Mechanism And Regulatory Network Of PTB1 On Rice Seed Setting Rate

The seed setting rate is one of the most important components of four major rice yield traits,which directly affects the yield of rice.Nowadays,a large number of genes or QTL associated with rice yield has been isolated and cloned in many aspects,such as panicle number,grain number per panicle and grain weight.At the same time,these rice yield related genes has started to be used in high-yield rice breeding by breeders,it showed great theoretical significance and utilization potentiality.However,gene associated with seed setting rate controlling is poorly understood,which made an obstacle for rice yield further improvement,it's own to various stress from the environment which significantly affected the seed setting rate.In angiosperms,a double fertilization followed by flowering and pollination.Plant sperm cell existence a defection with no flagella and resulted can't free-swimming,therefore pollen and pollen tube have emerged to petect and transport sperm cell to embryo sac.While the pollen grain landed on stigma,it experienced adhesion,recognition,hydration before germination and pollen tube formation,subsequently the pollen tube growth in style of papilla cells space and enter into the pistil,the pollen tube growth in stigma extension continues its downward and through the TTS to reach the micropylar.Embryo sac released attraction signals to guide pollen tube growth into ovule.Then pollen tube growth stopped and ruptured into two sperm cell.During the pollen tube transmission process,multi-signals involved in pollen tube growth and guidance,it followed by a lot of interations between male-female.A female-sterility gene PTB1(Pollen Tube Blocked 1)was been cloned from the mutant,and the candidate gene encodes a E3 ligase which may involved in ubiquitination,and the mutant shows growth defection in TTS.Our results suggested that PTB1 can control pollen tube growth to regulate rice seed setting rate.For further,we investigated the mechanism of PTB1 on regulating rice seed setting rate by a protein-protein interact methods.Furthermore,RNA-Seq of the pistil in different pollen tube growth period,we get a lot of pollen tube growth regulate factors,these results laying the foundation for a network of PTB1 regulation of pollen tube growth.The main results are as follows:1.The expression pattern of PTB1 gene was defined.qRT-PCR and GUS histochemical staining showed that PTB1 is a constitutively expressed gene,it highly expressed in pistil and pre-emergence inflorescence which is consistent with the function of pollen tube growth controlling.2.PTB1 is crucial for rice seed setting rate controlling.Different transgenic lines showed varieties seed setting rate,and the seed setting rate is significantly correlated to gene relative expression level and normal growthed pollen tube rate.We conclued that PTB1 expression positively regulates PT growth in a dose-dependent manner which positively regulate the rice seed setting rate.3.The reason of abnormal temperature leads to decreased rice seed setting rate own to PTB1.Deal with high or low temperature during rice booting stage resulted low seed setting rate,as well as the gene expression level decreased and the pollen tube growth abnormal under high and low temperature,it indicates that the expression level of PTB1 is the main reason which affects rice seed setting rate.However,methylation sequencing of gene promoter,there's no significant methylated site between WT and MT.4.A high quality yeast two hybrid library had been constructed.According to PTB1 expression pattern,a young panicles of rice cDNA library was constructed for yeast two hybrid.The total CFU=9.6×107 and gene insert size=1.3Kb.The library is also suitable for the screening of reproductive development related genes.5.Using PTB1 as bait to capture the prey in yeast library,we obtained 67 clones,it contained 9 candidate proteins which reading frame correctly.Among of them,OsPPI1 was captured 16 times with different protein frame or insert size,the other candidates was detected 1 or more times.6.The full-length protein of OsPPIl,OsPORB and MSP can interact with PTB1 in yeast and Nit cell.According to the prediction of protein functional domains,the PTB1 and OsPPI1 were truncated for finding the functional domain associated with PTB1-OsPPI1 interaction,we found that the trans-membrane of PTB1 and C-terminal of OsPPI1 is essential for their interaction.7.OsPPI1 is essential for pollen tubes growth in the style.Knocked out of OsPPI1 by using the strategy of CRISPR-Cas9,gene knocked out plants exhibit significantly seed setting rate decrease own to the abnormal pollen tubes growth in stigma.The conclusion suggests that PTB1 and OsPPI1 may belongs to a same pathway for pollen tube growth regulating.8.OsPPI1 can interact with OsHA7 in yeast,as well as the crucial function domain OsPPI1-N252 and OsHA7-c115 were confirmed for their interaction.We supposed that OsPPI1 may regulate PM-H+-ATPase activity to affect pollen tube growth.At the same time,Multiple genes involved in BR-signal transduction were expressed differentially between 202R and fs,confirmed that OsPPI1 response for BR-signal transduction.9.A large number of differencial expression genes were founded through RNA-Seq analysis.An interesting phenomenon of pollen tube growth in 0 to 30min and 30min to 60min after pollination may belongs to different biology process.10.Annotation of differencial expression genes shows that CaM/CML?CHX?CHIT/Pectin?Hsp and protease inhibitor gene family associated with pollen tube growth in stigma.As well as a part of plant hormone-related,Pathogenesis-related and stress-related genes occurred significantly up-regulated expression.A lot of these genes had been enriched in protein processing in endoplasmic reticulum and oxidative phosphorylation pathway,we suppose that the two pathway contained PTB1-OsPPI1-OsHAs regulates pollen tube growth.