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作物学报 ›› 2024, Vol. 50 ›› Issue (11): 2764-2774.doi: 10.3724/SP.J.1006.2024.42008

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻类病变早衰突变体lmes6的表型鉴定与基因定位

胡瑶洁1(), 刘亚萍1, 郑君妍1, 韩婷1, 马伯军1, 叶亚峰2, 刘斌美2,*(), 陈析丰1,*()   

  1. 1浙江师范大学生命科学学院, 浙江金华 321004
    2中国科学院合肥物质科学研究院, 安徽合肥 230031
  • 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:2024-07-12
  • 通讯作者: *刘斌美, E-mail: liubm@ipp.ac.cn; 陈析丰, E-mail: xfchen@zjnu.cn
  • 作者简介:E-mail: 2227126579@qq.com
  • 基金资助:
    国家自然科学基金项目(32071987);国家自然科学基金项目(32101697);国家自然科学基金项目(32272096);浙江省自然科学基金项目(LQ22C130005);浙江省自然科学基金项目(LZ23C130004);浙江省自然科学基金项目(LZ24C130016)

Phenotype identification and gene mapping of lesion mimic and early senescence mutant lmes6 in rice

HU Yao-Jie1(), LIU Ya-Ping1, ZHENG Jun-Yan1, HAN Ting1, MA Bo-Jun1, YE Ya-Feng2, LIU Bin-Mei2,*(), CHEN Xi-Feng1,*()   

  1. 1College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
    2Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei 230031, Anhui, China
  • Received:2024-02-05 Accepted:2024-06-20 Published:2024-11-12 Published online:2024-07-12
  • Contact: *E-mail: liubm@ipp.ac.cn; E-mail: xfchen@zjnu.cn
  • Supported by:
    National Natural Science Foundation of China(32071987);National Natural Science Foundation of China(32101697);National Natural Science Foundation of China(32272096);Natural Science Foundation of Zhejiang Province(LQ22C130005);Natural Science Foundation of Zhejiang Province(LZ23C130004);Natural Science Foundation of Zhejiang Province(LZ24C130016)

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摘要：

植物类病变与早衰的发生受激素、代谢、环境信号等多重因素影响, 其分子机制尚未完全清楚。从水稻辐射诱变库中筛选到一个类病变早衰突变体lmes6 (lesion mimic and early senescence 6), 与野生型相比, lmes6突变体叶片从分蘖期开始出现类病变、叶色发黄、叶绿素含量显著降低, 其穗长、每穗粒数、粒长及单株产量显著降低; 组织染色显示该突变体的叶片出现细胞程序性死亡及活性氧过量积累; 且该突变体增强了对水稻白叶枯病与细菌性条斑病的抗性。遗传分析表明lmes6的突变表型受单隐性核基因控制。利用图位克隆技术将目的基因精细定位在水稻7号染色体53 kb区间内, 经候选基因预测与PCR测序分析, 发现在lmes6突变体中一个编码Fd-GOGAT1 (依赖于铁氧还蛋白的谷氨酸合酶)的基因LOC_Os07g46460发生了一个碱基替换, 导致苯丙氨酸(F)变为亮氨酸(L), 是该基因的一个新的复等位基因, 与其他已报道的lc7、abc1、spl32等位突变相比, lmes6对水稻的生长发育、产量等方面抑制较小。对Fd-GOGAT1进行系统发育树分析及同源蛋白序列比对, 结果表明Fd-GOGAT1蛋白在单子叶植物中高度保守。

关键词: 类病变, 早衰, lmes6, 精细定位, 序列分析

Abstract:

The occurrence of lesion mimic and early senescence is affected by multiple factors, including hormones, metabolism, and ambient signals. However, the molecular mechanism underlying this process remains incompletely understood. In this study, we identified a lesion mimic and early senescence mutant, named lmes6 (lesion mimic and early senescence 6), through screening of a heavy ion beam radiation mutagenesis library. Comparative analysis with the wild-type control revealed that the leaves of the lmes6 mutant exhibited lesion mimic, chlorosis, and a significant reduction in chlorophyll content from the tillering stage. Additionally, the mutant displayed a notable decrease in panicle length, grain number per panicle, grain length, and yield per plant. Tissue staining showed that the mutant exhibited programmed cell death and excessive accumulation of reactive oxygen species in the leaves. Furthermore, the mutant displayed enhanced resistance to bacterial blight and bacterial leaf streak. Genetic analysis showed that the mutant phenotype was governed by a single recessive nuclear gene. Through map-based cloning techniques, the gene was precisely mapped to a 53 kb region on rice chromosome 7. Candidate gene prediction and PCR sequencing analysis identified a gene, LOC_Os07g46460, which encodes Fd-GOGAT1 (ferredoxin-dependent glutamate synthase 1) in the lmes6 mutant. A single base substitution in this gene resulted in the conversion of L-phenylalanine (F) to leucine (L), which is one of new multiple alleles of this gene. Interestingly, compared to the three allelic mutations of this gene reported, lc7, abc1, and spl32, the lmes6 mutant exhibited less impact on growth and yield of rice. Phylogenetic analysis and alignment of the homologous protein sequences of Fd-GOGAT1 revealed its high conservation among monocots.

Key words: lesion mimic, early senescence, lmes6, fine mapping, sequence analysis

表1

用于基因定位的分子标记PCR引物序列"

分子标记
Molecular marker		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
R7M30 ATGTCGCCTACGAGTTTTC TTCATGTGACCATTTGTGC
RM22017 GCTACCATGCTAGTCGTAATGC GCTAGTGTAAACTTTGGCATCG
RM22091 CCGTGAGGGCCATGTACAGACG TGCCACGTCAGCACTCTTTCTTCC
RM22144 GCAGAAGCAGGAGCCCAATATCC CTGTCTGTCCAGCACCAACACC
RM22171 TAGTACCGCCATTACCATTCATCC GACGGTGGGACTCCTAATTACAGC
Indel 1 CCACCTTCAATCCCTGCC AGTGAAGACCGCCCAGATC
Indel 2 GCACAGACACAACCTTTAGGA GTGTCAGGTTTTGGCTCGG
Indel 3 CTTACAATGCCCACAAAGGA TTGCTGGTAAAGAATGCTAATC
Indel 4 CGTTTGATCGTCCGTCTTAT AATTGCCATGACTCCTCCTC
Indel 5 CGCAGAGAGTTCAAGGAAGC AGACTGTTGCTAACCATCATGG

表2

水稻LMES6基因PCR扩增引物序列"

引物名称
Primer name		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
LMES6-1 TTCCCGCATCCACCACCA CATCTAGGGCTTGTATTGGTAC
LMES6-2 GATCTGAGATTCTTGGGCAATT TCACGCCCACGCCATACAG
LMES6-3 CAATGGCTTCACAAGGCAAGG TCACCGTGGGCATCTTCC
LMES6-4 CCCTGCCATCCCGATACTTC CCCTTCACCAGGCTTTGC
LMES6-5 TGAGCCTGCAACCTCCAT GGGCCATTACTGTGGACATCTT
LMES6-6 TTTCCTGGTGTTCCTGGTG GTGGACCGTGGTGTTATCGT

图1

水稻lmes6突变体的突变表型与抗病性 A: 分蘖期的植株表型; B: 成熟期的植株表型; C: 成熟期的剑叶表型; D: 染色前WT和lmes6的叶片表型; E~G: 叶片的TB、DAB和NBT染色; H: 接种Xoc和Xoo菌株的水稻叶片; I: 叶片的叶绿素含量测定; J: 接种Xoc菌株的叶片病斑占总叶面积的比例统计; K: 接种Xoo菌株叶片的病斑长度统计. WT: 野生型对照武运粳7号; lmes6: 水稻类病变和早衰突变体; **表示WT和lmes6间的t检验存在极显著性差异(P < 0.01); I~K中误差线表示标准差。标尺: 5 cm (A); 15 cm (B); 2 cm (C); 1 cm (D~H)."

图2

水稻lmes6突变体与其野生型对照的农艺性状比较 A: 株高; B: 剑叶长; C: 剑叶宽; D: 有效分蘖数; E: 穗长; F: 每穗粒数; G: 结实率; H: 粒长; I: 粒宽; J: 粒厚; K: 千粒重; L: 单株产量; M~O: WT 和lmes6 突变体的 10 粒种子的长度、宽度和厚度的照片; P: 穗长. *与**分别表示WT和lmes6间的t检验存在显著性差异(P < 0.05)和极显著性差异(P < 0.01); A~L中误差线表示标准差. 标尺: 1 cm (M~O); 5 cm (P)."

图3

突变体lmes6目的基因的精细定位与候选基因测序分析 A: lmes6基因精细定位的遗传图谱; n为定位分析所用到F2突变单株数, 标记下的数字为该标记检测到的重组数。B: 定位区间的所有注释基因; C: 候选基因LOC_Os07g46460突变位点的测序结果。"

图4

植物Fd-GOGAT1同源蛋白的系统进化树"

图5

水稻Fd-GOGAT1及其同源蛋白的序列比对 红色箭头或括弧表示序列发生变异的位点; lc7、abc1与lmes6基因为spl32基因的不同等位变异, 在Fd-GOGAT1蛋白的谷氨酰胺转移酶结构域, spl32突变体发生19个氨基酸的缺失, lc7突变体发生天冬酰胺(N)到丝氨酸(S)的替换; 在FMN结合结构域, lmes6突变体发生苯丙氨酸(F)到亮氨酸(L)的替换, abc1突变体发生赖氨酸(K)到谷氨酸(E)的替换."

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