High-Molecular-Weight Glutenin Subunit Genes in Decaploid Agropyron elongatum

Transcription

1 Acta Botanica Sinica 2004, 46 (4): High-Molecular-Weight Glutenin Subunit Genes in Decaploid Agropyron elongatum FENG De-Shun, CHEN Fan-Guo, ZHAO Shuang-Yi, XIA Guang-Min * (School of Life Sciences, Shandong University, Jinan , China) Abstract: Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences suggested that the primary structure of the subunits encoded by the seven genes was very similar to that of y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6 and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L. (CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most closely related to 1Dy; the AgeloG3 was to 1By; the AgeloG5, AgeloG6 and AgeloG7 were to 1Ay. Key words: Agropyron elongatum ; HMW glutenin subunit; coding sequence; PCR; evolution Agropyron elongatum (StStE e E b E x, 2n=70) is a decaploid species in the Agropyron genus and is a good genetic resource for common wheat. However, few studies have been conducted to compare the structure and function of homoeologous genes from wheat and A. elongatum. Common wheat cultivars have three Glu-1 loci presenting on the long arms of chromosomes 1A, 1B and 1D, respectively, encode high-molecular-weight (HMW) glutenin subunit (Payne et al., 1987), each complex Glu-1 locus consists of two closely linked genes which code different type of HMW glutenin subunits, one with greater molecular weight, designated the x-type and the other with lower molecular weight, designated the y-type (Harberd et al., 1986). They usually contain 3 5 HMW glutenin s ubunits, and encoded by genes at the Glu-1A, Glu-1B and Glu-1D loci. Complete amino acid sequences of these proteins have indicated that there are three distinct domains: a central hydrophobic repetitive domain flanked by non-repetitive N- and C- terminal hydrophilic domain. The central repetitive domain comprises numbers of hexa- and nona- peptide motifs in the x- and y- type subunits. Besides this, tripeptide motif is found in x-type subunits. Difference in subunit size results mainly from the number of hexapeptide and nonapeptide (Shewry et al., 1989). The composition of HMW glutenin subunits can influence the bread-making quality directly (Payne et al., 1981), for example, the subunits 1Dx5 and 1Dy10 encoded by Glu-1D locus are positive to excellent bread-making quality. Transgenic experiments indicated that overexpression of certain subunit-coding sequence in wheat improved baking quality (Barro et al., 1997). It was previously found that the coding sequences of HMW glutenin subunits are not interrupted by introns and are highly conserved at 5' and 3' terminal sequences, this makes it possible to clone unknown HMW glutenin subunits using PCR directly from genomic DNA (Shewry et al., 1989; D Ovidio et al., 1995). The isolated gene can also be used further to study the relationship between structure and function of protein through the expression of polypeptides in bacteria. Some coding s equences for HMW glutenin subunit have been cloned with PCR from common wheat, Triticum tauschii, Aegilops ventricosa and Aegilops umbellulata (D Ovidio et al., 1995; Mackie et al., 1996 ; Xie et al., 2001; Liu et al., 2002). Decaploid A. elongatum has many excellent characters such as high content of seed protein and high resistance to stres s. In China, a s eries of high quality hybrid cultivars were derived from sexual hybrid progenies between A. elongatum and common wheat, e. g. Xiaoyan 6 (Zhou et al., 1995), and some somatic hybrid lines were obtained from progenies of T. aestivum with A. elongatum (Xia et Received 23 Jul Accepted 20 Nov Supported by the Hi-Tech Research and Development (863) Program of China (001AA241032). * Author for correspondence. <Xiagm@ sdu.edu.cn>.

2 al., 2003). These somatic hybrid lines have higher protein content and higher quality than their parental wheat, with different HMW glutenin subunits which are absent in the parental wheat (Xiang et al., 2001; Zhao et al., 2003). But study on the origin of excellent HMW glutenin subunits indicated that the coding sequences of 1Bx14 and 1By15 in hybrid lines of Xiaoyan 6 did not originate from A. elongatum (Fan and Guo, 2000), and that of 1 4 subunits in high quality somatic hybrid lines showed different electrophoresis mobility from A. elongatum and parental wheat (Zhao et al., 2003). So it is necessary to investigate the origin of these subunits and other storage proteins in these hybrids through comparing their coding s equences between A. elongatum and wheat. Up to now, only the coding sequences of A, B, D, C, U and G genomes of wheat and its related species have been reported (Allaby et al., 1999; Liu et al., 2002). In this study, we investigated some of HMW glutenin subunit compositions in A. elongatum and the nuclear acid sequences encoding those subunits of this species. The results provided us not only a basic situation of the gene structure but also the possibility to compare amino acid sequence with that of wheat and other species, so as to deduce the evolutionary relationship (Allaby et al., 1999). 1 Materials and Methods 1.1 Plant materials Agropyron elongatum (Host) Nevski (Lophopyrum elongatum; Thinopyrum ponticum; StStE e E b E x, 2n=70) and Triticum aestivum L. (AABBDD, 2n=42) used in this study are stored in our laboratory. HMW glutenin subunits from hexaploid wheat cultivars Chinese Spring (2+12 and 7+8), Jinan 177 (2+12 and 7+9) and a high quality somatic hybrid line -12 between Jinan 177 and A. elongatum with some HMW glutenin subunits different from its parents were used as standards for accessing the electrophoresis mobility of the subunits in A. elongatum (Zhao et al., 2003). 1.2 Extraction and SDS-PAGE analysis of glutenin subunits Endospermic storage proteins prepared for SDS-PAGE analysis were described previously (Zhao et al., 2003). Three individual seeds were examined for investigating the composition of HMW glutenin subunits in each accession. Total protein was fractionated by SDS-PAGE on 10% separating gel (C=2.67%) and 3.7% stacking gel (C=2.67%). The stacking gel buffer is 0.5 mol/l Tris-HCl (ph 6.8) and the separating gel buffer is mol/l Tris-HCl (ph 8.3). The Tris -glycine buffer (0.025 mol/l Tris-HCl (ph 8.3), mol/l glycine, 0.001% SDS) s ys tem was adopted for electrophoresis. A constant current of 15 ma was used to run for 18 h at 4. After electrophoresis, the protein bands were s tained for 5 h with 0.1% (W/V) Coomassie brilliant blue G-250, 12.5% (W /V) trichloroacetic acid, and then distained with distilled water. 1.3 Amplification and cloning of the ORFs of glutenin genes from A. elongatum CTA B method was adopted for the extraction of genomic DNA from leaves of A. elongatum according to Murray and Thompson (1980). For amplifing the ORFs of glutenin genes, two primers s pecific for HMW glutenin genes were us ed: P1:(5'-ATGGCTA AGCGGC/TTA / GGTCCTCTTTG-3') and P2: (5'-CTATCACTGGCTA/GGCC GACAATGCG-3') (Xie et al., 2001; Liu et al., 2002). A high-fidelity polymerase LA GC Taq with GC buffer (TaKaRa, Dalian, China) was used instead of Taq polymerase in PCR in order to reduce the ris k of introducing errors into the sequence. Protocol for PCR of the ORFs: the denaturing step was at 95 for 3 min; basic cycling conditions were 36 cycles, each with a 40 s denaturing step at 94 and 4 min annealing and extension steps at 68, finally kept at 68 for 10 min. PCR products were separated in 1.0% agarose gel. 1.4 Sequencing the cloned ORFs and sequence analysis The purified PCR products were ligated into pucm-t vector (Sangon, Shanghai, China) and then transformed into Escherichia coli DH10B competent cells. Following the identification of positive clones, a set of subclones was prepared using the nes ted deletion method followed Sambrook et al. (1989). DNA sequencing was performed by commercial company (TaKaRa, Dalian, China and Gentech, Shanghai, China). For sequence analysis, programs of the NCBI and EBI networks were used. 2 Results 2.1 SDS-PAGE analysis Glutenin s ubunits in the endos perm tis sue of A. elongatum and wheat Chinese Spring and cv. Jinan 177, as well as somatic hybrid -12 were extracted and subjected to SDS-PAGE analysis. Figure 1 illustrates that the number of glutenin subunits in A. elongatum was much more than that in common wheat and hybrid (Fig.1). 2.2 Cloning and sequencing the ORFs of glutenin subunit genes from A. elongatum Using degenerate primers P1 and P2, we specifically amplified seven fragments in genomic PCR (Fig.2). They were named AgeloG1, AgeloG2, AgeloG3, AgeloG4, AgeloG5, AgeloG6 and AgeloG7. The first three fragments

3

4

5

6

7 FENG De-Shun et al.: High-Molecular-Weight Glutenin Subunit Genes in Decaploid Agropyron elongatum Barro F, Rooke L, Bekes F, Gras P, Tatham A S, Fido R, Lazzeri P A, Shewry P R, Barcelo P Transformation of wheat with high molecular weight subunit genes results in improved functional properties. Nat Biotechnol, 15: Belton P S On the elasticity of wheat gluten. J Cereal Sci, 29: Bustos A D, Rubio P, Jouve N Molecular characterization of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat. Theor Appl Genet, 100: Cassidy B G, Dvorak J, Anderson O D The wheat lowmolecular-weight genes: characterization of six new genes and progress in understanding gene family structure. Theor Appl Genet, 96: D Ovidio R, Porceddu E, Lafiandra D PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus. Theor Appl Genet, 88: D Ovidio R, Masci S, Porceddu E Development of a set of oligonucleotide primers sp ecific for genes at the Glu-1 complex loci of wheat. Theor Appl Genet, 91: Fan S-H, Guo A-G A study on the origin of HMW glutenin subunit 14 and 15 in Xiao Yan 6. Acta Univ Agric Boreali- Occidentalia, 28 (6):1 5. (in Chinese with English abstract) Forde J, Malpica J M, Halford N G, Shewry P R, Anderson O D, Greene F C, Miflin B J The nucleotide sequence of a HMW subunit gene located on chromosome 1A of wheat (Triticum aestivum L.). Nucleic Acids Res, 13: Harberd N P, Bartels D, Thompson R D DNA restrictionfragment variation in the gene family encoding high-molecular-weight (HMW) glutenin subunits of wheat. Biochem Genet, 24: Liu Z -J, Zhang X-M, Wan Y- F, Liu K-F, Wang D-W Characterization of high-molecular-weight glutenin subunits and t heir coding genes from Aegilops umbellulata. Acta Bot Sin, 44: Mackie A M, Sharp P J, Lagudah E S The nucleotide and derived amino acid sequence of a HMW glutenin gene from Triticum tauschii and comparison with those from the D genome of bread wheat. J Cereal Sci, 24: Murray M G, Thompson W F The isolation of high molecular weight plant DNA. Nucleic Acids Res, 8: Payne P I, Corfied K G, Holt L M Correlations between the inheritance of cert ain high-molecular-weight subunits of glutenin and bread-making quality in progenies of six crosses of bread wheat. J Sci Food Agr, 32: Payne P I, Nightingale M A, Krattiger A F, Holt L M The relationship between HMW glutenin subunit composition and the breadmaking qualit y of British-grown wheat varieties. J Sci Food Agr, 40: Rafalski J A Structure of wheat -gliadin genes. Gene, 43: Sambrook J, Fritsch E F, Maniatis T Molecular Cloning: a Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press. Shewry P R, Halford N G, Tatham A S The high-molecular-weight subunits of wheat, barley and rye: genetics, molecular biology, chemistry and role in wheat glutenin structure and functionality. Miflin B J. Oxford Surveys of Plant Molecular and Biology. Vol. 6. Oxford: Oxford University Press Shewry P R, Tatham A S, Fido R, Jones H, Barcelo P, Lazzeri P A Improving the end use properties of wheat by manipulating the grain protein composition. Euphytica, 119: Wan Y F, Wang D W, Shewry P R, Halford N G Isolation and characterization of five novel high-molecular-weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrical. Theor Appl Genet, 104: Xia G M, Xiang F N, Zhou A F, Wang H, He S X, Chen H M Asymmet ric somatic hybridizat ion bet ween wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevski. Theor Appl Genet, 107: Xiang F-N, Feng B-M, Xia G-M, Chen H-M Agronomic trait and protein component of F 2 hybrid originated from intergeneric somatic hybridization between Triticum aestivum and Agropyron elongatum. Acta Bot Sin, 43: Xie R L, Wan Y F, Zhang Y, Wang D W HMW glut enin subunits in multiploid Aegilops species: comp osition analysis and molecular cloning of coding sequences. Chin Sci Bull, 46: Zhao T-J, Quan T-Y, Xia G-M, Chen H-M Glutenin and SDS sedimentation analysis of the F 5 somatic hybrids between Triticum aestivum L. and Agropyron elongatum. J Shandong Univ (Nat Sci), 38(3): (in Chinese with English abstract) Zhou H-P, Li B, Li Z-S T he study of breeding blue-grain gene translocation of wheat. Acta Bot Boreali-Occidentalia Sin, 15: (in Chinese with English abstract) (Managing editor: ZHAO Li-Hui)