Silencing Of Genes At Glu-A1and Glu-B1Loci Of Triticum Dicoccum And Transferring Of Them Into Synthetic Hexaploid Wheat

Cultivated emmer wheat (T. dicoccum Schrank,2n=4x=28, AABB) was one of the hulled original tetraploid wheat species. The four HMW-GS genes in two cultivated emmer wheats PI94668and PI349045were all silent. To understand and utilize of these genes improving the quality of hexaploid wheat, we introduced the HMW-GS genes of invovled tetraploid wheat and Ae. tauschii into synthetic hexaploid wheat by the process of allohexaploidization. Using SDS-PAGE, the HMW-GS of six synthetic hexaploid wheats (SHW) and their parent invovled were analyzed. To elucidate the mechanisms underlying the HMW-GS gene silence in two cultivated emmer accessions PI94668and PI349045, we cloned and sequenced the HMW-GS genes from both accessions. Further, we measured the total protein content of five SHW lines and their parents invovled two T. dicoccum and six Ae. tauschii accessions. The main results are as follows:1. Composition analysis of HMW-GS of Ae. tauschiiThe HMW-GS compositions of six Ae. tauschii accessions were3.1t+11*t (AS60, AS85),2t+12t(AS2387, AS66,AS88), and2.1t+10t (AS2388), respectively.2. Comparative analysis of HMW-GS from SHW lines by SDS-PAGEIt was shown that the HMW-GS genes encode by Glu-Al and Glu-B1of T. dicoccum were still silent in the SHW and the Dx and Dy subunits of Ae. tauschii were also introduced. Insteadly, some new protein bands appeared and some protein bands disappeared were observed in the LMW-GS and prolamin regions of SHW.3. Molecular cloning and sequence comparisons of silent HMW-GS genes from T. dicoccumFour DNA fragments with1,830(Ay),2,157(By),2,373(Bx) and2,496bp (Ax) in lengths were respectively amplified using genomic DNA of PI94668and PI349045. The nucleotides were stored in GenBank under accession numbers JQ007586-JQ007593.Sequences alignment indicated that silence of the1Ay gene in two accessions were caused by four inframe stop codons at142,151,286and355sites downstream of the start codon (ATG). One, two and one inframe stop codons were occurred at the position of glutamine (Q) in undecapeptide (SYYPGQASPQ*), hexapeptide (PG*WQE and PG*GQQ), and nonapeptide (GHHPASLQ*), respectively. A premature stop codon located at139site of the coding sequence, which was located at the glutamine (Q) sites of hexapeptide (PG*GQQ) was responsible for the silence of two1Ax gene in two accessions. Similarly, the silence of1By in two accessions were resulted from a stop codon located at237site of the coding region at the glutamine (Q) sites of hexapeptide (PGQR*Q). The silence of1Bx genes in two accessions were also the result of inframe stop codons, but the number and position of inframe stop codons were not the same. The silence of1Bx in PI94668and PI349045were resulted from one and two stop codon at213site of the glutamine (Q) in hexapeptide (SGQG*Q), and186and213sites of the glutamine (Q) in hexapeptide (PGQK*Q and SGQG*Q), respectively.4. Determination of protein contentBy using FTIR spectrometer, the total protein content of24samples involved in five SHW lines and their two T. dicoccum and six Ae. tauschii parents were investigated. The results showed that the protein content of SHW lines were either equal to or higher than that of their parents.