Screening Of The Interaction Components Of Floral Develoment Genes CsWIP1 And CsPI In Cucumber

Cucumber,Cucumis sativus is an important vege Tab.crop,and is also a classical model plant for the study of sexual differentiation of dioecious plants.Flower Development and sexual differentiation are important issues in cucumber biology.Cs WIP1 and Cs PI genes play an important role in inducing pistil and inhibiting stamen development in cucumber flower development,especially in female flowers.In cucumber,the mutation of Cs WIP1 gene is monoecious,and pistils are found in flowers at all nodes of the plant.On the other hand,the down-regulated expression of Cs PI gene is closely related to the development of stamens in cucumber female flowers.However,the regulation mechanism of Cs WIP1 and Cs PI genes about flower development in cucumber is not clear,especially the interaction components of transcription factor Cs WIP1 and the upstream female flower development related factors of Cs PI genes have not been found.In this paper,the c DNA Library of Cucumber Terminal Bud was constructed,the yeast two hybrid bait plasmid was constructed with Cs WIP1,and the single hybrid bait plasmid was constructed with the promoter fragment of Cs PI,in order to elucidate the mechanism of regulatory differentiation of flower development genes and to provide reference for perfecting the network map of sexual differentiation.The experiment led to the following conclusions:1、The total RNA was extracted from terminal buds of cucumber 406,406a,GY14,9930.The double-stranded c DNA was synthesized by the SMART^TM technique.The product was purified on a CHROMA SPINTM+TE-1000 column and transformed it into the competent yeast strain Y187 with the linearized vector p GADT7-Rec.The c DNA Library of Cucumber Terminal Bud was constructed by SMART^TM technology.Using the high activity of homologous recombination enzymes in yeast cells,the two strains were homologous recombined in yeast to produce circular library plasmids with replicative activity,and then all the transformed yeasts grew in the SD/-Leu medium plates.Finally,the c DNA Library of Cucumber Terminal Bud was constructed successfully.The capacity of the library was more than 2×10⁶cfu independent clones,and the average length of the inserted fragments was about 1 kb.Also,the recombination rate was 98%,which indicated the high quality of the c DNA.It provides the material basis for screening the interacting proteins through Y1H and Y2H.It fits the requirement of library construction.2、The cucumber Cs WIP1 gene was successfully cloned and the recombinant bait plasmid p GBKT7-Cs WIP1 was constructed.The Bait plasmid was transferred into Y2H Gold yeast competent cells by Yeast Transformation Kit.The recombinant bait plasmid p Cs PI-Ab Ai was constructed and its minimum inhibitory concentration of Ab A^r was determined to be 150ng/m L.At the same time,the results showed that the bait plasmid had no toxic effect on the growth of yeast and no self-activation activity.Thus it can eliminate other potential problems for the screening process.3、The yeast two-hybrid interaction protein of Cs WIP1 gene was screened and 29genes were identified.Two of them（Csa7G024140.1 and Csa2G116270.1）were located in the nucleus and expressed during the development of pollen et al.organs.They were selected as candidate factors.The Csa7G024140.1 gene encoding bromodomain protein,its homologue At BET10 in Arabidopsis is a negative regulator of sugar and ABA signals and plays a important role in pollen germination and tube elongation.Csa2G116270.1 gene encodes homologous domain protein,and its homologous gene At2G23820 in Arabidopsis encodes a metal-dependent phosphate hydrolase,which plays a role in organism metabolism and biological regulation.The interaction between the two genes and p GBKT7-Cs WIP1 was preliminarily identified by blue-white spot analysis,and the functional prediction of the candidate genes could provide direction for further verification.A total of 120 candidate genes,including 4 ethylene-related transcription factors,1 MYB factor and 1 MADS-BOX factor,were screened by Yeast one-hybrid interaction analysis of Cs PI gene,the function of transcription factor gene was predicted to provide reference for follow-up research.