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作物学报 ›› 2018, Vol. 44 ›› Issue (03): 369-375.doi: 10.3724/SP.J.1006.2018.00369

• 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Vp15基因等位突变体的遗传分析与分子鉴定

王瑞1,2,*(), 张秀艳3,*(), 陈阳松2, 杜依聪2, 汤继华1, 王国英2, 郑军2,*()   

  1. 1 河南农业大学农学院, 河南郑州 450002
    2 中国农业科学院作物科学研究所, 北京 100081
    3 中国农业大学生物学院, 北京 100193
  • 收稿日期:2017-08-07 接受日期:2017-11-21 出版日期:2018-03-12 网络出版日期:2017-12-18
  • 通讯作者: 王瑞,张秀艳,郑军
  • 作者简介:

    第一作者联系方式: 王瑞, E-mail: 18612261636@163.com; 张秀艳, E-mail: xyzhang0818@163.com

  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0101002)和中国农业科学院创新工程专项经费资助

Genetic Analysis and Molecular Characterization of a New Allelic Mutant of Vp15 Gene in Maize

Rui WANG1,2,**(), Xiu-Yan ZHANG3,**(), Yang-Song CHEN2, Yi-Cong DU2, Ji-Hua TANG1, Guo-Ying WANG2, Jun ZHENG2,*()   

  1. 1 College of Agriculture, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 School of Life Science, China Agricultural University, Beijing 100193, China
  • Received:2017-08-07 Accepted:2017-11-21 Published:2018-03-12 Published online:2017-12-18
  • Contact: Rui WANG,Xiu-Yan ZHANG,Jun ZHENG
  • Supported by:
    This study was supported by the National Key Research and Development Program of China (2016YFD0101002) and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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摘要：

在玉米繁种过程中发现一个玉米穗发芽突变体(viviparous), 命名为vp-like4。经过连续多代自交发现该突变体性状能稳定遗传, 并且表现为隐性单基因控制。以vp-like4与自交系Mo17杂交构建F2遗传定位群体, 利用BSR-Seq方法, 将目的基因定位于玉米第5染色体173.8~175.6 Mb之间。通过基因组序列信息分析发现, 在此定位区间内存在一个已报道的Vp15基因。Vp15基因编码钼喋呤合酶小亚基, 参与类胡萝卜素裂解为ABA的过程。利用2个独立的vp15突变体vp15-umu1和vp15-DR1126的杂合体, 分别与vp-like4突变体杂合体做杂交进行等位测验, 发现杂交后代中正常籽粒与穗发芽籽粒比例符合3∶1分离比。基因组序列分析发现vp-like4突变体中Vp15基因在第2外显子末端及3°非翻译区有60个碱基的缺失, 与所报道的vp15突变体vp15-umu1、vp15-DR1126均在第2个外显子有Mutator转座子插入的突变方式不同。进一步通过RT-PCR检测发现, vp-like4突变体中Vp15基因的表达量显著降低。以上实验证据表明, vp-like4是一个新的Vp15基因等位突变体。

关键词: 玉米, 穗发芽, 突变体, Vp15, 基因定位

Abstract:

We identified a new maize viviparous mutant during seed reproduction, designated as vp-like4.This mutant phenotype was steadily inherited and genetically regulated by a single recessive gene. Using an F2 segregation population derived from vp-like4 and inbred line Mo17, we mapped the target gene in an interval from 173.8 to 175.6 Mb on chromosome 5 by the BSR-Seq strategy. Using genomic sequence database, we found that viviparous gene Vp15 is located in this mapping region. The maize Vp15 gene encodes the molybdopterin synthase small subunit, which is required in the process of catalyzing the reaction from carotenoid to ABA. The heterozygous plants from two independent vp15 mutants, vp15-umu1 and vp15-DR1126, were used to cross with vp-like4 heterozygous plants, showing a 3:1 segregation ratio for normal and viviparous kernels. The genomic sequence analysis revealed that vp-like4 mutant had a 60-bp deletion in the second exon and 3’-untranslated region of Vp15 gene, which is different from vp15-umu1 and vp15-DR1126 both mutated from a Mutator transponson inserting in the second exon of Vp15 gene. Further RT-PCR analysis revealed that the expression level of vp15 was significantly lower in vp-like4. Taken together, these evidences suggest that vp-like4 is a new allele mutant from vp15.

Key words: maize, viviparous, mutant, Vp15, gene mapping

表1

本研究所用的引物"

引物
Primer		 正向序列
Forward sequence (5°-3°)		 反向序列
Reverse sequence (5°-3°)
VP15-G-F1/R1 AAGGACGCGAGAGTTTTTGA GGTGAGCGAGTAACCCATGT
VP15-G-F2/R2 ACATGGGTTACTCGCTCACC CTTCCTGGTAGCGAGACGTG
VP15-G-F3/R3 GCTCGTTCACCATCCAGAAT AGCAAAGCCCTTTCTGAACA
VP15-G-F4/R4 GGGCTTTGCTAAGATGTCTCC GGACAATACATAACTGCTCCCTAAA
VP15-G-F5/R5 TCTTCCACTCTTCCTACCAGCTA AGGAGCCAACAACTGCTGAT
VP15-G-F6/R6 GAACAATCGAGCCTTCTTCG TCGTCCTCCTTGACCACTTC
VP15-G-F7/R7 CACCAACTGCAAGATCGAAA CCAACAGACGTCAACACCAG
VP15-CDS-F1/R1 GGAACTCTCTGATACCTGGCTTT CAGTTCCGTGATTAGCCGC
Zm-GAPDH-F/R CCCTTCATCACCACGGACTAC AACCTTCTTGGCACCACCCT

图1

vp-like4突变体穗发芽表型 A: vp-like4杂合突变体授粉后30 d果穗上正常籽粒和穗发芽籽粒; B:vp-like4杂合突变体授粉后60 d果穗上正常籽粒和穗发芽籽粒; C: 成熟的正常籽粒(WT)和穗发芽籽粒(vp)。标尺=1 cm。"

表2

vp-like4杂合突变体自交授粉后正常籽粒和穗发芽籽粒的分离情况"

年度
Year		 地点
Location		 植株基因型
Plant genotype		 籽粒表型Kernel phenotype
正常籽粒 Normal 穗发芽籽粒 viviparous 总数 Total χ2 (3:1)
2014 海南Hainan vp-like4/+ 355 127 482 0.398
325 111 436 0.027
2016 北京Beijing vp-like4/+ 129 47 176 0.189
225 70 295 0.190

图2

利用BSR-Seq方法对vp-like4突变体基因的初定位"

图3

利用杂合突变体对vp-like4和vp15进行等位测验 A, B: 以vp-like4杂合突变体混粉分别杂交vp15-umu1(A)和vp15-DR1126(B)的果穗上有穗发芽籽粒。C, D: 以vp15-umu1(C)、vp15-DR1126(D)分别混粉杂交vp-like4的果穗上有穗发芽籽粒。"

图4

Vp15基因结构示意图及3个突变体的突变位置"

表3

vp-like4与vp15突变体等位测验"

父母本基因型
Parental genotype		 籽粒表型 Kernel phenotype
正常籽粒Normal 穗发芽籽粒viviparous 总数Total χ2 (3:1)
vp-like4/+ × vp15-umu1/+ 217 68 285 0.141
vp-like4/+ ×vp15-DR1126/+ 123 40 163 0.002
vp15-DR1126/+ ×vp-like4/+ 262 83 345 0.116
vp15-umu1/+ ×vp-like4/+ 239 84 323 0.124

图5

实时定量PCR检测Vp15基因分别在3个突变体vp-like4、vp15-umu1、vp15-DR1126中的正常(WT)和穗发芽(vp)籽粒中的表达量"

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