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作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2088-2096.doi: 10.3724/SP.J.1006.2023.23059

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Miniature1基因等位突变体的鉴定与遗传分析

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()   

  1. 山东省农业科学院玉米研究所 / 小麦玉米国家工程研究中心 / 农业农村部黄淮海北部玉米生物学与遗传育种重点实验室, 山东济南250100
  • 收稿日期:2022-09-07 接受日期:2023-02-10 出版日期:2023-08-12 网络出版日期:2023-02-28
  • 通讯作者: 汪黎明,何春梅
  • 作者简介:E-mail: wqian456@163.com
  • 基金资助:
    国家自然科学基金项目(32101761);国家自然科学基金项目(31971964);山东省农业科学院农业科技创新工程项目(CXGC2022A02)

Characterization and genetic analysis of a new allelic mutant of Miniature1 gene in maize

WANG Juan(), XU Xiang-Bo, ZHANG Mao-Lin, LIU Tie-Shan, XU Qian, DONG Rui, LIU Chun-Xiao, GUAN Hai-Ying, LIU Qiang, WANG Li-Ming(), HE Chun-Mei()   

  1. Maize Research Institute, Shandong Academy of Agricultural Sciences / National Engineering Research Center of Wheat and Maize / Key Laboratory of Biology and Genetic Improvement of Maize in Northern Yellow-Huai River Plain, Ministry of Agriculture and Rural Affairs, Jinan 250100, Shandong, China
  • Received:2022-09-07 Accepted:2023-02-10 Published:2023-08-12 Published online:2023-02-28
  • Contact: WANG Li-Ming,HE Chun-Mei
  • Supported by:
    National Natural Science Foundation of China(32101761);National Natural Science Foundation of China(31971964);Agricultural Science and Technology Innovation Project of the Shandong Academy of Agricultural Sciences(CXGC2022A02)

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摘要：

籽粒发育是决定玉米产量和品质的关键因素, 但目前对其遗传调控机制的研究还相当不完善。我们在田间筛选到一个玉米籽粒发育突变体, 表现为籽粒灌浆不充分, 胚和胚乳变小, 成熟籽粒干瘪或“空果皮”。该突变表型受单个隐性核基因控制。通过图位克隆的方式将候选突变基因定位到2号染色体1.1 Mb的区间内, 进一步研究发现, 该区间内Miniature1 (Mn1)基因在第1个外显子处发生hAT转座子插入, 导致Mn1基因表达下调和无义突变。此插入突变与突变体籽粒灌浆缺陷表型完全连锁, 该突变体被命名为mn1-m2。通过与mn1-89突变体进行等位测验, 确认mn1-m2即为Mn1基因的等位突变体。因此, 本研究鉴定了一个新的mn1等位突变体, 其突变位点及方式与已知mn1突变体均不相同, 完善了玉米籽粒突变体的种质资源信息, 也为Mn1调控籽粒发育机制的解析提供新的遗传材料。

关键词: 玉米, 籽粒灌浆, Mn1, 突变体

Abstract:

Grain development is a key determinant for maize grain yield and quality, but the regulatory mechanisms have not been fully revealed. A maize mutant deficient in kernel development was found and selected in our field. The mutant exhibited an incomplete grain filling, reduced embryo and endosperm size, and shriveled mature grains and empty pericarp. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene. The candidate gene was preliminarily mapped to a 1.1 Mb interval on chromosome 2, and further a hAT transposon was inserted into the first exon of Miniature1 (Mn1) gene, which resulting in a frame-shift mutation and down-regulated expression of Mn1. The transposon insertion was fully linked to the defective kernel phenotype of the mutant, which was nominated as mn1-m2. Allelism test of mn1-m2 and mn1-89 suggested that mn1-m2 was a noval allelic mutant of Mn1 gene. In conclusion, we identified a new allelic mutant of Mn1 gene with different mutation site and type, which improved maize germplasm resources and provided new genetic materials for the analysis of the mechanism of Mn1 regulation on kernel development.

Key words: maize, grain filling, Mn1, mutant

表1

基因定位所用的分子标记"

引物名称
Primer name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
umc2249 AGAAGGTCGTCGTCCTGGAAC GCATAGACTCCCTGACAGCCAC
umc2030 CTTCAGCAACCGGAGACGAG GATGCAGTGTGCCAATAAAGATGA
umc2079 CGGCCTCGCTGTCTTCTAGC ATGATCACGTCGTGCTGGTAGTG
umc2125 CAAGGGTAAGGGCAAGATGGTAGT CTGAGGTCTACCTCGGCCATC
IDP1 TGCACATGTCTGGTACTCGC GTCATGCTGTCCAACACCG
IDP2 TGCGATATCTGTGTTTGCCTTG ACATCCATGCCATGAACTTTGC
IDP3 TGTAGCAGACACTCTCAAGCACAAC CCCTGGGAACCAAATCAGCCACA
IDP4 AGCAGGTCCGAGGAGTTTC CGGGAGACGGTTTGAATG
IDP5 GGAAACAGCAGTAGCAGAGAGA CTCCTAGTACGTGATTGCATCCA

表2

Mn1特异InDel标记及RT-PCR引物"

引物名称
Primer name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
InDel (P1/P2) TGTGATATGGCAGCTAAGAG CGAGGTCCTTGTAGTTGTAG
Mn1 RT-PCR CTGGGCTAACGAGTCCGACT CCACACCGTCCTTGGAATCG
FPGS RT-PCR ATCTCGTTGGGGATGTCTTG AGCACCGTTCAAATGTCTCC

图1

野生型及突变体mn1-m2表型分析 A: 野生型、杂合(+/mn1-m2)及纯合mn1-m2突变体的成熟果穗, 图中箭头为杂合型植株自交后分离出的突变型籽粒; B: 生长3周幼苗; C: 成熟期籽粒; D: 授粉后25 d籽粒的胚乳(左)和幼胚(右)的表型; E: 百粒重统计。A图标尺为5 cm, C~D图标尺为1 cm。"

图2

野生型及mn1-m2授粉后20 d籽粒的石蜡切片观察 A: 授粉后20 d野生型籽粒顶部胚乳观察; B: 授粉后20 d mn1-m2籽粒顶部胚乳观察; C: 为(A)图中红色方框区域的放大图; D: 为(B)图中红色方框区域的放大图。A~B图标尺为500 μm; C~D图标尺为100 μm。"

图3

mn1-m2基因的图位克隆 利用mn1-m2与B73之间的多态SSR和In-Del (IDP)标记, 通过放大定位F2群体, 结合对目标区间内基因测序, 最终定位到突变基因为Mn1, 编码细胞壁蔗糖转化酶。该基因在第1个外显子的第18个核苷酸后发生hAT转座子(166 bp)的插入。图中标红基因为Mn1。黑框代表外显子, 黑线代表内含子。n: 群体大小; Recombinants: 重组单株数。"

图4

mn1-m2突变体中mn1基因表达水平及hAT转座子插入的鉴定 A: 野生型及mn1-m2授粉后15 d籽粒中Mn1基因表达水平, FPGS为内参基因; B: In-Del分子标记引物P1和P2位于hAT转座子两侧的示意图; C: In-Del分子标记与mn1-m2突变表型完全连锁。野生型扩增产物大小为495 bp, mn1-m2扩增产物大小为661 bp。"

图5

mn1-m2与mn1-89突变体等位测验 A: Mn1基因结构及2个突变体的突变位置; B: 突变体mn1-m2, mn1-89及其杂交后代果穗的表型; C: mn1-m2背景野生型的籽粒; D: mn1-m2籽粒; E: mn1-m2与mn1-89杂交F1代籽粒; F: mn1-89籽粒; G: mn1-89背景野生型W22的籽粒。B图标尺为5 cm, C~G图标尺为1 cm。"

图6

RT-PCR分析Mn1基因在玉米不同组织中的表达水平 A: 分别提取野生型玉米幼穗、苞片、花丝、雄穗和旗叶组织RNA, 检测Mn1表达水平; B: 分别提取授粉后13、15、19、23、25和29 d籽粒的胚和胚乳RNA, 检测Mn1表达水平。FPGS为内参基因。"

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