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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (3): 572-579.doi: 10.3724/SP.J.1006.2022.13005

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-03-12 Published:2021-07-19
  • Contact: CHEN Hong-Yu E-mail:15839300945@163.com;chenhongyu@henau.edu.cn
  • 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)

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

Table 1

Primers for gene mapping in this study"

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

Table 2

Primers used in this study"

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

Fig. 1

Kernel phenotype of maize 5601Q mutant A: the self-cross mature ear of F1 plant; B: the comparison of single wild-type and mutant kernel; C: the longitudinal-sectional view of single wild-type and mutant kernel; D: the overall comparison of wild-type and mutant kernels. Bar: 1 cm."

Fig. 2

Kernel component and germination for WT and 5601Q A: 100-kernel weight of wild-type and mutant kernels; B: the starch content of wild-type and mutant kernels; C: the soluble sugar content of wild-type and mutant kernels; D: the statistical analysis of wild-type and 5601Q seed germination tests; E: the comparison of the phenotype between wild-type and 5601Q mutant seedings; * and ** represent significant difference between the mutant and wild-type at the 0.05 and 0.01 probability levels, respectively. Bar: 1 cm."

Table 3

Segregation ratio of mutant kernels in four F2 ears"

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

Fig. 3

Fine mapping of 5601Q mutant and structure schematic diagram of Bt2 gene A: the fine mapping of 5601Q mutant; N: the number of individuals for gene mapping; Recombinant: the number of recombinants; B: schematic diagram of Bt2 gene structure and mutation sites of two mutants."

Table 4

Allelism test of 5601Q with bt2"

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

Fig. 4

Allelism test of 5601Q with bt2 by heterozygous mutants A: 5601Q/+ × bt2/+ shrunken kernels appear on the ears of hybrids; bar: 1 cm. B: bt2/+ × 5601Q/+ shrunken kernels appear on the ears of hybrids; bar: 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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