Preliminary Study On Gene Expression Analysis Of GA20-ox In Castor (Ricinus Communis L.) And Transformation Of RNA Interference GA20-ox Gene

With the development of modern agriculture economy,the planting industrial structure on castor also turns to the development tendency that is dwarf variety.Under the same conditions,the dwarfed castor has better performance than high-stem ones in saving water,drought resistance,resistance to lodging and realizing mechanized harvesting.Thus dwarfed castor breeding has become a hotspot.In the process of plant growth,plant hormone,gibberellin?GA?is involved in the development of stalk internode and therefore affects the height of the plant.Gibberellin 20 oxidase?GA20-oxidase?is a key regulator of gibberellin biosynthesis and determines the synthetic amount of bioactive GAs.During this research,GA20-oxdiase was cloned and studied in order to provide the theory support for the dwarfed castor breeding.The bases sequence of castor GA20-oxidase gene?XM₀02510827.1?was cloned by homologous gene cloning method,which has been published in NCBI database.The results of Sequences analysis by bioinformatics software showed that the target gene has been obtained,marked as RcGA20-ox.Then GA20-oxidase gene expression quantities in different organs of castor were analyzed using RT-PCR,and the results indicated that the highest expression of GA20-oxidase gene occurred in castor seeds and tender leaves.Furthermore,GA20-oxidase gene expressions in tender leaves during different periods from four typical high-stems?"Zhongbei 4","Zhongbei 5","Tianbi 26","Zhebi 36"?and four dwarfed castor?"Bilv 2","Jiaxiang 2","CSR24.181","Zhebi 17"?were analyzed by RT-qPCR,and the results indicated the RcGA20-ox gene expression is one of the important factors affecting the plant height.With castor cDNA and intermediate carrier pHANNIBAL as templates,forward fragments,reverse fragments and introns were amplified,marked as GA20s,GA20a,Intron,respectively.To construct RNA interference carrier pBI-GA20ox-RNAi,pBI121 was double digested by Xba I and Sac I,then connected with the linear vector through three PCR fragments by using the In-Fusion technology.Then pBI-GA20ox-RNAi was transferred into Agrobacterium EHA105 by the method of DNA freeze-thaw.And the agrobacterium culture medium for infection was successfully prepared.The key steps in the castor high efficient regeneration system were optimized,using molecular level evaluation mechanism,combined the LECl gene expression and the actual budding efficiency of regeneration system.The optimum induction medium for adventitious bud was determined as follows:MS substrate+0.5mg/L 6-BA+0.2mg/L IBA.The effect of GA3 concentrations on adventitious bud elongation was studied,and the optimal result was determined as follows:MS substrate+0.5mg/L 6-BA+0.2mg/L IBA+O.1mg/L GA3.Under the optimized conditions,induced roots grew into complete plants and transplanted successfully.Using the self-built castor genetic transformation system,castor embryo was infected by Agrobacterium carrying RNA interference with vacuum assisted.Sixteen resistant plants were selected and the probability is 3.05%.Five transgenic plants were obtained by PCR detected and the probability is 0.95%.The conversion of GA20-ox gene was proved to be restrained under different levels by RT-qPCR.