作物学报 ›› 2014, Vol. 40 ›› Issue (08): 1340-1349.doi: 10.3724/SP.J.1006.2014.01340
杨帆1,陈其皎1,2,高翔1,2,*,赵万春1,2,*,强琴琴1,吴丹1,孟敏1
YANG Fan1,CHEN Qi-Jiao1,2,GAO Xiang1,2,*,ZHAO Wan-Chun1,2,*,JIANG Qin-Qin1,WU Dan1,MENG Min1
摘要:
醇溶蛋白是面筋的主要成分之一,对小麦品质具有重要影响。根据数据库中全长α-醇溶蛋白基因设计了一对通用引物,从5份一年生簇毛麦(Dasypyrum villosum)品系中共得到52条序列,长度在816~873 bp之间(GenBank登录号为KJ004676~KJ004727)。核酸序列分析表明,其中有8条假基因,有1条(KJ004680)缺失终止密码子。推导氨基酸序列显示,KJ004677、KJ004686、KJ004714及KJ004696含有1个额外的Cys,其中,前3条序列由于Tyr→Cys所致,而KJ004696则由于Ser→Cys突变。序列间的差异主要出现在N-端重复区和多聚谷氨酰胺I区,根据N端重复区多肽序列的差异将一年生簇毛麦α-醇溶蛋白分为5种类型。为了分析具有额外Cys的α-醇溶蛋白所具有的品质效应,选取KJ004708 (具有典型的6个Cys)和KJ004714 (具有1个额外的Cys)分别构建表达载体,IPTG诱导后均得到分子量约30 kD的蛋白,与理论值相符;目的条带经切胶串联质谱鉴定证明,这2个α-醇溶蛋白基因在大肠杆菌中正确表达。对表达的蛋白亚基进行纯化、复性和低温冷冻干燥,经4 g粉质仪分析表明,KJ004708和KJ004714均能改善面团的加工品质,其中具有1个额外Cys的KJ004714亚基对面粉品质的改善更为显著。
[1]Qualset C O, Zhong G Y, De Pace C, Mc Guire P E. Population biology and evaluation of genetic resources of Dasypyrum villosum. In: Damania A B ed. Biodiversity and wheat improvement. Chichester: John Wiley & Sons, 1993. pp 227–233 [2]De Pace C, Qualset C O. Mating system and genetic differentiation in Dasypyrum villosum (Poaceae) in Italy. Plant Syst Evol, 1995, 197: 123–147[3]Frederiksen S. Taxonomic studies in Dasypyrum (Poaceae). Nord J Bot, 1991, 11: 135–142[4]Nielsen J. Host range of the smut species Ustilago nuda and Ustilago tritici in the tribe Triticeae. Can J Bot, 1978, 56: 901–915[5]Chen X, Shi A N, Shang L M, Leath S, Murphy J P. The resistance of H. villosa to powdery mildew isolates and its expression in wheat background. Acta Phytopathol Sinica, 1997, 27: 17–22[6]Minelli S, Ceccarelli M, Mariani M, De Pace C, Cionini P G. Cytogenetics of Triticum • Dasypyrum hybrids and derived lines. Cytogenet Genome Res, 2005, 109: 385–392[7]Yildirim A, Jones S S, Murray T D. Mapping a gene conferring resistance to Pseudocercosporella herpotrichoides on chromosome 4V of Dasypyrum villosum in a wheat background. Genome, 1998, 41: 1–6[8]Jan C C, De Pace C, Mc Guire P E, Qualset C O. Hybrids and amphiploids of Triticum aestivum L. and T. turgidum L. with Dasypyrum villosum (L.) Candargy. Z P?anzenzücht, 1986, 96: 97–106[9]Smith J G, Kidwell K K, Evans M A, Cook R J, Smiley R W. Evaluation of spring sereal grains and wild Triticum germplasm for resistance to Rhizoctonia solani AG-8. Crop Sci, 2003, 43: 701–709[10]Zhong G Y, Dvorák J. Evidence for common genetic mechanisms controlling the tolerance of sudden salt stress in the tribe Triticeae. Plant Breed, 1995, 114: 297–302[11]Montebove L, De Pace C, Jan C C, Scarascia-Mugnozza G T, Qualset C O. Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L.) Candargy. Theor Appl Genet, 1987, 73: 836–845[12]Shewry P R, Parmar S, Pappin D J C. Characterization and genetic control of the prolamins of Haynaldia villosa: relationship to cultivated species of the Triticeae (rye, wheat and barley). Biochem Genet, 1987, 25: 309–325[13]Blanco A, Resta P, Simeone R, Parmar S, Shewry P R, Sabelli P, La?andra D. Chromosomal location of seed storage protein genes in the genome of Dasypyrum villosum (L.) Candargy. Theor Appl Genet, 1991, 82: 358–362[14]Liu C J, Chao S, Gale M D. Wsp-1, a set of genes controlling water-soluble proteins in wheat and related species. Genet Res, 1989, 54: 173–181[15]Li J M, Yang Z M, Tian H Q, Huang F, Gang P T. Somatic cell clone establishment and amphiploid synthesis in a Triticum aestivum • Haynaldia villosa intergeneric hybrid. Hereditas (Beijing), 1991, 13: 1–3[16]Mohammad P, Hossain M A, Khodarker N A, Shiraishi M. Study for morphological characteristics of species alien to wheat in Bangladesh. Sarhad J Agric, 1997, 13: 541–550[17]Okocha P I. Peculiarities of nucleo-cytoplasmic interactions in allocytoplasmic forms of wheat. Global J Pure Appl Sci, 1999, 5: 431–435[18]De Pace C, Snidaro D, Ciaf? M, Vittori D, Ciofo A, Cenci A, Tanzarella O A, Qualset C O, Scarascia Mugnozza G T. Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphytica, 2001, 117: 67–75[19]谷淑波, 于振文, 王东, 张永丽. 小麦贮藏蛋白对加工品质的影响及对环境的反应. 山东农业大学学报(自然科学版), 2009, 40: 309–312Gu S B, Yu Z W, Wang D, Zhang Y L. Effects of wheat storage protein on processing quality and reacting to environment. J Shandong Agric Univ (Nat Sci), 2009, 40: 309–312 (in Chinese)[20]Payne P I, Holt L M, Jackson E A, Law C N, Damania A B. Wheat storage proteins: their genetics and their potential for manipulation by plant breeding. Philos Trans R Soc Lond, 1984, 304: 359–379[21]Shewry P R, Halford N G. Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot, 2002, 53: 947–958[22] 朱西平, 李鑫, 李雅轩, 晏月明. 普通小麦及近缘粗山羊草α-醇溶蛋白基因的克隆、定位与进化分析. 作物学报, 2010, 36: 580–589Zhu X P, Li X, Li Y X, Yan Y M. Cloning, chromosomal location, and evolutionary analysis of α-gliadin genes from Aegilops tauschii and common wheat (Triticum aestivum L.). Acta Agron Sin, 2010, 36: 580–589 (in Chinese with English abstract)[23]Qi P F, Wei Y M, Yue Y W, Yan Z H, Zheng Y L. Biochemical and molecular characterization of gliadins. Mol Biol, 2006, 140: 713–723[24]Murray H G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucl Acids Res, 1980, 8: 4321–4325[25]van Herpen T W, Goryunova S V, van der Schoot J, Mitreva M, Salentijn E, Vorst O, Schenk M F, van Veelen P A, Koning F, van Soest L J, Vosman B, Bosch D, Hamer R J, Gilissen L J, Smulders M J. Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes. BMC Genomics, 2006, 7: 1[26]李光蓉, 任正隆, 刘成, 周建平, 杨足君. 多年生簇毛麦α-醇溶蛋白基因的分离与序列分析. 作物学报, 2008, 34: 1097–1103Li G R, Ren Z L, Liu C, Zhou J P, Yang Z J. Isolation and sequence analysis of α-gliadin genes from Dasypyrum breviaristatum. Acta Agron Sin, 2008, 34: 1097–1103 (in Chinese with English abstract)[27]Molberg O, Uhlen A K, Jensen T, Flaete N S, Fleckenstein B, Arentz-Hansen H, Raki M, Lundin K E, Sollid L M. Mapping of gluten T cell epitopes in the bread wheat ancestors: implications for celiac disease. Gastroenterology, 2005, 128: 393–401[28]Li G R, Liu C, Zeng Z X, Jia J Q, Zhang T, Zhou J P, Ren Z L, Yang Z J. Identification of α-gliadin genes in Dasypyrum in relation to evolution and breeding. Euphytica, 2009, 165: 155–163[29]Chen GX, Lv D W, Li W D, Subburaj S, Yu Z T, Wang Y J, Li X H, Wang K, Ye X G, Ma W, Yan Y M. The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly. Funct Integr Genomics, 2014, 14: 149–160[30]Li G R, Zhang T, Ban Y R, Yang Z J. Molecular characterization and evolutionary analysis of α-gliadin genes from Eremopyrum bonaepartis (Triticeae). J Agric Sci, 2010, 2: 30–36[31]张晓霞, 焦浈, 董振营, 李世明, 王燃, 凌宏清, 秦广雍, 王道文. 普通小麦品种小偃54中α/β-醇溶蛋白编码基因的克隆与序列分析. 作物学报, 2011, 37: 1497–1502Zhang X X, Jiao Z, Dong Z Y, Li S M, Wang R, Ling H Q, Qin G Y, Wang D W. Cloning and sequence analysis of α/β-gliadin genes from common wheat variety Xiaoyan 54. Acta Agron Sin, 2011, 37: 1497–1502 (in Chinese with English abstract)[32]李玉阁, 邢冉冉, 李锁平. 栽培一粒小麦α-醇溶蛋白新基因的克隆与序列分析. 麦类作物学报, 2012, 32: 387–392Li Y G, Xing R R, Li S P. Cloning and sequence analysis of new α-gliadin genes from Triticum monococcum. J Triticeae Crops, 2012, 32: 387–392 (in Chinese with English abstract)[33]Anderson O D, Litts J C, Greene F C. The α-gliadin gene family: I. Characterization of ten new wheat α-gliadin genomic clones, evidence for limited sequence conservation of flanking DNA, and southern analysis of the gene family. Theor Appl Genet, 1997, 95: 50–58[34]李敏, 高翔, 陈其皎, 董剑, 赵万春, 王明霞. 普通小麦中α-醇溶蛋白基因(GQ891685)的克隆、表达及品质效应鉴定. 中国农业科学, 2010, 43: 4765–4774 Li M, Gao X, Chen Q J, Dong J, Zhao W C, Wang M X. Cloning, prokaryotic expression and in vitro functional analysis of α-gliadin gene from common wheat. Sci Agric Sin, 2010, 43: 4765–4774 (in Chinese with English abstract)[35]李光蓉, 郎涛, 刘成, 周建平, 任正隆, 杨足君. 小麦新品种‘成电麦1号’α-醇溶蛋白基因的分离与序列分析. 中国农学通报, 2011, 27(1): 203–208Li G R, Lang T, Liu C, Zhou J P, Ren Z L, Yang Z J. Isolation and sequence analysis of α-gliadin genes from wheat cultivar Chengdianmai 1. Chin Agric Sci Bull, 2011, 27(1): 203–208 (in Chinese with English abstract)[36]Anderson O D, Greene F C. The α-gliadin gene family: II. DNA and protein sequence variation, subfamily structure, and origins of pseudogenes. Theor Appl Genet, 1997, 95: 59–65[37]刘千, 龙海, 魏育明, 颜泽洪, 郑有良. 小麦品种‘川农16’α-醇溶蛋白基因序列分析. 中国农业科学, 2008, 41: 2168–2173Liu Q, Long H, Wei Y M, Yan Z H, Zheng Y L. Sequence analysis of α-gliadin genes from wheat variety Chuannong 16. Sci Agric Sin, 2008, 41: 2168–2173 (in Chinese with English abstract)[38]Xie Z, Wang C, Wang K, Wang S, Li X, Zhang Z, Ma W, Yan Y. Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.). Theor Appl Genet, 2010, 121: 1239–1251[39]田纪春. 谷物品质测试理论与方法. 北京: 科学出版社, 2006. pp 338–340Tian J C. Theory and Method of Test in Grain Quality. Beijing: Science Press, 2006. pp 338–340 (in Chinese)[40]姜薇莉, 孙辉, 凌家煜. 粉质质量指数(FQN)对于评价小麦粉品质的实用价值研究. 中国粮油学报, 2004, 19(2): 42–48Jiang W L, Sun H, Ling J Y. Applicability of FQN in evaluation of wheat flour quality. J Chin Cereals Oils Assoc, 2004, 19(2): 42–48 (in Chinese with English abstract) |
[1] | 余国武, 青芸, 何珊, 黄玉碧. 玉米SSIIb蛋白多克隆抗体的制备及其应用[J]. 作物学报, 2022, 48(1): 259-264. |
[2] | 王珍, 姚梦楠, 张晓莉, 曲存民, 卢坤, 李加纳, 梁颖. 甘蓝型油菜BnMAPK1的原核表达、亚细胞定位及酵母双杂交文库筛选[J]. 作物学报, 2020, 46(9): 1312-1321. |
[3] | 苏亚春,王竹青,李竹,刘峰,许莉萍,阙友雄,戴明剑,陈允浩. 甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定[J]. 作物学报, 2017, 43(04): 510-521. |
[4] | 喻时周,杨成龙,郭建春,段瑞军. 海马齿甜菜碱醛脱氢酶基因克隆、高效表达及酶学特性分析[J]. 作物学报, 2016, 42(10): 1569-1574. |
[5] | 强治全,梁雅珺,于正阳,杜娅,张帅,朱维宁,张林生. 小麦wzy2-1基因的克隆及功能分析[J]. 作物学报, 2016, 42(08): 1253-1258. |
[6] | 王晓红, 朱攀攀, 梁燕梅, 韩淑梅, 赵爱春, 王传宏, 鲁成, 余茂德. 桑树多聚半乳糖醛酸酶抑制蛋白基因MaPGIP1的克隆及功能分析[J]. 作物学报, 2015, 41(09): 1361-1371. |
[7] | 成伟,郑艳茹,葛丹凤,程光远,翟玉山,邓宇晴,彭磊,谭向尧,徐景升*. 甘蔗转录激活因子ScCBF1基因的克隆与表达分析[J]. 作物学报, 2015, 41(05): 717-724. |
[8] | 白云凤,聂江婷,张忠梁,李平,张维锋,闫建俊,冯瑞云,张耀. 籽粒苋AhNAD-ME的序列特征与表达[J]. 作物学报, 2014, 40(12): 2192-2197. |
[9] | 杨华,高翔,陈其皎,赵万春,董剑,李晓燕. 簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定[J]. 作物学报, 2014, 40(04): 600-610. |
[10] | 谭秦亮,李长宁,杨丽涛,李杨瑞. 甘蔗ABA信号转导关键酶SoSnRK2.1基因的克隆与表达分析[J]. 作物学报, 2013, 39(12): 2162-2170. |
[11] | 谢登雷,崔江慧,常金华. 高粱中SbDREB2基因的克隆与表达分析[J]. 作物学报, 2013, 39(08): 1352-1359. |
[12] | 朱斌,陆俊杏,彭茜,翁昌梅,王淑文,余浩,李加纳,卢坤,梁颖. 甘蓝型油菜MAPK7基因家族及其启动子的克隆与表达分析[J]. 作物学报, 2013, 39(05): 789-805. |
[13] | 周凯, 宋丽艳, 叶武威*, 王俊娟, 王德龙, 樊保香. 陆地棉耐盐相关基因GhSAMS的克隆及表达[J]. 作物学报, 2011, 37(06): 1012-1019. |
[14] | 王明霞, 高翔, 陈其皎, 董剑, 赵万春, 李艳亮, 李敏. 小麦品种陕253 γ-醇溶蛋白基因的克隆、原核表达与功能鉴定[J]. 作物学报, 2011, 37(01): 79-86. |
[15] | 朱西平,李鑫,李雅轩,晏月明. 普通小麦及近缘粗山羊草α-醇溶蛋白基因的克降、定位与进化分析[J]. 作物学报, 2010, 36(4): 580-589. |
|