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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (3): 572-579.doi: 10.3724/SP.J.1006.2022.13005

所属专题： 玉米：遗传育种·种质资源·分子遗传学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一个新的玉米Bt2基因突变体的遗传分析和分子鉴定

徐宁坤(), 李冰, 陈晓艳, 魏亚康, 刘子龙, 薛永康, 陈洪宇*(), 王桂凤   

  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, 河南郑州 450002
  • 收稿日期:2021-01-19 接受日期:2021-06-16 出版日期:2022-03-12 网络出版日期:2021-07-19
  • 通讯作者: 陈洪宇
  • 作者简介:E-mail: 15839300945@163.com
  • 基金资助:
    国家自然科学基金项目(U1804235);国家自然科学基金项目(31771800);河南农业大学科技创新基金项目(KJCX2020A04)

Genetic analysis and molecular characterization of a novel maize Bt2 gene mutant

XU Ning-Kun(), LI Bing, CHEN Xiao-Yan, WEI Ya-Kang, LIU Zi-Long, XUE Yong-Kang, CHEN Hong-Yu*(), WANG Gui-Feng   

  1. College of Agronomy, Henan Agricultural University/Key Laboratory of Wheat and Maize Crops Science, Zhengzhou 450002, Henan, China
  • Received:2021-01-19 Accepted:2021-06-16 Published:2022-03-12 Published online:2021-07-19
  • Contact: CHEN Hong-Yu
  • Supported by:
    National Natural Science Foundation of China(U1804235);National Natural Science Foundation of China(31771800);Science and Technology Innovation Fund of Henan Agricultural University(KJCX2020A04)

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摘要：

玉米籽粒发育调控机制的研究对于玉米产量与品质性状的遗传改良十分重要。本研究鉴定了一个新的转座子插入的籽粒皱缩突变体5601Q, 遗传分析表明其籽粒缺陷稳定遗传且为单基因隐性突变。构建其B73背景的F2分离群体, 通过图位克隆将该突变定位于玉米4号染色体上60.19~62.58 Mb的区间。基因注释分析发现, 区间内存在一个已报道参与玉米籽粒发育的基因BRITTLE ENDOSPERM2 (Bt2), 其编码玉米胚乳淀粉合成途径中的一个限速酶——腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucose pyrophosphorylase, AGPase)的小亚基。籽粒储藏物质分析表明, 该突变体百粒重及淀粉含量显著降低, 但可溶性糖含量增加为野生型的4.67倍。利用本实验室已确定的Bt2基因突变体1774与5601Q进行等位测验, 确认了5601Q是Bt2的一个新的等位突变体。分子鉴定表明, 5601Q突变体中Bt2基因的第2个外显子存在一个Mutator 19转座子的插入。综上, 5601Q籽粒发育缺陷是由Bt2基因的突变导致, 本研究为解析玉米Bt2基因在胚乳储藏物质积累中的作用机制提供了新的种质资源。

关键词: 玉米, 籽粒突变, 图位克隆, AGPase, Bt2

Abstract:

The research of the molecular mechanism underlying maize kernel development is particularly important for the genetic improvement of maize yield and quality traits. In this study, we characterized a new shrunken kernel mutant 5601Q, which was generated by a random transposon insertion. Genetic analysis indicated that the kernel phenotype was stably controlled by a single recessive gene. F2 segregating population was constructed by crossing 5601Q into B73 inbred line, and the mutant gene was located in the genetic interval of 60.19-62.58 Mb on chromosome 4. Sequence annotation showed that the BRITTLE ENDOSPERM2 (Bt2) gene, previously reported to be involved in maize kernel development, was located in this region. Maize Bt2 gene encoded the small subunit of ADP-glucose pyrophosphorylase (AGPase), the first rate-limiting enzyme in the starch biosynthetic pathway of higher plants. Compared with wild type, 100-grain weight and starch content of mutant 5601Q decreased significantly, but the soluble sugar content increased dramatically 4.67 times. We confirmed that 5601Q was a new allele mutant of Bt2 by allelic test of Bt2 mutant 1774 and 5601Q. Sequencing analysis revealed that Mutator 19 transposon was inserted in the 2nd exon of Bt2 gene. In summary, our results indicated that the shrunken kernel in 5601Q was caused by the loss-of-function of Bt2 gene, which provided a new germplasm resource to elucidate the mechanism of maize Bt2 gene in endosperm storage substance accumulation.

Key words: maize, defective kernel mutant, gene mapping, AGPase, Bt2

表1

基因定位引物序列"

引物
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
K5 ATCCGGTACCACCAATTCCT CGTCTGCAGCATGAAACACT
K10 CACACTCCACCTGCATCACT GGCACCAACGAGGATTAGTC
YF100 GACCAACACTGCCACTTCCTTC AGTTGTAGTTGCTGCTCGTTCG
A3 TCCAAAAACAGAACGCAGTG ACTCAATGATCGGGCATAGG
A4 AGATGGCAATGGCTATGAGG CCAAGCAACAAGAGCAACAA
K10-15 CCAACTTGTAGCCACGAAAAGG GCCGATACCGTGACCCAAATC
A12 CCATGATAATTGCGCCTTTT ATTTGGGACATCAACATGCA

表2

本研究所用到的引物"

引物
Primer name		 正向序列
Forward sequence (5′-3′)		 反向序列
Reverse sequence (5′-3′)
Bt2-F1/R1 TCTTGCCTCCCTCGTCCTTCCTC GGACAAGGACAGCTCTGAAACTGA
Bt2-F2/R2 GTTATTGGTTTCCGTGGAGCTA TTTGCCAGATGCCAGCCATTGA
Bt2-F3/R3 CTCTCTTCCCTTCCAAAGAAAGGC TTCATAGTCCATCCGGTACAGG
Bt2-F4/R4 ACCTGTACCGGATGGACTATGA TTCCTTCCATCCTGCCTTGAGACA
Bt2-F5/R5 TGTCTCAAGGCAGGATGGAAGGAA GACCGTTGGAACATCAGTTTGG
Bt2-F6/R6 TGAATAGGCTTGCATGCTCT AATCCGCACTCCAAACCAGCAA
Tir 8.2 CGCCTCCATTTCGTCGAATCCSCTT

图1

玉米5601Q突变体籽粒表型 A: F1植株自交成熟果穗; B: 单粒野生型籽粒和突变体籽粒对比; C: 野生型和突变体籽粒纵切图; D: 整体野生型籽粒和突变体籽粒对比, 标尺为1 cm。"

图2

WT和5601Q籽粒成分测定和萌发测试 A: 野生型籽粒和突变籽粒的百粒重; B: 野生型籽粒和突变籽粒的淀粉含量; C: 野生型籽粒和突变籽粒的可溶性糖含量; D: 野生型和5601Q籽粒萌发测试的统计分析; E: 野生型和5601Q幼苗表型比较; *、**分别表示野生型与突变体在0.05和0.01水平差异显著, 标尺为1 cm。"

表3

F2果穗突变型籽粒的分离比例"

果穗
Ear		 野生型籽粒数
Wild-type		 突变体籽粒
Mutant		 χ2
1 418 144 0.08600
2 485 140 2.25300
3 176 142 1.61100
总计 Total 1279 426 0.00019

图3

5601Q突变体的精细定位和Bt2基因的结构示意图 A: 5601Q突变体的精细定位; N: 定位所用的群体数目; Recombinant: 发生重组的个体数目; B: Bt2基因结构示意图及2个突变体的插入位置。"

表4

5601Q与bt2的等位测试"

父母本基因型
Parental genotype		 籽粒表型 Kernel phenotype χ2
正常籽粒 Normal kernel 突变体籽粒 Mutant kernel 总数 Total
5601Q/+ × bt2/+ 155 55 210 0.102
bt2/+ × 5601Q/+ 162 55 217 0.015

图4

利用5601Q的杂合体与bt2杂合体进行等位测试 A: 5601Q/+ × bt2/+杂交后代果穗上出现籽粒皱缩籽粒, 标尺为1 cm; B: bt2/+ × 5601Q/+杂交后代果穗上出现籽粒皱缩籽粒, 标尺为1 cm。"

[1] 赵然, 蔡曼君, 杜艳芳, 张祖新. 玉米籽粒形成的分子生物学基础. 中国农业科学, 2019, 20:3495-3506.
Zhao R, Cai M J, Du Y F, Zhang Z X. Molecular biological basis of maize grain formation. Chin Agric Sci, 2019, 20:3495-3506 (in Chinese with English abstract).
[2] Dai D W, Tong H Y, Cheng L J, Peng F, Zhang T T, Qi W W, Song R T. Maize Dek33 encodes a pyrimidine reductase in riboflavin biosynthesis that is essential for oil-body formation and ABA biosynthesis during seed development. J Exp Bot, 2019, 19:5173-5187.
[3] Chen X, Feng F, Qi W, Xu L, Yao D, Wang Q, Song R. 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
[4] Wang G, Zhong M, Shuai B, Song J, Zhang J, Han L, Ling H, Tang Y, Wang G, Song R. Arabidopsis Arabidopsis. New Phytol, 2017, 214:1563-1578.
doi: 10.1111/nph.2017.214.issue-4
[5] Ren R C, Lu X, Zhao Y J, Wei Y M, Wang L L, Zhang L, Zhang W T, Zhang C, Zhang X S, Zhao X Y. Pentatricopeptide repeat protein DEK40 is required for mitochondrial function and kernel development in maize. J Exp Bot, 2019, 70:6163-6379.
doi: 10.1093/jxb/erz391
[6] Fu S N, Meeley R, Scanlon M J. Empty pericarp2 encodes a negative regulator of the heat shock response and is required for maize embryogenesis. Plant Cell, 2002, 14:3119-3132.
doi: 10.1105/tpc.006726
[7] Jose F, Gutierrez M, Mauro D P. Empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant growth in maize. Plant Cell, 2007, 19:196-210.
doi: 10.1105/tpc.105.039594
[8] Liu Y J, Xiu Z H, Meeley R, Tan B C. Empty pericarp5 encodes a pentatricopeptide repeat protein that is required for mitochondrial RNA editing and seed development in maize. Plant Cell, 2013, 25:868-883.
doi: 10.1105/tpc.112.106781
[9] Sun F, Wang X, Bonnard G, Shen Y, Xiu Z, Li X, Gao D, Zhang Z, Tan B. Empty pericarp7 encodes a mitochondrial E-subgroup pentatricopeptide repeat protein that is required for ccmFN editing, mitochondrial function and seed development in maize. Plant J, 2015, 84:283-295.
doi: 10.1111/tpj.12993
[10] Wang G, Sun X, Wang G, Wang F, Song R. Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics, 2011, 189:1281-1295.
doi: 10.1534/genetics.111.133967
[11] Mertz E T, Bates L S, Nelson O E. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science, 1964, 145:279-280.
doi: 10.1126/science.145.3629.279
[12] Yao D, Qi W, Li X, Yang Q, Song R. Maize opaque10 encodes a cereal-specific protein that is essential for the proper distribution of zeins in endosperm protein bodies. PLoS Genet, 2016, 12:e1006270.
doi: 10.1371/journal.pgen.1006270
[13] Feng F, Qi W, Lyu Y. Opaque 11 is a central hub of the regulatory network for maize endosperm development and nutrient metabolism. Plant Cell, 2018, 30:375-396.
doi: 10.1105/tpc.17.00616
[14] Holding D R, Otegui M S, Li B, Meeley R B, Dam T, Hunter B G, Jung R, Larkins B A. The maize Floury1 gene encodes a novel endoplasmic reticulum protein involved in zein protein body formation. Plant Cell, 2007, 19:2569-2582.
pmid: 17693529
[15] Coleman C E, Clore A M, Ranch J P. floury2 phenotype in transgenic maize floury2 phenotype in transgenic maize. Proc Natl Acad Sci USA, 1997, 94:7094-7097.
doi: 10.1073/pnas.94.13.7094
[16] Qi L, Wang J, Ye J, Zheng X, Xiang X, Li C, Wang Q, Zhang Z, Wu Y. The maize imprinted gene Floury3 encodes a PLATZ protein required for tRNAs and 5S rRNA transcription through interaction with RNA polymerase III. Plant Cell, 2017, 29:2661-2675.
doi: 10.1105/tpc.17.00576
[17] Wang G, Qi W, Wu Q, Yao D, Song R. floury4 as a novel semidominant opaque mutant that disrupts protein body assembly floury4 as a novel semidominant opaque mutant that disrupts protein body assembly. Plant Physiol, 2014, 165:582-594.
doi: 10.1104/pp.114.238030
[18] Fedoroff N V, Furtek D B, Nelson O E. Cloning of the bronze locus in maize by a simple and generalizable procedure using the transposable controlling element Activator (Ac). Proc Natl Acad Sci USA, 1984, 81:3825-3829.
doi: 10.1073/pnas.81.12.3825
[19] Theres N, Scheele T, Starlinger P. Bz2 locus of Zea mays using the transposable element Ds as a gene tag Bz2 locus of Zea mays using the transposable element Ds as a gene tag. Mol Gene Genet, 1987, 209:193.
[20] Chourey P S, Nelson O E. The enzymatic deficiency conditioned by the shrunken-1 mutations in maize. Biochem Genet, 1976, 14:1041-1055.
pmid: 1016220
[21] Hannah L C, Tuschall D M, Mans R J. Multiple forms of maize endosperm ADP-glucose pyrophosphorylase and their control by shrunken-2 and brittle-2. Genetics, 1980, 95:961-970.
pmid: 17249055
[22] Laughnan J R. sh2 factor on carbohydrate reserves in the mature endosperm of maize sh2 factor on carbohydrate reserves in the mature endosperm of maize. Genetics, 1953, 38:485-499.
pmid: 17247452
[23] James M G, Myers R A M. sugary1, a determinant of starch composition in kernels sugary1, a determinant of starch composition in kernels. Plant Cell, 1995, 7:417-429.
pmid: 7773016
[24] Shure M, Wessler S, Fedoroff N. Waxy locus in maize Waxy locus in maize. Cell, 1983, 35:225-233.
pmid: 6313224
[25] Kim K N, Fisher D K, Gao M, Guiltinan M J. Molecular cloning and characterization of the Amylose-Extender gene encoding starch branching enzyme IIB in maize. Plant Mol Biol, 1998, 38:945-956.
pmid: 9869401
[26] Correns C. Bastarde zwischen Maisrassen, mit besonderer Berücksichtigung der Xenien. Nature, 1901, 65:126.
[27] Ferguson J E, Rhodes A M, Dickinson D B. The genetics of sugary enhancer (se), an independent modifier of sweet corn (su). Heredity, 1978, 6:377-380.
doi: 10.1038/hdy.1952.46
[28] Gonzales J W, Rhodes A M, Dickinson D B. Carbohydrate and enzymic characterization of a high sucrose sugary inbred line of sweet corn. Plant Physiol, 1976, 58:28-32.
pmid: 16659614
[29] Preiss J, Danner S, Summers P S, Morell M, Barton C R, Yang L, Nieder M. Molecular characterization of the Brittle-2 gene effect on maize endosperm ADP glucose pyrophosphorylase subunits. Plant Physiol, 1990, 92:881-885.
pmid: 16667400
[30] Bae J M, Giroux M, Hannah L C. Cloning and characterization of the brittle-2 gene of maize. Maydica, 1990, 35:317-322.
[31] 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
[32] Dickinson D B, Preiss J. Presence of ADP-glucose pyrophosphorylase in Shrunken-2 and Brittle-2 mutants of maize endosperm. Plant Physiol, 1969, 44:1058-1062.
pmid: 16657157
[33] 李晓旭, 李家政. 优化蒽酮比色法测定甜玉米中可溶性糖的含量. 保鲜与加工, 2013, 13(4):24-27.
Li X X, Li J Z. Determination of the content of soluble sugar in sweet corn with optimized anthrone colorimetric method. Stor Proc, 2013, 13(4):24-27 (in Chinese with English abstract).
[34] Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8:4321-4325.
doi: 10.1093/nar/8.19.4321
[35] Smith-White B J, Preiss J. Comparison of proteins of ADP- glucose pyrophosphorylase from diverse sources. J Mol Evol, 1992, 34:449-464.
pmid: 1318389
[36] Greene T W, Hannah L C. Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions. Plant Cell, 1998, 10:1295-1306.
[37] Greene T W, Hannah L C. Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions. Proc Natl Acad Sci USA, 1998, 95:13342-13347.
doi: 10.1073/pnas.95.22.13342
[38] Wilson L M, Whitt S R, Ibáñez A M, Rocheford T R, Goodman M M, Buckler E S. Dissection of maize kernel composition and starch production by candidate gene association. Plant Cell, 2004, 16:2719-2733.
doi: 10.1105/tpc.104.025700
[39] Cossegal M, Chambrier P, Mbelo S, Balzergue S, Martin-Magniette M L, Moing A, Deborde C, Guyon V, Perez P, Rogowsky P. bt2 maize kernels bt2 maize kernels. Plant Physiol, 2008, 146:1553-1570.
doi: 10.1104/pp.107.112698 pmid: 18287491
[40] Gustafson J P, Shin J H, Kwon S J, Lee J K, Min H K, Kim N S. Genetic diversity of maize kernel starch-synthesis genes with SNAPs. Genome, 2006, 49:1287-1296.
doi: 10.1139/g06-116
[41] Tenaillon M I, Sawkins M C, Long A D, Gaut R L, Doebley J F, Gaut B S. Zea mays ssp. mays L.) Zea mays ssp. mays L.). Proc Natl Acad Sci USA, 2001, 98:9161-9166.
doi: 10.1073/pnas.151244298
[42] 乐素菊, 刘鹏飞, 曾慕衡, 王伟权, 王晓明. 超甜玉米bt2基因SNP位点的分析及分子标记辅助筛选. 西北农林科技大学学报(自然科学版), 2012, 40(11):73-78.
Yue S J, Liu P F, Zeng M H, Wang W Q, Wang X M. Analysis of SNP locus of bt2 gene in super sweet maize and molecular marker assisted screening. J Northwest Agric For Univ (Nat Sci Edn), 2012, 40(11):73-78 (in Chinese with English abstract).
[43] 单明珠, 周余庆, 李发民, 刘萌娟. 甜玉米籽粒含糖量性状的研究. 西北农林科技大学学报(自然科学版), 2006, 34(6):111-114.
Shan M Z, Zhou Y Q, Li F M, Liu M J. Study on the traits of sugar content in sweet corn. J Northwest Agric For Univ (Nat Sci Edn), 2006, 34(6):111-114 (in Chinese with English abstract).
[44] 于惠琳, 吴玉群, 胡宝忱, 尤丹, 王延波. 超甜玉米系与其野生型玉米系籽粒发育过程中糖分积累规律. 辽宁农业科学, 2019, (3):77-79.
Yu H L, Wu Y Q, Hu B Z, Yu D, Wang Y B. Sugar accumulation regularity of super sweet maize and its wild type maize during kernel development. Liaoning Agric Sci, 2019, (3):77-79 (in Chinese with English abstract).
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