| KTM3315A belongs to non-1B/1R K-type wheat male sterile line with cytoplasmic background of Aegilops kotschyiand contains fertility restoration gene against cytoplasm of Triticum timopheevii and Aegilops kotschyi respectively,Rf3 and rfv1,located on 1BS chromosome of Triticum macha wheat,which belongs to YM type Wheat male sterile line.There is great potential in the two-line hybrid breeding of wheat due to ecological sensitivity of the fertility.Some scholars have studied the physiology and morphology of KTM3315 A,but the molecular mechanism of the fertility conversion is still to be studied.The purpose of this study is to research the molecular mechanism of KTM3315 A by bioinformatics analysis,to provide a theoretical basis for the more effective use and improvement of KTM3315 A,and to contribute to the study of thermo-sensitive sterile wheat.In this study,lncRNA sequencing was performed using the anther of the thermo-sensitive male sterile line KTM3315 A during the critical period of fertility conversion on different temperature.Differentially expressed mRNAs were analyzed by bioinformatics analysis,and their functional enrichment analysis was performed to determine the candidate genes and key metabolic pathways of fertility conversion;By analyzing the sequencing results of lncRNA,based on the differentially expressed of the lncRNA target genes,the function of lncRNA were deduced by bioinformatics analysis;In addition,qRT-PCR was performed on fertility-related genes in three developmental stages.The following results were getting:1.A total of 6 samples of long-chain noncoding sequencing were performed,with a total of 72.72 Gb Clean Data.Each sample Clean Data reached 10.81 Gb and Q30 base was 95.52% or more.The clean Reads of each sample were aligned with the specified reference genome,respectively,and the efficiency was varied from 65.85% to 70.51%.According to the expression level of the genes in different samples,16,839 differentially expressed genes were identified;functional annotation and enrichment analysis were performed.The results showed that the COG functional classification of differentially expressed genes was mainly enriched in carbohydrate and amino acid transport and metabolismcategories.The differentiallyexpressed genes were enriched in starch and sucrose biosynthesis,phenylpropanoidbiosynthesis pathway,which laying the foundation for further research.2.By analyzing all the transcription factors(MYB、ER、HS、bHLH、WRKY、MAD-box、NAC、GLK2,and GATA)in the differentially expressed genes,it was found that the proportion of MYB transcription factor was the highest(15%),which involved in fertility conversion of line KTM3315 A.Moreover,we suggested that the MYB transcription factor interacts with jasmonic acid and phenylpropanoid biosynthetic pathway under fertile condition,and promotes the synthesis of pollen outer wall by promoting the up-regulation of key enzymes in the biosynthesis pathways of phenylpropanoid and jasmonates,which makes KTM3315 A fertile.3.Through the expression pattern screening,a Long chain nocoding gene,lncRNA-TaPCF,was obtained,which regulated positivelytwo transcription factors(B-box zinc protein 25 and OCS element-binding factors1),promoted the up-regulation of key enzymes in photosynthetic carbon fixation pathway,ensured the smooth synthesis of soluble sugar and starch and protein.Therefore,it is speculated that KTM3315 A under high temperature conditions can assure the synthesis of substances required for anther development,and leadthe fertility conversion of KTM3315 A.4.The results of quantitative PCR showed that the qRT-PCR results of differentially expressed genes were consistent with the sequencing results.It was also confirmed that the fertility transformation of KTM3315 A was accompanied by changes in the expression level of fertility transformation related genes.Quantitative validation of genes can provide more reliable theoretical support for subsequent transgenic validation. |
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