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作物学报 ›› 2024, Vol. 50 ›› Issue (11): 2712-2719.doi: 10.3724/SP.J.1006.2024.43008

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米穗发芽突变体vp2的基因克隆及功能研究

张馨月1,2(), 秦阳2, 李瑞3, 黄全生4, 王逸茹2,*(), 郑军1,2,*()   

  1. 1青岛农业大学农学院, 山东青岛 266109
    2中国农业科学院作物科学研究所, 北京 100081
    3甘肃农业大学农学院, 甘肃兰州 730070
    4新疆农业科学院核技术生物技术研究所, 新疆乌鲁木齐 830091
  • 收稿日期:2024-02-05 接受日期:2024-06-20 出版日期:2024-11-12 网络出版日期:2024-07-11
  • 通讯作者: *郑军, E-mail: zhengjun02@caas.cn; 王逸茹, E-mail: wangyiru@caas.c
  • 作者简介:E-mail: winnie990820@163.com
  • 基金资助:
    新疆维吾尔自治区重点实验室开放课题(2023D04070)

Cloning and functional analysis of viviparous mutant vp2 in maize

ZHANG Xin-Yue1,2(), QIN Yang2, LI Rui3, HUANG Quan-Sheng4, WANG Yi-Ru2,*(), ZHENG Jun1,2,*()   

  1. 1College of Agronomy, Qingdao Agricultural University, Qingdao 266109, Shandong, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Agriculture, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    4Institute of Nuclear Technology and Biotechnology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, Xinjiang, China
  • Received:2024-02-05 Accepted:2024-06-20 Published:2024-11-12 Published online:2024-07-11
  • Contact: *E-mail: zhengjun02@caas.cn; E-mail: wangyiru@caas.c
  • Supported by:
    Open Project of Key Laboratory, Xinjiang Uygur Autonomous Region(2023D04070)

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摘要：

玉米穗发芽是指籽粒在果穗上未脱离母体时胚芽便开始萌发生长, 穗发芽会严重影响玉米的产量和品质。因此高效挖掘和利用优良基因, 培育抗穗发芽玉米品种对我国农业生产非常重要。本研究中的玉米穗发芽突变体vp2具有明显的穗发芽表型且性状能稳定遗传, 受隐性单基因控制。通过分析vp2突变体基因组序列发现突变体中2个编码基因(Zm00001d015355和Zm00001d015356)存在缺失, Zm00001d015356编码对羟基苯丙酮酸双加氧酶(ZmHPPD1)。hppd1突变体表现穗发芽表型, 且与vp2杂交后代中正常与穗发芽籽粒符合3∶1遗传分离比, 结果表明Vp2编码ZmHPPD1基因。为进一步分析ZmHPPD1调控籽粒穗发芽的机制, 我们检测了突变体中内源激素和ABA合成通路代谢物含量的变化, 发现与正常籽粒相比, 穗发芽籽粒中ABA含量显著下降, 八氢番茄红素显著积累, 且紫黄质、玉米黄质和叶黄素含量显著降低。这表明ZmHPPD1通过影响八氢番茄红素向番茄红素的转化, 使ABA合成受阻, 导致玉米籽粒打破休眠提前萌发。以上研究结果为解析玉米穗发芽的机制提供了基因资源。

关键词: 玉米, 穗发芽, 植物激素, 脱落酸

Abstract:

Maize vivipary, the precocious germination of seeds on the ear, significantly impacts maize yield and quality. Developing vivipary-resistant maize varieties through the discovery of novel genes is crucial for agricultural production in China. In this study, the maize mutant vp2 exhibited a clear viviparous phenotype with stable inheritance, controlled by a single recessive gene. Genome sequence analysis of the vp2 mutant revealed deletions in two coding genes (Zm00001d015355 and Zm00001d015356), with Zm00001d015356 encoding p-hydroxypyruvate dioxygenase (ZmHPPD1). The hppd1 mutant also displayed a viviparous phenotype. Furthermore, test crosses between vp2 and hppd1 heterozygous plants showed a 3:1 segregation ratio between normal and viviparous kernels, suggesting that ZmHPPD1 is the candidate gene for vp2. To further investigate the mechanism by which ZmHPPD1 regulates maize vivipary, we analyzed endogenous hormone and metabolite content in the ABA synthesis pathway. The results indicated a significant decrease in ABA levels, a substantial accumulation of octahydro lycopene, and a notable reduction in purple xanthophyll, zeaxanthin, and lutein in viviparous kernels. ZmHPPD1 disrupts ABA synthesis by affecting the conversion of octahydro lycopene to lycopene, leading to the loss of dormancy and early germination of maize kernels. These findings provide valuable genetic resources for breeding vivipary-resistant maize.

Key words: maize, viviparous, plant hormones, abscisic acid

图1

vp2突变体穗发芽表型 A: vp2杂合突变体授粉后25 d果穗及籽粒表型, 标尺为1 cm; B: 18 d、21 d、25 d收获的正常籽粒(WT)和穗发芽籽粒(vp2)的纵向切片, 标尺为500 μm。"

表1

本研究所用的引物"

引物
Primer		 引物序列
Primer sequences (5′-3′)
vp2-F1 CGCAGCCAGATACAAACGTT
vp2-R1 GGGACATGGGAGCTCGAATT
TIR8.1 GAAGCCAACGCCAWCGCCTCYATTTCGTCGAAT
vp2-F2 ACTACGGGCTGAGCAGGTT
vp2-R2 TGAAGATTTGGAGCAGCACG
GAPDH-F CCCTTCATCACCACGGACTAC
GAPDH-R AACCTTCTTGGCACCACCCT

表2

vp2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"

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)
B73×vp2/+ 162 48 210 0.406
128 37 165 0.454

图2

Vp2编码对羟基苯丙酮酸双加氧酶ZmHPPD1 A: 利用BSR-Seq对vp2突变体基因进行初定位; B: B73和vp2突变体材料中基因差异; C: vp2突变体中ZmHPPD1的表达量。"

图3

利用杂合突变体hppd1对vp2进行等位测验 A: ZmHPPD1基因结构示意图及hppd1突变体的突变位置; B: hppd1中Mu转座子插入突变体的鉴定; C: hppd1杂合突变果穗及籽粒表型; D: hppd1突变体与vp2杂交果穗及籽粒表型。"

表3

hppd1杂合突变体自交授粉以及与vp2等位测验后正常籽粒和穗发芽籽粒的分离情况"

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)
hppd1/+ 96 31 127 0.003
128 44 172 0.008
hppd1/+×vp2/+ 171 64 235 0.512
156 57 213 0.264

图4

WT和vp2籽粒中内源激素含量分析 A: 脱落酸; B: 脱落酸葡萄糖酯; C: 水杨酸; D: 生长素; E: 独角金内酯; F: 茉莉酸; G: 细胞分裂素; H: 乙烯。**表示在0.01概率水平显著相关。"

图5

ABA合成通路上相关代谢物质的含量 A: 八氢番茄红素; B: α-胡萝卜素; C: 叶黄素; D: β-胡萝卜素; E: 玉米黄质; F: 紫黄质。*、**分别表示在0.05和0.01概率水平显著相关。"

图6

WT和vp2突变体的ABA生物合成途径 A: WT籽粒ABA生物合成途径; B: vp2突变体ABA生物合成途径。代谢通路中绿色表示物质含量下降, 红色表示物质含量上升。"

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