Analyze The Molecular Mechanisms Of OsMND1 In Regulating Meiosis Of Polyploid Rice

Polyploidy is an important manifestation of the evolution in species.From an evolutionary perspective,polyploid rice increases the risk of combination of favorable genes,which makes rice have the dual advantage of resistance and yield.But due to a doubling of gene dosage,meiosis of polyploid rice is confused and leads to low seed set rate and pollen fertility.Therefore,improving seed rate is critical for breakthrough of polyploid breeding.By employing the so-called strategy "breed super rice varieties via two superiority of distant hybridization and polyploidization",our lab has bred two lines of polyploid rice,which has high seed set rate and meiosis stablity,called PMeS lines(Polyploid Meiosis Stablity).OsMND1,involved in rice meiosis,is the homologous gene of yeast(S.cerevisiae)MND1(meiotic nuclear divisions 1)and has high homology with Arabidopsis.NMND1 plays a important role in homologous chromosome pairing,synapsis and recombination.Currently,this gene has't reported in detail about its functions in rice polyploid.Meiosis is a very important and special way of cell division,and it is significant for maintaining genomic stability and diversity.So it is whorth to carry out the regulatory mechanism of rice polyploid meiosis as the starting point to OsMND1.Clone and expression of OsMND1,constructing OsMNDl gene over-express vector and RNAi vector aim at OsMND1 gene have been finished before.Also we get OsMND1-overexpressed rice BLL-C-2x and OsMND1-RNAi DHN.In this study,the research materials are OsMND1-overexpressed rice BLL-C-2x,PMeS line HN-4x,OsMND1-RNAi DHN and non-PMeS line BLL-4X.The main finished expriments as follows:1.By bioinformatics prediction,we find that this gene does't have transmembrane domain and signal peptide,but has a helical-helical structure.This indicates that OsMND1 does not belong to the secreting protein and it has interaction with the other genes.By BLAST we know OsMND1 with DNA homology of yeast is up to 49.7%,protein homology is 41.7%,and with protien homology of other plants is more than 40%.And phylogenetic analysis explaines that this gene in vivo is very conservative.By Tissue-specific expression pattern analysis in PMeS line HN-4X,we found the gene is expressed mainly in meiosis spike and embryos.Subcellular localization analysis shows that the protien is mainly located in the nucleus.2.We make OsND1-overexpressed BLL-C-2x doubled and obtain positive plants of OsMND1-overexpressed rice BC+ by morphological,cytological and molecular(PCR,RT-PCR,southern blot)identification.BC+ compared with BLL-4x can increase seed rate and pollen fertility;Abortion statistics found main reason of abortion in BLL-4x is unfertilized,and in BC+ abortion happened mainly in the filling stage of embryonic development;After male meiocyte meiosis analysis and transverse section analysis,we find that BC+ can improve meiotic chromosome behavior and anther development of non PMeS BLL-4x.3.By PCR,RT-PCR,southern blot,we obtain positive plants of OsMND1-RNAi DHN.Compared with HN-4x in pollen fertility and seed rate,those of DHN are decreased.;Abortion statistics found main reason of abortion in DHN is unfertilized,and in HN-4x abortion happened mainly in the filling stage of embryonic development;Compared with HN-4x,DHN anther semi-thin sections show:OsMND1 interference cause the size of microsporocyte is inconsistent at meiosis.Tetrad microspores not closely fit whit tapetum,and microspores are stir dry and shrinkage in vacuolated pollen stage.Stain of chromosome are not deep and pollen grains are deformed at maturity;Meanwhile compared with PMeS strain HN-4x,meiotic behavior of OsMND1-RNAi HN-4x is adversely affected,including single line chromosome at zygotene,winding confusion chromosome at pachytene,and chromosomes can't properly dispersed at diakinesis,which lead chromosome to unequal segregation and backwardness.These results confirm the OsMND1 did participate in meiosis of polyploid Rice,and the gene may play a role in chromosome recombination process at the early stage.4.To further understand the mechanism of OsMND1,we analysis the expression of HOP2,DMC1,RAD51,ZEP1 by quantitative PCR in BC+ and DHN,the results is that the expression of HOP2,DMC1 and ZEP1 is up-regulated in BC+,and it is reduced in DHN.But the expression of RAD51 is little changed in BC+,and is up-regulated in DHN.This hints that OsMND1 is closely related to the meiosis.ZEP1 is the central element of synaptonemal complex,so this also explains that OsMND1 probably related with synapsis and pairing in meiosis.