| Glutenin is the special storage protein in seed of Triticeae, play an significant role in the determination of flour processing quality as the primary component of gluten protein of wheat grain. For the study of subunit function and quality breeding of wheat with good quality gene , it's important to clone the glutenin subunit gene and assess the molecular characters of glutenin. The information of glutenin from the wheat relative speices would be helpful to obtain more varieties of glutenin subunit and screen the useful gene for wheat improvement.Thinopyrum intermedium (Host) Barkwarth (= Agropyron f intermedium (Host)Beav or Elytrigia intermedia (Host) Nevski, 2n=6X=42, EjE^EiXX), is one of wheat related species, possess many desirable characteristics for wheat improvement. Since 1920s, a number of wheat Th. intermedium crossing groups have been done and a series of hybrids have been developed, many useful genes of Th .intermedium have been transferred to wheat.In present paper, Th .intermedium, T. aestivum-Th. intermedium partial amphiploids , wheat- disomic alien addition lines (TAI- I &TAI- II series) were used to study the constitution of glutenin subunit of Th. intermedium , identify the speciality subunits of Th. Intermedium, clone the glutenin subunit genes from Th. Intermedium, and analyze the molecular characterization of Th. intermedium genes. It would be essential research to find candidate genes from Th. intermedium for wheat quality breeding.The important results obtained as followings .1 We analyzed the glutenin SDS-PAGE profiles and studied the constitution of glutenin subunits of Th. Intermedium. By the comparison with wheat glutenin subunit bands, the difference between glutenin locus of wheat and Th. Intermedium were described.2 Six High-molecular-weight glutenin subunit genes of Th. Intermedium were cloned by Genomic PCR with a parr of degenerate primers. These genes were named as Ax-1, Ax-3, Ay-1, Ay-2, Ay-3 and Ay-4. They had similar primary structure with other reported HMW-GS genes, were high degree homology with x-type and y-type subunit genes of wheat respectively. There were in-frame stop codon in 4 novel y-type subunitgenes, indicated they were pseudogenes. The N-terminus of Ay-1 and Ay-3 contained 105 amino acid residues and had many mutation points, different from other y-type genes.3 Four novel Low-molecular -weight glutenin subunit genes have been cloned from genomic DNA of Thinopyrum intermedium. According to N-terminus sequence of amino acid deduced from nucleotide, these genes could be classified into two new types termed as Ai-M and Ai-Q type LMW-GS gene. The alignment of primary structure suggested four genes were different from previous published LMW-GS genes. Three of these pAL1306, pAL506 and pAL507 fitted into Ai-M type, which encoded polypeptides with N-terminal sequences of MESNIIISFLKPWL-. The pAL1231 encoded a Ai-Q type subunit, mature peptide had a N-terminal deletion, signal peptide followed by amino acids sequence of QQQLPQQ-.4 The SDS-PAGE and genomic PCR have been employed to characterize the glutenin and glutenin subunit locus of Triticum aestivwn-Thinopyrum intermedium alien addition series. The results indicated that the addition lines TAI-13 and TAI-25 carried a pair of Th. intermedium chromosomes with glutenin subunits gene loci, and the extra chromosomes belonged to the homoeologous group 1. The addition line TAI-11 contained the low-molecular -weight glutenin subunit gene loci, but the homoeologous of extra chromosome couldn't been determined.5 Then, four low-molecular-weight glutenin subunit (LMW-GS) genes (13003, 13006, 13045 and 13514) originated from Th. intermedium were isolated from immature endosperm of TAI-13 by RT-PCR. They belonged Ai-M type, Ai-Q type , and Ai-I type subunit genes respectively. The polypeptids coded by 13514 had the specific primary structure, which contained 9 Cys residue in molecular, and 26 repeat units, could formed 3 inter-molecular disulphide bonds by inference, and wa... |
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