Cloning,Prokaryotic Expression and Processing Quality Effective Analysis of Avenin-like Genes from Wheat
|School||Northwest University of Science and Technology|
|Course||Crop Genetics and Breeding|
|Keywords||Triticum aestivum.L avenin-like protein prokaryotic expression processingquality effect|
A new found storage protein,avenin-like, is caught a great attention for its number ofcysteine residues in recent years. It is apt to form inter-disulfide bonds or intra-disulfide bondswith neighboring storage proteins,which may broaden the protein-nets and develop elasticityin a dough,resulted changing the processing quality.Research in this field has just been started not long ago,during which both structure andquality effective on avenin-like are infrequent.It is quite necessary to further the study of it.The present study aimed at cloning, prokaryotic expression and functional analysises ofavenin-like genes from common wheat.The avenin-like genes were cloned by gene specificprimers, being designed according to conserved domains of published genes, followed bysequencing and bioinformatic anayzing. JN542444was cloned into the expression vector, andtransformed into host bacteria Escherichia coli Rosetta-gami B(DE3). After induced by IPTG,the fusion protein was purified by His-Trap affinity chromatography, and then been integratedinto the control flour to determine the quality effect utilization of10g Farinograph.Results are as follows:1. Twenty-two novel avenin-like genes （GenBank accession No. were from JN542443to JN542464）with855bp were isolated by gene-specific primers from12wheat cultivars, allof which encoding284amino acid residues.2. The number and the positions of Cys residues were strictly conservative in21genesof22, which possibly could form7intra-and4inter-disulfide bonds, except JN542452with17Cys residues.3. Three pseudogenes（JN542448, JN542455and JN542456）were resulted from asingle base substitution.4. An unique type of the avenin-like genes, which was identical to7cultivars of12inthis study and event the other cultivars HM027636in GenBank, was named “avenin-like I”.Phylogenetic and evolutionary analyses revealed that avenin-like subunits were leasthomology with ω-gliadin and closely related to LMW-GS in wheat storage proteins.SDS-PAGE resuslts showed the gene was successfully expressed in E. coli system.5. The farinograph data of purified protein revealed that “Avenin-like I” has a significant positive effect on wheat dough quality.6. Although lack of polymorphism in the tested cultivars, the “Avenin-like I” subunitcould improve wheat flour processing quality.