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

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

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)

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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, crk4是sh4/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。"

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