The Molecular Mechanism Of CML25 Involving Regulation 2n Pollen Strong Competitive Role To Produce Polyploidy Offsprings Of 'Y18' In Rose

CML25 protein belongs to the family of calmodulin-like proteins(CMLs),which regulates pollen germination and pollen tube growth by binding to Ca2+.There are competitions in different ploidy pollen during germination.The differential expression of CML25 gene in pollen germination at different times and in different ploidy pollen affects the process of pollen germination in the body,which may be the reason for the competitive strength of different ploidy pollen.Using colchicine to induce 2n pollen,gene cloning,protein structure analysis and function prediction,and green fluorescent protein(GFP)labeling and mapping technology,the CML25 gene was involved in the regulation of pollen competition,and the following conclusions were obtained:(1)'Y 18' as Chinese ancient rose diploid variety,naturally has 1.39%of 2n pollen,while tetraploid rose 'D2'((?))× 'Y18'((?))can produce tetraploid offspring with a ratio of 43.33%,indicating that 2n pollen has a strong effect.Different treatments of colchicine induced 2n pollen of 'Y18',and 0.1%colchicine had the best effect on 3 days,and the induction rate reached 3.68%.The ultrastructure of induced 2n pollen,natural 2n pollen and In pollen was observed.The induced 2n pollen was similar to the natural 2n pollen.The pollen strips were lighter than the In pollen strips.The pores were less dense,and there were almost no pores near the extreme.The differences are in line with the direction of evolution.(2)The transcriptome of 'Y18' pollen and germinated 6 h pollen tube were sequenced,and 151 genes related to pollen were screened,which were mainly involved in pollen development,pollen and stigma recognition,pollen germination and pollen tube growth process;There are 28 genes,of which the CML25 gene sequence has a pair of EF-Hand domains,which belong to calcium binding protein and participate in signal transduction mechanism and are hydrophilic proteins.Ca2+ may enter the cytoplasm from apoplasts through Cyclic nucleotide-gated ion channels(CNGCs)and act on CML proteins.(3)The qPCR results showed that the expression of CML25 gene changed before and after pollen germination,and the expression of pollen germination was significantly different at 8 h.At this time,pollen entered the stigma and showed strong competition,indicating that CML25 gene exercised its biological function at this stage.CML25 subcellular levels are localized to the pollen cytoplasm and cell wall.The GFP protein was labeled with CML25 protein,and it was found that the expression of CML25 protein in 2n pollen tube was higher at the same germination time,which was speculated to be related to the strong competition of 2n pollen.At 6 h after germination,the expression level of CML25 in the pollen tube gradually increased from the germination hole to the pollen tube tip,which was consistent with the Ca2+ distribution.Adding inhibitor bisphenol A(BPA),In and 2n pollen showed pollen tube bending,apical expansion,pollen tube wall rupture,fluorescence gradient disappeared in pollen tube,indicating that BPA changed Ca2+ concentration and CML25 expression,thereby inhibiting the growth of pollen tubes,leading to abnormal growth.In summary,the diploid rose and the tetraploid rose produce 43.33%of the tetraploid offspring,which shows the competitive strength of 2n pollen.After the induction of colchicine,the percentage of 2n pollen is increased to 3.96%,which provides for subsequent experiments.The basic material,subcellular localization,found that CML25 was mainly localized in pollen cell wall and cytoplasm,and the expression level in 2n pollen was higher than that in In pollen.During germination,CML25 mainly binds to Ca2+.Adding bisphenol A changes the Ca2+ concentration and inhibits the expression of CML25,thus affecting the growth of pollen tubes.