Study On The Interactions Between Determinant Factors Of Self-incompatibility In Brassica Oleracea L. By A Yeast Two-Hybrid System

Ever since Darwin's pioneering research, the evolution of self-incompatibility has been regarded as one of the most prevalent evolutionary transitions in many flowering plants. The response of self-incompatibility is a major mechanism to prevent inbreeding and promote much-line hybrid, maintaining genetic diversity, which consists of male and female specificity genes at the S locus. In the self-incompatibility (SI) response of crucifers, the recognition of self-related pollen with the stigma epidermis which is effected through the activity of two tightly linked and highly polymorphic genes encoded by the S locus.①The S locus cysteine-rich protein gene SCR [also designated SP11] encodes a small hydrophilic and positively charged peptide that is localized to the pollen coat.②The S locus receptor kinase gene SRK has 7 exons encoding a single-pass transmembrane serine threonine kinase, which contains the N terminal extracellular domain (S domain), transmembrane Domain and a serine/threonine kinase activity of the intracellular C-terminal domains. The extracellular S domain was encoded by the first exon which is pollen ligand binding site, determined the specific self-incompatibility Identification response, led to the stigma papilla cells SI signal transduction, eventually leading self-incompatibility response.In this study a yeast two-hybrid system was adopted to investigate their potential interaction domains between SCR/SP11 and the extracellular fragment of SRK (eSRK). The full length SCRB3, SCRB3-1 and SCRB3-2 amplified from Brassica oleracea L.'B3'were cloned into the pGBKT7 to construct the recombinant bait plasmids which were then transformed into yeast Y2HGold cells by PEG/LiAc method. The DNA fragments including eSRKB3, eSRKB3-1 and eSRKB3-2 were cloned into the pGADT7 to construct the recombinant activation domain (AD) plasmids which were transformed into yeast Y187 cells. And these recombinant plasmids are no toxic and without autonomous activation effect to yeast cells. The yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK. The transmembrane domain of SRK is presence or not which will have no effect on studying the interaction between SCR and SRK. Our result will be used to further study the the interaction mechanism between SCR and SRK. Our results showed that:1 The amplification of different truncated fragments SCRB3-s and Sequence AnalysisThe different Truncated fragments SCRB3-s were amplified from leaf genomic DNA and bud RNA in Brassica oleracea L.'B3'by PCR and RT-PCR. By using 1%(w/v) agarose gel electrophoresis, the SCRB3-s fragments length are identical with the theory value of primer extension, and we find that SCRB3 contains a 302bp intron. Then subcloned into pGBKT7 and constructed the recombinant bait plasmids. The DNA sequencing analysis showed that pGBKT7-SCRB3-s recombinant bait plasmids contain SCRB3-S fragments, and the insertion site and the reading frame are correct, we finded that the sequences SCRB3-S are identical with SP1128 sequences. Doing sequence alignment of 6 different S haplotypes (S6, S8, S13, S28, S54, Sf2) by Vector NTI Advance 10.0, the consistency and similarity during different S haplotypes revealed high polymorphism of SCR gene. The SCRB3 sequence encodes 85 amino acids which include a complete signal peptide and a mature peptide; SCRB3-1 protein contains 3 amino acids (VEA) of C terminal peptide and complete mature peptide; SCRB3-2 contains 58 amino acids.2 The amplification of different Truncated fragments eSRKB3-s and Sequence AnalysisThe different Truncated fragments eSRKB3-s were amplified from leaf genomic DNA and bud RNA in Brassica oleracea L.'B3'by PCR and RT-PCR. By using 1%(w/v) agarose gel electrophoresis,the eSRKB3-s s fragments length are identical with the theory value of primer extension, and there is no intron in the eSRKB3 gene. Then subcloned into pGADT7 and constructed the recombinant plasmids. The DNA sequencing analysis showed that pGADT7-eSRKB3-s recombinant plasmids contain eSRKB3-s fragments, and the insertion site and the reading frame are correct, we finded that the sequences eSRKB3-s are identical with SRK28 sequences. The analysis of BLAS online showed that.the eSRKB3 encodes 426 amino acids, contains B-Lectin domain, SLG domain and PAN_APPLE domain. The eSRKB3-1 did not include the signal peptide sequence, which encoding the 30 to 426 amino acids; eSRKB3-2 encodes the amino acids between 140 and 426, which contains the hypervariable region of eSRK sequence.3 The toxicity and Autoactivation detection of recombinant bait plasmidsThe transformants of Y2HGold [pGBKT7-SCRB3-s] and Y2HGold [pGBKT7] were cultured on SD/-Trp plates at 30℃. After three days, we can find white well clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-SCRB3-s were no toxic to yeast cell. In addition, Y2HGold [pGBKT7-SCRB3.S] could grow on SD/-Trp,SD/-Trp/x-a-gal plates and could not grow on SD/-Trp/x-a-gal/AbA plates, and the colonies did not turn blue on SD/-Trp/x-a-gal plates which indicates that pGBKT7-SCR B3-5 do not activate the reporter gene AUR1-C, and MEL1 in yeast cells.4 The toxicity and Autoactivation detection of recombinant AD plasmid pGADT7-eSRKB3-sThe empty vector pGADT7 and pGADT7-eSRKB3-s were transformed into Y187,the ransformants of Y187 [pGADT7] and Y187 [pGADT7-eSRKB3-s] grew well on the SD/-Leu plates, while the yeast strain Y187 which was not transformed recombinant plasmid did not grow on SD /-Leu medium. It is suggested that recombinant plasmid pGADT7-eSRKB3-s was successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187. In addition, the white clones of Y187 [pGADT7-eSRKB3-s] grew well in SD/-Leu plates and the colonies did not turn blue on SD/-Leu/x-a-gal plates. The results indicate that recombinant plasmid pGADT7-eSRKB3-s do not activate the reporter gene MEL1.5 The interaction detection between different truncated fragments of SCRB3.S and eSRKB3-sThe analysis of bioinformatics software shows that SCRB3 can mutually recognize with SRKB3 in the space to form a fusion protein.The recombinant plasmids were successfully transformed into the diploid yeast mating cell, the four group of Y2HGold[pGBKT7-SCRB3-2]xY187[pGADT7-eSRKB3-2], Y2HGold[pGBKT7-SCRB3-2]xY187[pGADT7-eSRKB3-1],Y2HGold[pGBKT7-SCRB3-1]xY187[pG ADT7-eSRKB3-2]andY2HGold[pGBKT7-SCRB3-1]xY187[pGADT7-eSRKB3-1] could emerge significantly blue clones, then remove blue clones on DDO/x/A plates to QDO/x/A plate, after four days, we can find the blue clones on the plate, which may indicate that activate the reporter gene AUR1-C, MEL1,ADE2 and HIS3. The resoults show that the yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK.The transmembrane domain of SRK is presence or not which will have no effect on studying the interaction between SCR and SRK. Key words:Brassica oleracea L. var capitata L.; self-incompatibility; S-locus receptor kinase (SRK); S-locus cysteine-rich protein (SCR); yeast two-hybrid...