Phenotypic Changes And Expression Analysis Of Overexpressing CsMADSs Gene In Arabidopsis Thaliana

MADS-box family genes are a class of genes encoding transcription factors and play an important role in plant growth and development.Such as vegetative absorption in plant roots,vernalization,flowering time,meristem differentiation,embryo development and fruit ripening.In recent years,MADS-box has been found to play an important role in anthocyanin biosynthesis,stress resistance,root structure,bud dormancy,and even leaf development.In this paper,CsMADSs(Cs MADS08,Cs MADS21)genes of cucumber were cloned,subcellular localization of transgenic gene in tobacco,construction of expression vector,agrobacterium mediated transformation of Arabidopsis thaliana,screening and molecular detection of transgenic plants,Staining Profile of transgenic Arabidopsis in different tissues,and the expression of CsMADSs gene in transgenic plants are detected,The results are as follows:1.Construction of pCAMBIA1305-CsMADSs over-expression vector.p MD19-T-CsMADSs plasmids were digested by Bam H I-Sal I,and p CAMBIA1305 was digested by Sal I-Bgl II,and connected by T4 ligase,and then transformed into Escherichia coli JM109.2.Subcellular localization of CsMADSs.PCAMBIA1305-CsMADSs were transformed into Agrobacterium tumefaciens by freeze-thaw method,fluorescence confocal results showed that Cs MADS08 was located in the cell membrane and the cell nucleus,while Cs MADS21 was located in the cell membrane.3.Phenotypic changes analysis of transgenic plants.Over-expression Arabidopsis thaliana plants were obtained by the floral dip method,Hgy and Gus were used to verify over-expression genes.Transgenic plants showed that leaves were purple.Over-expression of Cs MADS08 plants were short,rosette leaves and branches reduced.And a large number of stem leaves,reduce the number of pods.Phenotypic separation were detected in the T4 generation,part phenotype were just like the front,while the increasing branch numbers and pod numbers were found in the other plants.While in over-expression Cs MADS21 plants,rosette leaves and stem leaves were increased,the flowering period is one week in advance,more pods and plump and the earlier maturity stage.4.GUS staining of over-express plants.GUS staining was carried out on the organs of Cs MADS08 and Cs MADS21 plants,and the results showed that the roots were stained in over-express Cs MADS08 plants,while the roots,stems,flowers,leaves and pods were stained in over-express Cs MADS21 plants.5.Changes of anthocyanin content in over-expressing plants.Results showed that anthocyanin content was significantly different in the leaves of the over-expressed plants than that of the wild type.6.Quantitative expression of target genes was detected by real-time fluorescence quantitative analysis.The total RNA of each organ of T3 plant was extracted and the expression of CsMADSs gene was detected by fluorescent quantitative PCR.The results showed that the higher expression was detected in the stem of Cs MADS08 plants,while higher expression of flowers in Cs MADS21 plants.In summary,the phenotypic changes and expression analysis were studied in transgenic plants which expressing cucumber CsMADSs gene.This study provided a theoretical basis for further exploration of the function about the cucumber CsMADSs gene.