Molecular Cloning Of HMW Glutenin Subunits From Roegneria Kamoji Cv. Dujiangyan

High molecular weight glutenin subunits (HMW-GS) are important storage proteins in wheat. The compositions and contents of HMW-GSs play an important roles in determining the processing quality of wheat flours. Roegneria C. Koch is an important perennial genera in the tribe Triticeae (Poaceae). It includes more than 130 species all over the world, and about 70 species distributes in China. Roegneria kamoji Keng is a hexaploid species (2n=6x=42), containing the StYH genome. R. kamoji is an excellent wild grass, and wide distributed almost all over China, except Qinghai and Tibet. Roegneria kamoji cv. Dujiangyan is a new grass strains cultivated by Triticeae Research Institute, Sichuan Agricultural University. It is an excellent pasture with high yield.The type and composition of HMW-GS gene is an important indicator of wheat quality. Some new HMW-GS gene were cloned from Elymus, Pseudoroegneria, Leymus, Aegilops, Thinopyrum, Secale and Eremopyrum and other Triticeae species. At present there has not been reported about cloning and expression of R. kamoji.By studying the HMW-GS gene of R. kamoji cv. Dujiangyan can enrich our understanding of HMW-GS gene in Triticeae species and explore the evolution of wheat gluten polymer gene. HMW-GS gene of R. kamoji cv. Dujiangyan were polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) analyzed, cloned and sequenced, prokaryotic expression and clustering analysis in this study. The main results were as follows:1. HMW-GS gene compositions in R. kamoji cv. DujiangyanThrough the SDS-PAGE analysis of single seed from R. kamoji cv. Dujiangyan, only one HMW-GS band were detected. Compared with Chinese spring (CS), Chuanyu 12 (CY12), the band is faster than 1Dy12. We deduced that the molecular weight of R. kamoji cv. Dujiangyan HMW-GS is smaller than 1Dy12.2. Molecular cloning and amino acid sequence analysis of R. kamoji cv. DujiangyanDesigned the primers for PCR amplification of R. kamoji cv. Dujiangyan, obtained an about 1.2kb of HMW-GS gene fragment. Cloned and sequenced the 1.2kb DNA band and sequence analysis showed that HMW-GS of R. kamoji cv. Dujiangyan with typical HMW subunit gene characteristics:the 5’end of a signal peptide of 21 amino acids and 104 amino acids N-terminal; the 3’end of a 42 amino acids at the C-terminal. HMW-GS genes with known wild relatives of wheat and perennial species for comparison, analysis showed that:N-terminal of the gene and y-type subunits and perennial species containing Si genome has high homology relatively; C-terminal is the x-type subunits and containing St genome perennial species has high homology. and the C-terminal has the x-type specific element (LAAQLPAMCRL).3. Phylogenetic analysisCompared the N-, C-terminal amino acid sequence of HMW-GS from R. kamoji cv. Dujiangyan with previous reported 23 HMW-GSs from Triticum, Aegilops, Horde um, Elymus and other Triticeae species, two results were obtained. N-terminal clustered into two branches:the HMW-GS of perennial species with St genome and wheat y-type subunits, Glu-1-2 from Leymus racemosus, D-hordein from Hordeum chilense and Glu-W1-1, Glu-W1-2 from Australopyrum retrofractum clustered into a large branch, wheat x-type subunits clustered into another branch. HMWT-GS of R. kamoji cv. Dujiangyan gathered with Glu-1Stl, Glu-Stl from the Pseudoroegneria, 1Stl.3, Glu-S(2 from Elymus and Glu-1-2 from Leymus to a small branch with 94% support rate. The HMW-GS of R. kamoji cv. Dujiangyan and these genes have a certain genetic similarity in N-terminal. This results suggested that HMW-GS of R. kamoji cv. Dujiangyan might be expression from the St genome. From the C-terminal cluster, also clustered into two branches:HMW-GS of R. kamoji cv. Dujiangyan and wheat x-type subunits, lSt1.3, Glu-St2 from Elymus, Glu-Stl, Glu-lSt1 from Pseudoroegneria, D-hordein and Glu-1-2 from Leymus racemosus clustered into one branch. Wheat y-type subunits were clustered into a single branch. The HMW-GS from R. kamoji cv. Dujiangyan were gathered with the Glu-Stl from Pseudoroegneria spicata. Glu-lSt1 from Pseudoroegneria stipifolia, Glu-St2 from Elymus sibiricus, lSt1.3 from Elymus canadensis, as well as Glu-1-2 from Leymus racemosus. These results showed that the HMW-GS from R. kamoji cv. Dujiangyan have obvious genetic similarity with Glu-Stl, Glu-lSt1, Glu-St2,lS1.3 Glu-1-2. We deduced that the N-terminal of HMW-GS from R. kamoji cv. Dujiangyan similar to y-type subunits, C-terminal similar to x-type subunits.4. Bacterial expression of HMW-GS from R. kamoji cv. DujiangyanA pair of primers without the sequence encoding signal peptide was designed to amplify R. kamoji cv. Dujiangyan HMW-GS from PMD19-T. The amplified fragment was inserted into the pET-30a, then transformed into E.coli strain BL21 (DE) plySs, which induced by isopropyl-β-D-thiogalactoside (IPTG). The encoding gene was expressed highly in E.coli. The mobility of R. kamoji cv. Dujiangyan HMW-GS in vitro expressed was similar to that of seed by using SDS-PAGE.