Characterization and comparative analysis of the complete Haemonchus contortus β-tubulin gene family and implications for benzimidazole resistance in strongylid nematodes

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  Characterization and comparative analysis of the complete Haemonchus contortus β-tubulin gene family and implications for benzimidazole resistance in strongylid nematodes
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  BioMed   Central Page 1 of 13 BMC Plant Biology  Open Access Research article Characterization and comparative analysis of HMW glutenin 1Ay alleles with differential expressions Qian-TaoJiang  †1,2 , Yu-MingWei †1,2 , FengWang  1 , Ji-RuiWang  1,2 , Ze-HongYan 1,2  and You-LiangZheng* 1,2  Address: 1  Triticeae Research Institute, Sichuan Agricultural University, Ya'an, Sichuan, 625014, PR China and 2 Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Ya'an, Sichuan, 625014, PR ChinaEmail: Qian-TaoJiang-qiantaojiang@hotmail.com; Yu-MingWei-yuming71@hotmail.com; FengWang-wangfeng808@hotmail.com; Ji-RuiWang-wangjr7@163.com; Ze-HongYan-zhyan104@163.com; You-LiangZheng*-ylzheng@sicau.edu.cn* Corresponding author †Equal contributors Abstract Background: High-molecular-weight glutenin subunits (HMW-GSs) have been considered asmost important seed storage proteins for wheat flour quality. 1Ay subunits are of great interestbecause they are always silent in common wheat. The presence of expressed 1Ay subunits indiploid and tetraploid wheat genotypes makes it possible to investigate molecular information of active 1Ay genes. Results: We identified 1Ay subunits with different electrophoretic mobility from 141 accessionsof diploid and tetraploid wheats, and obtained the complete ORFs and 5' flanking sequences of 1Ay  genes including 6 active and 3 inactive ones. Furthermore, the 5' flanking sequences werecharacterized from 23 wild diploid species of Triticeae. All 6 active 1Ay possess a typical HMW-GSprimary structure and some novel characteristics. The conserved cysteine residue within therepetitive domain of y-type subunits was replaced by phenylalanine residue in subunits of 1Ay (Tu-e1), 1Ay (Tu-e2), 1Ay (Ta-e2) and 1Ay (Td-e). Particularly, 1Ay (Ta-e3) has an unusual largemolecular weight of 2202 bp and was one of the known largest y-type HMW-GSs. The translationsof 1Ay (Tu-s) , 1Ay (Ta-s) and 1Ay (Td-s) were disrupted by premature stop codons in their codingregions. The 5' flanking sequences of active and inactive 1Ay genes differ in a few base substitutionsand insertions or deletions. The 85 bp deletions have been found in promoter regions of all 1Ay  genes and the corresponding positions of 6 species from  Aegilops and Hordeum . Conclusion: The possession of larger molecular weight and fewer conserved cysteine residues areunique structural features of 1Ay genes; it would be interested to express them in bread wheat andfurther to examine their impact to processing quality of wheat. The 1Ay genes from T  . urartu arecloser to the genes from T  .  turgidum dicoccon and T  .  aestivum , than those from T  . monococcumaegilopoides . The 85 bp deletion and some variations in the 5'flanking region, have not interruptedexpression of 1Ay genes, whereas the defects in the coding regions could be responsible to thesilence of the 1Ay genes. Some mutational events in more distant distal promoter regions are alsopossible causes for the inactivation of 1Ay genes. Published: 6 February 2009 BMC Plant Biology   2009, 9 :16doi:10.1186/1471-2229-9-16Received: 18 November 2008Accepted: 6 February 2009This article is available from: http://www.biomedcentral.com/1471-2229/9/16© 2009 Jiang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.   2009, 9 :16http://www.biomedcentral.com/1471-2229/9/16Page 2 of 13 Background In wheat and its relatives, seed storage proteins are mainly composed of glutenins and gliadins [1]. High-molecular- weight glutenin subunits (HMW-GSs) are important stor-age proteins in endosperm of wheat and its related species[1]. HMW-GSs play a key role in determining wheat glu-ten and dough elasticity which promote the formation of the larger glutenin polymer [2,3]. The allelic variation inHMW-GS compositions has been reported to account for up to 70% of the variation in bread making quality among European wheats, even though they only account for about 10% of seed storage proteins [2,4]. Therefore,HMW-GS genes are important and useful in molecular modification to improve the wheat grain quality.HMW-GSs are encoded by the Glu-1 loci on the long arms of chromosomes 1A, 1B and 1D, and each locus consists of 2tightly linked genes encoding an x-type and a y-type subunit,respectively. Theoretically, hexaploid wheat could contain 6different HMW-GSs, however, gene silence resulted in varia-tion of HMW-GS number: from 3 to 5 subunits in hexaploidbread wheat and from 1 to 3 subunits in durum wheat [5,6]. Among all 6 HMW-GSs, 1Dx, 1Dy and 1Bx are always active,and 1Ax and 1By sometimes appear silent. In hexaploid wheat, the gene encoding 1Ay subunit is always silent. How-ever, 1Ay subunits have been reported in some diploid andtetraploid wheats [7]. Although the expressed 1Ay subunitsin 2 accessions of wheat have been reported [8,9], such sub-units have never been confirmed by further molecular char-acterization. To date, more than 20 HMW-GS alleles havebeen isolated from wheat and its related species [10-30], andthese information has greatly improved our understanding in structure, heredity and expression of HMW-GSs. However,our knowledge on 1Ay genes is still deficient. The expressionof 1Ay subunits in some wild diploid and tetraploid wheatsoffers an opportunity to isolate and analyze nucleotidesequences of active HMW glutenin 1Ay genes [7,19,31,32]. Triticum urartu (AA, 2n = 14), Triticum monococcum aegilo-poides (AA, 2n = 14) and Triticum turgidum dicoccon (AABB,2n = 28) are important species possibly involved in the evo-lution process of hexaploid wheat. These species possessmany excellent characteristics such as high content of seedprotein and high resistance to stripe rust, scab and stress, which could be potentially employed to improve the agro-nomic traits of common wheat [33].In this study, we reported the identification of expressed1Ay subunits from total 141 accessions of T  .  urartu , T  . monococcum aegilopoides and T  .  turgidum dicoccon , and thecharacterization of the coding and promoter regionsequences of 6 active and 3 inactive 1Ay genes. To further understand the control of this allele expression, we alsocharacterized the 5' flanking sequences of y-type HMW-GS genes from 23 wild diploid species of Triticeae. Theobjectives of this study are: 1) to compare promoter andcoding region structures of active and inactive 1Ay alleles,and further to understand the control of 1Ay gene expres-sion; 2) to compare the primary structure of 1Ay subunits with other known HMW-GSs and analysis the evolutionof Glu-A1-2 alleles; 3) to provide the basis of the genetic transformation of active 1Ay gene to verify their effect on wheat processing quality. Results SDS-PAGE profiles of HMW-GSs  The SDS-PAGE profiles of HMW-GSs showed that 1Ay sub-units were differentially expressed in T  .  urartu , T  . monococ-cum aegilopoides and T  . t  urgidum dicoccon , whereas 1Ax subunits were expressed in all accessions of these 3 species(Figure 1). In T  . urartu and T  . turgidum dicoccon , 1Ay subu-nits displayed an electrophoretic mobility similar to that of 1Dy12 subunit. 1Ay subunits from T  . monococcum aegilo-poides migrated slower than those of T  . urartu , showing asimilar electrophoretic mobility with 1By8. Interestingly,1Ay subunit in one accession (PI306526) of T  .  monococcumaegilopoides migrated slower than all y-type subunits and1Bx7. To our knowledge, the y-type HMW-GS with suchslower electrophoretic mobility has never been reported,indicating that this subunit might possess a molecular masslarger than other y-type subunits. We also found that for theexpression frequency of 1Ay subunits, diploid wheats arehigher than tetraploid wheats (Additional file 1). Characterization of 1Ay coding sequences from diploid and tetraploid wheats In genomic PCR, there is only one amplified fragment ineach of T  . urartu and T  . monococcum aegilopoides , whereas 4fragments were amplified in 2 T  . turgidum dicoccon acces-sions. The amplified fragments in T  . urartu and T  . monococ-cum aegilopoides ranged from1800 to 2202 bp (Figure 2).It is close to the size of those typical y-type HMW-GSgenes except for the fragment of 2202 bp. In T  . turgidumdicoccon accessions, the molecular weight of fragments isbetween 1.8 and 2.5 kb (Figure 2). All amplified products were cloned. By terminal sequencing and enzyme diges-tions, the ORFs representing different 1Ay alleles weredetermined. The full length sequences of 1Ay ORFs wereobtained by using the method of nested deletion. The 9sequences were named as 1Ay (Tu-e1) , 1Ay (Tu-e2) and 1Ay (Tu-s) to represent the ORFs of 1Ay subunits from T  . urartu ; 1Ay (Ta-e1) , 1Ay (Ta-e2) , 1Ay (Ta-e3) and 1Ay (Ta- s) to represent the ORFs of 1Ay subunits from T  . monococ-cum aegilopoides ; and 1Ay (Td-e) and 1Ay (Td-s) to repre-sent the ORFs of 1Ay subunits from T  . turgidum dicoccon (the letter e and s represent the expressed and silencedsubunits, the numbers represent different alleles.). Allsequences were deposited in NCBI database with Gen-bank accession numbers from:EU984503 to EU984511. The primary structures of deduced 1Ay proteins  After translating the DNA into protein sequences, analysisof amino acid sequence indicated that the ORFs of 6 active   2009, 9 :16http://www.biomedcentral.com/1471-2229/9/16Page 3 of 13 1Ay genes possess a typical primary structure shared by other published HMW-GSs, although these subunits dif-fer greatly in sizes (Figure 3 and Table 1). Each of thesededuced subunits consists of a signal peptide with 21amino acids (aa), a conserved N-terminal region, a centralrepetitive domain and a C-terminal region. The N-termi-nal regions of these 6 subunits contain 104 aa and the C-terminal regions have 42 aa. Central repetitive domains of these subunits are composed of a similar repeat structureto other known y-type subunits. The subunit 1Ay (Ta-e3)is composed of 732 aa, larger than all other known y-typeHMW-GSs. The difference between 1Ay (Ta-e3) and other  y-type HMW-GSs were entirely due to variations of thenumber of repeat motifs. Compared to other 1Ay subu-nits, 13 extra hexapeptides and 5 extra nonapeptides havebeen inserted into the repetitive domain of 1Ay (Ta-e3), which resulted in 123 aa increases in its molecular mass. All conserved cysteine residues presented in knownHMW-GSs from wheat and its relative grasses wereobserved in the aa sequences of 1Ay (Ta-e1) and 1Ay (Ta-e3). For 1Ay (Ta-e1) and 1Ay (Ta-e3), the distributions of the 7 cysteine residues are conserved with 5 in N-terminalregion, 1 at the end of central repetitive domain and 1 inC-terminal region. However, the conserved cysteine resi-dues at the end of the central repetitive domain of 1Ay (Tu-e1) , 1Ay (Tu-e2) , 1Ay (Ta-e2) and 1Ay (Td-e)  wasreplaced by phenylalanine residues (Figure 3, Table 1). The translation of the sequence of 1Ay (Tu-s) , 1Ay (Ta-s) and 1Ay (Td-s)  were disrupted by in-frame premature stopcodons (Figure 3). In the coding sequences of 1Ay (Tu-s) and 1Ay (Ta-s) , there is 1 stop codon located in the N-ter-minal and C-terminal region, respectively; and 4 stopcodons were located in the repetitive domain of 1Ay (Td- s) . If the premature stop codons were ignored, the resulted SDS-PAGE analysis of high-molecular-weight glutenin subunits (HMW-GSs) of diploid and tetraploid wheat species Figure 1SDS-PAGE analysis of high-molecular-weight glutenin subunits (HMW-GSs) of diploid and tetraploid wheat species . a Diploid accessions of T  .  urartu : (Tu1) PI428309, (Tu2) PI 428308, (Tu3) PI 428318, (Tu4) PI 428310; b, c : Diploid accessions of T  . monococcum aegilopoides : (Ta1) PI 427928, (Ta2) PI 427759, (Ta3) PI 428007, (Ta4) PI 427622, (Ta5–6) Citr 17665, (Ta7–8) PI 277123, (Ta9–10) PI 306526; d: Tetraploid wheat accessions of T  . turgidum dicoccon : (Td1–2) PI 355475, (Td3–4) PI 355477; CS: Chinese spring. The SDS-PAGE profiles of HWM-GSs showed 1Ay subunits were differentially expressed in some accessions of T  .  urartu , T  . monococcum aegilopoides and T. turgidum dicoccon while 1Ax subunits were expressed in all accessions (marked by tailed-arrows). The expressed 1Ay subunits were marked by solid and the hollow arrows indicated the area where the absent subunit band might have been.PCR amplification of HMW-GS ORFs Figure 2PCR amplification of HMW-GS ORFs . Lane1–3: PI 428309, PI 428318, PI 428308 ( T  .  urartu ); lane 4–7: PI 428007, PI 277123, PI 306526, PI 427928 ( T  .  monococcum aegilopoides ) and lane 8 and 9: PI355475, PI355477 ( T  .  turgidum dicoccon ); M is 1 Kb DNA ladder.   2009, 9 :16http://www.biomedcentral.com/1471-2229/9/16Page 4 of 13 peptides of 1Ay (Tu-s), 1Ay (Ta-s) and 1Ay (Td-s) wouldalso have typical characteristics of HMW-GSs. Structural features of the 5' flanking promoter regions of Glu-A1-2 alleles and those in 23 Triticeae species  The 5' flanking promoter regions of both active and inac-tive 1Ay from diploid and tetraploid wheat species wereamplified using the primers P3 and P4. In previous study,regulatory elements (TATA box, complete HMW enhancer, partial HMW enhancer, E motif and N motif)have been identified in the study of promoter activity in wheat endosperm [34,35]. D'Ovidio (1996) previously reported the sequence locations of 5' flanking promoter regions of 1Ay alleles in T  . urartu to the positions of -595bp upstream of translational start codon. In this study, weextended the sequences to the positions -845 bp to cover all recognized elements mentioned above. It's more scien-tific to carry out the promoter comparison using thesequences including all recognized elements. Althoughcomparative analysis of promoter could not directly decide difference in function, it would useful in identifica-tion of regulatory elements variations which are relevant to gene function and evolution. All characterized promoter regions of 1Ay  were aligned tothe homologous regions of 1Ay (Cheyenne) (from com-mon wheat cv. Cheyenne), 1By9 and 1Dy10 . The 5' flank-ing promoter regions of both inactive and active 1Ay from T  . urartu , T  . monococcum aegilopoides , T  . turgidum dicoccon and T  . aestivum  were compared. A few base substitutionsand insertions or deletions were found even though thealignment showed high similarity (Figure 4). The N motif,E motif, complete enhancer and TATA box were well con-served in all compared alleles. An 85 bp deletion, in which the partial HMW enhancer was also included, wasobserved in the 5' flanking promoter regions of all 1Ay  genes from diploid, tetraploid and hexaploid wheats when compared to 1By9 and 1Dy10 (Figure 4). Our inves-tigation in the region extended to -845 bp did not find any obvious basis for differential expression. The y-typeHMW-GS promoter regions are conserved out to -1200 bpeven though some of these genes diverged 4–5 million years ago and the non-coding sequences of wheat divergefast. Some potential regulatory elements might be in the -845 to -1200 bp region.In order to further understand the control of HMW-GS 1Ay gene expression, we also characterized the corre-sponding 5' flanking regions from 23 diploid species of  Triticeae. The length of entire 5' flanking regions in 23 Triticeae species varied from 845 to 915 bp (GenBank:EU4233–EU4242, EU4245–EU4257). Multiple sequence alignment showed the 5' flanking of 23 Triticeae speciesregions were conserved but have more variations thanthose of Glu-A1-2 alleles (Additional file 2). A few substi-tutions were found in the elements of E motif, N motif,Partial enhancer and Enhancer. Interestingly, the 85 bpdeletion was also found in the corresponding regions of y-type HMW-GS and D-hordein genes from six diploid spe-cies of  Aegilops umbellulata (U),  Ae . uniaristata (N), Hor-deum bogdanii (H), H  . brevisubulatum (H), H  . bulbosum (I)and H  .  spontareaum (H) (Figure 5). Evolutionary analyses of Glu-A1-2 alleles  The phylogenetic analysis was conducted to investigatethe evolutionary relationships among the alleles encodedby Glu-A1-2 , Glu-B1-2 and Glu-D1-2 (Figure 6). The 5'flanking sequences plus the sequences encoding the signalpeptides and N-terminal domain were chosen to con-struct the phylogenetic tree under several principles for the sequence selections [36]. Firstly, we found that theregulatory elements that control the tissue specificity andexpression level of different HMW-GS genes are well con-served in HMW-GS alleles from 23 diploid species. Sec-ondly, the sequences encoding signal peptides and N-terminal domain are also relative conserved. Therefore, Table 1: Summary of primary structure properties of expressed 1Ay subunits compared to those of previously reported y-type subunits. Number of amino acid residuesNumber of cysteine residuesN-terminaldomainRepetitivedomainC-terminaldomainTotalN-terminalDomainRepetitiveDomainC-terminaldomainTotal1Ay (Tu-e1)1044384559750161Ay (Tu-e2)1044174556650161Ay (Ta-e1)1044614561051171Ay (Ta-e2)1044174556650161Ay (Ta-e3)1045834573251171Ay (Td-e)1044174556650161By91045354568451171Dy101044784562751171Dy12104490456395117All y-type HMW-GS genes of hexaploid wheat have a conserved cysteine residue at the end of repetitive domain. However, this cysteine residue was found not always present in wild wheats.   2009, 9 :16http://www.biomedcentral.com/1471-2229/9/16Page 5 of 13 Comparison of the primary structure of 1Ay subunits from different wheat species Figure 3Comparison of the primary structure of 1Ay subunits from different wheat species. Signal peptide was under-lined; N-terminal and C-terminal regions were boxed, respectively . Conserved cysteine residues were indicated by solid arrows while the substitutions of cysteine residues with phenylalanine residue (F) were marked by hollow arrows. The in-frame stop codons were represented by asterisks and boxed.
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