Cloning And Function Analysis Of GA2-oxidase Gene In Dwarf Pear Of ’Zhongai1’

‘Zhongai1’ is the first Chinese pear of ‘Jinxiang’ rootstock cultivar with independent intellectualproperty rights released by Research Institute of Pomology, Chinese Academy of Agricultural Sciences.It can cause dwarfing and compacting, and the25-year-old tree height was only about1.5m, while theaverage shoots length was only0.1m every year.‘Zhongai1’ also had a good affinity with scion androotstock when used as inter-rootstock, which can result in the efficient dwarf of pear varieties, earlyfruiting, early full-production and high fruit quality by grafting. Previous studies showed that thecontent of GA of ‘Zhongai1’ was markedly lower than other vigorous varieties as ‘Duli’,‘Zaosu’,‘Jinfeng’ and the maternal plant ‘Jinxiang’ and so on in tender leaves. GA2-oxidase was a keyregulation enzyme in metabolism pathway. It can change the bioactive GAs into deactivate GAs, andreduce the content of endogenous level of active GAs when highly expressed in plants. In this study,GA2-oxidase was isolated from ‘Zhongai1’ by homologous clone way, and its temporal and spatialexpression among ‘Zhongai1’,‘Zaosu’ and ‘Jinxiang’ were detected by real-time fluorescentquantitative reverse transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR,for the purpose of exploring the relationship between GA2-oxidase gene expression and ‘Zhongai1’dwarf mechanism, and providing the theoretical basis in fruit dwarfing and breeding. The specificresults as followed:1. Isolated GA2-oxidase gene full-length cDNA sequence from the young leaves of ‘Zhongai1’,GenBank accession number was JF441168. The sequence analysis result showed that the GA2-oxidasegene was1014bp, encoding a protein of337amino acids residue. The amino acid sequence had asimilarity between71.87%and97.63%with those of reported plants such as Chinese white poplar,upland cotton and apple.2. Using GA2-oxidase gene full-length specific primers to amplify in ‘Zaosu’ and the maternalplant ‘Jinxiang’ separately, and obtained their GA2-oxidase full-length cDNA sequences. Sequencealignment showed that the cDNA sequences only had few nucleotide bases difference in three pearvarieties, but their encoding amino acids sequences were completely consistent. Therefor, theGA2-oxidase had no gene structural differences in the three varieties, and the gene function was similarin ‘Zaosu’,‘Jinxiang’ and ‘Zhongai1’.3. Accorrding to the semi-quantitative RT-PCR and real time fluorescent quantitative RT-PCR, theGA2-oxidase gene expression was tested among ‘zhongai1’,‘Zaosu’ and ‘Jinxiang’ in tender leaves andshoot phloem in various growth stages. Three parts of the phloem as top, middle and bellow position ofthe shoot were analyzed, and the new shoot growth state were observed in detected periods at the sametime. The results indicated that：GA2-oxidase gene appear an expression peak in ‘zhongai1’ in themiddle growth stage, but in the other tested periods, the gene had no obvious expression differenceamong three varieties and its expression was just at a lower level. After the stage of peak expression, the new shoot of ‘Zhongai1’ gradually stopped growing, while the other two varieties continued growth.During the middle and later growth stage, the expression of GA2-oxidase gene in the middle position ofshoot phloem was significantly larger in ‘Zhongai1’ than in ‘Zaosu’ and ‘Jinxiang’, while in the topand bellow position three varieties had no obviously difference in all growth stage. In all tested growingperiods, GA2-oxidase gene always had the same expression intensity fluctuation in three varietiesbetween tender leaves and the top position of shoot phloem. Thus, the main reason of causing ‘Zhongai1’ dwarf may be concluded that, in the middle growth stage, the GA2-oxidase highly expressed in tenderleaves, resulting in the cessation of growth of new shoots prematurely. In later growth period, the genestrongly expressed in the middle position of shoot phloem, leading to its internodes shorter.4. Based on pCAMBIA1304, the restriction enzyme cutting site BglⅡ and BsteⅡwere used toconstruct overexpression vector pCAMBIA1304-GA2ox, for laying a foundation for verification onfunction of pear GA2-oxidase gene through transgenic technology.