Expression And Functional Verification Of TaGA-3,a Gene Related To CMS In Wheat

As one of the important food crops in the world,wheat,like other crops,shows heterosis in many aspects,such as yield,quality and agronomic characters.Heterosis is a common phenomenon in biology,and it is an important and effective way to improve crop yield and quality.Cytoplasmic male sterility（Cytoplasmic male sterility,CMS）is an important ways to make use of heterosis,which is widely used in wheat heterosis and hybrid seed production.As a kind of trace signal molecules widely existing in plants,plant hormones can regulate plant physiological responses at a low concentrations.At present,the study of fertility transformation from the balance of plant endogenous hormones has become an important topic in the field of male sterility.Therefore,the main purpose of this study is to explore the relationship between genes regulated by gibberellin and wheat fertility.The karyotypes and heterocytoplasms of five isonuclear alloplasmic male sterile lines（IAMSLs）materials（K87B1-706A,U87B1-706A,Va87B1-706A,C₆87B1-706A,Ju87B1-706A）and their homotypic maintainer lines（87B1-706B）were identified by molecular markers.The anthers of six materials were analyzed by transcriptome sequencing and q RT-PCR analysis,and a gibberellin regulating family protein gene（Ta GA-3）was obtained.Sequence analysis,phylogenetic tree analysis,subcellular localization,exogenous gibberellin spraying and barley stripe mosaic virus-mediated gene silencing（BSMV-VIGS）were used to explore the function of candidate gene,and the following main results were obtained:1.In this study,five isonuclear alloplasmic male sterile lines and their homotypic maintainer lines were used as materials to identify the genetic background and alloplasmic cytoplasm by molecular markers.In the identification of genetic background,it was found that each pair of SSR primers amplified genomic DNA bands with the same band pattern and size,indicating that their genetic background was the same,which indicated that they all had a 706 karyotype;Using the same method to identify chloroplast DNA,it was found that the same chloroplast SSR primers were polymorphic in amplifying chloroplast DNA of six materials,indicating that their cytoplasmic sources were different.2.Eight differentially expressed genes（DEGs）which were differentially expressed in anthers and involved in hormone（auxin,gibberellin,jasmonic acid）signal pathways were screened by transcriptome sequencing（RNA-sep）and bioinformatics analysis.Auxin and gibberellin genes were determined by quantitative analysis of anthers（mixed at different stages）.By spraying gibberellin and auxin at the same concentration（500 ng/μl）on male sterile lines and restorer lines,the seed setting rate showed that the seed setting rate of restorer lines decreased in different degrees,among which the seed setting rate of Ju type restorer line（Ju87B1-706R）decreased the most,which was used as the experimental material in the later stage.The spatio-temporal expression patterns of three gibberellin homologous genes in Ju87B1-706R and Ju87B1-706A were analyzed,and the candidate gene Ta GA-3 was obtained for further study.3.The phenotypes of stamens and anthers of Ju87B1-706R and Ju87B1-706A were observed.It was found that at the trinucleate stage,the filaments of Ju87B1-706R elongated,the anthers dehisced and pollen grains dispersed,and the anthers of Ju87B1-706A did not dehisce.Potassium iodide staining showed that the shape of pollen grains of Ju87B1-706A was irregular and insufficiently stained,so it belonged to the classic failure and staining failure.DAPI staining showed that pollen grains of Ju87B1-706R had two fusiform seminal nuclei and a round vegetative nucleus about to decompose at trinucleate stage.The pollen grains of Ju87B1-706A adhered together at each stage,and three round nuclei appeared at trinucleate stage.These results show that Ju87B1-706A is a male sterile line material,which completely accords with the characteristics of male sterility.4.Using Ju87B1-706A and Ju87B1-706R as templates,the gene Ta GA-3 was successfully isolated.Sequence analysis showed that there was no difference in the sequence between Ju87B1-706A and Ju87B1-706R.Genetic analysis of the gene shows that the gene is closely related to AK336180.1,but the function of AK336180.1 was not been reported.The results of subcellular localization showed that the gene was located on the nucleus,cell membrane or cell wall.When Ju87B1-706R was sprayed with different concentrations of gibberellin（50 ng/μl,500 ng/μl,1000 ng/μl）,it was found that with the increase of spraying concentration,the content of endogenous gibberellin and the expression of target gene increased gradually,but the seed setting rate decreased gradually,indicating that the dynamic balance of endogenous hormones had effects on gene expression and seed setting rate.5.The BSMV-VIGS test was carried out on the restorer line Ju87B1-706R sprayed with GA（1000 ng/μl）.After spraying GA,it was found that the florets and anthers of GA-CK,GA-γand GA-γ-PDS became thin,shriveled,non-dehiscent,and even transparent,while the florets and anthers treated with CK（without GA）and GA-γ-Ta GA-3 were normal and could be stained dark by potassium iodide.Two fusiform seminal nuclei appeared in DAPI staining at trinucleate stage,showing fertility.However,potassium iodide staining and DAPI staining of GA-CK,GA-γand GA-γ-PDS showed sterile phenotype,which was consistent with the results of phenotypic observation,and the results were verified again by quantitative analysis and seed setting rate.These results showed that gibberellin related gene Ta GA-3 was indeed related to fertility.