Molecular Mechanism Of The Rice Indica-japonica Hybrid Fertility Gene S5 And Analysis Of The Rice Peptide Transporter Family

Rice is one of the most important food crops in the world,and the yield improvement of rice can actively alleviate the food shortage problem.Using the great heterosis between indica and japonica subspecies of rice is an effective way to improve the rice yield.However,we need to solve the problem that the rice indica-japonica interspecific hybrids general are low fertility or sterility through the cloning and mechanism research of the indica-japonica hybrid fertility and the wide compatibility genes.In 2008,our lab cloned the S5(ORF5)gene which controls the indica-japonica hybrid female gametophyte(embryo-sac)fertility.Our recent genetic analysis confirmed that three closely linked genes:ORF3,ORF4 and ORF5 within the S5 loci are essential for the function of S5 and they have different genotypes among indica,japonica and wide compatibility varieties(ORF3+/-,ORF4+/-and ORF5+/-/n).ORF5+ can cause indica-japonica hybrid sterility only when the genotype of ORF4 is ORF4+r while ORF3+ can partially restore the fertility of indica-japonica hybrids.The main purpose of this study is to figure out the molecular mechanism that how these three genes of the S5 loci causing the indica-japonica hybrid embryo-sac abortion from the protein characteristic,protein interaction and gene expression analysis.ORF3+/-proteins are members of heat shock protein 70(HSP70)family,they both contain the signal peptide and the HSP70 domain.However,they only have the N-terminal ATPase domains of HSP70,while the C-terminal binding domains are completely missing.Yeast BiP(Binding protein)gene temperature-sensitive mutant function complementary showed that ORF3+/-could not restore the yeast mutant phenotype.We expressed the ORF3+/-proteins by prokaryotic expression and preliminary purificated them.We obtained the CDSs of ORF4+/-by RT-PCR.ORF4+/-encode unknown proteins,which have low homology with other proteins.ORF4+/-proteins both have the signal peptide and ORF4+protein contains a transmembrane domain at the C terminus.The transmembrane topology of ORF4+ was determined by the split-ubiquitin system.The result showed that the C-terminal of ORF4+ was located within the plasma membrane.Growth curve showed that the prokaryotic expression of ORF4+/-is toxic to E.coli.We used the Pichia pastoris expression system with the pPIC9 vector and SMD1168 strain to carry out the secretion expression of ORF5 in the culture medium,and high protease activity was obtained.Through optimizing the methods of ion exchange and gel filtration chromatography,the mature ORF5+ was purified.A preliminary enzymology of ORF5+ was carried out using the medium supernatant of Pichia pastoris expression,the result showed that the optimum temperature of ORF5+ was 3 7?,and optimum pH was 7.0.Transcriptional activation test in the yeast two-hybrid showed that propeptides of ORF5+/-had transcriptional activation.Yeast two-hybrid,bacterial two-hybrid and BiFC detection results all indicated that there are no interactions among ORF3+/-,ORF4+/-and ORF5+/-/n proteins.In order to find out the pathways S5 involved in,yeast two-hybrid system was used to obtain the interacting proteins of ORF3,ORF4 and ORF5.We screened the cDNA library of rice young panicle at the pollen-mother cell formation stage and some interaction proteins of ORF5-or ORF4-were identified.The ovaries at the functional megaspore stage of BallilaORF5+(ORF3-ORF4-ORF5+)transgene-negative and-positive plants were used for microarray analysis.Through the functions of some differentially expressed genes,we thought that ORF5+ might be involved in the programmed cell death(PCD).We also found that the expression of some endoplasmic reticulum(ER)stress-responsive genes,including OsbZIP50,increased significantly in the Ballila0ORF5+ plants.The expression of ORF3 rosed by 3.06-fold(P=0.0006)in the BallilaORF5+ plants,and a cis-regulatory element response to the ER stress was identified within the promoter of ORF3.Some previous researches showed that the expression of ORF3 was regulated by OsbZIP50.The RT-PCR result showed that OsIRE1-mediated mRNA splicing of OsbZIP50 existed in the BallilaORF5+ plants.Ovaries at functional megaspore stage of BallilaORF5+ plants were used to verify the expression of ER stress-responsive genes by qPCR analysis.The analysis showed that the expression patterns of most genes were consistent with the microarray result.These results indicated that ORF5+ had induced ER stress in the ovary of Balilla plant,and ORF3 was an ER stress-responsive gene.Ovaries at mature embryo-sac stage of BallilaORF5+ plants were used to analyze the expression of the ER stress-responsive and PCD-related genes by qPCR.The result showed that comparing with the functional megaspore stage,the expression levels of the ER stress-responsive genes at mature embryo-sac stage increased significantly.The expression of OsBI1,a modulator gene of ER stress-induced PCD increased significantly as well as the PCD-related genes:OsCP1,OsKODl and Hsr203j.These results indicated that the ORF5+-induced ER stress might provoke abnormal PCD and then caused the embryo-sac abortion.qPCR results also showed that in the ovaries at mature embryo-sac stage of BalillaORF3+ORF5+(ORF3+ORF4+ORF5+)homozygous plants,the expression of above-mentioned ER stress-responsive and PCD-related genes were maintained at the level of wild-type Balilla(ORF3-ORF4+ORF5-)plants.This result suggested that ORF3+ might act as a suppressor of the ORF5+-induced ER stress.Based on these results,combined with the function genetic validation of ORF3,ORF4 and ORF5,and the protein subcellular localizations,we hypothesize the molecular mechanism of S5:ORF5+ is secreted to the cell wall and hydrolyzes a substrate causing an ER stress signal which is transduced into the cell by plasma membrane-localized ORF4+.The ER stress subsequently actuates the OsIRE1-mediated splicing of OsbZIP50 mRNA producing OsbZIP50-S,a transcription factor that turns on expression of ER stress-responsive genes.When the genotype of ORF3 is ORF3-,ER stress induces PCD-related genes causing anomalous PCD,which leads to embryo-sac abortion,despite the presence of OsBI1.The ER stress is prevented in the presence of ORF3+ thus producing normal female gametes.Thus,ORF3,ORF4 and ORF5 are components involved in different stages of the ER stress-induced programmed cell death pathway regulating the indica-japonica hybrid embryo-sac fertility.This research studied a comprehensive molecular mechanism of S5 for the first time,and reveals a new molecular mechanism for the regulation of indica-japonica hybrid fertility.This study also provides a reference for the later research on the molecular mechanism of indica-japonica hybrids fertility and reproductive isolation of other species.The in-depth molecular mechanism research of S5 and the cloning of more and more indica-japonica hybrid sterility genes will provide the theoretical guidance to the rice breeding and may make it possible to use the indica-japonica subspecies heterosis to increase the rice yield.Peptide transporter(PTR)family whose member can transport di-/tripeptides and nitrate is important for plant growth and development.Although the rice(Oryza sativa L.)genome has been sequenced for a few years,a genomic survey,characterization and expression profile analysis of the PTR family in this species has not been reported.In this study,we report a comprehensive identification,characterization,phylogenetic and evolutionary analysis of 84 PTR family members in rice(OsPTR)as well as their whole-life expression patterns.Chromosomal distribution and sequence analysis indicate that 66.7%of OsPTR members are involved in the segmental and tandem duplication events.It suggests that genome duplication might be a major mechanism for expansion of this family.Phylogenetic analysis suggested that the OsPTR proteins could be classified into five major subfamilies.Three highly conserved motifs were identified in most of the OsPTR members.Meanwhile,expression profile of OsPTR genes has been analyzed by using Affymetrix rice microarray and qPCR in two elite hybrid rice parents,Minghui 63 and Zhenshan 97.Based on hierarchical clustering,expression pattern of OsPTR genes could be divided into five major groups.Seven genes are found to exhibit either preferential or tissue-specific expression during different development stages of rice.Under phytohormone(NAA,GA3 and KT)and light/dark treatments,14 and 17 OsPTR genes are differentially expressed respectively.Ka/Ks analysis of the paralogous OsPTR genes indicates that purifying selection plays an important role in function maintenance of this family.These investigations add to our understanding of the importance of OsPTR family members and provide useful reference for selecting candidate genes for functional validation studies of this family in rice.