| Glutenin polymers are formed by high (HMW-GS) and low molecular weight glutenins subunits(LMW-GS) on the basis of their mobilities in the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) in wheat(Triticum aestivum L.). It has been demonstrated that the variation in HMW-GS and LMW-GS composition has a strong impact on wheat bread-making quality. However, the deeply study on LMW-GS is far lower than HMW-GS. So, it's very importace to develop our own intellectual property about LMW-GS.In this paper, the SDS-PAGE separation condition was optimazed, then interrelated subunit may be for wheat bread-making quality was isolated in Zhongyou 9507, and identified them by LC-MS. In addition, we analyzed the codon usage of glutenin subunits genes in wheat, to explore HMW-GS genetic resources efficiently and clone new LMW-GS genes. Soon after, it isolated nucleotide sequence of interrelated subunit by RT-PCR and RACE. On this basis, we choose Xinchun 8 as the experimental material, then isolated the major subunit on wheat bread-making quality by optimized SDS-PAGE technonlgy, and identified them by LC-MS. The main results are as follows:The LMW-GS extraction buffer containing 0.3mol/L NaI and 1.4% 4-VP can obtain LMW-GS availably, it's the best condition under T=14%C=3%,tris-boric acid buffer or tris-glycine buffer to separate glutenin subunits effectively. It provide a theoretical basic step for studying LMW-GS in breeding quality wheat cultivar.It locked 1 interrelated subunit in Zhongyou9507, named ZY9507-L1, its apparent molecular weight is 34kDa. It obtained 5 peptides by LC-MS, named ZL1-1,ZL1-2,ZL1-3,ZL1-4,ZL1-5 orderly. At the same time, Query coverage is 66% between ZL1-1(Peptide is GIIQPQQPAQLEVIR)and amino acid sequence of known wheat LMW-GS(ACJ76961.1) in NCBI. Query coverage is 92% between ZL1-2 (Peptide is SVVHSIIMQQEQR)and amino acid sequence of known wheat LMW-GS(ABB04903.1) in NCBI. There is little similarity between other two peptides and known LMW-GS in NCBI.The value of Effective Numberof Codons (ENC) of the other 12 glutenin subunits genes was between 40-50, except the 1Dy10 gene was less than 40.The GC3 contents were all between 0.38-0.45. The 1Bx14 Gene preferred to use A or T at the end of codon and the remaining 12 subunit genes preferred to use G or C . The distance coefficient of 1Dy10 was larger with rest of 12 genes, but it was small between x-type or y-type subunit genes of each chromosomes. The results showed that the glutenin subunit genes of wheat possessed a strong codon usage bias clearly. The codon usage of glutenin subunits genes was similar, and there were optimal(one or two) for each amino acid codon. Except for 1By9 and 1Dy10, the other 12 genes had the smallest differences in frequency of utilizing codon. The results would help to explore HMW-GS genetic resources efficiently and clone new LMW-GS genes.According to the LC-MS and characterization on codon usage of glutenin subunits genes in Wheat, we designed the specific primers on the basis of ZL1-1 peptide, it extracted the total RNA in Zhongyou 9507 by Trizol, then it gained first strand cDNA by RT-PCR.And It gained 3'nucleotide sequence of ZL1-1 finally. It showed that, it gained two new genes, named ZL1-1P and ZL1-1I respectively. The coding regions of ZL1-1P is 620bp, encoding the proteins with 186 amino acid residues. The coding regions of ZL1-1I is 693bp, encoding the proteins with 209 amino acid residues. There have homology among ZL1-1P,ZL1-1I and the known wheat LMW-GS in NCBI. It indicated that the genes is the LMW-GS preliminary.It locked 1 major subunit which is about wheat bread-making quality in Xinchun 8, named XC8-L1, molecular weight is 46kD. It obtained 215 peptides by LC-MS, named XL1-1,XL1-2,XL1-3,XL1-4,XL1-5,……,XL1-215 orderly. At the same time, Query coverage is 100% among XL1-1(Peptide is AIIYSIVLQEQQQVR),XL1-2(Peptide is VNVPLYR),XL1-3(Peptide is TTTSVPFGVGTGVGAY),(Peptide is LEVMTSIALR)and amino acid sequence of wheat LMW-GS(ACZ59817.1) in NCBI, Query coverage is 100% among XL1-5(Peptide is TTTNVPFGVGTGVGSY) and XL1-6(Peptide is VNVPLYR)and amino acid sequence of wheat LMW-GS(ACA63873.1) in NCBI, Query coverage is 100% among XL1-7(Peptide is QIAQLEVMTSIALR) and XL1-8(Peptide is QIPEQSR)and amino acid sequence of wheat LMW-GS(ADX68840.1) in NCBI, Query coverage is 100% between XL1-9(Peptide is GTFLQPHQIAR)and amino acid sequence of wheat LMW-GS(ADX68849.1) in NCBI.It will laid a theorwtical foundation for establishing relevant functional gene cloning system,obtaining our own intellectual property LMW-GS genes. |
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