Isolation And Primary Function Identification Of Noval MhNCED And MhCCD Genes In Malus Hupehensis Rehd

The precursor of abscisic acid (ABA)--C15 compound xanthoxin derived from cleavage of the 11, 12 and 11', 12'double bond of 9-cis-violaxanthin and 9-cis-neoxanthin, this enzyme was called 9-cis-epoxycarotenoid dioxygenases (NCEDs) that catalyze the rate-limiting in ABA synthesis. Carotenoid cleavage dioxygenases(CCDs) cleavages the 9, 10 and 9', 10'double bound of various carotenoids, that can contribute to the diversity of apocarotenoids, which play important roles in many aspects of plat growth and development. In this thesis, a series of studies based on the materials of Malus hupehensis (Pamp) Rehd. and have been conducted on the gene isolation, sequence comparison, expression analysis, under stress and in different development stages and tissues. The main results are as follows:1. Isolation and characterization of MhNCED and MhCCD geneAccording to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from Malus hupehensis (Pamp) Rehd. roots. By RT-PCR and RACE-PCR, we isolated NCED and CCD genes from Malus hupehensis (Pamp) Rehd. roots and named MhNCED and MhCCD, with the accession number of GeneBank EU716329 and EU871633, respectively. The full-length cDNA of MhNCED is 2182bp, and the ORF of MhNCED encodes a 607-amino acid polypeptide. The predicted MW and PI were 66.90 kD and 6.56. the full-length cDNA of MhCCD is 1930bp, and the ORF of MhCCD encodes a 544-amino acid polypeptide. The predicted MW and PI were 61.35 kD and 6.30.2. MhNCED and MhCCD mRNA levels are signified differnt in different tissues and stages in Malus hupehensis (Pamp) Rehd. seeding. MhNCED up regulated and MhCCD down regulated the germination of Malus hupehensis (Pamp) Rehd. seeds.3. The response of MhNCED and MhCCD to the stress in Malus hupehensis (Pamp) Rehd.The MhNCED gene expression was higher than CK under the CuCl2 and CdCl2 stress, and both able to immediately induced the increase of ABA content. The analysis of Real time PCR showed that CuCl2 and CdCl2 treatment contribute to expression increased of MhNCED and MhCCD and reached up to the hightest value at 7 h and 12 h, respectively, then decrease to contral level .4. MJA and SNP pretreatment and stratification were effective to break Malus hupehensis (Pamp) Rehd. embryonic dormancy and can induced MhCCD high expression. With the extension of time stratification MhCCD expression was increased and reached up to maximum in germination seeds.5. Construction of expression vectorIn order to study the function of endogenous MhNCED and MhCCD in plants, construction vectors pBI121-MhNCED and MhCCD pBI121-3'segment containing 3'segment in antisence orientation driven by the constitutive cauliflower mosaic virus 35S promoter were assembled and introduced into Malus hupehensis (Pamp) Rehd., and gained transgene Malus hupehensis (Pamp) Rehd. in antisense.