Molecular Cloning And Sequence Analysis Of Glutenin Subunit Genes From Dwarfing Polish Wheat (Triticum Polonicum L.)

Dwarfing polish wheat (Triticum polonicum L., 2n=4x=28) was found in Tulufan,Xingjiang, China by Prof. Chi Yen and Jun-Liang Yang. It was the only one polish wheatthat has trait of dwarfing in China. The cytological studied showed that the dwarfing polishwheat has normal genome of A and B. In SDS-PAGE analysis of seed protein extracts, twoprotein bands with electrophoretic mobilities comparable to those of hexaploid wheat highmolecular weight glutenin subunits (HMW-GS) were found from dwarfing polish wheat,namely 1Ax-dp and 1Bx-dp. In order to study this HMW glutenin subunit combination, thegenes coding 1Ax-dp and 1Bx-dp were cloned and sequenced completely. Two lowmolecular weight glutenin subunits (LMW-GS) genes have also been cloned. The resultswere listed as follows:1. HMW-GS from dwarfing polish wheat were separated by SDS-PAGE. There aretwo expressed HMW-GS, 1Ax-dp and 1Bx-dp. The electrophoretic mobility of the 1Ax-dpSubunit was similar to that of the 1Dx2 subunit, and was situated between 1Ax1 and 1Dx5subunit. The mobility of the 1Bx-dp subunits was similar to that of the 1Bx7 subunit.2. In genome PCR experiments using primers P1 and P2, three DNA fragmentsranging from approximately 1.8 to 2.5 kb were amplified from dwarfing polish wheat. ThePCR products from this wheat were derived from 1ax (1Ax-dp), 1Bx (1Bx-dp) and 1Ay(1Ay-dp) (GeneBank accession numbers of 1Bx-dp and 1Ay-dp. AY722709å’ŒAY722710),respectively, 1Ay (1Ay-dp) was a silent gene With an in-frame internal stop codon. Thecomplete ORF of x-type HMW glutenin genes, 1Ax-dp and 1Bx-dp, were 2508 bp and2385 bp long, encoding 836 and 795 amino acids, respectively. The silent 1Ay-dp gene was1806 bp in size. Sequences comparison indicated that they had identical primary structure with the published HMW-GS. Phylogenetic analysis suggested that 1Ax-dp was mostclosely related to 1Ax1 and 1Ax2~* from Triticum aestivum, while 1Bx-dp was most closelyrelated to 1Bx7 from T. aestivum, and was also related to 1Bx23 and 1Bxl4 from T.aestivum.3. Two low-molecular-weight glutenin subunit (LMW-GS) genes, designatedLMW-DP1 and LMW-DP2 (GeneBank accession number: EF371504å’ŒEF371505), wereisolated from the dwarfing polish wheat. The complete ORFs of LMW-GS genes,LMW-DP1 and LMW-DP2, were 819 bp and 831 bp in size, encoding 273 and 277 aminoacids, respectively. LMW-DP1 contains 8 cysteines with one in N-terminal region andseven in C-terminal region. LMW-DP2 also contains 8 cysteines with one in repetitivedomain and seven in C-terminal region. Sequence comparison indicated that they hadprimary structure identical with the published LMW-GS. The main differences betweenLMW-DP1, LMW-DP2 and known LMW-GS are the insertion or deletion of amino acidsin the repetitive domain. Both the amino acid sequences and the phylogenetic treesuggested that LMW-DP1 should be included in group 2 and LMW-DP2 should beincluded in group 4.