Prokaryoti Expresson, Evolutionary Analysis Of Low Molecular Weight Glutenin And α-Gliadin Genes From Psathyrostachys Huashanica

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is endemic to China and its distribution is limited to Huashan Mountain in the Shaanxi Province of China. This wild species possesses many beneficial properties such as good quality, tolerance to salinity and drought, and a resistance to stripe rust, take-all fungus and powdery mildew. In the present study, the LMW-GS andα-glidain genes were isolated and characterized from the genomic DNA of P. huashanica by AS-PCR. The expression vectors of LMW-GS andα-glidain genes from P. huashanica were constructed and then the recombinant plasmids were expressed in a prokaryotic expression system after its transformation into BL21(DE3) pLysS host strain. Moreover, the evolutionary relationship of the LMW-GS andα-glidain genes encoded by Ns genome from P. huashanica were investigated by analyzing phylogenetic tree and estimating divergence time. The main conclusions of this study are as follows.1. Seven novel LMW-GS genes, LG-Ns-5 (HM475144), LG-Ns-6 (HM475145), LG-Ns-7 (HM475146), LG-Ns-8 (HM475147), LG-Ns-9 (HM475148), LG-Ns-10 (HM475149), and LG-Ns-11 (HM475150), were isolated and characterized from the genomic DNA of P. huashanica by AS-PCR. Molecular structure analysis indicated that these seven genes have the typical structure of LMW-GS genes. Each gene sequence identified contains eight conserved cysteine residues, with the exception of LG-Ns-9 that had an extra cysteine residue in the conserved domain III. Two genes (LG-Ns-6 and LG-Ns-7) have complete coding sequences, while the others are likely pseudogenes with one or two internal stop codons in the coding region. The secondary structure prediction of LG-Ns-6 and LG-Ns-7 showed that the repetitive domains only possessed numbers of coils, whereas the C-terminal domains were not only rich inα-helixes but also strands. The phylogenetic analysis of indicates that the LMW-GS genes from P. huashanica can be classified into two subgroups with the divergence time of 7.30 MYA (million years), suggesting that the LMW-GS genes encoded by the Ns genome diverged into two subclasses during the evolutionary process. The divergence between LMW-GS genes encoded by the Ns genome and those from the wild species related to wheat such as Triticum and Aegilops, occurred at about 12.72 MYA. The LMW-GS genes from Ns genome appeared to be more related to the LMW-GS genes from Leymus mollis with NsXm genomes and their divergence occurred at about 4.83 MYA. The sequence coding for the mature protein (without the signal peptide encoding sequences) of LMW-GS gene LG-Ns-7 from P. huashanica were ligated into the expression vector pET-28a (+) to produce the recombined plasmid pET28a-Glu-Ns. The results of SDS-PAGE and Western blot analysis confirmed that the new fusion protein could be the expressed product of the LG-Ns-7 gene. Moreover, the apparent molecular weight of the newly synthesized fusion protein corresponded to the deduced molecular weights of the LG-Ns-7 gene product, which suggested that the gene LG-Ns-7 was successfully expressed in the E. coli expression host BL21 (DE3) plysS strain.2. Molecular structure analysis of four obtainedα-glidain genes from P. huashanica revealed that all the genes had the typical structure ofα-gliadin genes and contained 8 or 9 cysteine residues, respectively. In particularly, The variations of the structure were detected in the repetitive domain and the polyglutamineⅡdomain of GQ139526 and GQ13952. Phylogenetic analysis showed that theα-gliadin genes encoded by the Ns genome were most closely related to theα-gliadin genes from Leymus mollis and Elymus ciliaris. Theα-glidain genes GQ139526 and GQ139527 from P. huashanica may be a new type ofα-glidain gene family. The analysis of evolutionary relationship revealed that the divergent times ofα-glidain gene FJ713595 with those encoded by the Ns genome was about 12.05 MYA. The divergence between theα-glidain genes GQ139526 and GQ139527 from P. huashanica and theα-glidain genes HQ416920 and HQ416921 occurred at about 2.66 MYA.The coding sequence of the mature protein of theα-glidain gene GQ139527 (Gli-Ns-5) from P. huashanica was inserted into pET-28a(+) to construct expression vector pET28a-Gli-Ns. The recombinant plasmids were successfully expressed in a prokaryotic expression system after its transformation into BL21(DE3) pLysS host strain. The results of SDS-PAGE and Western-blot demonstrated that the fusion protein could express normally in the prokaryotic expression system. The deduced molecular weight (37.5 kDa) of Gli-Ns-5 gene was consistent with that of its expression proteins.Cloning and molecular characterization, prokaryotic expression, phylogenetic analysis, and the divergence time estimates of the LMW-GS andα-glidain genes encoded by Ns genome of P. huashanica could provide a reference for revealing the functional properties of these genes, and investigating the evolutionary characters of Ns genome. Moreover, this study could also provide new candidate genes for wheat quality improvement.