A Preliminary Study On The Function Of TaWin1 Gene In Wheat Flower Development

Stamen is an important floral organ in the plant body,which its growth and development directly affect the breed of plants and the yield of crops.Male sterility is a common phenomenon in the plant kingdom that the stamens are abnormally developed and cannot produce fertile offspring.For the research of crop hybrid breed,male sterility is key to cultivate high-quality and high-yield varieties.Wheat(Triticum aestivum L.)is one of the three major grain crops in the world.Therefore,study the development of pistil and stamen is important to improve the wheat quality and yield.The homologous transformation sterility-1 mutant(HTS-1)is a new mutant surprisingly discovered by Professor Peng Zhengsong in the process of breeding wheat tristil(TP)near-isogenic line CSTP.In the floret of HTS-1,all or part of the stamens are homeosis to pistils,generally,so the number of pistils is 4-6.Therefore,the mutant is completely or partially sterile,and the average setting percentage is only 15.3% in the natural state.According to the genetic analysis,the mutant is controlled by at least two recessive genes(Pis1and hts),and is nothing to do with cytoplasmic inheritance.Pis1 gene is located on 2D chromosome and controls the three pistils character of wheat.The hts is based on a 7.2Mb section that has been positioned on chromosome 4A.In combination with previous transcriptome analysis,a gene with an abnormally high expression level in the pistillody(PS),namely the TaWin1 gene,is found in this position section.Therefore,overexpression speculating the TaWin1 gene will cause wheat stamen homologous transformation to pistil.In order to further explore the function of TaWin1,In this study,three pistil near-isogenic lines CSTP,CM28 TP and wheat stamens were homologously transformed into pistil mutant HTS-1 as experimental material.The function of TaWin1 gene is analyzed by gene clone,exogenous Ethrel and1-methylcyclopropene treatment,Real-time PCR detection and transgenic arabidopsis.The main results are as follows:1.The sequence similarity of TaWin1 gene in CM28 TP and HTS-1 is 99.77%,which the only difference was that two thymines(T)are inserted into the ORF downstream of TaWin1 gene in HTS-1.Therefore,the fragment size of TaWin1 gene in CM28 TP and HTS-1 is 883 bp and 885 bp,respectively and its ORF length is 408 bp.In the whole coding area,the amino acid sequence of TaWin1 has the highest similarity of96.38% with A.tauschii Win1,64.23% and 59.03% with anther O.brachyantha Win1 and S.bicolor Win1,58.82% with S.Bcolor Win1,38.57% with Arabidopsis thaliana subsp.Lyrata Win1,and 62.77% with O.sativa Win1.According to phylogenetic tree analysis,TaWin1 protein is the same class as A.tauschii Win1,anther O.brachynthaWin1,O.sativa Win1,S.bicolor Win1 and Z.mays Win1.Tawin1 has the closest relative with A.tauschii Win1,which further confirms that TaWin1 is the same gene family with Win1.Real-time PCR analysis shows that TaWin1 gene has the highest expression in pistillody stamens(PS)of HTS-1,with about 194 times of normal pistil(P)and 86 times of normal stamen(s)than the pistil(P)and stamen(S).2.CSTP and HTS-1 are treated by ethephon and 1-methylcyclopropylene(1-MCP)respectively,and characteristics of its stamens changed obviously,the pistillody rate of untreated HTS-1 is 59.61% and that of CSTP is 0.30,the pistillody rate of HTS-1treated by 1-MCP is 35.27% and The pistillody rate of CSTP treated by ethephon is40.53%.The pistillody of HTS-1 treated with 1-MCP was 24.34% lower than that of untreated HTS-1,and the pistillody rate of CSTP treated by ethephon was 40.23%higher than that of CSTP without any treatment.Real-time PCR is used to analyze the expression of key genes in ethylene pathway.The results showed that the expression levels of ACO2,CTR1,and EIN2 in the young ears of HTS-1 are higher than those of CSTP.After 1-MCP treatment,the expression of HTS-1 decreases,and the expression of CSTP treated by ethephon increases.The expression of ETR1 and SAM genes in the young ears of HTS-1 is significantly higher than that of CSTP,and the expression of HTS-1 decreases after 1-MCP treatment.The expression levels of ETR and SAM genes are lower in CSTP treated ethephon by and untreated CSTP young ears,and the differences are not significant.The expression levels of ACS in HTS-1 is higher than that of CSTP(about 6 times),and the expression levels of HTS-1 increases significantly after 1-MCP treatment.The expression levels of ACS in the young ears of ethephon treated CSTP is higher than that of untreated CSTP.Among the 6 genes tested,the expression difference of EIN2 gene is the most obvious.The expression of EIN2 gene in1-MCP-treated by HTS-1 decreases about 15 times compared with untreated HTS-1.In CSTP treated by ethylene,its expression is up-regulated about 16 times compared with untreated CSTP.The expression of TaWin1 gene in the young ears of HTS-1 is higher than that of CSTP.After 1-MCP treatment,the expression of TaWin1 gene in the young ear of HTS-1 is increased by 9.5 times.However,the expression of TaWin1 gene in young ears of CSTP treated by Ethrel had no significant change.3.By transgenic Arabidopsis thaliana technology,further analyze the function of TaWin1 gene.It is found that the overexpression of TaWin1 gene in Arabidopsis thaliana leads to a series of phenotypic changes,such as obvious shortening of filament and pedicel length,degeneration of filament,degradation of filament,etc.Real-time PCR was used to analyze the expression of AtACO2,AtACS and AtSAM in transgenicArabidopsis thaliana and wild Arabidopsis thaliana,which it is found that except for the AtACO2 gene,the other two key enzyme genes AtACS and AtSAM are up-regulated expression in transgenic Arabidopsis thaliana.This indicates that TaWin1 promotes ethylene synthesis in Arabidopsis thaliana to some extent.