[1]孙彦波,李忠峰,王俊,等.基于TILLING技术筛选大豆GmAGL15基因突变体[J].大豆科学,2019,38(06):906-913.[doi:10.11861/j.issn.1000-9841.2019.06.0906]
　SUN Yan-bo,LI Zhong-feng,WANG Jun,et al.Screening AGL15Mutant in Soybean by TILLING[J].Soybean Science,2019,38(06):906-913.[doi:10.11861/j.issn.1000-9841.2019.06.0906]
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基于TILLING技术筛选大豆GmAGL15基因突变体

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第38卷 期数: 2019年06期 页码: 906-913 栏目: 出版日期: 2019-11-20
Title:
Screening AGL15Mutant in Soybean by TILLING
作者:
孙彦波1; 2; 李忠峰3; 王俊1; 邱丽娟1; 2
（1.长江大学 农学院，湖北 荆州 434025； 2.中国农业科学院 作物科学研究所/国家农作物基因资源与遗传改良重大科学工程/农业部种质资源利用重点实验室，北京 100081； 3.河南农业大学 农学院，河南 郑州 450002）
Author(s):
SUN Yan-bo1; 2;  LI Zhong-feng3;  WANG Jun1;  QIU Li-juan1; 2
(1.College of Agriculture, Yangtze University, Jingzhou 434025, China; 2.Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture, Institute of Crop Sciences, Chinese Academy of Agricultural Science/National Key Facility for Gene Resources and Genetic Improvement, Beijing 100081, China; 3.College of Agriculture, Henan Agricultural University, Zhengzhou 450002, China)
关键词:
大豆; 转录因子; AGL15; EMS; TILLING
Keywords:
Soybean;  Transcription factor;  AGL15;  EMS;  TILLING
DOI:
10.11861/j.issn.1000-9841.2019.06.0906
摘要:
AGL15是MADS（MCM1-AGAMOUS-DEFICIENS-SRF）基因家族中的一员，MADS蛋白通过与下游基因启动子的顺式作用元件特异性结合调控植物种子中贮藏物质的积累。为验证大豆中AGL15基因的功能，利用TILLING技术在EMS诱变的中品661的M5中筛选出8个GmAGL15突变体。共检测到10个突变位点，其中6个突变位点发生在基因的编码区导致氨基酸发生改变，4个突变位点发生在基因的内含子。1个SNP导致该基因第2个外显子的第54个碱基由C变为T，并使该基因编码的第79位氨基酸由丙氨酸突变为缬氨酸；5个SNP发生在该基因的第6个外显子的112位碱基上，使得碱基由T变为A，导致该基因编码的第568位氨基酸由亮氨酸突变为甲硫氨酸。大豆GmAGL15的突变分布频率为1/805 kb。考种发现5个非同义突变体和1个内含子突变体的蛋白质或脂肪含量与野生型ZP661相比差异显著。本研究发现的等位变异有助于阐明GmAGL15在大豆种子中的作用机制，并为大豆的遗传改良提供宝贵的种质资源。
Abstract:
AGL15is a member of the MADS (MCM1-AGAMOUS-DEFICIENS-SRF) gene family. MADS proteins regulate the accumulation of storage substances in plant seeds by binding specifically to cis-acting elements of downstream gene promoters. To verify the function of AGL15gene in soybean, eight GmAGL15mutants were screened from M5 of ZP661 mutant induced by EMS with TILLING. Ten mutation sites were detected, six of which occurred in the coding region of the gene, resulting in changes in amino acids, and four occurred in the intron of the gene. One SNP changed from C to T at the 54th base of the second exon, and mutated the 79th amino acid from alanine to valine. Five SNPs occurred on the 112 base of the sixth exon, which changed the base from T to A, resulting in the mutation of the 568th amino acid from leucine to methionine. The mutation frequency of soybean GmAGL15was 1/805 kb. The protein or fat contents of five non-synonymous mutants and one intron mutant were significantly different from those of wild type ZP661. Allelic variation found in this study is helpful to elucidate the mechanism of GmAGL15in soybean seeds and provide valuable germplasm resources for soybean genetic improvement.

参考文献/References:

［1］朱浩哲, 黄初女, 王光达, 等. 浅谈大豆蛋白质品质改良［J］. 吉林农业科学, 2006, 31(1): 4-8. (Zhu H Z, Huang C N, Wang G D, et al. Talking about the improvement of soybean protein quality［J］. Journal of Jilin Agricultural Sciences, 2006, 31(1): 4-8.)［2］Wobus U, Weber H. Seed maturation: Genetic programmes and control signals［J］. Current Opinion in Plant Biology, 1999, 2(1): 33-38.［3］Lotan T , Ohto M , Yee K M , et al. Arabidopsis LEAFY COTYLEDON1is sufficient to induce embryo development in vegetative cells［J］. Cell, 1998, 93(7): 1195-1205.［4］Luerssen H, Kirik V, Herrmann P, et al. FUSCA3, encodes a protein with a conserved VP1/ABI3_like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana［J］. The Plant Journal, 1998, 15(6): 10.［5］Stone S L, Kwong L W, Yee K M, et al. LEAFY COTYLEDON2encodes a B3 domain transcription factor that induces embryo development［J］. Proceedings of the National Academy of Sciences, 2001, 98(20): 11806-11811.［6］Finkelstein R, Reeves W, Ariizumi T . Molecular aspects of seed dormancy［J］. Annual Review of Plant Biology, 2008, 59(59): 387-415.［7］Heck G R, Perry S E, Nichols K W, et al. AGL15, a MADS domain protein expressed in developing embryos［J］. The Plant Cell, 1995, 7(8): 1271-1282.［8］Messenguy F, Dubois E. Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development［J］. Gene, 2003, 316(1): 1-21.［9］Nardeli S M, Artico S, Aoyagi G M, et al. Genome-wide analysis of the MADS-box gene family in polyploid cotton (Gossypium hirsutum) and in its diploid parental species (Gossypium arboreum, and Gossypium raimondii)［J］. Plant Physiology & Biochemistry, 2018, 127: 169-184.［10］Holdsworth M J, Leónie B, Soppe W J J. Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination［J］. The New Phytologist, 2008, 179(1): 33-54.［11］Harding E W, Tang W, Nichols K W, et al. Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15［J］. Plant Physiology, 2003, 133(2): 653-663.［12］Mccallum C M, Comai L, Greene E A, et al. Targeting induced local lesions in genomes (TILLING) for plant functional genomics［J］. Plant Physiology, 2000, 123(2): 439-442.［13］Hdrich N, Gibon Y, Schudoma C, et al. Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity［J］. Journal of Plant Physiology, 2011, 168(12):1395-1405.［14］Lai K S, Kaothien-Nakayama P, Iwano M, et al. A TILLING resource for functional genomics in Arabidopsis thaliana accession C24［J］. Genes & Genetic Systems, 2012, 87(5): 291-297.［15］Martín B, Ramiro M, Martínezzapater J M, et al. A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis［J］. BMC Plant Biology, 2009, 9(1):147.［16］Till B J, Reynolds S H, Weil C, et al. Discovery of induced point mutations in maize genes by TILLING［J］. BMC Plant Biology, 2004, 4:12-19.［17］Peter C, Tai T H, Jennifer C, et al. Discovery of chemically induced mutations in rice by TILLING［J］. BMC Plant Biology, 2007, 7:19-30.［18］Uauy C, Paraiso F, Colasuonno P, et al. A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat［J］. BMC Plant Biology, 2009, 9:115-128.［19］Dong C M, Daltonmorgan J, Vincent K, et al. A modified TILLING method for wheat breeding［J］. Plant Genome, 2009, 2(1):39-47.［20］Suzuki T, Eiguchi M, Kumamaru T, et al. MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice［J］. Molecular Genetics & Genomics, 2008, 279(3): 213-223.［21］Stephenson P, Baker D, Girin T, et al. A rich TILLING resource for studying gene function in Brassica rapa［J］. BMC Plant Biology, 2010, 10(1): 62.［22］Cooper J L, Till B J, Laport R G, et al. TILLING to detect induced mutations in soybean［J］. BMC Plant Biology, 2008, 8(1): 9.［23］Bilyeu K D, Dierking E C. New sources of soybean seed meal and oil composition traits identified through TILLING［J］. BMC Plant Biology, 2009, 9(1): 1-11.［24］Lakhssassi N, Zhou Z, Liu S, et al. Characterization of the FAD2gene family in soybean reveals the limitations of gel-based TILLING in genes with high copy number［J］. Frontiers in Plant Science, 2017, 8: 324.［25］Jiang G Q, Yao X F, Liu C M. A simple CEL I endonuclease-based protocol for genotyping both SNPs and InDels［J］. Plant Molecular Biology Reporter, 2013, 31(6): 1325-1335.［26］Anai T. Potential of a mutant-based reverse genetic approach for functional genomics and molecular breeding in soybean［J］. Breeding Science, 2012, 61(5): 462-467.［27］Dreni L, Zhang D. Flower development: The evolutionary history and functions of the AGL6subfamily MADS-box genes［J］. Journal of Experimental Botany, 2016, 67(6): 1625-1638.［28］Zeng X, Liu H, Du H, et al. Soybean MADS-box gene GmAGL1promotes flowering via the photoperiod pathway［J］. BMC Genomics, 2018, 19(1): 51.［29］Perry S E, Zheng Q, Zheng Y. Transcriptome analysis indicates that GmAGAMOUS-Like 15may enhance somatic embryogenesis by promoting a dedifferentiated state［J］. Plant Signaling & Behavior, 2016, 11(7): e1197463.［30］Chen N, Veerappan V, Abdelmageed H, et al. HSI2/VAL1 silences AGL15to regulate the developmental transition from seed maturation to vegetative growth in Arabidopsis［J］. The Plant Cell, 2018, 30(3): 600-619.［31］Wang H, Tang W, Zhu C, et al. A chromatin immunoprecipitation (ChIP) approach to isolate genes regulated by AGL15, a MADS domain protein that preferentially accumulates in embryos［J］. The Plant Journal, 2002, 32(5): 13.［32］汪潇琳, 陈艳萍, 喻德跃. MADS-box基因GmAGL15在大豆种子发育过程中的表达［J］. 作物学报, 2008, 34(2): 330-332.(Wang X L, Chen Y P, Yu D Y. Expression of the MADS-Box gene GmAGL15in seed development of soybean［J］. Acta Agronomica Sinica, 2008, 34(2): 330-332.)［33］Fernandez D E, Heck G R, Perry S E, et al. The embryo MADS domain factor AGL15acts postembryonically: Inhibition of perianth senescence and abscission via constitutive expression［J］. The Plant Cell, 2000, 12(2):183-197.［34］王有伟, 苗燕妮, 江鹏, 等. 水稻产量、蛋白质及食味特性的关联研究［J］. 中国农学通报, 2017, 33(5): 1-5. (Wang Y W, Miao Y N, Jiang P, et al. Correlation studies on yield, protein and palatability of rice［J］. Chinese Agricultural Sicence Bulletin, 2017, 33(5): 1-5.)［35］Wilcox J R, Cavins J F. Backcrossing high seed protein to a soybean cultivar［J］. Crop Science, 1995, 35(4): 1036-1041.［36］郭小红. 不同年代育成大豆品种农艺性状和产量的比较［D］. 沈阳: 沈阳农业大学, 2017. (Guo X H. Comparison on agronomic traits and yield of soybean cultivars developed in different years［D］. Shenyang: Shenyang Agricultural University, 2017.）

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备注/Memo

基金项目：大豆种质资源保护与利用（2019NWB036）；国家农作物资源共享平台（NICGR2019）。第一作者简介：孙彦波（1991-），男，硕士，主要从事大豆突变体筛选和鉴定研究。E-mail:1012080725@qq.com。通讯作者：邱丽娟（1963-），女，博士，研究员，主要从事大豆基因资源发掘与利用研究。E-mail:qiulijuan@caas.cn。

更新日期/Last Update: 1900-01-01