The Study Of Cloning And Anti-transformation For ACC Oxidase Gene In Yali (Pyrus Bretschneideri Rehd.) And Density-increasing Of Chinese Jujube's Genetic Linkage Map

Pear (Pyrus L.) and Chinese jujube (Ziziphus jujuba M.) are both important economic tree species in China, they are widely cultivated in our country and are important parts in China's agricultural economy. To deepen the molecular biology study and use biotechnology for genetic improvement has important significance in speeding up the breeding process of the two fruit trees. So, in this study, focusing on pear and Chinese jujube, we carried out a series of expriments, such as gene cloning, genetic transformation, density-increasing of genetic linkage map and QTL analysis. The main results as follows:1. The full-length cDNA sequence of ACC oxidase gene was isolated from Yali fruits for the first time. It was 1235 bp long including 65 bp of 5'UTR, and 225 bp of 3'UTR and an ORF of 942 bp, encoding a 35.36 kDa protein with 314 amino acid residues. Base on the ORF, the genomic gene of ACO in Yali was also isolated, it had four extrons and three introns, which located in nt 106-347, nt 575-750 and nt 1085-1297. All the introns had GT at 5'end and AG at 3'end.2. Reaserch the deduce amino acids of ACO genes from Yali by bioinformatic analysis, it showed that this protein contained many conserved domains, and there were few differences among ACOs from different kinds of plants. The ACO in Yali had the homologous functional domain with 2OG-Fe(II) oxygenase super family, and was similar with Isopenicillin N synthase and related dioxygenases(PcbC), so it proved the ACO in Yali was belonged to Isopenicillin synthase family, which does not use 2-OG as the acidic substrate.3. Predicted the obtained Yali ACC oxidase amino acid sequence to secondary structure and tertiary structure, the results showed that the Yali ACO was a hydrophilic soluble protein but had 3 N-myristoylation sites, so it may be a lipid-anchored membrane protein which had a amide - myristic acid anchor hook to connect the membrane.4. Southern and Northern blotting were employed in this study to detect the copys of obtained ACO gene in Yali's genomic and it's expression system. The results showed this gene was single copyed in genomic and just expressed in reproductive organs, it is likely this gene only actived in the system II ethylene biosynthesis, but the system I ethylene biosynthesis was controlled by other ACO genes, so we can conclude that there were at least two ACO genes in Yali, and there were very different in nucleotide sequence.5. The anti-sense expression vector for obtained Yali ACO gene was constructed and transfromed in to Yali plants by using Agrobacterium-mediated method. Finally, 29 kanamycin resistant plantlets were obtained, identified them with PCR and Southern blot method, results showed the foreign gene had only been successful integrated into 4 of the 29 kanamycin resistant plants, the copies of the foreign gene in the 4 plant's genome were between 1~2.6. The ISSR-PCR reaction system of Chinese jujube was estabblished after analysised the effect of 6 main factors in PCR reaction using the single factor method. The optimal ISSR-PCR reaction system was determined as follows: 1×PCR Buffer,2.0 mmol·L-1 Mg2+,0.25 mmol·L-1dNTP,1.5U Taq polymerase,1.25μmol·L-1primer and 50ng template DNA in total 25μL reaction volume. The optimal annealing temperature was 58℃.7. By using 18 ISSR primiers, the research obtained 44 ISSR markers to increase density of the genetic linkage map for Chinese jujube. Finally, a high-density genetic linkage map of Chinese jujube was constructed, which consists of 15 linkage groups, covers 1338.81 cM, includes 388 AFLP markers, 35 RAPD markers and 34 ISSR markers. The average interval distance is 2.92 cM. Compared with the previous linkage map, the total length of map has increased 24.41 cM, the average interval distance has reduced 0.18 cM, and 3 distance >10cM between adjacent marker were filled.8. The putative QTLs controlling needle length in Chinese jujube, including trunk long needle length, trunk short needle length, branch long needle length and branch short needle length were identified based on the newly constructed genetic linkage map. Totally 34 QTLs were obtained, but only 10 QTLs of it were located on the same position as the QTLs analysis result base on previous linkage map, so it proved the density of genetic linkage map has impact on the accuracy of QTLs anlysis, and it also indicated these 10 QTLs have higher credibility that the tightly linked markers of it could be used in marker-assisted breeding.