Gene Cloning And Functional Analysis Of Gibberellin 2-ODDs Family Oxidase Gene In Walnut

Gibberellin(GAs)is one of the five major hormones regulating the growth and development of plants,which widely exists in bacteria,fungi and plants.GAs biosynthesis and metabolism is a multi-step enzymatic reaction process involving many enzymes.The key enzymes involved mainly come from three gene families:terpene synthase gene family(TPSs),cytochrome P450 monooxygenase family(P450s)and 2-oxoglutarate-dependent dioxygenases(2-odds),which play an important role in the final stage.GA20ox,GA3ox and GA2ox oxidases belong to 2-ODDs,regulating the normal growth and development of plants and responding to abiotic stress.Walnut is one of the world famous"four nuts".With the development of modern processing industry,people’s demand for it is increasing.However,the high tree height,poor stress resistance,lack of high-quality rootstocks and other factors seriously restricted the planting range of walnut and affected the healthy development of walnut industry.In this paper,walnut as the research object,through gene cloning and vector construction of gibberellin oxidase gene,genetic transformation,PEG simulated drought stress function analysis,transgenic micro grafting and other research,in order to obtain dwarf and drought resistant walnut rootstock,and provide theoretical basis for the sustainable development of walnut industry.The main results of this study are as follows:(1)The over expression and interference vectors of gibberellin 2-ODDs family oxidase gene in walnut were successfully constructedIn this study,we successfully constructed 35S::JrGA3ox::GFP overexpression vector by PCR amplification and homologous recombination.The conserved fragments of JrGA3ox and JrGA2ox genes were cloned and connected with the interference vectors of PTCK303.The interference vectors of PTCK303-JrGA3ox and PTCK303-JrGA2ox were successfully constructed.(2)Genetic transformation system of gibberellin 2-ODDs family oxidase gene in walnut mediated by Agrobacterium tumefaciensAgrobacterium tumefaciens mediated genetic transformation was carried out with the over expression vectors 35S::JrGA20ox::GFP,35S::JrGA3ox::GFP,35S::JrGA2ox::GFP and interference vectors PTCK303-JrGA20ox,PTCK303-JrGA3ox and PTCK303-JrGA2ox as acceptors.GFP fluorescence identification showed that the positive rates of JrGA20ox,JrGA3ox and JrGA2ox were 91%,88%and 89%,respectively,Gus staining showed that the positive rates of JrGA20ox,JrGA3ox and JrGA2ox interfering with the E3 generation of somatic embryos were 87%,83%and 86%.The development of positive somatic embryos experienced four stages:spherical embryo,heart-shaped embryo,torpedo type embryo and cotyledon type embryo;PCR electrophoresis detection and quantitative analysis of regenerated lines obtained from somatic embryo germination showed that over expression and interference positive regenerated lines of JrGA20ox,JrGA3oxand JrGA2ox genes were successfully obtained.(3)Regulation of plant height by gibberellin 2-ODDs family oxidase gene expression in WalnutThe phenotype,plant height and internode length of JrGA20ox-RNAi,JrGA3ox-RNAi and JrGA2ox-OE lines were analyzed.The results showed that the plants of JrGA20ox-RNAi,JrGA3ox-RNAi and JrGA20ox-OE showed semi dwarf phenotype.Compared with WT,the plant height decreased by33.4%,28.8%and 35.9%,and the internode length decreased by 30.3%,28.2%and 50.0%,respectively.Compared with WT,plant height increased by 46.5%,60.6%,37.7%,and internode length increased by70.4%,48.7%,28.9%,respectively.(4)Negative response of gibberellin 2-ODDs family oxidase gene JrGA20ox to drought stress in WalnutUnder PEG simulated drought stress,JrGA20ox-RNAi interfered with NBT,DAB,Evans blue staining,H2O2,O2~·ˉ,The contents of relative conductivity,MDA,stomatal opening and natural water loss rate were significantly lower than those of wild type and JrGA20ox-OE overexpression lines;SOD,pod,CAT activity,proline and chlorophyll content were significantly higher than those of wild type and JrGA20ox-OE overexpression lines;The relative expression levels of SOD,POD,LEA,P5CS and GAI were significantly higher than those of wild-type and JrGA20ox-OE overexpression lines,while the expression levels of VPE were significantly lower than those of wild-type and JrGA20ox-OE overexpression lines.The results showed that the expression of JrGA20ox gene was inversely proportional to the drought resistance of walnut plants.(5)Negative regulation of gibberellin 2-ODDs family oxidase gene JrGA20ox on drought stress in grafted plants of walnutThe over expression of JrGA20ox-OE and the interference of JrGA20ox-RNAi were used as rootstocks,and the wild-type plants were used as scions for micro grafting.After 15 days of grafting,the grafted plants healed well,and the survival rate of micro grafted plants reached 90%.The results showed that the expression of JrGA20ox gene was affected by grafting.Under drought stress,compared with the wild-type self-rooted grafted plants,the leaves of grafted plants with JrGA20ox-RNAi interference lines as rootstocks were greener,and the plants grew stronger.The expression levels of drought resistance genes SOD,LEA,P5CS in scions were significantly increased,and the expression levels of VPE were significantly decreased,which indicated that reducing the expression level of JrGA20ox gene could significantly enhance the drought resistance of grafted plants.