Cloning And Variation Analysis Of Hmw-gs Gene Of Aegilops Tauschii× Secale Cereale Amphiploids And Its Parents

Plant distant hybridization and polyploidization are the main pathways for the formation of new species,and they are also important drivers of plant evolution.Because of the interaction between parent genomes,most of the allopolyploidy is accompanied by extensive,rapid genetic and epigenetic variation.HMW-GS is one of the important storage proteins in wheat endosperm.Its composition and content determine dough elasticity and bread processing quality.Aegilops tauschii and Secale cereale contain many excellent agronomic traits and are widely used in genetic improvement of wheat.In this study,Aegilops tauschii × Secale cereale amphiploids and its parents was used to obtain HMW-GS gene sequence by targeted deletion subcloning,sequence alignment to analyze the sequence variation of HMW-GS gene in amphidiploid,it provides a theoretical basis for further study of functional gene sequence changes induced by introduction of foreign genomes during distant hybridization.The results of the study are as follows:According to the high degree of high conservation at both ends of the HMW-GS gene sequence,PCR amplification of Ae.tauschii × S.cereale amphiploids and its parents using primers.Two bands were detected in Ae.tauschii and S.cereale,and three bands were detected in the amplification.The maximal and minimum bands of the molecular weight have the same size with the Dx5 and Dy10.5 subunits of the parent A.tauschii,the middle band was a new glutenin band which could not be found from its two parents,while Rx2 and Ry6.5 subunits of S.cereale could not be found in the amplification.Because the molecular weight of HMW-GS gene is high and there are a lot of repeat sequences inside,we need to use subcloning method to obtain the full-length sequence of HMW-GS gene.The HMW-GS gene in Ae.tauschii was tentatively named At-Dx5,At-Dy10.5,and the HMW-GS gene in S.cereale was tentatively named Sc-Rx2,Sc-Ry6.5,and the HMW-GS gene in diploid was tentatively named as AS-Dx5,AS-Rx7*,AS-Dy10.5.The full length of At-Dx5 and At-Dy10.5 gene sequences were 2514 bp and 1968 bp,the AS-Dx5 and AS-Dy10.5 gene sequences with the same mobility were also 2514 bp and 1968 bp.Sequence alignment of At-Dx5 and AS-Dx5,At-Dy10.5 and AS-Dy10.5 using by ClustalX2 and Bioedit,the results showed that the HMW-GS gene(x-type and y-type)as the maternity of Ae.tauschii has little variation in amphidiploids,but only a few SNP differences.The full length of HMW-GS gene AS-Rx7* in amphiploids is 2046 bp,the results of nucleotide comparison with Sc-Rx2 and Sc-Ry6.5 subunit genes in S.cereale show that,the nucleotide sequence of AS-Rx7 * gene between 1-948 bp and 2305-2196 bp was basically consistent with the Rx subunit gene of S.cereale,and part of the 948-2305 bp gene was identical to the Ry subunit gene of S.cereale.so we speculated that the new subunit was produced by the unequal exchange of the x-and y-subunit genes of S.cereale.Comparison of AS-Rx7* subunit and known Rx subunit amino acid,the AS-Rx7* contains a cysteine residue more than other Rx subunits in the central repeats near the N-terminal region,so we speculate that the subunit has the potential to improve the quality of flour processing.Evolutionary relationships between the Rx subunits of the amphidiploid and the x-subunits and y-subunits of the parents,from which it is obvious that the AS-Rx7* gene and the Sc-Rx2 gene were gathered together,indicating that the Rx subunit of amphiploids and of S.cereale has a closer relationship.AS-Rx7* subunit successfully expressed in prokaryotic cells,its band mobility was basically consistent with that of RX subunit of seed protein.