356. The length of the genome sequence is 2200 bp. The spliced length of mRNA was 933 nucleotides (nts) and it encoded 311 amino acids. The length of 5' untranslated region of CsACO4 is 795 bp. The core region of the deduced promoter was located at -51- -11, TATA box at -47--39, and no CAAT box. Nine primary response elements concerning signal transduction were found in the upstream of TATA box, they are wound response elements, heavy metal response elements, drought response elements and auxin response elements.7. Northern blotting showed that the gene expressed among female flowers of gynoecious and monoecious genotypes, it could not express in other tissues or organs. This implied that the gene might be correlated with the female behavior positively.8. Plant expression vector of RNAi was constructed and genetic transformation was done using the Agrobacterium-mediated method. Five transgenic plants were identified by southern blotting. The morphological exploration showed that the inserted target fragment inhibited the endogenous gene expression and it contributed to the male differentiation in the lower nodes.9. The cauliflower ACO gene of BoACO was cloned using the method of homologous sequence with the GenBank accession number: AY676466. The size of BoACO is 1202 bp of nucliotides. Three exons and two introns were identified in this sequence. The spliced length of mRNA is 756 nts and encodes 252 amino acids. This gene expressed in the flowers and leaves, the levels in flower was higher than in the leaves.10. Plant expression vector of RNAi was constructed and genetic transformation was performed using the Agrobacterium-mediaied method. Three transgenic plants wereidentified by southern blotting. The aboundence of the endogenous mRNA in the transgenic plants was lower than in the wild type based on Northern blotting. As well the activity of ACO enzyme was inhibited significantly.