TaMYB305,a R2R3-MYB Transcription Factor,Regulates Anther And Pollen Development In Thermo-Sensitive Male-Sterile Wheat With Aegilops Kotschyi Cytoplasm

Wheat is one of the main food crops in China.With the growth of population and the reduction of cultivated land,increasing the yield per unit area has become an important task to ensure national food security and promote the sustainable development of agriculture.The utilization of wheat heterosis can greatly improve the yield and quality of wheat,but it is seldom used in production at present and there are many limiting factors.The K-type thermo-sensitive male sterile line with Aegilops kotschyi cytoplasm（K-TCMS）wheat material KTM3315A selected by our research team has stable genetic characteristics,and its fertility is controlled by temperature.This material is completely male sterile at low temperature（<18℃）,while its fertility can be restored at high temperature（≥20℃）.This material can realize"one line for two purposes"in production and is widely used as an ideal material for exploring the mechanism of wheat male sterility.Therefore,it is particularly important to deeply study the mechanism of temperature-sensitive wheat fertility conversion,excavate and control the related gene resources of wheat fertility conversion,and provide theoretical basis and technical support for creating new and efficient wheat varieties.The MYB transcription factor family is one of the largest transcription factor families found in plants.It widely regulates plant life processes,especially plays a crucial role in the growth and development of anthers.In the early stage,our research team screened a fertility related gene-MYB305 through gene family analysis and RNA-seq technology.In this study,using K-TCMS wheat KTM3315A as the material,the full-length gene sequence of TaMYB305 was cloned under AF and AS conditions.The function of TaMYB305 was verified using subcellular localization and GUS tissue localization techniques,and barley stripe mosaic virus induced gene silencing technology（BSMV-VIGS）was used to verify the function of TaMYB305,and the following main research results were obtained:1.Under fertility and sterility conditions,the full length of TaMYB305 gene was successfully cloned using the c DNA from the anthers of K-TCMS line wheat KTM3315A as a template.Gene sequence analysis showed that the length of TaMYB305 was 999 bp,and there were 16 SNPs in the nucleotide sequence of TaMYB305.The CDS sequence of TaMYB305 encodes 333 amino acids,with 7 amino acid differences under fertile and sterile conditions.Therefore,we speculate that the sequence difference may be the inducement of fertility conversion of KTM3315A at different temperatures.2.We extracted RNA from different tissues（stems,leaves,anthers）,fertile anthers（mononuclear,binuclear,and trinuclear）at different stages,and sterile anthers（mononuclear,binuclear,and trinuclear）at different stages and performed reverse transcription（RT）.Through real-time fluorescent quantitative PCR analysis,it was found that the expression of TaMYB305 was mainly concentrated in anthers,and the expression amount under fertile conditions was higher than that under sterile conditions,which showed that the expression of TaMYB305 in KTM3315A anthers was highly specific.In order to verify the expression mode of TaMYB305 in detail,the promoter of TaMYB305 was fused with the GUS reporter gene,and the PTaMYB305::GUS vector was constructed and transformed into Arabidopsis（Columbia-0）.β-Glucosidase（GUS）histochemical staining showed that a strong GUS（blue）signal was observed in the anthers and mature pollen grains of transgenic plants,which was consistent with the quantitative results of q RT-PCR.It shows that TaMYB305 plays an important role in the development of anthers and pollen.3.Subcellular localization of protein is very important for studying the function of protein.To determine the specific location of TaMYB305 in the cell,we constructed TaMYB305:e GFP fusion expression vector and infected 4-week-old tobacco（Nicotiana benthamiana）leaves.The instantaneous expression of 35S:e GFP protein in tobacco leaves showed that there were strong green fluorescence signals in the plasma membrane and nucleus,and 35S:e GFP was in the plasma membrane and nucleus,which showed that the subcellular localization experiment was successful.The transient expression of HY5:m Cherry（nuclear marker）protein in tobacco leaves showed that there was a red fluorescence signal in the nucleus,and HY5:m Cherry was in the nucleus.The instantaneous expression of TaMYB305:e GFP protein in tobacco leaves showed that the green fluorescence signal of TaMYB305 completely overlapped with the red fluorescence signal of HY5:m Cherry,which indicated that TaMYB305 was in the nucleus.4.Barley stripe mosaic virus was used to silence the VIGS gene of TaMYB305,and the phenotypic observation showed that the leaves of BSMV:TaMYB305 showed stripe disease spots.To identify the vitality of pollen,potassium iodide staining and DAPI staining were carried out.The results of I₂-KI staining showed that the anthers of TaMYB305 silenced plants were dyed yellow brown instead of black,indicating that TaMYB305 silencing led to less starch accumulation in pollen,that is,the vitality of pollen decreased.DAPI staining showed that at the trinuclear stage,the pollen mature cells from the control plant contained two fusiform sperm nuclei,while the pollen from the TaMYB305 silent plant contained two abnormal round sperm nuclei.These results showed that the cytological characteristics of the anthers of TaMYB305 silenced plants were consistent with that of KTM3315A under sterile conditions,which indicated that TaMYB305 silencing led to the pollen abortion of KTM3315A under fertile conditions,that is,TaMYB305 played an important role in the pollen development of KTM3315A wheat.