A Study On The GA20-oxidase Gene Silencing Induced Dwarfism Of Plant

Dwarfed and bonsai's woody flowering plants are important in landscaping and upholstery,and most of them are arbors by now.GA20-oxidase,which is one of the most important endogenesis factors for controlling high growth of woody flowering plants,can influence high growth of plants by adjusting the content of GA.In this work,antisense RNA and RNA interference were used to suppress expression of the GA20-oxidase gene in tobacco.This study could be a foundation work for dwarfism of woody flowering plants.The main research work and the results are as follows:1.The NtGA20ox gene was cloned from Nicotiana tabacum cDNA,and pBIantiW plasmid of antisense RNA and pBIRNAiW plasmid of RNA interference were constructed with marker gene NPTII;The PttGA20ox gene was cloned from pGEX-4T-2 plasmid,pBIbarantiG plasmid of antisense RNA and pBIbarRNAiG plasmid of RNA interference were constructed with marker gene bar.The four plasmids were transformed to Agrobacterium tumefaciens LBA4404.2.Regeneration and genetic transformation system for Nicotiana tabacum W38 and transgenic tobacco GW(transformed over expression gene PttGA20ox) were established and optimized. Subculture medium was MS + 6-BA 1.0 mg/L + NAA 0.1 mg/L + agar 5.5 g/L;Rooting medium was 1/2 MS + NAA 0.1 mg/L + agar 5.5 g/L;Differentiation medium was MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + agar 4.8 g/L.3.The most effective genetic transformation procedure was established.The results of different level reagents tests showed that the best reagents concentrations for adventitious bud regeneration were Kan 75 mg/L,Glu 0.5 mg/L,for segment propagation and rooting were Kan 150 mg/L,Glu 1.0 mg/L,and the best concentration of Cef was 250 mg/L for all.The genetic transformation test showed that pre-culture time,late selection and Acetosyringone were unconspicuous for the transformation; and leaves immersed in the bacterium solution(OD₆₀₀=0.6) for 10-15 min,co-cultured for 2 d can get the highest frequency of transformation.4.It showed that target gene had been integrated in genomic DNA of transgenic plants from the result of adventitious bud regeneration and molecular detection for transformed tobacco AW (transformed antisense NtGA20ox gene),RW(transformed inverted repeat NtGA20ox gene),AG (transformed antisense PttGA20ox gene),and RG(transformed inverted repeat PttGA20ox gene).5.Growth analysis showed that the transgenic plants were dwarfism as the controls.The stems of tobacco AW and RW were thicker than the untransformed control plants,their internodes were lower, and leaves were bigger,their rooting time was deferred and roots were less than control.The transgenic plants AG and RG showed a similar physiological index to the tobacco AW and RW, except the change for stems were unobvious compared with their control GW.6.Cytology comparison showed that the leaves cells of the transgenic plants were more compact than controls,and the length of stems cells was smaller than controls.7.The endogenous hormone contents of ABA and ZR of the transgenic plants were similar to the controls,but the contents of IAA,GA₃ and GA₄ were lower than controls.In the experiment of sprinkling extrinsic GA,all the transgenic plants came back to length growth as controls;it showed that the decreased contents of GA by gene transform could be reversed.