| Photoperiod and thermo sensitive genic male sterile(PTGMS)rice is the basis of two-line hybrid rice,and the mechanism of photothermo regulation of male fertility has always been the basic research focus of two-line hybrid rice.Our previous study found that lipids and flavonoids were involved in the regulation of male sterility in Photoperiod Sensitive Male Sterile Rice D52 S and Nongken 58S(NK58S).Based on previous studies,this study used photoperiod and thermo sensitive genic male sterile rice PA64 S as material,and and different fertility rice materials were obtained through different temperature treatments.The fertility phenotype analysis,cytological observation,transcription level,protein level,metabolism levels explore the regulatory role of lipids and their derivatives in fertility regulation of PA64 S.The main findings are as follows:1.Under the light of 14.0h,PA64 S was treated at 21 ℃ and 28 ℃ respectively when spikelets just entered the the formation stage of pistil and stamen,and the pollen staining rate and self pollination rate of the treated plants were counted.The results of 2017-2019 showed that the average pollen staining rate of PA64 S treated at 21 ℃ was 37.27%,and the average seed setting rate was 33.42%.The PA64 S pollen treated at 28℃ was almost completely aborted,with the pollen staining rate of only 0.22% and a seed setting rate of only 0.28%.These results indicate that the fertility of PA64 S is regulated by temperature,and PA64 S has obvious sensitivity to temperature response.2.The anthers of PA64 S treated at 21℃ and 28℃ at different developme.ntal stages were observed through transmission electron microscopy.The results showed that under the condition of sterility,the anther wall of sterile PA64 S at stage 11 was still a typical four-layer structure.There were complete nuclear organelles in tapetum cells,and neither tapetum nor middle layer was condensed and degraded.PCD occurred in tapetum cells of fertile PA64 S anthers at the same time.At the 12 th stage of anther development,sterile PA64 S anther wall was still four layers structure,tapetum had no obvious degradation,pollen exine had obvious irregular accumulation phenomenon,and the pollen at this time had been seriously atrophic and sterile.At the same time,fertile PA64 S tapetum was almost completely degraded,containing mature pollen full of starch grains,and the three-layer structure of pollen outer wall was clear.3.The development of anther cuticle and pollen exine of the two materials were observed by SEM.Compared with sterile PA64 S,the cuticle on the surface of fertile PA64 S anther was more regular,and the accumulation of sporopollenin was more compact.At the same time,it can be observed that the pollen grains in sterile PA64 S were atrophic and sterile.The results showed that the accumulation patterns of anthers and pollen walls of PA64 S plants with different fertility were different under 21 ℃ and 28℃,that is to say,there were differences in lipid metabolism involved in cuticle and sporopollenin.4.By LC-MS,lipid group analysis of PA64 S spikelets with different fertility was performed.It was found that in fertile PA64 S,sterols(including Ac Hex Cm E,Ac Hex Si E,and Ac Hex Zy E)were all up-regulated,and the content of Ac Hex Zy E was fertile PA64 S.2.1 times.The content of OAHFA and PEt of fertile PA64 S was also significantly higher than that of sterile PA64 S.Both phospholipids,PE and PG,also showed the same expression pattern.It is speculated that the differences in the content of lipid components in fertile PA64 S and sterile PA64 S affect the composition pattern of anthers and pollen walls.5.The transcriptome sequencing of PA64 S spikelets with different fertility was carried out at 21℃ and 28℃,and the differential genes(DEGs)expressed significantly in sterile PA64 S and fertile PA64 S were screened by padj<0.05 combined with |differential multiple|>1.The results showed that 1789 DEGs were screened in the tetrad stage of anther development,and 5084 DEGs in the following microspore release stage.Further analysis of GO enrichment of stage 9 showed that DEGs were mainly involved in cell wall tissue or biogenesis,hormone response,lipid response,and a series of other metabolic processes.This suggests that more genes were involved in the regulation of fertility,and lipid metabolism and hormone response pathway were involved in the regulation of fertility of PA64 S.6.The related genes involved in the synthesis,transport and PCD of anther cuticle and sporopollenin were analyzed by q PCR.The genes related to lipid synthesis and flavonoid synthesis were up-regulated in fertile materials,and the expression increased with the development of anthers.The results showed that the genes involved in sterile PA64 S lipid synthesis,flavonoid synthesis and PCD process of anther wall were abnormally expressed at the transcription level,which further affected the formation and transportation of ubbelonite and sporopollenin necessary for anther development and pollen formation.7.The results showed that ACOS12 and PKS2,which were related to fertility,were also expressed in PA64 S spikelets.The expression of ACOS12 protein increased with the development of the stage,and the content of ACOS12 protein in sterile spikelets was significantly higher than that in fertile ones.8.The results showed that the motif of the core response element of gibberellin existed in the CYP704B2 and PKS1 promoters involved in the synthesis of anther lipids and the EAT1 promoters involved in the degradation of tapetum.There are also gibberellin responsive elements in the promoter of ABCG26.At the same time,the content of gibberellin and the content of GAMYB,the positive regulator of gibberellin in rice,were determined.The results showed that the content of GAMYB gene was consistent with the genes related to lipid metabolism,which was also up-regulated in fertile materials.The above results indicate that gibberellin may involve in the regulation of PA64 S lipid metabolism genes.In this study,we analyzed spikelets of PA64 S with different fertility from different levels.The synthesis and transportation of lipids during PA64 S anther development affects the lipid accumulation of the pollen exine and the anther cuticle of PA64 S,further affecting the maturity of the pollen exine and the anther cuticle and the fertility of PA64 S pollen.The lipid content and type of PA64 S spikelets are also one of the factors affecting pollen fertility.In addition,gibberellin may involve in the development of PA64 S anther,and and then affect pollen fertility.This study provides basic information for further exploring the mechanism of fertility regulation of photoperiod and thermo sensitive genic male sterile rice. |
