| Wheat is one of the most important food crops in China,and it is very vital to ensure the food security.With the improvement of people’s living standard,the market demand for high-quality wheat is increasing gradually.The processing quality of wheat is mainly determined by gluten protein.Low molecular weight glutenin subunits are one of the three major components of wheat gluten,which affecting the extensibility and viscosity of dough.Although several LMW-GS have been isolated by researcher in China and and all over the world,there are still few reports on their detailed functions,which seriously limits the application of related genes.It is of great significance to reveal the LMW-GS genes associated with processing quality and to analyze the related mechanism.Furthermore,it will be benefit to further improvement of the wheat quality.In the present study,two weak gluten varieties,Zhengmai 004 and Yangmai 20,which had a great difference in Stability time,were used for dissecting the low molecular weight glutenin subunit genes which effect on wheat gluten quality by proteomics combined with biotechnology and dough rheology analysis.The main results are as follows.(1)The dough rheological properties of two weak gluten varieties,Zhengmai 004 and Yangmai 20,were analyzed by Farinograph and Extensograph.It was found that the stability time of Zhengmai 004 was 0.9 min,which was only 1 / 3 of Yangmai 20.In order to elucidate the molecular basis of their divergence,polyacrylamide gel electrophoresis was conducted to display and identify the glutenin subunits of Zhengmai 004 and Yangmai 20.The high molecular weight glutenin subunits of the two varieties were identical,however the subunits of LMW-GS in B-subunits were different.There was a 42 k D additional protein band detected in Zhengmai 004 comparing with Yangmai 20 at.By mass spectrometry analysis,combined with molecular weight information,the protein with the Genbank number of AGO17749 with the function annotation of LMW-GS was aligned and anchored in NCBI.(2)Based on the nucleotide sequence,primers were designed to amplify the reading frame sequence of AGO17749 in the two varieties.A 1200 bp fragment was amplified from Zhengmai 004,but no amplification product was found in Yangmai 20.Sequence analysis showed that there were three sequences in Zhengmai 004:LMWZM004-1,LMWZM004-2 and LMWZM004-3.The sequence similarity between LMWZM004-1 and the coding gene AY453155 in Glu-A3 b locus is 94%;The sequence similarity between LMWZM004-2 and the coding gene AY453155 of Glu-A3 a locus is99%,However,LMWZM004-3 contains several stop codons and cannot encode a complete protein,it is a pseudogene.Amino acid sequence analysis showed that the first amino acid of LMWZM004-1 and LMWZM004-2 were Isoleucine,that is the typical character of LMW-i.Besides,they contained signal peptide,repeat region and C-terminal.Compared with LMW-m and LMW-s,they lacked N-terminal.Phylogenetic analysis showed that LMW-GS encoded by LMWZM004-1 and LMWZM004-2 were clustered in the branch of LMW-i,while LMWZM004-1 and LMWZM004-2 were located in the branch of Glu-A3 b and Glu-A3 a,respectively.(3)According to unique sequence of LMWZM004-1 compared with other LMWGS genes,the specific primers for LMWZM004-1 were designed.SDS-PAGE detection and genotyping detection with LMWZM004-1 specific primer in 185 newly bred wheat lines demonstrated existence of LMWZM004-1 allele was consistent with the expression of 42 k D additional protein band detected in Zhengmai 004.Semiquantitative RT-PCR analysis showed that LMWZM004-1 was only expressed in grains,But not in grains of 7 days after pollination(DAP),only detected in grains of 28 days and 35 days after pollination.Further more,it expressed in the embryo not the endosperm of the grain with 28 DAP.(4)LMWZM004-1 gene specific primers were used forgenotyping 163 varieties in the natural populations and 185 newly bred wheat lines.Combined with the dough rheological analysis,it was found that LMWZM004-1 negatively correlated with Stability time,Development time,Extension area,Maximum resistance and Dough extensibility,significantly.Among the newly developed lines from the south of Huang Huai,compared with the strains with no LMWZM004-1,the Stability time of the strains containing LMWZM004-1 was reduced by 21.13%,and the Development time was reduced by 16.19%.In the natural population materials,compared with the varieties lacking of LMWZM004-1,the Extension area decreased by 27.86%,the Maximum resistance decreased by 23.24%,and the Dough extensibility decreased by 5.79%.In Further analysis of nine newly bred strong gluten wheat lines,only 11.11% of those lines contained LMWZM004-1 allele,which was much lower than the varieties in Henan,Hebei,Beijing and Yunnan.Those results indicated that LMWZM004-1 specific molecular marker could be used in wheat breeding. |
