Molecular Cloning Of Y-type Genes And The Mechanism Of "Expression-silence" For Glu-A1 In Triticum Dicoccoides

Triticum dicoccoides(2n=4x=28,AABB) is the donor ancestor species of T.aestivum L. (2n=6x=42,AABBDD).It had been considered as useful genetic resources for the improvement of wheat.It has been reported that HMW-GSs of T.dicoccoides had high genetic diversity,and the subunit lAy which was unexpressed in common wheat was expredssed in T.dicoccoides.In the present paper,gene cloning in combination with chromosome engineering was used to study the coding region of lAy gene in T.dicoccoides, in order to discuss the mechanism of "expression-silence" for lAy gene.The main results are as follows:1.Characterization and isolation of the HMW-GS lAy in T.dicoccoidesThe T.dicoccoides D1 and D100 were detected by SDS-PAGE.The results showed that the two shares distinctly expressed three patterns and a correspondingly thin pattern which was suspected as subunit lAy.In order to detect the thinner pattern,genomic PCR reaction was carried out by using universal primers and DNA fragments that represented the complete coding region sequences of the lAy genes were respectively obtained from D1 and D 100.The size of the amplified fragment was approximately between 1.8 to 2.0kb.The DNA fragments amplified from the two shares were separately cloned and sequenced.The size of the inserts for D1 and D100 were 1812bp and 1830bp respectively.Being compared,the DNA sequence of the lAy genes showed the highest homology with that of previously published 1Ay(AY260548) in T.dicoccum.2.The sequence character of the lAy HMW-GS gene in T.dicoccoidesIn comparison with the expressed lAy(AY245578) in T.urartu.(2n=2x=14,AA),D1 presented one in-frame premature stop codon at 208bp(TGA),and D100 presented three in-frame premature stop codons at 451bp(TAA),856bp(TAA),1353bp(TGA),respectively. According to the expressed lay sequences in T.urartu.,the primary structure of the translated lAy subunit contained a signal peptide(21 amino acid residues),an N-terminal domain(104 amino acid residues),a C-terminal domain(42 amino acid residues),and a central repetitive domain(441 amino acid residues).If the in-frame premature stop codons were ignored,the hypothetic protein of D1 and D100 would also resemble the expressed lAy,which contained the signal peptide,N- and C-terminal domains and central repetitive domain.But,the lAy in D1 had a deletion of 18 nucleotides(CCAGGACAAGGGCAACAA) at 805bp downstream from the start sequence.Accordingly,its amino acids sequence also had a deletion of a six-proteide(PGQGQQ).The length of the lay in D 100 was the same as AY245578.3.The molecular mechanism for the silence of layIn previous studies,premature stop codons in silent lay gene were all presented in central repetitive domain.But it was discovered that the only premature stop codon in D 1 was presented in N-terminal domain the first time in this study.The silence of lay in D100 was caused by premature stop codon(451bp) varied from the trinucleotide AAA into TAA via substitution of A(adenine)→T(thymine),which is quite rare.These results undoubtedly implied the specificity of the mechanism for the silence of lAy gene in T.dicoccoides.The sequence analysis of lAy gene in offsprings of T.dicoccoides and common wheat suggested that chromosome engineering process will lead to the appearance of new premature stop codons in offspring,and its location is relatively stable.According to these results,it is possible that the lay gene having been considered as being never expressed in common wheat is due to the forming of new premature stop codons in the evolution process from einkorn wheat to common wheat.4.The molecular evidence of wheat HMW-GS trait geneticsThe cloning and sequence analysis of lay gene in F4 and F₅ of D1×Chuannong16 were conducted,it was found that not only their sequence identity in the two generations was as high as 99.58 percent,but also the number and location of premature stop codons were exactly the same.This result undoubtedly provided molecular evidence for HMW-GS belonging to quality traits being stably inherited...