Effect Of The Expression Of 5-methylcytosine DNA Glycosylase On The Hordeins Composition Of Barley Seed

Barley?Hordeum vulgare,2n=14?,self-pollination,gramineae,hordeum,is one of the major crops in China.Similar to the nutrient composition of wheat,but the content of gluten?an elastic protein?is less,and the content of?-glucan and soluble fiber is slightly higher.In China,it is mainly distributed in the Yangtze River Basin,the Yellow River Basin and the Tibetan Plateau.Barley grain solute protein is one of the main storage proteins in the seed endosperm,and its content not only has a certain impact on beer brewing,feed processing,but also can become an important source of antigen for celiac disease.Three foreign cultivated species were used in these study experimental materials:Steptoe and Morex from the United States and Clipper from Australia.Using the barley hordeins promoter region with different methylation mechanism to construct 5-methylcytosine DNA glycosylase?DME?silencers and transforming barley,the main conclusions are:1.Using Morex as a material to amplify high molecular weight hordein Hor3-1.By using a different fidelity enzyme to determine the site of 4 base differences were due to species speccifcity.After that,the CpG island prediction was performed on the obtained sequence,the GC frequency of the sequence was between 0.4¹.2,most of the sites were higher than the threshold of 0.6,and the GC content of CpG islands was between 40%⁵0%,below the significant level.By predicting the cis-regulatory elements,in addition to the TATA-box and CAAT-box,an element involved in endosperm-regulated expression was discovered,the Prolamin box,which bound to the transcription factor of the Dof-like protein.2.By means of bioinformatics,it was analyzed that the gene of barley HvDME?FM164415.1?contained 17 exons and 16 introns,and its 5946 base pair coding sequence could be translated into 1981 putative amino acids.This was essentially the same as the 1982amino acid of wheat TaDME?AEF38424?.Both protein secondary structures contained common conserved motifs:the N-terminal lysine-rich region,the A-domain,the DNA glycosylase conserved domain,the EndIII₄Fe-4Se domain,and the B-domain.The helix-hairpin-helix structure in the conserved domain of the DNA glycosylase was bifunctional and played an important role in the function of DME enzyme.3.Using the wheat pHMW-DMEhp vector preserved in this laboratory as a target of transformation,the hairpin fragment constructed with wheat TaDME-5A had 93%similarity in the barley DME nucleotide sequence,which laid the foundation for the use of wheat DME fragments to produce functions in barley.Then,the barley endosperm-specific pHor3-DMEhp expression vector was constructed by restriction enzyme ligation.4.In order to improve the induction rate of barley immature embryos and the differentiation rate of callus,Clipper and Steptoe were used as transformation materials,and the genetic transformation conditions of barley gene guns were gradually optimized.The optimized conditions were:the size of recipient embryos must be suitable,preferably between1² mm;the concentration of 2.5 mg�L^-1Dicamba should be added to the callus induction medium;the concentration of 0.5 mol�L^-11 mannitol hypertonic treatment of callus was required before bombardment.5.After gene gun transformation,among the 56 viable seedlings,six positive seedlings were selected by designing specific primers and PCR methods:two were Clipper and four were Steptoe,and the T₀ generation transformation ratio was 10.71%.Then the SDS-PAGE of the T₁ generation of positive plants revealed a missing band in the B component of the P₁E₈1-DME in the Clipper.While in Steptoe,the target bands of P₁E₄1-DME and P₁E₁₈1-DME were darker than wild plant.These results suggest that the initial success of the down-regulation of the B component,and provided materials for further identification.