Study On The Function Of ClGA2ox12 Gene In Cunninghamia Lanceolate (Lamb.) Hook

Gibberellin 2-oxidases(GA2oxs)are the major enymes of GA metabolism.GA2oxs take bioactive GAs and their precursors as substrates,irreversibly converting bioactive GAs into non-bioactive form.Then,the content of bioactive GAs is decreased to affect plant growth and development.To date,functional studies of GA2oxs were mainly focused on angiosperm,and rarely reported in gymnosperms.Cunninghamia lanceolate(Lamb.)Hook is one of fast-growing tree species which widely cultivated in south China.Researches have shown that GAs play the critical role in wood formation and tree growth.However,the molecular mechanism remains unclear.In an affert to further investigate the mechanism,this stufy chose C.lanceolate as research material,and cloned 2 GA2ox genes ClGA2ox12.1 and ClGA2ox12.2.the function of the two genes in wood formation and plant growth and development was studied:(1)Based on the transcriptome data of C.lanceolate,we obtained 5 potential sequences of ClGA2oxs:ClGA2oxs-1 to ClGA2ox-5.qPCR analysis revealed that ClGA2oxs-1 expression was increased among the 5 ClGA2oxs in C.lanceolate stem after exogenous GA(GA3)application,indicating ClGA2oxs-1 is able to be induced by GA.Phylogenetic analysis showed that ClGA2ox-1 is homologous with the Pinus tabuliformis PtGA2ox12.Therefore,ClGA2ox-1 was named ClGA2ox12.The cloning results of ClGA2oxl2 in both C.lanceolate stem and leaf showed 2 groups of ClGA2ox12 genes with relatively obvious differences.Moreover,the two groups of ClGA2ox12s corresopond in stem and leaf.Therefore,the two sequences are namly ClGA2ox12.1 and ClGA2ox12.2.(2)The coding sequences of both ClGA2ox12.1 and ClGA2ox12.2 are 942 bp,encoding 313 amino acids.Phylogenetic analysis showed that the two ClGA2ox12 genes scarcely belong to 3 classes of GA2oxs of angiosperm including Arabidopsis thaliana and Oryza sativa,but are ranged in the same class of GA2oxs of with PtGA2ox12.Sequence alignment and protein domain analysis showed that the amino acid sequences coding by ClGA2ox12.1 and ClGA2ox12.2 contain two conserved domains,DIOX_N and 20G-FeⅡ_Oxy,suggesting that both ClGA2ox12.1 and CIGA2ox12.2 have the common structural characteristics of the GA2oxs protein family.Analysis of physicochemical properties of amino acid sequences showed that molecular weight of the ClGA2ox12.1 and ClGA2ox12.2 are 35669.8Da and 35518.6Da,theoretical PIs of that are 5.74 and 5.39,respectively.Moreover,ClGA2ox12.1 and ClGA2ox12.2 are predicted hydrophilic and non-secretory protein,with no transmembrane-helix and signal peptide.(3)The expression partern of ClGA2ox12.1 and ClGA2ox12.2 showed that the two genes are both expressed in root,stem,leaf and flower of C lanceolate,and hightest expressed in root.Moreover,ClGA2ox12.1 expresion level in stem and leaf was found to be significantly highter than that in flower.However,the CIGA2ox12.2 expresion level among stem,leaf and flower showed no apparent differences.(4)The pCAMBIA1300-ClGA2ox12.1-GFP and pCAMBIA1300-ClGA2ox 12.2GFP vectors were constructed for subcellular localization.The two vectors were transferred into Agrobacterium EHA105 and positive cloning was obtained.ClGA2ox12.1 and ClGA2ox12.2 genes were transferred into tobacco(Nicotiana benthamiana)leaf through Agrobacterium-mediated transformation method.The results of confocal microscopy observation showed that ClGA2ox 12.1 and ClGA2ox12.2 were localized to cytoplasm.this indicates that the two proteins function in cytoplasm.(5)The pI121-ClGA2ox12.1-GUS and pBI121-ClGA2ox12.2-GUS vectors were constructed for gene overexpression in Chinese white poplar(Populus tomentosa).The two vectors were transferred into Agrobacterium EHA105 and positive cloning was obtained.ClGA2ox12.1 and ClGA2ox12.2 genes were transferred into Populus tomentosa through Agrobacterium-mediated transformation of stem.Transgenic plants werr obtained after screening.Plant height of transgenic poplars of ClGA2ox12.1 and ClGA2ox12.2 decreased by 36.6%±16.8%and 38.9%±9.33%,respectively,relative to wild-type.Transgenic poplars exhibted noticeable dwarf,suggesting that ClGA2ox12.1 and ClGA2ox12.2 negatively regulate the plant height of Populus tomentosa.Compared to wild-type,transgenic plants exhibited that lighter staining of lignified cells,lower lignin contet and deferred xylem vessel differentiation of in stems.Quantitative PCR analysis showed that lignin biosynthesis genes,PtoCCR2(Cinnamoyl CoA reductase),PtoCAD1(Cinnamyl alcohol dehydrogenase),were significantly downregulated in transgenic plants relative to wild-type.This indicates that ClGA2ox12.1 and ClGA2ox12.2 regulate lignin biosynthesis.Moreover,compared to wild-type,GA biosybthesis gene PtoGA20ox1 was upregulated but GA metabolism gene PtoGA2ox1 was downregulated,indicating that exogenous gene expression of ClGA2ox12.1 and ClGA2ox12.2 induces the feedback regulatory mechanism of GA genes of poplar.Based on the above research results,this study showed that ClGA2ox12.1 and ClGA2ox12.2 regulated plant height,lignin formation and xylem development.This work provided scientific basis for further investigation of molecular mechanisms of GAs governing wood formation,and provided technical support for genetic improvement of C.lanceolata wood quality.