Functional Analysis Of CsMADSs And Genetic Transformation To Cucumber

As an important transcription factor,MADS-box gene plays an important role in plant growth and development.Previous studies showed that over expression of CsMADS08 in Arabidopsis reduced the number of lateral branches,and a large number of rosette-like stem leaves appeared,which may be involved in the formation of leaves and lateral buds.However,earlier flowering,more and fuller pods,and earlier maturity were appeared in over-expressing CsMADS21 plants,which might be involved in the development of flower and fruit.In order to further study the function of CsMADSs,agl8 mutant was used as the material and CsMADSs were introduced by Agrobacterium-mediated method,and the resistant plants were obtained by screening.The phenotypic changes and recovery of the transformed plants were observed,and the expression of downstream regulatory genes were analyzed by fluorescence quantitative analysis;meanwhile,cotyledons in vitro was transformed by Agrobacterium mediated method,and transformed plants were regenerated by screening.The main results are as follows:1.Homozygous test of agl8 mutant.The leaf DNA of the mutant agl8 was extracted and verified by three primers.The results showed that all the mutants were homozygous.2.Phenotype analysis of CsMADS08.Using inflorescence infection method,the pCAMBIA1305-CsMADSs was transferred into agl8 mutant,and the genetic transformation plants were obtained by hygromycin screening and molecular detection,and the character and phenotype of each organ in the offspring were observed.The results showed that only CsMADS08 transformants were obtained,while no CsMADS21 transformants were obtained by the same method.The root length of CsMADS08 transformation plant did not change significantly,the plant height decreased,the number of branches and stem leaves were more than the mutant plants,the stem leaves were smaller,round,and not curled;the first flower of the transformation plant bloomed 4 days earlier than that of the mutant;the pod of the transformation plant was longer and fuller than that of the mutant,but the length was shorter than the wild pod,and the pod of the transformation plant would not crack in advance.3.Paraffin sections of cauline leaves.The mature stem leaves in the same part of wild,mutant and transformation plants were taken and the cross section of leaves was observed by paraffin section.The results showed that in the same size field,the epidermal cells of the transformed leaves were larger than those of the mutants,the cells were arranged orderly,the number of vascular bundle cells increased,and the leaves became thicker.4.Expression analysis of CsMADS08.The expression of CsMADS08 in different organs was detected by real-time fluorescence quantitative PCR.The results showed that CsMADS08 was highly expressed in all organs of the transformed plant,and the highest expression was in the stem leaves.5.Analysis of the expression of downstream regulatory genes.The expression of SHP1,SHP2,ALC and IND in different organs of wild,mutant,and transformed plants was detected by real-time fluorescence quantitative PCR.The results showed that compared with wild-type plants,all four genes were up-regulated in other organs of agl8 mutants except for SHP2 and ALC were down-regulated in the flowers of agl8 mutant;compared with the mutant plants,the expression level of four genes in the transformed plants was higher than that in the mutant plants,and they were down regulated in other organs,especially in the stem leaves.6.Genetic transformation of cucumber.The cucumber cotyledons were infected with pCAMBIA1305-CsMADSs agrobacterium solution,and the regenerated seedlings were obtained by the culture and regeneration of buds and roots and the selection of hygromycin.The genomic DNA of the regenerated seedlings was extracted,and the hygromycin resistance gene was amplified by PCR.The positive plants were tested and transferred to the culture medium and cultured in the incubator.The results showed that the positive transformants of CsMADS08 and CsMADS21 were obtained.The above results provide important theoretical basis for further exploring the function of CsMADSs and cultivating new varieties of cucumber.