Gene Cloning And Prokaryotic Expression Analysis Of GASA6 From Jatropha Curcas L.

Jatropha curcasL.(Euphorbiaceae:Jatropha)has been regarded as a kind of emerging energy materials for biodiesel because of its high oil content in seed(50%~60%)andthe production of seed is limited by the flower development.GASA(GA-Stimulated in Arabidopsis)gen family is reported to function at the downstream of DELLAregulated gibberellinsignaling pathway,and plays important roles in thegrowth and development ofplant.In this study,we obtained an mRNA segmentof a GASA6 homologous in the transcriptome of flower development,and got full-length cDNA of it by RACE-PCR,then nanmed it as JcGASA6.To get an insight into the functions of JcGASA6 in the flower development of J.curcas,we analyzed the gen expression in different development stages offlower,subcellular localization and the product of prokaryotic expressionof it.The obtained results were as follows: 1.Cloning and sequence analysis of JcGASA6 gen inJatropha curcasJcGASA6 gen was cloned using RT-PCR combined RACE-PCR.The results showed that cDNA of this gene has an ORF of 327 bp with a full-lengthof 859 bp,encodinga protein with 108 amino acids.In addition,theprotein encoded by this gen has a signal peptide a function domain of GASA family.2.Epression of JcGASA6 in different developmental stages of Jatropha flowesRT-qPCR technique was used to analyze the expression of JcGASA6 in nine important period of flower development,including the stage before sex differentiation,the stage of megasporocyte formation,the stage of megasporocyt meiosis,the stage of embryo sac genesis,the stage ofthe maturation of embryo,the stage of microsporocyte genesis,the stage of the formation of single nucleus pollen,stage of the maturation of pollen.The results showed that the expression of this genewas significantly different between female flowers and male flowers.In male flowers,the gene expression increasedduring the whole sex development of male flower,and reached its maximum level at the stage of maturation of pollen period.However,in female flowers,expreesion level of it was firstly increased and then declined after the stage of megasporocyt meiosis.3.The subcellular localization of JcGASA6proteinTransient expression vectorpBWA(V)HS-GASA6-osgfp was constructed and used to analyze the subcellular localization of JcGASA6 protein.The results indicated that the protein is located intonoplast.4.Prokaryotic Expression of JcGASA6Prokaryotic expression vector of JcGASA6 was constructed and then expressed in BL21.The optimal conditions for expression of JcGASA6 were as follows: IPTG concentrationwas 0.75 mmol/L,induction timewas16 hours.Solubility analysis found most of protein preducted by JcGASA6 in E.coli BL21 was existed in the forme as inclusion body.Its molecular weight is 10.971 KDa and isoelectric point is 9.24,as identified by protein mass spectrometry.JcGASA6 protein is a kind of hydrophilic protein,and its secondaryconstruct was characterized by two alpha helix,abeta-corner,irregular curl anda transmembrane segment.Like GASA family in Arabidopsis,in the conservation domain of JcGASA6,there were 12 conservative cysteine sites,forming six disulfide bonds.