| The development of female gametophyte is the precondition of sexual reproduction,which is the necessary life process for the formation of mature female gametophyte and the alternation of generations.It is demonstrated that abnormal development of female gametophyte often leads to female sterility of plants,which affects the production.However,the present research on female sterility is mainly focused on angiosperms,the investigation of gymnosperms remains to be revealed.In previous study,the No.28 female sterile line of Pinus tabulaeformis was found in the xingcheng Seed Orchard,Liaoning province.In our previous studies,it is showed that the ovule abortion is due to the cessation of division during the free nuclear mitosis of the female gametophyte,and significant differences of the expressions of PtRPL 7a(60 s ribosomal protein gene),PtRab(Rab small GTP binding protein gene made)and PtDnaJ(DnaJ-like protein gene)during the process of female gametophyte development were observed.Furthermore,by using fluorescence in situ hybridization technique,obvious spatial and temporal specificity of the transcript expressions of these genes were odserved in ovule.In present study,to further explore the causes of the suspension of free nuclei mitosis in female gametophyte,we focus on the biological functional of the three genes in ovules at free nuclei mitosis stage by heterologous expression on the the basis of ovules of 14 fertile line and No.28 sterile line.In addition,we constructed a miRNA database of different stages of free nuclear division in female gametophyte,as well as screened and identified related miRNAs and their candidate target genes.The main results in this study as follows:1.Real-time quantitative PCR analysis showed that the expression of PtRPL7a,PtRab and PtDnaJ genes at different stages of free nuclear division of Pinus tabuliformis ovules were significantly different.The expression of PtRPL7a gene in the ovule of the fertile line increased with the development of the female gametophyte,while there was no significant variation in the ovule of sterile line.The expression of PtRab gene in the ovule of the fertile line gradually increased with the development of the female gametophyte,while almost no expression was observed in the ovule of the sterile line.The expression of PtDnaJ gene in the ovule of the sterile line increased with the development of the female gametophyte,while its expression gradually decreased in the ovule of the sterile line.2.The sequence alignment analysis results showed there is high similarity between the sequences of three genes and the homologous genes in the Picea asperata To further explore the functions of three target genes,we amplified the coding regions of three target genes by rapid-amplification of cDNA ends(RACE),and constructed recombinant plasmids for heterologous transformation of Arabidopsis thaliana.Next,we analyzed the phenotype of the three transgenic plants.The results showed that the expression of PtRab gene in overexpressing Arabidopsis thaliana lines was low.The expression levels of PtRPL7a and PtDnaJ genes were obviously increased in overexpressing Arabidopsis thaliana lines,with the folds change of 44.91 and 99.00.However,no obviously differences were observed in these three transgenic plants compared with wild-type,respectively.The embryo sac development of the three transgenic plants needs further observation.3.In this study,Solexa sequencing of eighteen small RNA libraries obtained by us from the early,middle and late stages of free nuclear division in fertile lines and sterile lines of Pinus tabulaeformis ovules.A total of 257,597,230 high-quality sequences were generated,and 530 known miRNAs families and 899 novel potential miRNAs were identified by Blast and Patmatch software.Notebly,we found that the expressions of 20 known miRNAs were significant differences between fertile lines and sterile lines.Furthermore,the GO function annotation and KEGG pathway analysis showed that the target genes were mainly enriched in biological processes such as cell processes,signal transduction,metabolism and phytohormone signal transduction and intrinsic pathway.4.Based on Real-time PCR experiments,the expressions of miR160-x,miR390-x,miR8569-y and miR156-x and their corresponding five target genes during the development of female gametophytes in fertile line and sterile line ovules were confirmed.The results showed that the expression of miR160-x,miR390-x,miR8569-y and miR156-x in the fertile line ovules were obviously higher than those in the sterile line.Additionally,the expression of miR 160-x in the ovules of the fertile lines decreased with the development of female gametophytes,while gradually increased in the ovules of the sterile lines.The expression of miR390-x,miR8569-y and miR156-x were highest in the middle of free mitosis of the fertile ovule.Converse,the expression of miR390-x gradually increased in the ovule of the sterile line,while the expression of miR8569-y and miR156-x gradually decreased in the ovules of the sterile lines.Except for HIPP26,the remaining miRNAs and their target genes were negatively regulated.In addition,the qRT-PCR data showed a high degree of agreement with the expression profiles obtained by sRNA sequencing and transcriptome sequencing.In conclusion,based on the in-depth analysis of three target genes in the female gametophyte development of Pinus tabulaeformis,we found that the expression levels of these genes were significantly different between the fertile line and the sterile line ovule.Moreover,the phenotype analysis results of three overexpressing plants showed that no obviously differences were observed in these three transgenic plants compared with wild-type.In addition,we studied and identified miRNAs and their regulated target genes during the development of female gametophyte,and described the expression patterns and biological functions of candidate miRNAs.These results revealed the possible regulatory networks associated with female gametophyte development and provided new insight for understanding regulation mechanism of free nuclear division of female gametophytes.In all,these results lay foundation for the better understanding the vital regulatory functions of miRNAs in female gametophytes development and facilitate the theory of xylophyta development biology. |
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