The Functional Analysis Of OguCMS-related Genes In Cabbage(Brassica Oleracea Var.capitata)

Ogura cytoplasmic male sterile (OguCMS) is the most widely used malesterile type in cabbage(Brassica oleracea var. Capitata). Early expression pattern analysisindicated that BoMF1expression down regulated5.2times and BoMF2express up regulated12.4times in OguCMS anther of cabbage. On the basis of OguCMS-related genes BoMF1﹑BoMF2and orf138from Brassica oleracea in our laboratory previously, the sequences werecloned and analyzed. In order to study the gene function, the plant expression vectorpBI121-BoMF1and pBI121-BoMF2were constructed and transformed into Arabidopsis thaliana.Meanwhile, in order to identify the self activation function and self toxic effect, the Yeast baitvector pGBKT7-orf138-X(X=0,1,2) were constructed and transformed into Yeast AH109.The main results were as follows:1.BoMF1and BoMF2were cloned from cabbage OguCMS sterility gene. By Blast analysis,the gene had88%and78%homology with At2g63060and At2g63060in Arabidopsis. At thesame time, the over expressing vector PBI121-BoMF1and PBI121-BoMF2of plant werestructured and transformed into Arabidopsis thaliana and transgenic rate were1.6%and1.45%. According to the transgenic plant phenotype of Arabidopsis thaliana T2generation,we found it appeared partial sterility characters: BoMF1transgenic plant appeared novelpetal-like floral structures at bud base, short and aberrant stamens, and aborted seed; BoMF2transgenic plant appeared short and aberrant stamens, the phenomenon of abortive seeds wereobviously, like loose seeds and missing seeds.2.In this study, the pGBKT7-orf138-X(X=0,1,2) bait vector of the Yeast two-hybrid BDregion were structured and transformed into the Yeast cell AH109to analysis the activationand toxicity of bait plasmid. Results showed that AH109[pGBKT7-orf138-X (X=0,1,2)] evengrown milk white clonies in SD/-Trp/x-a-gal plates, and no clonies in SD/-Ade/-Trp/x-a-gal、SD/-His/-Trp/x-a-gal plates, which indicated pGBKT7-orf138-X(X=0,1,2)couldn’t activateyeast reporter genes MEL1and ADE2, HIS3autonomously. So they didn’t haveself-activation capacity. Meanwhile, after being cultured in SD/-Trp liquid medium for16hthe OD600of them were1.059、1.200、1.151respectively, even reached0.8. This indicatedthe bait plasmid pGBKT7-orf138-X(X=0,1,2) had no toxicity to yeast strain. The aboveresults show that those bait plasmids can be used to fish interacting proteins from cDNAlibrary.