Cloning And Prokaryotic Expression Of An Avenin-Like Gene From Wheat Variety "Shaan 253"

According to the conventional classification, the storage proteins of wheat can be falled into two main classes: glutenin and gliadin.Wheat storage proteins are not only an important source of vegetable proteins, but also the decisive factor of the quality of flour processing. After realizing the importance of wheat quality ,the study of wheat storage proteins becomes more and more extensive and in-depth in recent years. The rapid development of molecular biology and genomics provides a brand new avenue for wheat quality improvement. A great deal of researches have been undertaken both at home and abroad and have made significant progress in this field. Biotechnology has shown a great potential in improving the wheat quality, processing quality and nutritional quality, etc. Related research focus on discoverying coding genes of wheat storage protein, positioning and transgenic technology, etc. In recent years, a new class of protein which has a certain degree of homology with oat storage protein (avenin) called avenin-like is found in the endosperm of wheat.As a special storage protein type, the discovery and cloning of avenin-like gene are not only for the perfection of seed storage proteins types,but also for the comprehensive improvement of quality and provide more diversified screening indices. At the same time,the expression and functional identification of avenin-like gene will provide a richer wheat breeding molecular markers. After sequence alignment, Kan revealed that the a-type avenin-like protein corresponds to the low molecular weight gliadin which was previously discovered by Salcedo,Anderson,Clarke,Peter and Harsch.And the study of this type of subunit was carried out by both proteomics and protein fragments methods.Although.the cloning and expression analysis of closely related species of the genus Triticum aestivum's b-type avenin-like protein has carried out and found a rare type containing 19 cysteine residues,the overall study of the subunit still not go far enough, especially the information of the hexaploid common wheat.The quantity of cysteine residues of these types of proteins far exceeds the previously reported amount in both wheat storage protein and oat storage protein. With so many cysteine residues ,it may form more abundant disulfide bonds within or between molecules, which may have an impact on processing quality of flour.There are four main issues in the current study of wheat storage protein subunits:(1) Lack of effective means of protein separation.The family of wheat storage proteins with wide variation are relatively large.Meantime, each wheat storage protein subunits can not be separated by the current purification technology and glutenin and gliadin can not be complete separated by it also;(2) Difficult to meet the required mount of proteins used for structural and functional research in vitro;(3) The lack of convenient, simple, practical methods which can be used to study the function of storage proteins in a short time;(4) The effects of different genetic backgrounds, cultural environment on the quality of storage protein were difficult to eliminate,which made the conclusions be repeatability.In order to isolate a large number of avenin-like storage protein subunits,this study isolated oat storage protein genes from the wheat variety "Shaan 253".New avenin-like genes were cloned using PCR method from the wheat variety "Shaan 253" and then the cloned avenin-like genes were subcloned into the expression vector.It were transformed into host bacteria Escherichia coli Rosetta-gami B (DE3) after the digestion and sequence identification. The fusion protein was induced by IPTG and then detected using Western blot.The results showed that 3 avenin-like genes were obtained(Accession No.: GQ903577,GQ903578,GQ903579). Sequence analysis showed that GQ903579 was a pseudogene. And GQ903578 was expressed in prokaryotic system. Results are as follows:1. avenin-like genes were obtained from the wheat variety "Shaan 253"( Accession No.:GQ903577,GQ903578,GQ903579). Sequence analysis showed that the full– length of all the 3 avenin-like genes were 855bp ,which share the highest similarity between 99.5%-99.7% with barley (Hordeum vulgare) avenin-like gene family members. The 3 genes were initially identified as avenin-like subunit genes.2. The alignment of avenin-like gene coding sequence showed that the main difference of the coding sequence lies in the base substitutions in the repeat sections and there was also no base insertion and deletion (InDel). Clear preference and regional were observed in the polymorphism of nucleotides: Signal peptide was the most conservative region; the main substitute types in N terminal were C→G and A→G; Repeat region had the highest mutation frequency.The highest transversion frequency was A→T,followed by T→C,G→C,A→G;C terminal was relative stability and the transversion frequency of A→C,G→C were very high.3. The alignment of amino acid sequence showed that GQ903577 and GQ903578 encoding proteins of 284 aa,which speculated relative molecular weight was about 33 kD. Mismatch between the two proteins was due to the replacement of A→V in the 50th aa. The space between GQ903577 and GQ903579 was caused by nonsense mutations(Q→*) occurred in 158th aa of GQ903579. From the perspective of triplets, the first base conversion (CAA→TAA) of Glu codon occurred in GQ903579 led to the emergence of stop codon.So, GQ903579 was speculated to a avenin-like pseudogene and could not encode a functional mature protein.4. Subcloned into the pre-built high-expression vector. Positive plasmid pET32a-avenin-S253 was induced by IPTG and then was expressed in the host bacteria Rosetta gami B (DE3) plysS. Western blot and SDS-PAGE analysis indicated that there were a fusion protein with a 33kD,which general consistent with the calculated molecular weight by DNAMAN.