Analysis Of The Function Of TaMYB5-likee Gene And Its Roles In Pollen Abortion Induced By SQ-1 In Wheat

Wheat is an important food crop for human beings.The increase of yield per unit area has been the focus of agricultural scientists all over the world,especially breeding scientists.With the development of biotechnology,the utilization of wheat heterosis has made great progress,among which,the fastest progress has been made in the preparation of hybrid wheat male sterility induced by chemical hybridization agents.However,the creation of excellent sterile lines is still the bottleneck restricting the application of chemical hybrid wheat in large areas.Therefore,in order to further explore the molecular mechanism of wheat pollen abortion and serve the innovation of new chemical wheat hybrids,This study intends to focus on the interaction protein gene TaMYB5-like of differentially expressed gene TaRVE6 excavated from wheat physiological male sterile line 1376（PHYMS-1376）and normal fertile line 1376（CK-1376）induced by chemical hybridization agent SQ-1.In order to further analyze the biological function of TaMYB5-like and explore the molecular mechanism of TaMYB5-like in the process of pollen abortion induced by SQ-1 in wheat.This study mainly obtained the following results:1.TaMYB5-like encoded protein is a transcription suppressor,localized in the nucleus,composed of 272 amino acids,molecular weight of 29.36 KDa,PI=6.18.Overexpression of TaMYB5-like gene in Arabidopsis Thaliana and knockout of TaMYB5-like gene in Arabidopsis Thaliana homologous gene At MYB5 were found:Compared with the control,the plant height of OE-TaMYB5-like Arabidopsis Thaliana was significantly decreased by3cm（P<0.05）,while the plant height of atmyb5-like mutant was slightly increased,but the difference was not significant.Compared with control Arabidopsis WT,the seeds of OE-TaMYB5-like overexpressed plants increased significantly（P<0.05）,while the mutant atmyb5-like plants were smaller than wild type seeds,but the difference was not significant.The results of different tissue expression patterns showed that TaMYB5-like was expressed in the root,stem,leaf and grain of wheat,but the expression level was highest in the grain and stem,suggesting that TaMYB5-like might be related to plant height and grain size.Compared with CK-1376,TaMYB5-like expression was significantly increased in anthems of PHYMS-1376 plants at late uninucleate stage,binucleate stage and trinucleate stage.However,Alexandrite dye solution was used to stain Arabidopsis pollen of WT,OE-TaMYB5-like and atmyb5-like,all of which showed red,indicating fertility.These results indicated that TaMYB5-like gene was involved in the process of pollen abortion induced by SQ-1 in wheat.2.The self-activation effect of TaMYB5-like protein was detected,and the self-activation region was found to be at the C-terminal.Subsequently,inhibitors 3-AT and Ab A were used to inhibit the full length of TaMYB5-like and TaMYB5-like N+SANT.It was found that TaMYB5-like lost its self-activating effect in full length at 50 mmol·m L^-1Ab A and 1000 mg·m L^-13-AT,and at 600 mg·m L^-13-AT and 30 mmol·m L^-1Ab A TaMYB5-like-N+SANT lost transcriptional autoactivation.Then,under the above inhibition background,the yeast two-hybrid technology was used to screen the TaMYB5-like interacting proteins,and the full-length TaMYB5-like proteins were inhibited respectively.TaZnf＿C2H2,TaH2B4A and TaPX1A were screened by TaMYB5-like-N+SANT inhibition and TaMYB5-like-SANT+C-terminal interception.By domain analysis,it was found that TaZnf＿C2H2,TaH2B4A and TaPX1A contained Znf＿C2H2,H2B and PX domains,respectively.The subcellular localization of TaZnf＿C2H2,TaH2B4A and TaPX1A proteins showed that except for TaPX1A,TaZnf＿C2H2 and TaH2B4A were all located in the nucleus.The expression patterns of TaZnf＿C2H2,TaH2B4A,TaPX1A and TaMYB5-like genes in the anthers of PHYMS-1376and CK-1376 wheat tetrad,early uninucleate stage late uninucleate stage,binucleate stage and trinucleate stage were compared,respectively.The expression pattern of TaZnf＿C2H2 gene was basically consistent with that of TaMYB5-like gene.The expression patterns of TaH2B4A,TaPX1A and TaMYB5-like genes were inconsistent,which indicated that TaMYB5-like may cooperate with TaZnf＿C2H2 protein,but TaH2B4A and TaPX1A may be interfered by other factors.Complex network involved in SQ-1 induced pollen abortion of wheat physiological male sterility.3.The promoter analysis of TaMYB5-like gene showed that the promoter contained several cis-acting elements such as CAAT-box,Sp1,I-box,MYC and MYB,and its key acting elements were between（-）850-（-）1000bp,containing three elements,MYC,Sp1 and DRE.The maximum Ab A concentration of DRE using Pab Ai-hybrid system could not inhibit the autoactivation.When MYC and Sp1 used Y187-p HIS2 system,self-activation could be inhibited under the background of 150 m M 3-AT.Then the interaction of Sp1 and MYC was screened by yeast single hybridization.Six candidate genes were obtained（Gene ID:NP＿001392433.1,XP＿044383704.1,WP＿166488593,GBC10048.1,XP＿044448301.1,XP＿044362145.1）.