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作物学报 ›› 2025, Vol. 51 ›› Issue (3): 598-608.doi: 10.3724/SP.J.1006.2025.42028

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻MKKs家族基因成员OsMKK4的不同等位基因型及自然变异对籽粒的影响

刘建国1,2(), 陈冬东2, 陈玉玉2, 易琴琴2, 李清2, 徐正进1,*(), 钱前2,*(), 沈兰2,*()   

  1. 1沈阳农业大学水稻研究所, 辽宁沈阳 110866
    2中国水稻研究所, 浙江杭州 311401
  • 收稿日期:2024-06-06 接受日期:2024-10-25 出版日期:2025-03-12 网络出版日期:2024-11-12
  • 通讯作者: *沈兰, E-mail: shenlan@caas.cn; 钱前, E-mail: qianqian188@hotmail.com; 徐正进, E-mail: xuzhengjin@126.com
  • 作者简介:E-mail: liujianguo316@163.com
  • 基金资助:
    浙江省自然科学基金探索项目(LY22C130005);国家自然科学基金项目(32171987)

Effects of different alleles and natural variations of OsMKK4, a member of the rice MKKs family gene, on grains

LIU Jian-Guo1,2(), CHEN Dong-Dong2, CHEN Yu-Yu2, YI Qin-Qin2, LI Qing2, XU Zheng-Jin1,*(), QIAN Qian2,*(), SHEN Lan2,*()   

  1. 1Rice Research Institute of Shenyang Agricultural University, Shenyang 110866, Liaoning, China
    2China National Rice Research Institute, Hangzhou 311401, Zhejiang, China
  • Received:2024-06-06 Accepted:2024-10-25 Published:2025-03-12 Published online:2024-11-12
  • Contact: *E-mail: shenlan@caas.cn; E-mail: qianqian188@hotmail.com; E-mail: xuzhengjin@126.com
  • Supported by:
    Zhejiang Provincial Natural Science Foundation Exploration Project(LY22C130005);National Natural Science Foundation of China(32171987)

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摘要：

MAPK信号通路中的MKKs家族基因在水稻生长发育和防御信号传导中起承上启下的作用, 并参与调控多种生物学过程。本研究通过生物信息学分析, 鉴定了水稻中的MKKs基因, 并构建了它们的系统发育树, 揭示了MKKs家族基因的保守性和多样性。利用基因编辑技术, 编辑日本晴水稻品种中OsMKK4基因, 成功获得了6种该基因不同突变类型的材料, 其粒长、粒宽和千粒重均较野生型显著降低。单倍型分析结果表明, OsMKK4基因的单倍型有明显的籼粳分化, 主要为Glu14粳型和Leu14籼型。含有Glu14粳型稻的平均粒长较低, 平均粒宽和千粒重较高。此外, 将Kasalath中带有Leu14单倍型的OsMKK4基因导入日本晴, 导致DHX (CSSL)的粒长、粒宽和千粒重较日本晴显著降低; 与Kasalath相比, 代换系的粒宽和千粒重显著增加。本研究探索了OsMKK4基因的重要单倍型对水稻籽粒的影响, 为更精确的分子设计育种提供基因资源和策略。

关键词: 水稻, MAPK, OsMKK4, 粒型, 单倍型

Abstract:

The MKK gene family in the MAPK signaling pathway plays a pivotal role in connecting processes related to rice growth, development, and defense signaling, regulating various biological processes. In this study, we identified MKK genes in rice through bioinformatics analysis and constructed a phylogenetic tree, revealing both the conservation and diversity within the MKK gene family. Using gene editing technology, we edited the OsMKK4 gene in the Nipponbare rice variety and successfully obtained six lines with different mutation types in this gene. Grain length, grain width, and 1000-grain weight in these mutant lines were significantly reduced compared to the wild type. Haplotype analysis revealed clear differentiation in OsMKK4 gene haplotypes between indica and japonica rice, primarily divided into the Glu14 japonica type and Leu14 indica type. On average, japonica rice containing Glu14 exhibited shorter grain length but higher grain width and 1000-grain weight. Furthermore, introducing the OsMKK4 gene with the Leu14 haplotype from Kasalath into Nipponbare resulted in significantly reduced grain length, grain width, and 1000-grain weight in the DHX (CSSL) compared to Nipponbare. However, compared to Kasalath, the grain width and 1000-grain weight of the substitution line increased significantly, particularly in grain width. This study highlights the

influence of key OsMKK4 gene haplotypes on rice grain traits, providing valuable genetic resources and strategies for more precise molecular breeding design.

Key words: rice, MAPK, OsMKK4, grain size, haplotype

表1

引物序列信息"

引物名称
Primer name		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
OsMKK4g1 GGCAAGCGACGTGAGGTCCCGCTG AAACCAGCGGGACCTCACGTCGCT
OsMKK4g2 GGCAGTGCCCACCCCGCCGAATT AAACAATTCGGCGGGGTGGGCAC
OsMKK4-JC GCGAGCCCACCAGAAAACGC TCGTCGTGGTTCCCGTAGAGCA
Hyg ACCAGACACGAGACGACTAA ATCGGTGCGGGCCTCTTC

图1

水稻MKKs家族基因及编码蛋白的系统发育与结构分析 A: MKKs不同物种间系统进化树; B: MKKs水稻系统进化树; C: MKKs保守基序; D: MKKs的保守结构域; E: MKKs启动子顺式作用元件; F: MKKs基因结构。"

图2

OsMKK4 gRNA靶位点示意图及突变蛋白的氨基酸变化 A: OsMKK4靶位点示意图。OsMKK4的2个gRNA靶点位置和碱基序列, 红色字体为PAM序列。B: 黄色高亮为PAM序列; 红色小写字母为插入碱基; +表示插入; -表示缺失; *表示翻译的终止。C: 测序结果中红色方框表示突变位点。D: 突变蛋白三级结构分析。NPB: 日本晴; osmkk4 1-1、osmkk4 1-2、osmkk4 2-1、osmkk4 2-2、osmkk4 2-3和osmkk4 2-4: OsMKK4的6种突变类型。"

图3

OsMKK4基因编辑新种质的农艺性状比较分析 A: 日本晴与日本晴背景编辑新品系的植株形态比较, 标尺为10 cm; B: 不同突变新品系粒长和粒宽比较, 标尺为1 cm; C: 不同突变新品系株高的统计分析; D~F: 分别为千粒重、粒长与粒宽的统计分析, 数据为平均值。±标准差(n ≥ 9)。*表示P < 0.05水平差异; **表示P < 0.01水平差异。缩写同图2。"

图4

OsMKK4基因自然变异与单倍型网络分析 A: OsMKK4基因结构、蛋白质结构及5种变异位置; B: 8种单倍型的网络图, 黑线代表2种单倍型之间突变数量; C: 1072份材料中8种单倍型的千粒重数据统计; D: 1072份材料中8种单倍型的粒长数据统计; E: 1072份材料中8种单倍型的粒宽数据统计(n ≥ 10)。相同字母表示在0.05水平上差异不显著, 不同小写字母表示在0.05概率水平差异显著。Hap: 单倍型; XI、GJ、Aus、Bas和admix: 根据起源区域不同而划分的不同水稻类型。"

图5

OsMKK4的自然变异与籽粒性状差异相关 A: 水稻亚种氨基酸序列分布; B~D: 氨基酸序列组合的千粒重、粒长、粒宽数据统计(n ≥ 10)。相同字母表示在0.05水平上差异不显著, 不同小写字母表示在0.05概率水平差异显著。缩写同图4。"

图6

自然变异染色体代换系的籽粒性状考察 A: 3种品系基因及氨基酸序列的比对; B: 3种品系基因测序结果; C: 3种品系的粒长和粒宽比较, 标尺为1 cm; D~F: 3种品系千粒重、粒长和粒宽数据统计(n ≥ 9)。*表示P < 0.05水平差异; **表示P < 0.01水平差异。DHX: 日本晴为受体亲本、Kasalath为供体亲本的水稻染色体片段代换系。Kasalath: 一种水稻基因组的重要代表性籼型常规稻品种。缩写同图2。"

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