Studies On The Function Of TaAFP-Ba/b Gene In Rice And Wheat

TaAFPs is the homologous gene of AtAFP,which regulates the germination in embryo and seed dormancy as a negative regulator of ABA signaling.Previous studies found that the TaAFP-Ba and TaAFP-Bb alleles of TaAFP-B affected the transcript expression level and the stability of m RNA.Compared with TaAFP-Ba,TaAFP-Bb had a4 bp deletion in the 5?UTR region.In order to further study the structural influences of allelic variation in 5?UTR of TaAFP-Ba/b,the biolistic bombardment and agrobacterium-mediated transformation were used to study the functions of TaAFP-Ba/b F,PTaAFP-Ba/b and TaAFP-Ba/b S.The results were as follows:(1)The transcript expression levels of TaAFP-Ba in wheat seeds were all higher than those of TaAFP-Bb in different days after pollination.(2)The fluorescence intensity of td Tomato ER,translation level of GUS and GUS activity in wheat leaves were all p ITV1-TaAFP-Ba F > p ITV1-TaAFP-Bb F >p ITV1#1534.These results indicated that the 4bp deletion in 5'UTR of TaAFP-B influenced the translation level of marker gene and the inserted fragment of TaAFP-Ba F enhanced the gene expression,while the deleted fragment of TaAFP-Bb F reduced the gene expression.(3)The promoter of PTaAFP-Ba/b might be constitutive,and the GUS activity of transgenic PTaAFP-Ba-GUS rice was higher than that of PTaAFP-Bb-GUS,indicating that the promoter activity of PTaAFP-Ba was higher than that of PTaAFP-Bb in different tissues of wheat.(4)By dd PCR identification,seven,six,three and five lines with single copy T-DNA insertion fragment were obtained,respectively,from the four kinds of transgenic rice of TaAFP-Ba S,TaAFP-Bb S,TaAFP-Ba S-GFP and TaAFP-Bb S-GFP,the ratio of single-copy number being 77.78%,66.67%,75% and 55.56%.The transcript expression levels of TaAFP-B in different tissues were seed> root > stem > leaf(30 DAG),and increased significantly in all tissues of transgenic rice compared with the control.Beside,the transcript expression level in TaAFP-Ba S/-GFP was significantly lower than in TaAFP-Bb S-GFP,and the fusion protein expression level in TaAFP-Ba S-GFP was also lower than in TaAFP-Bb S-GFP.The average GI% values of TaAFP-Ba S and TaAFP-Bb S in transgenic rice were 58.47 and 65.52,respectively,their difference reaching to the significant level(P=0.0011).The average GI% values of TaAFP-Ba S-GFP and TaAFP-Bb S-GFP were 66.11 and 69.08 respectively,which were also different(P=0.0312).(5)Three and four lines of single copy number were detected in transgenic wheat of TaAFP-Ba S and TaAFP-Bb S,and the ratio was 50% and 44.4%,respectively.The transcript expression levels of TaAFP-B in different tissues and transgenic types were consistent with rice.However,the average GI% values of TaAFP-Ba S and TaAFP-Bb S were 59.79 and 42.58(P=0.0007),respectively.(6)The trends of transcript expression levels in TaAFP-B in transgenic rice and wheat were the same.The average GI% values in TaAFP-Ba S/-GFP lines of rice were lower than in TaAFP-Bb S/-GFP lines.However,the average values in TaAFP-Ba S lines of wheat were higher than in TaAFP-Bb S,which was consistent with the GI% of common wheat and contrary with the transcript expression level trend,which might be related to the overexpression of single copy gene in allohexaploid wheat,leading to the epigenetic silencing.