Cloning Of JcGAST1 Gene And Its Expression Analysis In Flower Development Of Jatropha Curcas

Jatropha curcas(Jatropha curcas L.),it is a deciduous shrub of Euphorbiaceae.Because of its high oil content,it has become the main green and pollution-free energy plant in China,and it is also recognized as the most likely alternative fossil energy and the tree with great development potential in the world.However,because of the low seed yield of this plant,the planting of J.curcas brings little economic benefit which greatly prevents the exploitation and expansion of this energy crop,and the small ratio of female to male flowers is thought as one of the critically limited factors for the low seed yield of J.curcas.Previous studies shown that gibberellin plays an important role in regulating flower development and can regulate the ratio of female to male flowers.Transcriptome analysis showed that JcGAST1 which is a gibberellin-singnal related gene was differently expressed between female and male flowers,and would be involved in flower development in J.curcas.This paper studied the JcGAST1 from the aspect of molecular biology so as to get an insight into the role of it in the flower development of J.curcas.In this study,we chose JcGAST1 as the research object.Its full length of c DNA was cloned,and not only constructed its prokaryotic expression system,identifiedits prokaryotic expression product,analyzed and predicted the structural characteristics of JcGAST1 protein,but also studied the expression of JcGAST1 gene in different stages of flower development.And we also constructed the expression vector to verify the function of JcGAST1 gene in tobacco flower development.This study maybe lay a foundation for further study on the role of JcGAST1 gene in the flower development of J.curcas and the mechanism of gibberellin affecting in flower development.The main results of this study are as follows:1.The full-length c DNA cloning of JcGAST1 gene from J.curcas.In this experiment,the partial fragments of JcGAST1 gene was used as templates which selected from the data of J.curcas flower transcriptome,using RACE-PCR technology to amplify 5'-terminal c DNA and 3'-terminal c DNA.Through DNAMAN software to splice the 5'-terminal c DNA and 3'-terminal c DNA to obtain the full length of JcGAST1 gene.The full length of JcGAST1 gene is 586 bp,and the ORF is 348 bp which encodes 115 amino acids.The gene contains a GASA domain and signal peptide,and its belongs to the GAST family.2.The Prokaryotic expression of JcGAST1 gene.The prokaryotic expression vector p ET-30a/JcGAST1 of JcGAST1 gene was transformed into E.coli BL21(DE3)competent cells,used IPTG to induced it,and the SDS-PAGE electrophoresis showed that JcGAST1 gene was expressed successfully.The results of mass spectrometric identification also showed that the expressed product of JcGAST1 gene was GAST1 protein,its molecular weight was 10.746 k Da,and its p I was 9.44.Otherwise,it has 8 peptides matched with GAST1 protein,and the coverage rate was 74%.In addition,JcGAST1 protein has no transmembrane structure,and contains a signal peptide domain and GASA functional domain.Through the analysis of the physical and chemical properties of the protein,it is found that it is a stable protein and a hydrophilic protein.And through multiple alignment analysis,it was found that the protein had a conserved region of 70 amino acids at the C-terminal and 12 conserved cysteines in the conserved region of 60 amino acids at the C-terminal.The phylogenetic tree analysis also found that the affinity of J.curcas was the closest to Hevea brasiliensis.3.The expression Analysis of JcGAST1 gene in different stages of flower development.Through the previous research in the laboratory,the flower development of J.curcas was divided into nine key stages.There are sex undifferentiated period,megasporocyte stage,the meiosis of megasporocyt stage,embryo sac stage,mature embryo stage,microsporocyte stage,microspore tetrad stage,single nucleus pollen stage,and pollen maturation stage.In this experiment,the expression of JcGAST1 gene in flower development was analyzed by q RT-PCR,with the undifferentiated period of J.curcas as control.The results showed that JcGAST1 gene has significant differences in nine key stages of J.curcas flower development.During the development of male flowers,the expression of JcGAST1 gene increased significantly from the microsporocyte stage to the maximum at the single nucleus pollen stage,and then decreased at the pollen maturation stage.During the development of female flowers,the expression of JcGAST1 gene in the meiosis of megasporocyt stage did not change significantly during the development of female flowers,but then began to decrease in the embryo sac stage,until the expression of JcGAST1 gene decreased to the lowest in the mature embryo stage.In the nine critical stages of J.curcas flower development,the expression of JcGAST1 gene in male flower was higher than it in female flower,especially in microspore tetrad stage and single nucleus pollen stage.It indicated that JcGAST1 gene may be involved in the development of microspore tetrad stage and single nucleus pollen stage.4.The expression of JcGAST1 protein in different stages of flower development.Using Western Blot technique,analyzed the expression of JcGAST1 protein in different stages of flower development.The results showed that the band of JcGAST1 protein was clear in the undifferentiated stage,the microspore tetrad stage and the single nucleus pollen stage,especially in the microspore tetrad stage,indicated that the expression of JcGAST1 protein was the best in the microspore tetrad stage,which may be involved in the development of the microspore tetrad stage.5.Analysis of genetic transformation of JcGAST1 gene in Nicotiana tabacum.The expression vector pBWA(V)HS/JcGAST1 was constructed with pBWA(V)HS vector as the framework and transformed into tobacco K326 by agrobacterium tumefaciens-mediated method.The molecular detection of transgenic tobacco showed that JcGAST1 gene was successfully expressed in transgenic tobacco.In addition,compared with wild type tobacco K326,transgenic tobacco had larger leaves(for example,longer and wider leaves),more compact plant type and larger crown width.Otherwise,the length of stamens in transgenic tobacco flowers was longer than that in pistils,while in wild-type tobacco K326,pistils were longer than stamens,indicated that JcGAST1 gene not only participated in the development of male flowers and promoted the growth of filaments,but also participated in the vegetative growth of plants,especially the growth and development of leaves.