Cloning And Functional Analysis Of GA2OX Gene In ’Nantongxiaofang’ Persimmon (Diospyros Kaki Linn. CV. Nantongxiaofangshi)

’Nantongxiaofangshi’(Diospyros kaki Linn. cv. Nantongxiaofangshi) is a rare dwarf cultivar being found in Nantong during ’the Fruit Tree Resources Survey of Jiangsu Province’in 1982. It has no obvious main trunk, and central leader is weak. The trees are spreading, and show the significant dwarfness character. The height of adult tree is only about 3.5-4 meters, which is approximately equal to 60% that of the standard-type growing under the same conditions. In this study, two GA2ox genes associated with tree stature were cloned from ’Nantongxiaofangshi’leaves. We detected the expressions of two genes by real-time quantitative RT-PCR and constructed vector and preliminarily analysised its function through overexpressing in transgenic tobacco plants. The main researchs are as follows:1. Total RNA was extracted from leaves of ’Nantongxiaofangshi’ persimmon by improved CTAB method. Degenerate primers were used to cloned two genes of GA2oxs family. The full-length cDNA sequences were obtained by RACE, named DkGA2ox1 and DkGA2ox2 respectively. The two amino acid sequences shared 73-77% in homology comparing with Populus tomentosa(JX102472.1), oleander AY594292.1), tobacco(AB125232.1), petunia (GU059939.1), apple (FJ571521.1), pear (JF441168.1) and grape (JQ608472.1). The conserved structural domain analysis revealed that DkGA2ox1 and DkGA2ox2 had the typical functional domains of GA2ox protein, containing Fe2+ binding sites (DkGA2ox1: His-205, Asp-207, His-262; DkGA2ox2:His-204, Asp-206, His-261) and 2-oxoglutarate binding sites(DkGA2ox1:Arg-272, Ser-274; DkGA2ox2:Arg-271, Ser-273), as well as the 2OG-Fe (Ⅱ)-Oxy protein domains. The cDNA sequence was 1318bp and 1267 bp, containing 5’untranslated region (UTR) with lengthes of 198bp and 61bp,3’UTR with lengthes of 97bp and 172bp, and coding region with lengthes of 999bp and 1005bp which encoded 332 and 334 amino acids respectively. The protein were stable proteins, had no signal peptide, transmembrane domains, significant hydrophobic region. Phylogenetic analysis indicated that DkGA2ox1 and DkGA2ox2 belonged to C19-GAs.2. After construction of transient expression vector and onion epidermal cell transformation, subcellular localization assays showed that the GA2ox1 protein was located in the nucleus and cytoplasm. The quantitative RT-PCR results showed that the highest expression level of GA2oxl and GA2ox2 were detected in florescence, and all higher than that in ’dafangshi’during all the 7 phenological periods. The expression of gibberellin 2-oxidase genes in ’Nantongxiaofangshi’were related with the dwarf trait.3. Plant expression vector pYH4215-DkGA2oxl and pYH4215-DkGA2ox2 were constructed and transferred into tobacco via the method of Agrobacterium-mediated. By Hyp screening, GUS staining, PCR and RT-PCR,2 DkGA2ox1 and 2 DkGA2ox2 transgenic positive lines were detected. The morphological traits were observated on transgenic lines. Morphological identification showed that the heighs of the transgenic lines 1-1,1-3,2-1, 2-4 were 59.31%,41.54%,63.52% and 32.11%% of control. The internode lengthes of the transgenic lines were 11.91%-64.99% of control. The leaves lengthes of the transgenic lines were 52.94-76.74% of control. The leaves widens of the transgenic lines were wider than control by 4.37%-30.16%. Besides, GA1+3 content decreased by 41%-70%; net photosynthetic rate increased by 11.37%-33.5%; chlorophyll content was higher than WT by 9.10%-32.79% associating with the darker leaves. qRT-PCR showed that the expression levels of both NtGA20ox and NtGA3ox were raised.