Cloning And Functional Analysis Of The TaTAC1 Gene Regulating Tiller In Bread Wheat(triticum Aestivum L.)

Tiller is a branch formed during growth and development of cereal crops,which is an important indicator for plant architecture establishment and closely related to crop yield.Tillering traits are one of the important contents of plant architecture research,in which tiller angle regulates the tillering gap,which is crucial for light interception and competition between adjacent plants,and tiller number is the key factor of crop yield formation.By selecting and breeding wheat varieties with suitable tillering charaters can greatly improve plant architecture and increase its yield.Understanding the molecular genetic mechanism of tiller and exploiting favorable alleles will be helpful to improve plant architecture and breed new high-yield wheat varieties.In this study,a mutant of tetraploid wheat with upright and compact plant architecture was used as the experimental material.Using BSA + 660 K SNP chip technology and bioinformatics method,a gene controlling tiller angle of wheat was successfully cloned.The gene was highly cogenetic with a rice tillering regulation gene Os TAC1,so it was named TaTAC1.Based on the investigation of TaTAC1 mutant,transgene and related genetic population,its morphological characteristics,genetic localization,spatiotemporal expression pattern and promoter transcriptional activities were preliminarily analyzed.The main research results were as follows:1.In the pre-study period,a mutant with a significantly reduced tiller angle and a compact erect strain was screened from the tetraploid wheat Kronos EMS mutant library.The mutant was backcrossed with the wild type and self-crossed.Then the extreme phenotype plants from BC2F2 generation were selected to sequence in a mix pool,and all the genes in the localization segment were analyzed by using the 660 K SNP chip of wheat,combined with bioinformatics technology,and the key genes were sequenced and aligned,and the target gene TaTAC1 was finally determined.2.The three alleles TaTAC1-A1,TaTAC1-B1 and TaTAC1-D1 of the tiller regulating gene TaTAC1 on chromosomes 5A,5B and 5D,respectively,were isolated from hexaploid wheat Chinese spring(CS).The full lengths of the three alleles are: 1335 bp,1336 bp and 1335 bp,respectively.They are composed of four exons and three introns,including a 783 bp open reading frame and encoding a protein of 260 amino acids,with predicted protein sizes of 29132 Dα,28912Dα and 29012 Dα,respectively.The 5’ and 3’ untranslated regions(UTR)sequences of three alleles of TaTAC1 were obtained by RACE,in which the 5’-UTR lengths were: 46 bp,45 bp and156 bp,and the 3’-UTR lengths were 361 bp,362 bp and 335 bp,respectively.Then assembled the CDS of the three alleles with their respective 5’-UTR and 3’-UTR to obtain the complete c DNA sequence with the full length of 1190 bp,1190 bp and 1274 bp,respectively;3.Subcellular localization of the fusion protein was performed by constructing the co-expression vector of TaTAC1-GFP,and the results showed that TaTAC1 was expressed in the cytoplasm,cell membrane and nucleus of tobacco epidermal cells,indicating that TaTAC1 was a constitutively expressed protein.4.The real-time quantitative PCR(q RT-PCR)detection of different tissue sites if CS was performed to analyze the temporal and spatial expression pattern of TaTAC1.The results showed that TaTAC1 gene was highly expressed in lamina joint and leaf-sheath pulvinus,indicating that it may mainly regulate the tiller angle and leaf angle of wheat.Further analysis of the expression levels of TaTAC1 alleles: TaTAC1-A1,TaTAC1-B1 and TaTAC1-D1 was analyzed by the lamina joint of CS and tetraploid wheat Kronos(KWT),The results showed that the TaTAC1-A1 allele was domonantly expressed,speculating that TaTAC1-A1 might be the main functional allelic genotype of TaTAC1.5.By phenotypic observation and quantitative analysis of TaTAC1-A1 transgenic wheat T3 plants and control Lunxuan987(LX987)plants revealed that T3 had more tiller number,larger tiller angle and leaf angle than the control LX987 during the whole growth period.This results further demonstrate the regulation of TaTAC1 gene on tiller.6.The determination of endogenous hormones in the base tissues of T3 and LX987 plants showed that the content of auxin(IAA)and cytokinin(CTK)in T3 was significantly higher but the strigolactone(SL)was lower than that of the control plants.Therefore,it is speculated that TaTAC1 may affect the tiller of wheat through the regulation of endogenous plant hormones.7.By isolating the promoter of TaTAC1-A1 allele,we found a 242 bp insertion/deletion polymorphism in the promoter region of cultivar wheat with larger tiller angle and smaller tiller angle.The allelic variation with 242 bp deletion upstream of the start codon was named TaTAC1-A1 a,and the allelic variation of the 242 bp insertion upstream of the initiation codon was named TaTAC1-A1 b,and the ploymorphic marker was developed accordingly.Then,the polymorphism analysis was performed on 153 wheat genetic populations.It was found that wheat varieties with the TaTAC1-A1 a allele had a larger tiller angle,while wheat varieties with the TaTAC1-A1 b allele showed a smaller tiller angle.It is speculated that the difference in tiller phenotype caused by TaTAC1 maybe come from the 242 bp insertion/deletion polymorphism of the TaTAC1-A1 allele in the promoter region.8.GUS staining and GUS activity assay were performed on the two promoter variants of TaTAC1-A1 allele using the GUS reporter system.The results showed that the transcriptional activity of the TaTAC1-A1 b promoter was higher than that of TaTAC1-A1 a,further verified the differences in the transcriptional activity of the two variant promter types.