Screening And Functional Study Of Differentially Expressed Genes Of Female Gametophyte During The Period Of Free Nuclei Mitosis In Pinus Tabulaeformis Carr.

Female sterility is a common phenomenon in the plant kingdom,which will lead to a decline in the seed setting rate and affect the reproduction and utilization of germplasm resources.Therefore,strengthening the research on plant female infertility has important theoretical and practical significance,but the current reports are only limited to angiosperms such as Arabidopsis thaliana,and systematic research has not been carried out in gymnosperms.In this study,the free nuclei of the ovules of No.28 sterile line(STE)and No.15(FER)fertile line Pinus tabulaeformis were used as materials.Functional analysis of differential genes was carried out based on transcriptome sequencing of the next generation sequencing(NGS)and single-molecule real-time sequencing(SMRT),and the molecular regulation mechanism of mitotic stopped during free nuclei mitosis was studied.Meanwhile,the expression patterns of related genes in ovules of Pinus tabulaeformis were detected and the key genes were identified for biological function verification,interaction gene exploration and tissue localization,so as to reveal the molecular regulation mechanism of female sterility in ovule of Pinus tabulaeformis.In addition,this paper also analyzed the alternative splicing of the Pinus tabulaeformis transcripts and the distribution and structural characteristics of microsatellites.In this study,based on NGS,a total of 18 c DNA libraries of 6 samples from the ovules of the fertile and sterile lines of Pinus tabulaeformis were constructed.Two libraries of fertile line and sterile line were constructed based on the SMRT.The main achievements is as follows:1.Based on NGS,a total of 142.7G Clean Data was obtained from 18 c DNA libraries,and 98038 Unigenes were obtained after assembly.After functional annotation of the differentially expressed genes(DEGs)between FER and STE at each ovule developing stages,the DEGs involved in the signal transduction of plant hormones were furrther analyzed.The results revealed that the signal pathways related to auxin,cytokinin and gibberellin may be blocked in the ovule of sterile line.Based on the results of NGS,the characteristics and distribution of the microsatellites of the ovules of Pinus tabulaeformis were analyzed.In addition,the expression trend of DEGs related to plant hormones to verify the accuracy of the results of NGS by q RT-PCR.2.NGS(Hi Seq TM 4000 platform)has obtained 961,064,004 reads,and SMRT(Pac Bio RS II platform)has obtained a total of 19.07 G data.A total of 40828 transcripts were obtained in fertile lines and 44195 transcripts in sterile lines.The transcriptome sequencing data of NGS was matched to the data of SMRT for accurate qualitative and quantitative analysis of the transcripts.The trend analysis of DEGs was carried out to screen genes with completely opposite expression trends in fertile and sterile lines during ovule development.These genes were involved in pathways such as auxin response,energy metabolism,signal transduction,cell division,and stress response.q RT-PCR was used to verify the accuracy of the corrected second-generation data.3.SMRT has the advantage of ultra-long read length,which is more suitable for analyzing the structure of the transcript.Based on the data of SMRT,the microsatellite information can be analyzed more accurately,and the number of SSRs detected based on SMRT was significantly higher.By analyzing the variable shearing of ovules from fertile lines and sterile lines,it was found that there were significant differences in the number of variable shear events between the two lines.And it was found that the differential genes AGPL1,b HLH66 and TUBA with opposite expression trends had different transcripts in the two lines.The occurrence of variable shearing events may also be one of the inducements of ovule abortion.4.Through WGCNA analysis,the modules specifically expressed in fertile and sterile lines were obtained.We analyzed the GO and KEGG annotation results of the genes in these modules and constructed the network regulation map.Combined the results of paired comparison between two lines the key genes that may affect the development of the ovules of Pinus tabulaeformis were screened and functionally verified.5.The full CDS of Ptrplv,Pt H4 and Pt Dna J were amplified by RACE technology,and the physicochemical properties of the encoded amino acids were analyzed by bioinformatics analysis.In addition,overexpression vectors of these three genes were constructed.After analyzing the vegetative growth phenotypes of Arabidopsis overexpressing Ptrplv and Pt Dna J,it was found that the phenotypes of increased leaf area and longer root system were indeed obtained,but the genes interacted with Ptrplv and Pt Dna J were not found yet.Fluorescence in situ hybridization was used to analyze the spatial and temporal expression of Ptrplv and Pt Dna J,and it was found that they were significant different expressed in different periods between two lines.In the fertile lines,the expression of these two genes was mainly concentrated in the surrounding diploid tissues and tapetum,while the fluorescence signal detected in the female sterile lines was significantly less.Ptrplv did not even detect the fluorescence signal in the whole process of female gametophyte development of female sterile lines.In conclusion,this study used NGS and SMRT sequencing technology to enrich the molecular regulation network of female sterility of Pinus tabulaeformis,and analyzed the differences in variable shearing events between the fertile and sterile lines of Pinus tabulaeformis,as well as the differences distribution characteristics in microsatellites.Two genes related to ovule development were screened for functional verification,and the temporal and spatial expression of these two genes were explored.This paper provides new ideas and directions for digging the abortion mechanism of gymnosperms.