Screening Interactive Proteins Of Ogura Cytoplasmic Male Sterility Related Gene Orf138 In Cabbage(Brassica Oleracea Var.Capitata) And The Functional Analysis

Cabbage C ytoplasam Male Sterility(CMS) gene orf138 full- length and its upstream and downstream motif yeast carrier bait p GBK T7-1138,pGBKT7-142,pGBKT7-43138, were constructed base on the open reading frames of O guCMS-related genes orf138 through molecular cloning technology from Brassica oleracea in our laboratory previously. Yeast bait vector pGBKT7-1138, pGBKT7-142, pGBK T7-43138 were transformed into Yeast Y2 HGold and then activation and toxic effects were verified. The yeast two hybrid cDNA library of the cabbage(Brassica oleracea var.capitata) Ogura cytoplasmic male sterility(OguCMS) line that our lab constructied were used for screening by mating method. we successfully obtained interactio ns proteins from the yeast two hybrid cDN A library. Furthermore,we verified the interaction proteins between orf138 and ethylene response transcription factors RAP2-12 by methed of bimolecular fluorescence complementation(BIFC). The main conclusions here are as follows:1. Base on cDNA template of cabbage O guCMS sterile buds,the orf138 gene were cloned. By analyzing structure domain orf138, the N-terminal domain structure of 142 and C-terminal domain structure were cloned. At the same time, we build bait carriers of pGBK T7-142,pGBK T7-43138,pGBKT7-1138. After transformed into yeast Y2 HGold and then activation and toxic effects were verified. The results show that the bait ait carriers of pGBK T7-142、 pGBK T7-43138、pGBKT7-1138 were no the fuction of activation and no toxic effects for Y2 HGold.It can be used for screening of cDNA library.2. Completed the large number of screening work of the bait pGBKT7-1138,pGBK T7-43138,pGBK T7-142, respectively, obtained interactions of proteins from the yeast two hybrid c DNA library. The bait of p GBK T7-1138 obtain four matches candidate protein genes, seven matches candidate protein genes for p GBK T7-142 and three for pGBKT7-142.3. According to the positive candidate clones owned obtained by mating method,the single cDN A library plasmids interaction with bait were separated and extracted.We confirmed that the encodes proteins of orf138 full- length interaction with ADP-ribosylation factor1- like,Cox19- like CHCH family protein, ethylene-responsive transcription factor RAP2-12, glutathione S-transferase DHAR1. The sites of interaction with orf138 were also verified. Finally,Combine the data of experiences and bioinformatics analysis,the function of positive candidate genes were predicted.4. The plant bimolecular fluorescence complementation expression vector, pSPYN E-1138, pSPYN E-142, pSPYNE-43138 and pSPYCE-1138 D were constructed successfully. The experience of Bimolecular fluorescence complementation show that interactions between orf138 and candidate genes of ethylene response transcription factors RAP2-12 were verified,as well as their interactio ns structure domain may be the membrane protein of 142. A new clue related gene orf138 cabbage OguCMS function mechanism research was provided.