中国农业科学 ›› 2018, Vol. 51 ›› Issue (19): 3639-3650.doi: 10.3864/j.issn.0578-1752.2018.19.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇




  1. 1新疆农业大学农学院/新疆农业大学生物技术重点实验室,乌鲁木齐 830052;2新疆农业大学科学与技术学院,乌鲁木齐 830091;3中国农业科学院作物科学研究所/国家小麦改良中心,北京100081
  • 收稿日期:2018-04-21 出版日期:2018-10-01 发布日期:2018-10-01
  • 通讯作者: 耿洪伟,Tel:13579873801;
  • 作者简介:战帅帅,Tel:17690762406;
  • 基金资助:

Arabinoxylan Feruloyl Transferase Gene Cloning and Development of Functional Markers in Common Wheat

ZHAN ShuaiShuai1, BAI Lu2, XIE Lei1, XIA XianChun3, REN Yi1Lü  WenJuan1, QU YanYing 1, GENG HongWei1   

  1. 1 College of Agronomy, Xinjiang Agricultural University/Laboratory of Agricultural Biological Technology, Urumqi 830052; 2College of science and technology, xinjiang agricultural university, urumqi 830091; 3Institute of Crop Science, Chinese  Academy of Agricultural Sciences/National Wheat Improvement Center, Beijing 100081
  • Received:2018-04-21 Online:2018-10-01 Published:2018-10-01

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