MicroRNA Identification And Expression Analysis Of Fertility Transformation In Wheat Thermo-Sensitive Male Sterile Line BNS366

MicroRNAs are a class of non-coding endogenous small RNA molecules and widespread around eukaryotes,which mainly play a negative regulatory role in gene expression at the post-transcriptional level.Many studies have shown that miRNAs are widely involved in the growth and development of plants.Some specific miRNAs are involved in the anther development of plants,which is an important cause of abnormal anther development and male sterility.At present,most of the studies on miRNA in anther development are focused on rice,cotton,rape and other crops.There are few reports on the mechanism of miRNA regulation of infertility in wheat which is a heterohexaploid plant with the complex genome.Therefore,it is of great significance to study the miRNA regulating wheat male sterility for analyzing the mechanism of wheat male sterility.In this study,the anthers of wheat thermo-sensitive male sterile line BNS366 under low temperature sterile conditions and normal fertile conditions were used as materials.The miRNAs expressed during anther development of wheat BNS366 were identified by Small RNA sequencing technology,and the significant differential expression miRNAs and their target genes were screened and analyzed by bioinformatics methods.The main results are as follows:1.The fertility performance of wheat sterile line BNS366 at different sowing dates:BNS366 was sown in the field in October,November,December and mid-January,respectively.The seed setting rates of the four sowing dates were 14.76%,28.83%,42.5%and 84.1%,respectively.At the same time,the pollen of wheat BNS366 in different sowing dates was observed by cytology.In the first sowing date,the pollen grains were shriveled,and the I2-KI staining was yellowish brown,and most of them were sterile.At the IV sowing date,most of the pollen was stained blue-black,and the pollen was fertile.2.Small RNA sequencing of different fertility anthers of wheat BNS366:Four libraries of NN1(fertile pollen mother cell stage),NN2(fertile tetrad stage),SL1(sterile pollen mother cell stage)and SL2(sterile tetrad stage)were constructed from sterile and fertile pollen at different stages.A total of 54,318,314 raw reads were obtained by Small RNA sequencing.After filtration,the libraries of SL1,SL2,NN1 and NN2 had 11,858,471,13,455,460,13,823,035 and 14,434,405 clean reads,respectively.A total of 929 miRNAs were identified,including 81 known miRNAs and 848 new miRNAs.3.Screening of differentially expressed miRNAs:through the significant analysis of expression levels,21 differentially expressed miRNAs were screened from 81 known miRNAs,and 8 new miRNAs with the largest differential expression folds were selected from 848 new miRNAs.A total of 1068 target genes were predicted as the target genes for these 29 differentially expressed miRNAs,of which 25 target genes regulated by 13 miRNAs were highly expressed or specifically expressed in wheat spikes.4.qPCR analysis of miRNA and target genes:Real-time quantitative PCR analysis showed that five miRNAs,miR9662a,miR5062,miR9662b,miR9653a and miR9672b,and their potential target genes TraesCS6B02G028000,TraesCS5D02G192700,TraesCS6B02G043100,TraesCS3B02G251700 and TraesCS7D02G383200 showed opposite expression patterns in different fertility of BNS366 anthers.Among them,TraesCS3B02G251700 and TraesCS7D02G383200 maintained high expression levels during early panicle,flower development and meiosis,suggesting that they may play an important role in the fertility transformation of wheat BNS366.5.Functional analysis of miRNA:Promoter cloning and Arabidopsis genetic transformation of seven reported fertility-related miRNAs(miR395b,miR5062,miR9672b,miR9676,miR531,miR9653a,miR9670)were performed.By predicting the promoter elements,it was found that most of the promoters of these genes contained the low temperature response element LTR or the abscisic acid response element ABRE.At the same time,seven miRNAs were transformed into Arabidopsis thaliana,and the positive transformation materials were screened.Subsequently,the fertility regulation mechanism of BNS366 was analyzed by identifying the tissue expression patterns of these miRNAs.In summary,the expression profiles of anther miRNAs in thermo-sensitive male sterile wheat BNS366 under sterile and fertile conditions were analyzed by Small RNA sequencing technology in this study.Some miRNAs and their target genes that may be involved in the regulation for the fertility conversion of BNS366 fertility were found in the anther development,which provided a reference for the subsequent analysis of miRNA on the infertility mechanism of thermo-sensitive male sterile wheat BNS366.