Interaction Analysis Of Stigma S-domain Receptor-like Kinase With Pollen PCP-B In Brassica Oleracea

Brassicaceae plants have a more sophisticated dry stigma than wet stigma,which lack water and small molecular secretions on the stigmatic papillae cell surface,can specifically recognize compatible and incompatible pollen landing on the stigma.Only compatible pollen can induce papilla cells to secrete water and other nutrients to promote pollen hydration and germination,while incompatible pollen can not induce this secretory process,pollen hydration and germination are inhibited.This highly regulated secretory process not only provides the first checkpoint in pollen selection during early interaction of dry stigmas with compatible or incompatible pollens and accepting compatible,but also provides an ideal model system for studying the cell-cell signal transduction mechanism during double fertilization.Therefore,further exploring the molecular mechanism of the interaction between pollen and stigma can help us realize the artificial regulation of compatible and incompatible pollination,and promote seed production,which have great scientific value and practical significance.At present,little is known about the molecular mechanism of exocyst mediated secretion of dry stigma papilla cells induced by compatible pollen,but the signal transduction mechanism in the early process of sporophytic self-incompatibility in Brassica crops has been well studied,Exo70A1,GLO1 and PLD α 1 are essential factors for dry stigma to accept compatible pollen in Brassica napus,and the polar secretion mediated by the exocyst complex in dry stigma papilla cells is involved in the response of stigma acceptance compatible pollen and rejection incompatible pollen.After the mature pollen landing on the dry stigma surface,pollen must and have to induce the stigma to release water and other substances,and pollen finish hydration and restore its metabolic activity,then can complete the subsequent physiological process.Therefore,to isolate and identify the key interacting factors from pollen and stigma which causing polar secretion of dry stigma papilla cells has become a breakthrough to explore the compatible signaling pathway.Previous studies have shown that PCP-B family proteins are the key pollen factors regulating compatible pollen hydration on the dry stigma,but its kinase receptors in the stigma have not been isolated,and its regulatory mechanism is still unclear.Therefore,isolation and identification of the regulatory elements of compatible pollen hydration on dry stigma can help us to elucidate the molecular mechanism of dry stigma specifical acceptance of compatible pollen and the early events of pollen-stigma interaction,which have great significanceIn this study,we analyzed the tissue expression of BoPCP-B and S-domain receptor-like kinase in Brassica oleracea by semi-quantitative RT-PCR,selected the BoPCP-B genes expressed in anthers and S-domain receptor-like kinase genes expressed in stigmas.The S-domain receptor-like kinase interacting with BoPCP-B were screened by yeast two hybrid,then the GST Pull-down techniques were used to further clarify the protein interaction between PCP-B and S-domain receptor-like kinase.Finally,the intracellular interacting proteins of S-domain receptor-like kinase were identified by c DNA library screening,which lays the foundation for further analysis the function of S-domain receptor-like kinase interacting with PCP-B in pollen hydration in B.oleracea.The main results are as follows:(1)Screening of anther expressed PCP-B and stigma expressed S-domain receptor-like kinaseWe used c DNAs of the leaves,stigmas,sepals,anthers and petals on the day of flowering of B.oleracea ‘A4’as materials.We used to semi-quantitative RT-PCR to screen BoPCP-B genes expressed in anther and S-domain receptor-like kinase genes expressed in stigma,Three PCP-B genes(BoPCP-B4 、 BoPCP-B15 and BoPCP-B22)and ten SD receptor kinases(BoSD-RLK10、BoSD-RLK1、BoSD-RLK34、BoSD-RLK41、BoSD-RLK32、BoSD-RLK9、BoSD-RLK47、BoSD-RLK3、BoSD-RLK6 and BoSD-RLK37)were detected in the above five tissues.The results showed that three PCP-Bs were expressed in the anther,and had the highest expression level in petals than other tissues.Among the ten SD receptor kinases,there were eight receptor kinases were expressed in the stigma,however,BoSD-RLK6 only expressed in the sepals,and BoSD-RLK37 was not expressed in the five tissues.Among the eight receptor kinases expressed in stigma,BoSD-RLK47 and BoSD-RLK34 were specifically expressed in the stigma,BoSD-RLK3,BoSD-RLK9 and BoSD-RLK10 were highly expressed in stigma,and BoSD-RLK1 was expressed in all 5 tissues,and the expression level was relatively low.(2)Yeast two-hybrid screening of stigma S-domain receptor-like kinase interacting with anther PCP-BAccording to the expression analysis of BoPCP-B and S-domain receptor-like kinase in pollen and stigma,we cloned three BoPCP-Bs(BoPCP-B4,BoPCP-B15 and BoPCP-B22)and eight SD-receptor kinase genes(BoSD-RLK10,BoSD-RLK1,BoSD-RLK34,BoSD-RLK41,BoSD-RLK32,BoSD-RLK47,BoSD-RLK3 and BoSD-RLK9),and analyzed their sequences,the results showed that BoSD-RLK47 had base deletion while BoSD-RLK3 had four base addition,which led to a change in its coding frame,the other gene sequences were consistent with the corresponding target genes’ sequence.The coding sequences of three BoPCP-Bs(BoPCP-B4,BoPCP-B15 and BoPCP-B22)were subcloned into p GBKT7 plasmid,and the extracellular domain coding sequences of six receptor kinases(BoSD-RLK10,BoSD-RLK1,BoSD-RLK34,BoSD-RLK41,BoSD-RLK32 and BoSD-RLK9)were subcloned to p GADT7 plasmid,to detect the interaction by yeast two-hybrid technique.The results show that among the 18 combinations,a total of four combinations interact with each other,they are BoSD-RLK34×BoPCP-B15,BoSD-RLK34×BoPCP-B4,BoSD-RLK10×BoPCP-B15 and BoSD-RLK10×BoPCP-B4.Among them,there are three combinations firstly appeared in each repetition,they are BoPCP-B4 and BoSD-RLK34,BoPCP-B15 and BoSD-RLK34,BoPCP-B15 and BoSD-RLK10.(3)GST Pull-down detection of anther PCP-B interacting with stigma S-domain receptor-like kinaseTo further verify the results of yeast two-hybrid,we used GST Pull-down to further detect the three combinations firstly appeared in each repetition.The coding sequences of BoPCP-B4 and BoPCP-B15 were subcloned into p GEX-6P-1 to construct the vectors p GEX-6P-1 × BoPCP-B4 and p GEX-6P-1 × BoPCP-B15,the coding sequences of BoSD-RLK34 and BoSD-RLK10 were subcloned into p ET-43.1a(+)to construct the vectors p ET-43.1a × BoSD-RLK34 and p ET-43.1a × BoSD-RLK10.The recombinant plasmids were transferred to BL21 to express the target protein by IPTG induction.After in vitro incubation and Western blotting,the results showed that there was interaction between BoPCP-B4 and BoSD-RLK34,BoPCP-B15 and BoSD-RLK34,BoPCP-B15 and BoSD-RLK10.(4)Bioinformatics analysis of BoSD-RLK34 and BoSD-RLK10 encoding sequencesUsing the highly self-incompatibility ‘A4’ stigma c DNAs of Brassica oleracea‘A4’as template to clone the coding sequences of BoSD-RLK34 and BoSD-RLK10.Sequence analysis showed that the BoSD-RLK10 coding length was 1977 bp,encoding658 amino acids,and the BoSD-RLK34 coding length was 1950 bp,encoding 649 amino acids.BoSD-RLK34 and BoSD-RLK10 were all hydrophilic proteins with transmembrane structure.CDD online analysis of conserved domains showed that both proteins have extracellular conserved domains S＿locus＿glycop and PAN＿AP＿plant,intracellular DUF3403 domain and PKc＿like domain containing serine/threonine kinase.Compared with SRK,the most significant difference is the lack of a conserved intracellular domain DUF3660.Protein structure predictions indicated that BoSD-RLK34 and BoSD-RLK10 were mainly random coil.Haplotype specificity analysis of BoSD-RLK34 and BoSD-RLK10 showed that both genes had no haplotype specificity.(5)Screening the interaction proteins of BoSD-RLK10 kinase domain in stigmaThe intracellular cloning sequence of BoSD-RLK10 was subcloned into p GBKT7,and yeast expression vector p GBKT7×BoSD-RLK10 was constructed.We screened the interacting proteins of BoSD-RLK10 kinase domain using yeast two-hybrid from B.oleracea stigma two hybrid c DNA library.The results show that there was a total of 65 candidate protein points,and 35 proteins were identified by PCR and sequencing,which may involve in multiple biological-processes.Among them,equilibrative nucleotide transporter 7,protein transparent testa 12 and coatomer subunit alpha-1have transport activity and are involved in protein transport,and protein transparent testa 12 is involved in anther cracking,flavonoid metabolism and pollen development,coatomer subunit alpha-1 participate in the vesicle-mediated protein transport process.