欢迎访问作物学报,今天是

作物学报 ›› 2023, Vol. 49 ›› Issue (10): 2613-2620.doi: 10.3724/SP.J.1006.2023.33004

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米籽粒突变体crk4的基因克隆与等位性分析

李萌园1(), 张文成2, 高勇1, 秦永田2, 薄仕榕1, 宋琨洋1, 汤继华1, 付志远1()   

  1. 1河南农业大学农学院 / 省部共建小麦玉米作物学国家重点实验室 / 河南粮食作物协同创新中心, 河南郑州 450046
    2鹤壁市农业科学院, 河南鹤壁 458030
  • 收稿日期:2023-01-14 接受日期:2023-04-17 出版日期:2023-10-12 网络出版日期:2023-04-24
  • 通讯作者: 付志远, E-mail: fuzhiyuan2004@163.com
  • 作者简介:E-mail: 18839774957@163.com
  • 基金资助:
    河南省重点研发与推广专项(科技攻关)项目(232102111080)

Map-based cloning and allelic analysis of gene controlling maize kernel mutant crk4

LI Meng-Yuan1(), ZHANG Wen-Cheng2, GAO Yong1, QIN Yong-Tian2, BO Shi-Rong1, SONG Kun-Yang1, TANG Ji-Hua1, FU Zhi-Yuan1()   

  1. 1National Key Laboratory of Wheat and Maize Crop Science / Collaborative Innovation Center of Henan Grain Crops / College of Agronomy, Henan Agricultural University, Zhengzhou 450046, Henan, China
    2Hebi Academy of Agricultural Sciences, Hebi 458030, Henan, China
  • Received:2023-01-14 Accepted:2023-04-17 Published:2023-10-12 Published online:2023-04-24
  • Contact: E-mail: fuzhiyuan2004@163.com
  • Supported by:
    Key Technology Research and Development Program of Henan Province(232102111080)

摘要:

籽粒突变体是克隆籽粒发育关键基因并解析其遗传调控机制的重要材料。crk4 (crumpled kernel 4)是育种选系过程中发现的籽粒突变体, 与野生型相比, 其籽粒灌浆差、粒重和发芽率显著降低。遗传分析表明该突变由单个隐性核基因控制, 图位克隆将该基因定位于玉米5号染色体614 kb的物理区间内, 该区间包含11个在籽粒中表达的蛋白编码基因。序列分析表明, Zm00001d017427基因的第2个外显子上存在一个由C碱基缺失造成的crk4特异的终止突变。Zm00001d017427编码金属-烟酰胺转运蛋白(Metal-nicotianamine transporter, Sh4-shrunken4/YSL2), 是已报道的籽粒突变体ysl2的等位基因。等位性测验结果表明, crk4sh4/ysl2的一个新的等位突变体。crk4的鉴定为阐明Sh4基因调控玉米籽粒发育的分子机制提供了新的种质资源。

关键词: 玉米, 籽粒突变体, crk4, 图位克隆, sh4/ysl2

Abstract:

Kernel mutants are the important materials for cloning genes related to grain development and analyzing their genetic regulation mechanism. Crk4 (crumpled kernel 4) is a kernel mutant identified in the course of maize breeding and selection. Compared with the wild type, crk4 showed significantly lower in grain filling, grain weight, and germination rate. Genetic analysis showed that the mutant was controlled by a single recessive nuclear gene, which was mapped to a 614 kb physical distance on chromosome 5 by map-based cloning. In the 614 kb interval, 11 protein-coding genes were expressed in kernel. Sequence analysis revealed that there was a crk4-specific termination mutation in the second exon of Zm00001d017427 gene, which is caused by the deletion of C base. Zm00001d017427 encoded the metal-nicotianamine transporter (Sh4-shrunken4/Ysl2), which had been reported as the target gene of kernel mutant ysl2. The allelism test indicated that crk4 was a new allele mutant of ysl2/sh4. The identification of crk4 provided a new germplasm for elucidating the molecular regulation mechanism of Sh4 on seed development in maize.

Key words: maize, kernel mutant, crk4, map-based cloning, sh4/ysl2

表1

基因定位引物"

引物名称
Primer name
正向引物序列
Forward sequence (5'-3')
反向引物序列
Reverse sequence (5'-3')
S09840
S09979
ACGCCAGTGCTAATGGAATCG
ATCGCCCTTGAGGTGAAACAAC
GCAACACAACACGCCAACAC
AGTCGAAAGCCAACCAAAGGAG
5-14 CATTTTGAGCGCTTTGATGA AGCAGCTCCTGTCGTGAAGT
5-32 GCCAGCGTCTTCTCTGATTC CTTGGTATGTTTCGCCAGGT
303 TCGGGTGGGATGGTTGGGGT GCCGTGGCGCTGAGAACAGT
499 ACTGCGACACTTGACGATGGGT GTGCCGGAGCAAACGAGCCT
5-38 GGTTGGGAGGATGATATCGA TCTCTCACTCTCAGGCAGCA
5-41 TGAGTCCGATGCTCACAGAG CTTTAGCGACGGCACTTACC
592-6 AAAAGCTTATACGGTTACAAGGT TGTTTACTGACATATGATCTCCAA
873-1 GGAAAGCGAAGCAGGTAA GTGGCTCAATCTGGAAACAT
5-284 CCCGAAGCTCGGTGCCAGGA GCAAGGACTTCAGTTGTTATGTTGCTC
S10043 GATTTCCTTGATCCTGGGTGAG TGTGTATGCAAATGGACTTAGC
S10049 TGGCATGGAGTCCACCAATTAC CACGGTGCCCAAACAAAAGAG
S10084 GGGTTGGGTTCAGGTGTTTCC GTCAGAGCACAAGGTGGGATG
S10118 AGAGCAGAGCGGAGCAGAC GTGTCAAGCAAAGCGTGTGTG

图1

crk4的表型分析 A: Mo17×crk4 F3分离果穗, 红色箭头表示crk4籽粒, 标尺为1 cm; B: 野生型和crk4的代表性籽粒比较, 标尺为1 cm; C: 野生型和crk4籽粒的粒长、粒宽、粒厚比较; D: 野生型和crk4的百粒重比较, 5次重复; E: 野生型和crk4籽粒的纵切比较, 标尺为1 cm; F: 野生型和crk4籽粒的横切比较, 标尺为1 cm; G: 野生型和crk4籽粒的芽率比较。柱状图均以平均值±标准误差表示, *: 差异显著性水平P < 0.05; **: 差异显著性水平P < 0.01; ***: 差异显著性水平P < 0.001。"

表2

F2分离果穗的籽粒表型统计"

果穗
Ear
正常籽粒个数
No. of normal kernels
突变籽粒个数
No. of mutant kernels
籽粒总数
Total kernels
实际比值
Observed ratio
卡方值
χ2 test
374-1 329 116 445 2.84:1 0.270<χ20.05
374-2 271 85 356 3.19:1 0.240<χ20.05
374-3 296 98 394 3.02:1 0.003<χ20.05

图2

crk4的图位克隆 A: crk4的图位克隆, 红色数字代表交换单株的数目, 黑色数字代表群体大小; crk4最终定位在5号染色体的614 kb的范围内, 11个基因在籽粒中表达, 其中红色箭头表示目的基因; B: CRK4基因结构示意图, 其中黑框表示外显子, 黑线表示内含子, 红色箭头表示crk4的突变位点, 黑色箭头依次表示等位突变体ysl2, sh4的突变位点; C: CRK4蛋白的结构域示意图。"

表3

候选区间内的基因注释"

基因名称 Gene name 注释Annotation
Zm00001d017422 Homeobox-leucine zipper protein ATHB-6
Zm00001d017423 Origin recognition complex subunit 2
Zm00001d017424 ATP-dependent zinc metalloprotease FTSH 7 chloroplastic
Zm00001d017425 Cytochrome b5 (isoform A-like)
Zm00001d017427 Probable metal-nicotianamine transporter YSL16
Zm00001d017429 Yellow stripe 1
Zm00001d017432 Uncharacterized LOC100275088
Zm00001d017435 Uncharacterized LOC100275801
Zm00001d017438 Uncharacterized LOC100381739
Zm00001d017441 Cyclin-P4-1
Zm00001d017444 Probable WRKY transcription factor 51

表4

候选基因测序引物"

引物名称
Primer name
正向引物序列
Forward sequence (5'-3')
反向引物序列
Reverse sequence (5'-3')
17427-1 TTGGCCAGAGAAAGCAACGA ATAAATGGCGGCGACCTCTC
17427-2 CAGGATTCCCCAGGAGAGGA CCAGGATTCGGGTGGATAGC
17427-3 ATGGATTCGGCTCTCATCGC CTCGCCAACGTGATGAAGGA
17427-4 CTTGGGTACGTCAGCTTGTA GGCGACTTTTGCTTCTACGTC
17432-1 ACTCCCTGCTCAGAAGCACT CCCAACTTCGTAGGCACGAT
17432-2 TTTGTAGCAAGACCAACAAGCAAAA TGTGCGAAATTGTGAATGACCA
17432-3 TCCAGACCATACGAGGTCAGT TGCTTCTGAGCAGGGAGTTT
17432-4 TGCCAGGTTTCAATGACCTCT CATTTGGCAGAATCCACGGC
17432-5 CGTCCACAGAGGGGACAAAC GCTCGGTGTCTTTTCAGGGA

图3

crk4与ysl2的等位测验 A: ysl2/+×crk4/+杂交果穗上的籽粒性状, 标尺为1 cm; B: crk4/+×ysl2/+杂交后代果穗上的籽粒性状, 标尺为1 cm; C: ysl2/+×crk4/+和crk4/+×ysl2/+杂交果穗上的籽粒分离统计; D~E: 正反交果穗上突变籽粒的测序结果, D为crk4中的突变位点检测, E为ysl2中的突变位点检测。"

[1] Russell S D. Double fertilization. In: Russell S D, Dumas C, eds. International Review of Cytology. Academic Press, 1992. pp 357-388.
[2] Sosso D, Luo D P, Li Q B, Sasse J, Yang J L, Gendrot G, Suzuki M, Koch K E, McCarty D R, Chourey P S, Rogowsky P M, Ross-Ibarra J, Yang B, Frommer W B. Seed filling in domesticated maize and rice depends on sweet-mediated hexose transport. Nat Genet, 2015, 47: 1489-1493.
doi: 10.1038/ng.3422 pmid: 26523777
[3] Chourey P S, Nelson O E. The enzymatic deficiency conditioned by the shrunken-1 mutations in maize. Biochem Genet, 1976, 14: 1041-1055.
doi: 10.1007/BF00485135 pmid: 1016220
[4] Bhave M R, Lawrence S, Barton C, Hannah L C. Identification and molecular characterization of shrunken-2 cDNA clones of maize. Plant Cell, 1990, 2: 581-588.
pmid: 1967077
[5] Qiao Z Y, Qi W W, Wang Q, Feng Y N, Yang Q, Zhang N, Wang S S, Tang Y P, Song R T. ZmMADS47 regulates zein gene transcription through interaction with opaque2. PLoS Genet, 2016, 12: e1005991.
[6] Zhang X, Mogel K J H V, Lor V S, Hirsch C N, Vries B D, Kaeppler H F, Tracy W F, Kaeppler S M. Maize sugary enhancer1 (se1) is a gene affecting endosperm starch metabolism. Proc Natl Acad Sci USA, 2019, 116: 20776-20785.
doi: 10.1073/pnas.1902747116 pmid: 31548423
[7] Schmidt R J, Burr F A, Burr B. Transposon tagging and molecular analysis of the maize regulatory locus opaque-2. Science, 1987, 238: 960-963.
pmid: 2823388
[8] Zhang Z Y, Dong J Q, Ji C, Wu Y R, Messing J. NAC-type transcription factors regulate accumulation of starch and protein in maize seeds. Proc Natl Acad Sci USA, 2019, 116: 11223-11228.
doi: 10.1073/pnas.1904995116 pmid: 31110006
[9] Li C B, Yue Y H, Chen H J, Qi W W, Song R T. The ZmbZIP22transcription factor regulates 27-kD γ-zein gene transcription during maize endosperm development. Plant Cell, 2018, 30: 2402-2424.
doi: 10.1105/tpc.18.00422
[10] Li X J, Zhang Y F, Hou M M, Sun F, Shen Y, Xiu Z H, Wang X M, Chen Z L, Sun S S M, Small I, Tan B C. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa). Plant J, 2014, 79: 797-809.
doi: 10.1111/tpj.2014.79.issue-5
[11] Chen X Z, Feng F, Qi W W, Xu L M, Yao D S, Wang Q, Song R T. Dek35 encodes a PPR protein that affects cis-splicing of mitochondrial nad4 intron 1 and seed development in maize. Mol Plant, 2017, 10: 427-441.
doi: 10.1016/j.molp.2016.08.008
[12] Dai D W, Luan S C, Chen X Z, Wang Q, Feng Y, Zhu C G, Qi W W, Song R T. Maize dek37 encodes a P-type PPR protein that affects cis-splicing of mitochondrial nad2 intron 1 and seed development. Genetics, 2018, 208: 1069-1082.
doi: 10.1534/genetics.117.300602
[13] Ren R C, Wang L L, Zhang L, Zhao Y J, Wu J W, Wei Y M, Zhang X S, Zhao X Y. Dek43 is a P-type pentatricopeptide repeat (PPR) protein responsible for the cis-splicing of nad4 in maize mitochondria. J Integr Plant Biol, 2020, 62: 299-313.
doi: 10.1111/jipb.v62.3
[14] Qi W W, Lu L, Huang S C, Song R T. Maize dek44 encodes mitochondrial ribosomal protein L9 and is required for seed development. Plant Physiol, 2019, 180: 2106-2119.
doi: 10.1104/pp.19.00546
[15] Qi W W, Yang Y, Feng X Z, Zhang M L, Song R T. Mitochondrial function and maize kernel development requires dek2, a pentatricopeptide repeat protein involved in nad1 mRNA splicing. Genetics, 2017, 205: 239-249.
doi: 10.1534/genetics.116.196105
[16] Klepek Y S, Geiger D, Stadler R, Klebl F, Landouar-Arsivaud L, Lemoine R, Hedrich R, Sauer N. Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-Symport of numerous substrates, including myo-inositol, glycerol, and ribose. Plant Cell, 2005, 17: 204-218.
doi: 10.1105/tpc.104.026641
[17] Buttner M. The monosaccharide transporter(-like) gene family in Arabidopsis. FEBS Lett, 2007, 581: 2318-2324.
doi: 10.1016/j.febslet.2007.03.016
[18] Schulz A, Beyhl D, Marten I, Wormit A, Neuhaus E, Poschet G, Büttner M, Schneider S, Sauer N, Hedrich R. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J, 2011, 68: 129-136.
doi: 10.1111/j.1365-313X.2011.04672.x
[19] Lalonde S, Wipf D, Frommer W B. Transport mechanisms for organic forms of carbon and nitrogen between source and sink. Annu Rev Plant Biol, 2004, 55: 341-372.
pmid: 15377224
[20] Curie C, Panaviene Z, Loulergue C, Dellaporta S L, Briat J F, Walker E L. Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature, 2001, 409: 346-349.
doi: 10.1038/35053080
[21] Roberts L A, Pierson A J, Panaviene Z, Walker E L. Yellow stripe1 expanded roles for the maize iron-phytosiderophore transporter. Plant Physiol, 2004, 135: 112-120.
doi: 10.1104/pp.103.037572 pmid: 15107503
[22] Schaaf G, Ludewig U, Erenoglu B E, Mori S, Kitahara T, von Wirén N. ZmYS1functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. J Biol Chem, 2004, 279: 9091-9096.
doi: 10.1074/jbc.M311799200
[23] Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa N K. OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant J, 2004, 39: 415-424.
doi: 10.1111/tpj.2004.39.issue-3
[24] Li J K, Fu J J, Chen Y, Fan K J, He C, Zhang Z Q, Li L, Liu Y J, Zheng J, Ren D T, Wang G Y. The U6 biogenesis-like 1 plays an important role in maize kernel and seedling development by affecting the 3' end processing of U6 snRNA. Mol Plant, 2017, 10: 470-482.
doi: S1674-2052(16)30263-5 pmid: 27825944
[25] He Y H, Yang Q, Yang J, Wang Y F, Sun X L, Wang S, Qi W W, Ma Z Y, Song R T. Shrunken4 is a mutant allele of ZmYSL2 that affects aleurone development and starch synthesis in maize. Genetics, 2021, 218: iyab070.
doi: 10.1093/genetics/iyab070
[26] Zang J, Huo Y Q, Liu J, Zhang H R, Liu J, Chen H B. Maize YSL2 is required for iron distribution and development in kernels. J Exp Bot, 2020, 71: 5896-5910.
doi: 10.1093/jxb/eraa332 pmid: 32687576
[27] Yen M R, Tseng Y H, Saier Jr M H. Maize yellow stripe1, an iron-phytosiderophore uptake transporter, is a member of the oligopeptide transporter (OPT) family. Microbiology, 2001, 147: 2881-2883.
pmid: 11700339
[28] Tsai C Y, Nelson O E. Mutations at the shrunken-4 locus in maize that produce three altered phosphorylases. Genetics, 1969, 61: 813-821.
doi: 10.1093/genetics/61.4.813 pmid: 17248442
[29] Doehlert D C, Kuo T M. Sugar metabolism in developing kernels of starch-deficient endosperm mutants of maize. Plant Physiol, 1990, 92: 990-994.
doi: 10.1104/pp.92.4.990 pmid: 16667416
[30] Roschzttardtz H, Conéjéro G, Divol F, Alcon C, Verdeil J L, Curie C, Mari S. New insights into Fe localization in plant tissues. Front Plant Sci, 2013, 4: 350.
doi: 10.3389/fpls.2013.00350 pmid: 24046774
[31] Hannah L C, Boehlein S. Maize kernel development. In: Larkins B A, ed. Biosynthesis in Maize Endosperm. Boston, MA: CABI, 2017. pp 149-159.
[1] 杨文宇, 吴成秀, 肖英杰, 严建兵. 基于Adaptive Lasso的两阶段全基因组关联分析方法[J]. 作物学报, 2023, 49(9): 2321-2330.
[2] 艾蓉, 张春, 悦曼芳, 邹华文, 吴忠义. 玉米转录因子ZmEREB211对非生物逆境胁迫的应答[J]. 作物学报, 2023, 49(9): 2433-2445.
[3] 黄钰杰, 张啸天, 陈会丽, 王宏伟, 丁双成. 玉米ZmC2s基因家族鉴定及ZmC2-15耐热功能分析[J]. 作物学报, 2023, 49(9): 2331-2343.
[4] 白岩, 高婷婷, 卢实, 郑淑波, 路明. 近四十年来我国玉米大品种的历史沿革与发展趋势[J]. 作物学报, 2023, 49(8): 2064-2076.
[5] 王兴荣, 张彦军, 涂奇奇, 龚佃明, 邱法展. 一个新的玉米细胞核雄性不育突变体ms6的鉴定与基因定位[J]. 作物学报, 2023, 49(8): 2077-2087.
[6] 王娟, 徐相波, 张茂林, 刘铁山, 徐倩, 董瑞, 刘春晓, 关海英, 刘强, 汪黎明, 何春梅. 一个新的玉米Miniature1基因等位突变体的鉴定与遗传分析[J]. 作物学报, 2023, 49(8): 2088-2096.
[7] 韦金贵, 郭瑶, 柴强, 殷文, 樊志龙, 胡发龙. 水氮减量密植玉米的产量及产量构成[J]. 作物学报, 2023, 49(7): 1919-1929.
[8] 李荣, 勉有明, 侯贤清, 李培富, 王西娜. 施氮对还田秸秆腐解及养分释放、土壤肥力与玉米产量的影响[J]. 作物学报, 2023, 49(7): 2012-2022.
[9] 梅秀鹏, 赵子堃, 贾欣瑶, 白洋, 李梅, 甘宇玲, 杨秋悦, 蔡一林. 热诱导转录因子ZmNF-YC13调控热胁迫应答基因提高玉米耐热性[J]. 作物学报, 2023, 49(7): 1747-1757.
[10] 常丽娟, 梁晋刚, 宋君, 刘文娟, 付成平, 代晓航, 王东, 魏超, 熊梅. 转基因玉米ND207转化事件特异性定性PCR检测方法及其标准化[J]. 作物学报, 2023, 49(7): 1818-1828.
[11] 张振博, 贾春兰, 任佰朝, 刘鹏, 赵斌, 张吉旺. 氮磷配施对夏玉米产量和叶片衰老特性的影响[J]. 作物学报, 2023, 49(6): 1616-1629.
[12] 刘佳, 邹晓悦, 马继芳, 王永芳, 董志平, 李志勇, 白辉. 谷子MAPK家族成员的鉴定及其对生物胁迫的响应分析[J]. 作物学报, 2023, 49(6): 1480-1495.
[13] 李璐璐, 明博, 高尚, 谢瑞芝, 王克如, 侯鹏, 薛军, 李少昆. 不同熟期玉米品种籽粒田间脱水特征差异性分析[J]. 作物学报, 2023, 49(6): 1643-1652.
[14] 王玉珑, 于爱忠, 吕汉强, 吕奕彤, 苏向向, 王鹏飞, 柴健. 绿洲灌区麦后复种绿肥并还田对翌年玉米根系性状及水分利用效率的影响[J]. 作物学报, 2023, 49(5): 1350-1362.
[15] 李慧, 王旭敏, 刘苗, 刘朋召, 李巧丽, 王小利, 王瑞, 李军. 基于夏玉米产量和氮素利用的水氮减量方案优选[J]. 作物学报, 2023, 49(5): 1292-1304.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] 李绍清, 李阳生, 吴福顺, 廖江林, 李达模. 水稻孕穗期在淹涝胁迫下施肥的优化选择及其作用机理[J]. 作物学报, 2002, 28(01): 115 -120 .
[2] 王兰珍;米国华;陈范骏;张福锁. 不同产量结构小麦品种对缺磷反应的分析[J]. 作物学报, 2003, 29(06): 867 -870 .
[3] 杨建昌;张亚洁;张建华;王志琴;朱庆森. 水分胁迫下水稻剑叶中多胺含量的变化及其与抗旱性的关系[J]. 作物学报, 2004, 30(11): 1069 -1075 .
[4] 王永胜;王景;段静雅;王金发;刘良式. 水稻极度分蘖突变体的分离和遗传学初步研究[J]. 作物学报, 2002, 28(02): 235 -239 .
[5] 王丽燕;赵可夫. 玉米幼苗对盐胁迫的生理响应[J]. 作物学报, 2005, 31(02): 264 -268 .
[6] 田孟良;黄玉碧;谭功燮;刘永建;荣廷昭. 西南糯玉米地方品种waxy基因序列多态性分析[J]. 作物学报, 2008, 34(05): 729 -736 .
[7] 胡希远;李建平;宋喜芳. 空间统计分析在作物育种品系选择中的效果[J]. 作物学报, 2008, 34(03): 412 -417 .
[8] 王艳;邱立明;谢文娟;黄薇;叶锋;张富春;马纪. 昆虫抗冻蛋白基因转化烟草的抗寒性[J]. 作物学报, 2008, 34(03): 397 -402 .
[9] 郑希;吴建国;楼向阳;徐海明;石春海. 不同环境条件下稻米组氨酸和精氨酸的胚乳和母体植株QTL分析[J]. 作物学报, 2008, 34(03): 369 -375 .
[10] 邢光南, 周斌, 赵团结, 喻德跃, 邢邯, 陈受宜, 盖钧镒. 大豆抗筛豆龟蝽Megacota cribraria (Fabricius)的QTL分析[J]. 作物学报, 2008, 34(03): 361 -368 .