Study On The Interactions Class Ⅱ SCR, SRK And THL From Brassica Oleracea L. By A Yeast Two-Hybrid System

The purpose of self-incompatibility is an important mechanism of evolution to prevent self-pollinating. The SI molecular processes of Brassica oleracea as follows: S-locus cysteine-rich protein (namely the S-locus protein11), existing in the pollen, recognized with the S receptor kinase gene on the stigma, subsequently thioredoxin-like protein was released from SRK, then realized self-incompatibility. There are three important elements in the signal transduction of Brassica oleracea, including S-locus receptor kinase extracellular domain, S locus cysteine-rich protein/S-locus protein11and class of thioredoxin proteins. In the present study, we investigated the interaction relationship between SCR, THL and SRK by the yeast two-hybrid system.We acquired two important encoding sequences from Brassica oleracea by PCR technology. SCR2(class Ⅱ) and THL1were subcloned into pGBKT7, eSRK2(class Ⅱ)、 SRKJ (class Ⅰ, SRK6) and eSRK28(class Ⅰ) were subcloned into pGADT7.The yeast two-hybrid system was used to verify the interaction between eSRK2-SCR2, eSRK28-SCR2, SRKJ-THL1. The results indicated that the recombinant expression vectors did not show autoactivation and toxicity. The experimental group Y2HGold [pGBKT7-SCR2] xY187[pGADT7-eSRK2] grown blue clones and activated the reporter gene AUR1-C,MEL1, HIS3on the SD/-Trp-Leu-His/x-a-gal/AbA nutritional plate. The experiment of Y2HGold [pGBKT7-SCR2] xY187[pGADT7-eSRK28] indicated that there wasn’t interactions between different types of SRK-SCR. The experiment of Y187[pGADT7-SRKJ]xY2HGold[pGBKT7-THL1] showed clones and activated the reporter genes of AUR1-C, MEL1, HIS3and ADE2. Our studies have shown that there are interactions between class Ⅱ SCR, SRK and THL1. We have found that the interaction between class Ⅰ eSRK and SCR were higher than class Ⅱ eSRK and SCR, the same to THL and SRKJ. All above mentioned provides some insights into the molecular mechanism of the interaction and determination of the interaction motif between class Ⅰ type and class Ⅱ-type. The main results are as follows:1The amplification and sequence analysis of class Ⅰ and class Ⅱ SCR2and eSRK2Amplified eSRK2located between74bp-1321bp of the first exon and encoding416amino acids. We compared the SRK amino acid sequence of Type Ⅰ and Ⅱ by Vector NTI Advance and found that The application of Vector NTI Advance analysis of the Type Ⅰ and Ⅱ amino acid sequence and found that the similarity between the class Ⅱ eSRK higher than the class I type. The sequence similarity between S-2, S-5and S-15in class Ⅱ type ranged from93%to97.3%. The protein sequence similarities bewteen S-1、 S-7、S-8、S-14、S-11、S-24and S-29in class-Ⅰ were lower than in class Ⅱ. Comparsion analysis found that the amino acid sequence of type Ⅰ and Ⅱ showd great differences in the amino acid loci219-330and446-460. There is partial deletion exist in class type I.In this study, we amplified the second exon SCR2encoding65amino acids. Vector NTI Advance analysis showed that different SCR amino acid sequence had higher similarity with Arabidopsis. The amino acid similarity of haplotype SCR in the class-Ⅰ type ranged from20%to76%. The protein sequences similarity between different haplotype SCR in class-Ⅱ were63-94%. There are four cysteine residues in class-Ⅰ SCR is highly conserved, another four were not while all of the eight cysteine residues showed highly conserved.2The analysis of toxicity and autoactivation detection of recombinant bait plasmids and the toxicity detection of recombinant plasmid pGADT7-SRKThe colonies of pGBKT7-SCR2plasmid appeared in the SD/-Trp and SD/-Trp/-x-a-gal plates. Plasmids of pGBKT7-THL1grown well in SD/-Trp and SD/-Trp/-x-a-gal plates and the size, desity of colonies were similar to pGBKT7. It indicated that transferred proteins were not detrimental to yeast.The colonies of pGBKT7-SCR2and pGBKT7-THLl not appeard in the Trp/SD/-x-a-gal/AbA plate. Plasmids of pGBKT7-SCR2and pGBKT7-THLl in yeast cells did not activate the reporter gene AUR1-C and MEL1and did not happen of its own transcriptional activation.Colonies of pGADT7-SRKJ and pGADT7-eSRK2grown well in SD/-Leu plate. The size and growth time of control pGADT7were similar to pGADT7-eSRK2and pGADT7-SRKJ. That suggested that there are no toxic effects of expression of the protein of yeast. 3The detection of interaction between SCR2and eSRK2, eSRK28and SCR2, SRKJ and THL1Plasmids of Y187[pGADT7-SRKJ]×Y2HGold[pGBKT7-THL1] and Y2HGold [pGBKT7-SCR2×Y187[pGADT7-eSRK2] can grow blue colonies on the plate of DDO/x/A, and activated the reporter genes AUR1-C and MEL1. Plasmids of Y2HGold [pGBKT7-SCR2]xY187[pGADT7-eSRK28] not appeared blue colonies on the DDO/x/A, plate, and there wasn’t any interactions between them. In order to detect the possibility of interaction between each other, we put the bule colonies from the DDO/x/A plate on TDO/x/A and QDO/x/A. We found that there were bule colonies on TDO/x/A, while QDO/x/A not. These results indicated that reporter gene of HIS3was activated, and ADE wasn’t. While, colonies of Y187[pGADT7-SRKJ] xY2HGold[pGBKT7-THL1] and Y2HGold [pGBKT7-SCR28] xY187[pGADT7-eSRK28] showed blue on plates of TDO/x/A and QDO/x/A. These suggested that reporter genes of HIS3and ADE were activated. The fusion strains of Y2HGold[pGBKT7-SCR2]xY187[pGADT7-eSRK28] not grown in deficiency plates, and proved that there wasn’t any interaction between different haplotypes.