| Wheat is the main food source in the northern region.Hybrid wheat breeding is one of the effective ways to use heterosis to improve wheat yield.F-type thermo-photo-sensitive cytoplasmic male sterile(FPTCMS,referred to as FA)It is the main way of wheat heterosis utilization.Wheat cannot produce normal pollen during the anther maturity period,which provides an ideal material for the study of cytoplasmic male sterility,anther growth and development and pollen development.Our research group has carried out research on the F-type photothermosensitive cytoplasmic male sterile line,and found that the material is greatly affected by light and temperature,and its sterility is very stable,which has great application value in wheat two-line hybrid breeding.There are few studies on the cytology and molecular mechanism of pollen abortion in F-type thermo-photo-sensitive cytoplasmic male sterility line.In view of this,in this study,the F-type thermo-photo-sensitive cytoplasmic male sterile line(FA)and the control material Beijing 411 is the test material.The phenotypic and cytological observation of the sterile line and the control material,the pistil and stamen at the trinucleate stage,as well as the tapetum and microspores at different developmental stages,were carried out by microscopy,and the FA at each stage was related physiologically.Determination of indicators,in order to understand the characteristics of FA abortion;using transcriptome sequencing technology to carry out bioinformatics analysis of sterile lines and conventional materials during the critical period of abortion tetrad anthers,using real-time fluorescence quantitative analysis to verify the transcriptome Sequencing results;according to previous research and quantitative results,the candidate gene TaINV-6D was mined from the transcriptome sequencing data,and clone analysis,physicochemical property analysis and preliminary bioinformatics analysis were performed on it;the following main research results were obtained:(1)The results of phenotypic cytological observation and determination of physiological indexes showed that the pistils of sterile lines developed normally and could receive foreign pollen normally;the anthers of FA in sterile environment were at the trinucleate stage.Compared with FA in the fertile environment and Jing 411 in different fertility environments,it showed obvious shrinkage and non-scattering powder.The results of I2-KI staining in the trinucleate stage showed that the FA in the sterile environment was obviously aborted.;Tissue section and determination of reactive oxygen species and POD showed that the tapetum degraded in advance in the tetrad stage of FA anther in sterile environment,and the characteristics of abortion were obvious.(2)Transcriptome sequencing was performed on the tetrad stages of FA and control material Jing 411 anther in different fertility environments.After removing the background differences of material differences and environmental differences,a total of 204 differentially expressed genes were excavated.Combined with the comparative analysis of the reference genome,the differentially expressed genes were found to be significantly enriched in the phenylpropane biosynthesis pathway,fructose metabolism and mannose metabolism through GO,KOG,and KEGG analysis.A total of 11 fertility-related genes were excavated from these pathways.The results of real-time quantitative PCR showed that the transcriptome results were highly accurate and could be used to analyze the dynamic changes of FA fertility-related genes.(3)According to the transcriptome screening results,quantitative results and previous studies,the sucrose invertase gene was screened out as a fertility candidate gene.Cloning,sequencing,physicochemical properties and bioinformatics analysis were performed.Based on transcriptome data and quantitative results,it was found that TaINV-6D(TraesCS6D02G261200),a sterility-related gene encoding sucrose invertase located on chromosome 6D,was significantly down-regulated in FA under sterile conditions.The 534bp CDS sequence of TaINV-6D was cloned from anthers of FA in sterile environment.Sequence analysis showed that TaINV-6D encodes 180 amino acids and has a typical PMEI conserved domain.The subcellular localization predicts that it is located on the cell membrane,and it can be preliminarily speculated that the gene may be the cell wall sucrose invertase gene.The promoter sequence of the gene contains light-responsive elements and low-temperature response cis-acting elements,and it is speculated that it may be involved in biological processes such as light and temperature stress.qRT-PCR results showed that the gene was expressed relatively high in glume and stamen.The above results speculate that TaINV-6D may be involved in the fertility regulation of FA.The results of this study lay a foundation for further research on the function of sucrose invertase gene in wheat. |
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