Cloning And Functional Study Of OsPKS2 In Controlling Rice Pollen Wall Development

Rice is one of the most important food crops in the world.More than 50%of the world's population is rice-based.Currently,our most important mission is to increase rice production.The promotion and application of hybrid rice in production has made a significant contribution to rice production in China,even the whole world.The male sterility in rice fertility is the basis for effective utilization of heterosis.In the male reproductive development of rice,the development of anthers and pollen act as important roles.At the same time,the normal pollen wall formation process is an irreplaceable part in the development of anthers.During the growth and development of rice,pollen wall protects male gametes from various environmental stresses and pathogen attack,and promotes pollen germination.The sporopollenin biosynthesis in tapetum cells is crucial for the formation of pollen wall.In recent years,much progress has been made in the study of sporopollenin metabolism in the development of the pollen wall of Arabidopsis,a monocotyledonous plant.However,little is known about the molecular mechanism of sporopollenin synthesis in rice pollen wall formation.Therefore,in order to know more about pollen development in rice,this study screened mutant library of indica rice varieties induced by EMS,thus we obtained a male sterile mutant material called ospks2-1.Then conducted a series of analysis and identification.Below are key research findings:1.Phenotypic analysis of mutant ospks2-1.ospks2-1 showed normal vegetative growth,inflorescence and flower morphology,but it had smaller and yellower anthers than 9311.The number of pollen grains of ospks2-1.anther is less than 9311,and pollen grains cannot be stained by I₂KI solution.Anther semi-thin sections,scanning electron microscopy,and transmission electron microscopy revealed that the mutant male sterility phenotype was mainly caused by defects in pollen wall formation.2.Mapped clones of OsPKS2.We used the MutMap method to isolate and identify SNP mutations that caused the mutant male sterility phenotype.This mutation is located in the coding region of the LOC_Os07g22850 and this mutation results in amino acid substitutions from Ser163?AGC?to Asn?AAC?of its encoded protein.Further linkage analysis revealed that the mutation was co-segregated from the male sterile phenotype in the F2 population.Successful knockout mutants obtained by knockout of the LOC_Os07g22850 gene performed the same as the ospks2-1 sterile phenotype.Therefore,we determine that LOC_Os07g22850 is OsPKS2.3.Phylogenetic analysis of OsPKS2 related proteins.OsPKS2 encodes a plant-specific type III polyketide synthase.We analyzed its related proteins in monocotyledonous plants and found that OsPKS2 is a homologous protein of Arabidopsis PKSB/LAP5 and is present in both monocotyledons and twins.The leaf plants are conserved among them,thus suggesting that the biological function of OsPKS2 in pollen wall formation may be similar to that of PKSB/LAP5.4.OsPKS2 expression pattern analysis.QPCR,GUS histochemical staining and in situ hybridization showed that OsPKS2 was specifically expressed in the tapetum during pollen development,further suggesting that it may be involved in rice pollen development.The results of subcellular localization showed that OsPKS2 protein was mainly located on the endoplasmic reticulum.This is similar to the protein localization of PKSB/LAP5 in Arabidopsis,further suggesting that they may have similar functions.5.Expression analysis of pollen development related genes in ospks2-1.We used QPCR to analyze the expression of pollen development-related genes in 9311 and ospks2-1.Anthers,the results suggest a possible feedback regulation of sporopollenin precursor metabolism in rice pollen wall development.In addition,the pollen development pathway involved by OsPKS2 may be independent of the pollen development pathway regulated by OsDEX1.In conclusion,our results demonstrate that OsPKS2 is a key gene controlling male fertility in rice and it participates in the synthesis of sporopollenin precursors in tapetum cells to regulate pollen wall formation.