Cloning And Prokaryotic Expression And In Vitro Functional Analysis Of α-gliadin Genes From Dasypyrum Villosum

Gliadin is one of the major components in gluten. It is the main protein in ripe wheatseed and has a great effect on wheat quality. According to the full lengths of α-gliadin genesdeposited in NCBI database, a conserved primer pair was designed. Through the PCRamplification, connection, conversion and other methods,52α-gliadin genes have beencloned in five Dasypyrum villosum lines (GenBank accession numbers: KJ004676toKJ004727). By the alignment analysis on online BLAST in NCBI, these52α-gliadin geneshad very high similarity rates with those which had been cloned and published fromDasypyrum breviaristatum. Among them, the similarity rate could be up to94%. The lengthsof these52α-gliadin genes were all between816bp and873bp. The most common sequencelength was858bp, showing19times in total.1. With the nucleotide sequence analysis for the total52genes, the results show thatthere were eight genes (KJ004720~KJ004727) belong to the pseudogenes because of thepremature termination. Among these, the deletion of cytosine (C) at the position of642bp inKJ004725could result in the reading frame shift mutation, while the cause of other7pseudogenes was all because of the nucleotide substitution. Among these7pseudogenes,except two bases in the codon of KJ004726was substituted by adenine (A) at the same time,the rest pseudogenes were all caused by the substitution from cytosine (C) to thymine (T). Inaddition, KJ004680had none termination codon.2. Deduced amino acid sequence anaylsis shows that there had43complete α-gliadinprotein sequences. All their lengths were between271and290aa, and they all had the typicalstructures of α-gliadin protein sequence. Besides, the extra Cys which KJ004677, KJ004686and KJ004714contained were all from the Tyrâ†'Cys mutation, whereas, the extra Cys inKJ004696was from the Serâ†'Cys mutation. Amino acid variation mainly occured inN-terminal repetitive region and polyglutamine domain I. Variation in N-terminal repetitiveregion formed five groups in the43α-gliadins. Using these43α-gliadin amino acid sequencesand other α-gliadin amino acid sequences from other species to construct a polygenetic tree, the result indicates that the α-gliadin protein sequences in this study could be classified into aseparate cluster.3. There are four toxic peptide loci in the sequence of α-gliadin which can cause theceliac disease. More specifically, they are Gliα-α (QGSFQPSQQ), Gliα-α2(PQPQLPYPQ),Gliα-α9(PFPQPQLPY) and Gliα-α20(FRPPQQPYPQ). After the sequence alignmentanalysis with these four toxic peptide loci, the results indicate that all the43α-gliadinsequences did not have the complete sequence of the Gliα-α2, Gliα-α9and Gliα-α20loci,besides, they also had a deletion of Gliα-α locus. According to the alignment analysis in thesefour toxic peptide loci of different α-gliadin sequences from different species, we can find thatdifferent α-gliadin sequences from different species had much variation in the distribution ofthese toxic peptide loci.4. To study the effects of an extra Cys on dough quality, the prokaryotic expressionvectors for KJ004708(with the typical six Cys residues) and KJ004714(with an extra Cys)were constructed, and proteins of about30kD were obtained in Escherichia coli BL21(DE3)under the induction, of which in agreement with the predicted molecular weight. Theseexpressed proteins were verified by matrix-assisted laser desorption-ionization time-of-flightMALDI-TOF/TOF tandem mass spectrometry analysis. The result shows that the sequencesof these α-gliadins were as same as the deduced amino acid sequence, and their C.I.%all got100%. The results indicate that the two α-gliadins were expressed correctly in E. coli.5. After purification, renaturation and lyophilization process, the functions of theexpressed proteins were tested with4g Farinograph. The results indicate that KJ004708andKJ004714had different effects on flour quality. KJ004708reduced the gluten strength, but itdramatically improved the dough development time and farinograph quality number. However,KJ004714with an extra Cys could had a dramatic positive effect on all the seven farinographparameters, so it dramatically improved the gluten both in the strength and elasticity.