Agrobacterium-mediated Transformation Of Apple With FT And Construction Of CDNA Library From Jujube Early Flower Buds

1. Apple was one of the most major fruit species in the world. It had a long juvenile phase that influenced the progress of the varietal improvement and economic benefit in early times. In order to obtain early flower plants, our research was using transgene technology to transform the gene that promote flowering into apple genome. The FT gene of Arabidopsis promotes flowering was used to transform apple cultivar Gala by leaves, through optimizating of Agrobacteria-mediated genetic transformation conditions to established the genetic transformation system, and using the PCR detection.The results are as follows:This experiment research the effects of antibiotics (Cefotaxime Cefazolin Carbenicillin Ampicillin) on inhibition of Agrobacterium tumefaciens. Results indicate: Proliferation of LBA4404 was suppressed completely by cefotaxime and carbenicillin, The medium containing 400 mg/L cefotaxime and 600 mg/L carbenicillin can suppressed completely , Cefazolin almost can not suppressed in proliferation of A. tumefaciens while ampicillin can not.This experiment research the effects of antibiotics (Kanamycin Cefotaxime Cefazolin Carbenicillin) on callus differentiation and adventitious bud regeneration from apple leaves and inhibition of Agrobacterium tumefaciens for affect the transformation efficiency. Result indicates that the adventitious buds regeneration was completely suppressed in the medium containing 5 mg/L kanamycin as selection pressure. The medium containing 500 mg/L cefotaxime and 600 mg/L cefazolin has the highest regeneration rate of apple leaves, and the medium containing 400 mg/L can suppress the regeneration of adventitious buds. In the transformation system of Gala, 500 mg/L of Cefotaxime and 5 mg/L Kanamycin were added to as the suitable selection pressure in de-bacteria culture. We have obtained 2 transformed shoots from 300 kanamycin resistant shoots by PCR and the transformation ratio was 0.66%. 2. Chinese jujube (Zuzuphus jujuba Mill.) is an specific tree in china, have resistances against coldness, drought, early fructification figures. The flower differentiation of jujube has the feature of differentiated in the same year, short duration, fast development and a long endurance period. In order to understand the gene distribution and expression of jujube flower buds formation, we constructed of cDNA Library from jujube fruit bearing shoot.The cDNA library were only linked 7 times, we form choose 1338 positive clones to extract of plasmid DNA, The 566 plasmids carried gene fragments which were digested, we choose 469 clones which size were larger than 500bp, they were compared with other sequences in GenBank through internet by BLAST. There are 238 express sequence tags (ESTS) were identified, of them, 229 ESTs contain regions with high identity to their homolog including some repeated fragments to other genes, in Genbank while 9 ESTs were unknown functions, The ESTs with significant protein homology were sorted into 12 functional categories. In the 229 ESTs, occupying 19% of protein synthesis have 46 ESTs containing a largest ratio, primary metabolism have 26 ESTs, occupying 11% ;cell growth have 25 genes, also contain a large ration occupying 10%;disease defense have 23 ESTs occupying 10%; ion transduction have 2 ESTs containing the least ratio, only occupying 1%; besides unclassified gene 45 occupying 19%. The Results indicated in the early differentiation of flower buds, activity is most vigorous, need for quantitative substance metabolism and energy metabolism, the most activity is the protein synthesis. Some defense-relate genes were identified with cDNA library, they are possible related to resistances against coldness, drought etc in jujube.