小麦阿拉伯木聚糖阿魏酸酰基转移酶基因的克隆与功能标记开发

doi:10.3864/j.issn.0578-1752.2018.19.002

摘要/Abstract

摘要： 【目的】克隆小麦阿拉伯木聚糖阿魏酸酰基转移酶（arabinoxylan feruloyl transferase，AFT）基因，开发与FAX含量紧密连锁的功能标记，提高对FAX含量预测的准确性，为小麦加工品质的改良提供依据。【方法】应用FR846233为探针，通过同源克隆的方法获得小麦FAX含量基因gDNA全序列，使用DNAMAN软件比较高、低FAX含量品种间的序列差异性；基于序列差异使用Primer5.0软件设计特异性引物，开发与FAX含量紧密连锁的功能标记并利用一套中国春缺体-四体系和3AL、3AS双端体系进行染色体物理定位；同时结合PCR验证的方法，利用253份来自于中国主要冬麦区的小麦品种（系）对功能标记的实用性进行验证，采用IBM SPSS statistics 19.0软件进行FAX含量与基因型间的相关性分析等数据统计分析。【结果】2对特异性引物B1、B2最终扩增出长度分别为800 bp及710 bp的片段，B1和B2扩增的PCR片段有80 bp重叠，将片段拼接得到位于3A染色体上AFT基因TaBahd-A1。TaBahd-A1序列由1 429个碱基对组成，得到等位变异TaBahd-A1a和TaBahd-A1b，2个等位变异都拥有一个1 266 bp的开放阅读框（open reading frame，ORF），都具有2个外显子和1个内含子，内含子符合典型GT-AG结构，等位变异序列之间相似度为98.08%，具有24个单核苷酸多态性（single nucleotide polymorphisms，SNPs）位点和3个插入缺失（insertion-deletion，InDel）InDel位点，可编码421个氨基酸残基，预测分子量为45.2 kD。基于107 bp SNP处开发了2个互补显性标记AFTA2和AFTB2。AFTA2在TaBahd-A1a材料中能扩增出692 bp片段，与高FAX含量相关，在具有TaBahd-A1b等位变异的材料中未能扩增出片段。AFTB2则只能在TaBahd-A1b等位变异类型的材料中扩增出438 bp片段，并与低FAX含量相关，在TaBahd-A1a等位变异类型的材料中未能扩增出片段。同时利用一套中国春缺体-四体系和双端体材料将AFTA2和AFTB2定位在小麦3AL染色体。用功能标记AFTA2和AFTB2检测253份中国冬小麦材料，结果表明，不同基因型的FAX含量差异达到显著水平（P＜0.05）。从不同麦区来看表现各有不同，其中在北部冬麦区不同基因型的FAX含量在北部冬麦区材料间差异不显著；而在黄淮麦区，含有TaBahd-A1a的品种FAX含量显著高于含有TaBahd-A1b的品种（P＜0.05）。TaBahd-A1等位变异分布频率表明，TaBahd-A1a是与高FAX含量相关优异等位变异，且北部冬麦区TaBahd-A1a的频率（71.3%）显著高于黄淮冬麦区（60.2%）。【结论】基于TaBahd-A1序列成功开发1对显性互补标记AFTA2/AFTB2并定位于3AL染色体，标记与FAX含量相关可用于FAX含量的遗传改良。

关键词: 普通小麦, 阿魏酰阿拉伯木聚糖, 基因克隆, 功能标记开发

Abstract: 【Objective】 The arabinoxylan feruloyl transferase (AFT) gene was cloned from wheat, and the functional markers linked to the content of FAX were developed to improve the accuracy of predicting the content of FAX , in order to provide the basis for the improvement of wheat processing quality. 【Method】 The complete gDNA sequence of wheat FAX gene was obtained by homologous cloning method，using FR846233 as a probe. The sequence differences between high and low FAX content varieties were compared by the DNAMAN software; based on sequence difference, specific primers were designed with Primer5.0 software to develop functional markers closely linked to FAX content and a set of Chinese spring Nulli-tetrasomic lines and ditelosomic line 3AS, and 3AL were used for chromosome physical mapping; and the practicability of functional markers were verified by using 253 wheat varieties (lines) from the main winter wheat regions in China, combined with the method of PCR verification. IBM SPSS statistics 19.0 software was used to analyze the correlation between FAX content and genotypes. 【Result】Two pairs of specific primers B1 and B2 finally amplified fragments of 800 bp and 710 bp, respectively. And 80 bp overlaps of PCR fragments amplified by B1 and B2 were spliced to obtain the AFT gene that is located on chromosome 3A. The TaBahd-A1 sequence is consists of 1429 base pairs and allelic variants TaBahd-A1a and TaBahd-A1b are obtained. The two allelic variants possess a 1266 bp open reading frame, two exons and one intron. The introns conformed to the typical GT-AG structure. The similarity between the alleles was 98.08%, with 24 SNPs and 3 InDels, which could encode 421 amino acid residues and the predicted molecular weight was 45.2 kDa. Two complementary dominant markers AFTA2 and AFTB2 were developed based on the 107 bp SNP. AFTA2 was able to amplify a 692 bp fragment in the TaBahd-A1a material, which was associated with a high FAX content, but not in the material with TaBahd-A1b allelic variation. AFTB2 could only amplify a 438 bp fragment in TaBahd-A1b type material and correlate with low FAX content, but not in TaBahd-A1a type material, and AFTA2 and AFTB2 were located in 3AL chromosome of wheat by a set of Chinese spring Nulli-tetrasomic lines. Using the functional markers AFTA2 and AFTB2 to detect 253 Chinese winter wheat materials, the results showed that the difference in FAX content of different genotypes reached a significant level (P < 0.05), and there was no significant difference in FAX content in Northern China Plain Winter Wheat Region; but in the Yellow & Huai River Valley Winter Wheat Region, The FAX content of the TaBahd-A1a-containing variety was significantly higher than that of the TaBahd-A1b- containing variety (P<0.05). Therefore, the complementary dominant markers AFTA2 and AFTB2 are related to FAX content and can be effectively used for genetic improvement of FAX content. The frequency of TaBahd-A1 allele variation indicates that TaBahd-A1a is an excellent allelic variant associated with high FAX content, and the frequency of TaBahd-A1a in the Northern China Plain Winter Wheat Region (71.3%) is significantly higher than that in the Yellow & Huai River Valley Winter Wheat Region (60.2%). 【Conclusion】 The results suggested that these two STS markers are closely related to FAX content related gene and could be used for the improvement of wheat processing quality for wheat-based products.

Key words: common wheat, feruloyl arabinoxylan, gene cloning, functional marker development

战帅帅，白璐，谢磊，夏先春，任毅，吕文娟，曲延英，耿洪伟. 小麦阿拉伯木聚糖阿魏酸酰基转移酶基因的克隆与功能标记开发[J]. 中国农业科学, 2018, 51(19): 3639-3650.

ZHAN ShuaiShuai, BAI Lu, XIE Lei, XIA XianChun, REN Yi, Lü WenJuan, QU YanYing, GENG HongWei. Arabinoxylan Feruloyl Transferase Gene Cloning and Development of Functional Markers in Common Wheat[J]. Scientia Agricultura Sinica, 2018, 51(19): 3639-3650.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.19.002

https://www.chinaagrisci.com/CN/Y2018/V51/I19/3639

参考文献

[1] 何中虎, 晏月明, 庄巧生, 张艳, 夏先春, 张勇, 王德森, 夏兰芹, 胡英考, 蔡民华, 陈新民, 阎俊, 周阳. 中国小麦品种品质评价体系建立与分子改良技术研究. 中国农业科学, 2006, 39(6): 1091-1101.

HE Z H, YAN Y M, ZHUANG Q S, ZHANG Y, XIA X C, ZHANG Y, WANG D S, XIA L Q, HU Y K, CAI M H, CHEN X M, YAN J, ZHOU Y. Establishment of quality evaluation system and utilization of molecular methods for the improvement of Chinese wheat quality. Scientia Agricultura Sinica, 2006, 39(6): 1091-1101. (in Chinese)

[2] 刘志勇, 王道文, 张爱民, 梁翰文, 吕慧颖, 邓向东, 葛义强, 魏珣, 杨维才. 小麦育种行业创新现状与发展趋势. 植物遗传资源学报, 2018, 19(3): 430-434.

LIU Z Y, WANG D W, ZHANG A M, LIANG H W, LÜ H Y, DENG X D, GE Y Q, WEI X, YANG W C. Current status and perspective of wheat genomics, genetics, and breeding. Journal of Plant Genetic Resources, 2018, 19(3): 430-434. (in Chinese)

[3] GENG H W, XIA X C, ZHANG L P, QU Y Y, HE Z H. Development of functional markers for a lipoxygenase gene TaLox-B1 on chromosome 4BS in common wheat. Crop Science, 2012, 52: 568-576.

[4] 何中虎, 夏先春, 陈新民, 庄巧生. 中国小麦育种进展与展望. 作物学报, 2011, 37(2): 202-215.

HE Z H, XIA X C, CHEN X M, ZHUANG Q S. Progress of wheat breeding in China and the future perspective. Acta Agronomica Sinica, 2011, 37(2): 202-215. (in Chinese)

[5] 周素梅, 王强, 张晓娜. 小麦中功能性多糖阿拉伯木聚糖研究进展. 核农学报, 2009, 23(2): 297-301.

ZHOU S M, WANG Q, ZHANG X N. Research advances in functional polysaccharide-wheat arabinoxylan. Journal of Nuclear Agricultural Sciences, 2009, 23(2): 297-301. (in Chinese)

[6] DELCOUR J A, VANHAMEL S, HOSENEY R C. Physicchemical and functional properties of rye non-starch polysaccharides: II. The impact of a fraction containing water-soluble pentosans and proteins on gluten-starch loaf volumes. Cereal Chemistry, 1991, 68: 72-76.

[7] IZYDORCZYK M S, BILIADERIS C G. Cereal arabinoxylans advances in structure and physicochemical properties. Carbohydrate Polymers, 1995, 28: 33-48.

[8] 袁小平, 王静, 姚惠源. 小麦麸皮阿魏酰低聚糖对红细胞氧化性溶血抑制作用的研究. 中国粮油学报, 2005, 20(1): 13-16.

YUAN X P, WANG J, YAO H Y. A study on the inhibition of erythrocyte oxidative hemolysis in wheat bran. Journal of Chinese cereals and oils, 2005, 20(1): 13-16. (in Chinese)

[9] BONTPART T, CHEYNIER V, AGEORGES A, TERRIER N. BAHD or SCPL acyltransferase? What a dilemma for acylation in the world of plant phenolic compounds. New Phytologist, 2015, 208: 695-707.

[10] 刘亚男, 夏先春, 何中虎. 普通小麦TaDep1基因克隆与特异性标记开发. 作物学报, 2013, 39(4): 589-598.

LIU Y N, XIA X C, HE Z H.Characterization of dense and erect panicle 1 gene (TaDep1) located on common wheat group 5 chromosomes and development of allele-specific markers. Acta Agronomica Sinica, 2013, 39(4): 589-598. (in Chinese)

[11] 高华利, 王黎明, 柴军琳, 董普辉, 王春平, 李兴锋. 小麦籽粒品质性状基因功能标记的开发及应用. 中国粮油学报, 2016, 31(8): 152-157.

GAO H L, WANG L M, CHAI J L, DONG P H, WANG C P, LI X F. Development and application of gene functional markers of grain quality traits in wheat. Journal of the Chinese Cereals and Oils Association, 2016, 31(8): 152-157. (in Chinese)

[12] BURR S J, FRY S C. Feruloylated arabinoxylans are oxidatively cross-linked by extracellular maize peroxidase but not by horseradish peroxidase. Molecular Plant, 2009, 2: 883-892.

[13] MARTINANT J P, CADALEN T, BILLOT A, CHARTIER S, LEROY P, BERNARD M, SAULNIER L, BRANLARD S G. Genetic analysis of water extractable arabinoxylans in bread wheat endosperm. Theoretical and Applied Genetics, 1998, 97: 1069-1075.

[14] RAO R S P, MURALIKRISHNA G. Non-starch polysaccharide- phenolic acid complexes from native and germinated cereals and millet. Food Chemistry, 2004, 84: 527-531.

[15] RAO R S P, MURALIKRIAHNA G. Structural characteristics of water-soluble feruloyl arabinoxylans from rice (Oryza sativa) and ragi (finger millet, Eleusine coracana): variations upon malting. Food Chemistry, 2007, 104: 1160-1170.

[16] ORDAZ-ORTIZ J J, DEVAUX M F, SAULNIER L. Classification of wheat varieties based on structural features of arabinoxylans as revealed by endoxylanase treatment of flour and grain. Agriculture and food chemistry, 2005, 53: 8349-8356.

[17] 张岐军, 钱森和, 张艳, 何中虎, 姚大年. 中国软质小麦品种戊聚糖含量的遗传变异及其与饼干加工品质的关系. 中国农业科学, 2005, 38(9): 1734-1738.

ZHANG Q J, QIAN S H, ZHANG Y, HE Z H, YAO D N. Variation of pentosans in Chinese soft wheat cultivars and correlations with cookie quality. Scientia Agricultura Sinica, 2005, 38(9): 1734-1738. (in Chinese)

[18] 钱森和, 张艳, 王德森, 何中虎, 张歧军, 姚大年. 小麦品种戊聚糖和溶剂保持力遗传变异及其与品质性状关系的研究. 作物学报, 2005, 31(7): 902-907.

QIAN S H, ZHANG Y, WANG D S, HE Z H, ZHANG Q J, YAO D N. Variation of pentosans and solvent retention capacities in wheat genotypes and their relationship with processing quality. Acta Agronomica Sinica, 2005, 31(7): 902-907. (in Chinese)

[19] 刘雨雨, 莫婷, 王晓晖, 史社坡, 刘晓, 屠鹏飞. 植物来源BAHD酰基转移酶家族研究进展. 中国中药杂志, 2016, 41(12): 2175-2182.

LIU Y Y, MO T, WANG X H, SHI S P, LIU X, TU P F. Research progress of plant BAHD acyltransferase family. China journal of Chinese material medical, 2016, 41(12): 2175-2182. (in Chinese)

[20] MITCHELL R A C, DUPREE P, SHEWRY P R. A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan. Plant Physiology, 2007, 144: 43-53.

[21] OHYANAGI H, TANAKA T, SAKAI H, SHIGEMOTO Y, YAMAGUCHI K, HABARA T, FUJII Y, ANTONIO B A, NAGAMURA Y, IMANISHI T, IKEO K, ITOH T, GOJOBORI T, SASAKI T. The rice annotation project database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Nucleic Acids Research, 2006, 34: 741-744.

[22] TANAKA T, ANTONIO B A, KIKUCHI S, MATSUMOTO T, NAGAMURA Y, NUMA H, SAKAI H, WU J, ITOH T, SASAKI T, AONO R, FUJII Y, HABARA T, HARADA E, KANNO M, KAWAHARA Y, KAWASHIMA H, KUBOOKA H, MATSUYA A, NAKAOKA H, SAICHI N, SANBONMATSU R, SATO Y, SHINSO Y, SUZUKI M, TAKEDA J, TANINO M, TODOKORO F, YAMAGUCHI K, YAMAMOTO N, YAMASAKI C, IMANISHI T, OKIDO T, TADA M, IKEO K, TATENO Y, GOJOBORI T, LIN Y C, WEI F J, HSING Y, ZHAO Q, HAN B, KRAMER M R, MCCOMBIE R W, LONSDALE D, O’DONOVAN C C, WHITFIELD E J, APWEILER R, KOYANAGI K O, KHURANA J P, RAGHUVANSHI S, SINGH N K, TYAGI A K, HABERER G, FUJISAWA M, HOSOKAWA S, ITO Y, IKAWA H, SHIBATA M, YAMAMOTO M, BRUSKIEWICH R M, HOEN D R, BUREAU T E, NAMIKI N, OHYANAGI H, SAKAI Y, NOBUSHIMA S, SAKATA K, BARRERO R A, SATO Y, SOUVOROV A, SMITH-WHITE B, TATUSOVA T, AN S Y, AN G, OOTA S, FUKS G, MESSING J, CHRISTIE K R, LIEBERHERR D, KIM H, ZUCCOLO A, WING R A, NOBUTA K, GREEN P J, LU C, MEYERS B C, CHAPARRO C, PIEGU B, PANAUD O, ECHEVERRIA M. The rice annotation project database (RAP-DB): 2008 update. Nucleic Acids Research, 2008, 36: 1028-1033.

[23] MATSUMOTO T, TANAKA T, SAKAI H, AMANO N, KANAMRI H, KURITA K, KIKUTA A, KAMIYA K, YAMAMOTO M, IKAWA H, FUJII N, HORI K, ITOH T, SATO K. Comprehensive sequence analysis of 24,783 barley full-length cDNA derived from 12 clone libraries. Plant Physiology, 2011, 156: 20-28.

[24] LI Y, ZHAO D H, JUN Y, ZHANG Y L, XIA, X C, TIAN Y B, HE Z H, ZHANG Y. QTL mapping of grain arabinoxylan contents in common wheat using a recombinant inbred line population. Euphytica, 2016, 208: 205-214.

[25] 吕文娟, 刘金栋, 白璐, 曲延英, 任毅, 闻伟鄂, 耿洪伟. 小麦籽粒阿魏酰阿拉伯木聚糖含量QTL定位. 分子植物育种, 2018, 16(5): 1530-1538.

Lü W J, LIU J D, BAI L, QU Y Y, REN Y, WEN W E, GENG H W. QTL mapping and genome-wide association study of Ferulic acid Arabiaxylan content in wheat grain. Molecular Plant Breeding, 2018, 16(5): 1530-1538. (in Chinese)

[26] GRIENENBERGER E, BESSEAU S, GEOFFROY P, DEBAYLE D, HEINTE D, LAPIERRE C, POLLET B, HEITZ T, LEGRAND M. A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines. The Plant Journal, 2009, 58: 246-259.

[27] BEAUGRAND J, CONIER D, DEBEIRE P, CHABBERT B. Arabinoxylan and hydroxycinnamatecontent of wheat bran in relation to endoxylanase susceptibility. Journal of Cereal Science, 2004, 40: 223-230.

[28] 杨莉, 黄玉莲, 常萍, 阎俊, 张业伦, 夏先春, 田宇兵, 何中虎, 张勇. 小麦阿拉伯木聚糖含量的QTL分析及其与品质性状的关系. 作物学报, 2014, 40(9): 1695-1701.

YANG L, HUANG Y L, CHANG P, YAN J, ZHANG Y L, XIA X C, TIAN Y B, HE Z H, ZHANG Y. QTL mapping for arabinoxylans content and its relationship with processing quality in common wheat. Acta Agronomica Sinica, 2014, 40(9): 1695-1701. (in Chinese)

[29] 李冰, 张照贵, 王佳佳, 李斯深. 小麦GDH1基因克隆及其功能标记开发. 山东农业科学, 2014, 46(10): 6-11.

LI B, ZHANG Z G, WANG J J, LI S S. Development of wheat GDH1 gene cloning and its functional markers. Shandong agricultural science, 2014, 46(10): 6-11. (in Chinese)

[30] HUANG S, MORRISON W R, Aspects of protein in Chinese and British common (hexaploid) wheats related to quality of white and yellow Chinese noodles. Journal of Cereal Science, 1988, 8:177-187.

[31] 李亚青, 毛新国, 赵宝存, 葛荣朝, 沈银, 黄占景. 小麦糖原合成酶激酶基因（TaGSK1）的染色体定位. 华农学报, 2006, 21(5): 39-41.

LI Y Q, MAO X G, ZHAO B C, GE R Z, SHEN Y, HUANG Z J. Chromosome localization of the gene of wheat glycogen synthase kinase (TaGSK1). Acta agricultural boreali sinica, 2006, 21(5): 39-41. (in Chinese)

[32] HESSLER T G, THOMSON M J, BENSCHER D, NACHIT M M, SORRELLS M E. Association of a lipoxygenase locus, Lpx-B1, with variation in lipoxygenase activity in durum seeds. Crop Science, 2002, 42: 1695-1700.

[33] SUN D J, HE Z H, XIA X C, ZHANG L P, MORRIS C F, APPELS R, MA W J, WANG H. A novel STS marker for polyphenol oxidase activity in bread wheat. Molecular Breeding, 2005, 16: 209-218.

[34] CARRERA V, ECHENIQUE W, ZHANG M, HELGUERA F, MANTHEY A, SCHRAGER0 A, PICCA G, CERVIGNI J, DUBCOVSKY. A deletion at the Lpx-B1 locus is associated with low lipoxygenase activity and improved pasta color in durum wheat. Journal of Cereal Science, 2007, 45: 67-77.

[35] SOMERS D J, ISAAC P, EDWARDS K. A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics, 2004, 109: 1105-1114.

[36] D’AURIA J C. Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology, 2006, 9: 331-340.

[37] YU X H, GOU J Y, LIU C J. BAHD superfamily of acyl-CoA dependent acyltransferases in Populus and Arabidopsis: bioinformatics and gene expression. Plant Molecular Biology, 2009, 70: 421-442.

[38] PENG M, GAO Y Q, CHEN W, WANG W S, SHEN S Q, SHI J, WANG C, ZHANG Y, ZOU L, WANG S C, WANG J, LIU X Q, GONG L, LUO J. Evolutionarily distinct BAHD N-acyltransferases are responsible for natural variation of aromatic amine conjugates in rice. Plant Cell Advance Publication, 2016, 28: 1533-1550.

[39] TREMMEL B K, LANG L, TOROK K, TOMOSKOZI S, VIDA G, SHEWRY P R, BEDO Z, RAKSZEGI M. Development and characterization of wheat lines with increased levels of arabinoxylan. Euphytica, 2017, 213: 291-306.

[40] WEI B, JING R L, WANG C S, CHEN J B, MAO X G, CHANG X P, JIA J Z. Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers, gene mapping based on SNPs. Molecular Breeding, 2009, 23: 13-22.

[41] SMITH P J, WANG H T, WILLIAM S, MARIA J, BREEANNA R. Designer biomass for next-generation biorefineries: leveraging recent insights into xylan structure and biosynthesis. Biotechnology for Biofuels, 2017, 10: 286-300.

[42] EUDES A, MOUILLE M, ROBINSON D S, BENITES V T, WANG G, ROUX L, TSAI Y L, BAIDOO E E K, CHIU T Y, HEAZLEWOOD J L, SCHELLER H V, MUKHOPADHYAY A, KEASLING J D, DEUTSCH S, LOQUÉ D. Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast. Microbial Cell Factories, 2016, 15: 198-214.