Isolation, Heterogenous Expression And Genetic Transformation Of Novel HMW Glutenin Subunit Genes From Aegilops Tauschii

The wheat storage proteins, especially the compositions and content of high molecular weight glutenin subunits (HMW-GS) play important roles in the flour processing and bread-making quality. Previous investigations showed that the genetic foundation of hexaploid wheat was limited whereas there were extensive allelic variations of HMW-GS in wheat related species, such as Aegilops tauschii. Therefore, molecular cloning and phylogenetic analysis of novel HMW glutenin subunit genes from wheat related species would not only provide potential elite gene resources for wheat quality improvement, but also offer useful information and evidence for further understanding the origin and evolution of hexaploid wheat. This work included gene cloning, heterogenous expression, post-translational modifications, phologenetic analysis of novel Glu-1 allelles from Aegilops tauschii, plant binary expression vector construction and transformation of tobacco with cloned glutenin subunits genes. The main results obtained were as followings:1. Isolation and heterogenous expression of novel HMW glutenin subunit genes in Aegilops tauschiiTwo pairs of novel HMW glutenin subunits in Aegilops tauschii (2n=2x=14, D^tD^t) TD81 and TD130 were identified by SDS-PAGE, RP-HPLC and MALDI-TOF-MS and designated as 1Dx5^*t +1Dy10.1^t and 1Dx5.1^*t + 1Dy12.1^*t, respectively. The coding genes of 1Dx5^*t, 10x5.1^*t, and 1Dy12.1^*t were isolated by AS-PCR and the complete ORFs were obtained. The 1Dx5^*t consists of 248lbp encoding a mature protein of 827 residues with the deduced M_r of 85,782Da while 1Dx5.1^*t comprises 2526bp encoding 842 residues with 87,663Da, and 1Dyl2.1^*t comprises 1947bp encoding 648 residues with 67,347.6Da. The nucleotide acid sequences and deduced amino acid sequences had typical characters of HMW glutenin subunits. The average numbers of variants per repeat unit and the mean frequency of variant units of subunit 1Dx5^*t were relatively lower, indicating that its repeat units was closer to the consensus.The deduced M_r^s of mature proteins encoded by 1Dx5⁹(*t) and 1Dx5.1^*t were consistent with those determined by MALDI-TOF-MS, while the deduced M_r of 1Dy 12.1^*t was much lower than that determined by MALDI-TOF-MS. Three novel genes Wx5^*t, Wx5.1^*t and IDy 12.1^*t had been submitted to GenBank with accession numbers DQ681076, DQ681077 and DQ681079. Two pairs of differential primers were designed containing no signal encoding domain to amplify Wx5(*t), Wx5.1^*t and 1Dy 12.1^*' genes, the PCR products were cloned into the bacterial expression vector pET30a and the hybrid vectors were transformed into E. coli strain BL21 (DE3)pLysS. Expression of the two novel ORFs of 1Dx5^*t and 1Dx5.1^*t and N-terminal sequencing confirmed the authenticities of the two novel genes. Interestingly, several hybrid clones of 1Dx5^*' gene expressed an ilk smaller protein relative to the authentic subunit present in seed proteins, which was confirmed to resulted from a long deletion of 180bp through illegitimate recombination as well as a in-frame stop codon formation. The derived molecular weight of the small protein was well consistent with the result of SDS-PAGE analysis. Our investigations provided the direct evidence for variable recombinant clones and brought out the possible mechanism of illegitimate recombination for this variation, which may provide useful information for further insights into glutenin gene variations and their evolutionary direction. It was interesting that the expressed protein of 1DyH.1^*t gene ORP moved slightly faster than lDy12.1^*t subunit from TD130 on SDS-PAGE.2. Phylogenetic analysis of Glu-1D allellesThe network, neighbor-joining tree and homological free were constructed to analyse the phylogenetic evolution of Glu-1D alleles. The results demonstrated that the 1Dx5^*t, 1Dx2^t, 1Dx1.6^t and 1Dx2.2^* represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in the associated star-like phylogeny. The results of network, neighbor-joining tree and homological tree analysis happened to have the same view, which suggested that there were more than one independent origins of hexaploid wheats. A possibly evolutionary way of Glu-D-1-1 alleles could be drawn from our analysises: the 1Dx2.2^* , deriving from a more primitive gene, was present in Ae. tauschii, and then transferred by rare outcrossing into common wheat. A mutational event, probably by illegitimate recombination, resulted in a large DNA fragment loss of 1Dx2.2^* and the generation of 1Dx2 gene. In certain recent evolutionary time of common wheat, a large fragment duplication in the repetitive domain of 1Dx2, presumably resulting from unequal crossing over as well as illegitimate recombination, was responsible for generating the 1Dx2.2 gene.3. Identification of post-translational modifications (PTMs) of 1Dy12.1^*t subunitBecause of the evident difference between the deduced M_rs and that determined by MALDI-TOF-MS and the anomalous electrophoretic behavior on SDS-PAGE, the reversed-phase high-performance liquid chromatography following by electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) were performed to explore the post-translational modifications in 1Dy12.1^*t subunit. In theory, 1Dy12.1^*t subunit contained 13 Arg and 7 Lysresidues, and absence of Arg-Pro bonds, thus the production of 21 tryptic peptides was expected. The ESI-MS analysis of 1Dy12.1^*t tryptic digest resulted in the identification of 12 incomplete unique peptides, which was less than 50% of that in theory. Although the results gave the highest sequence coverage of 19.8%, less than the accepted coverage, the certain post-translational modifications such as glycosylation and phosphorylations can not be ruled out. Moreover, the 1Dy subunit of another accession TD87, which moved slightly faster on SDS-PAGE and had a largely smaller M_rs determined by MALDI-TOF-MS than 1Dy12.1^*t subunit, had the absolutely same encoding sequeces as 1Dy 12.1^*t gene. We also found that although the expressed protein of 1D12.1^*t gene ORF in E coli moved slightly faster than 1Dy12.1^*t subunit from TD130, the expressed protein of the gene in tobacco endosperm had the same mobility and immunity as 1Dy12.1^*t subunit from TD130. So we speculated that certain post-translational modifications were most possibly exsisted in 1Dy12.1^*t subunit of TD130.4. Construction of plant binary expression vector containing glutenin subunit genes and transformation of tobaccoTwo kinds of plant binary expression vectors pBI121PVP1-Glu and pBINHP-GluT were constructed based on analysis of nucleotide acid sequences character. A series of identification of midterm and final vectors by suitable different restriction enzymes showed that the vectors construction was absolutely successful and correct. Especially the vector pBINHP-GluT, which placed the target gene under the regulation of the verified endosperm-specific promoter, which appended the restriction sites XbaI and KpnI beside 5' and 3' of the target glutenin subunit gene, the XbaI and KpnI sites have not been found in all published HMW-GS genes and most LMW-GS genes, furthermore, which placed the all operation domains between the left and right borders of T-DNA, had great predominance in plant transformation. The results of transformation of tobacco lamina mediated by Agrobacterium tumefaciens showed that in the endosperm of tobacco gene 1Dy 12.1^*t expressed a protein which had similar mobility with 1Dy12.1^*t subunit extracted from TD130, furthermore, westhern blotting showed that the expressed protein and extracted 1Dy12.1^*t subunit from TD130 all strongly hybridize with the multiclonal antibodies against HMW glutenin subunits, which confirmed the authenticities of the novel gene 1Dy 12.1^*t and the validity of the plant binary expression vector.