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作物学报 ›› 2022, Vol. 48 ›› Issue (5): 1119-1128.doi: 10.3724/SP.J.1006.2022.12022

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

OsSAMS1在水稻稻瘟病抗性中的功能研究

杨德卫1,2(), 王勋1(), 郑星星1, 项信权1, 崔海涛1, 李生平1,*(), 唐定中1,*()   

  1. 1福建农林大学农学院 / 福建省作物设计育种重点实验室 / 福建农林大学植物免疫研究中心, 福建福州 350002
    2福建省农业科学院水稻研究所; 福建福州 350018
  • 收稿日期:2021-04-02 接受日期:2021-09-09 出版日期:2022-05-12 网络出版日期:2021-10-18
  • 通讯作者: 李生平,唐定中
  • 作者简介:杨德卫, E-mail: dewei-y@163.com;
    王勋, E-mail: 1448293617@qq.com;第一联系人：**同等贡献
  • 基金资助:
    福建省属公益类项目(2020R11010016-3);福建省自然科学基金项目(2019J01424);福建省科技重大专项专题资助(2020NZ08016)

Functional studies of rice blast resistance related gene OsSAMS1

YANG De-Wei1,2(), WANG Xun1(), ZHENG Xing-Xing1, XIANG Xin-Quan1, CUI Hai-Tao1, LI Sheng-Ping1,*(), TANG Ding-Zhong1,*()   

  1. 1College of Agriculture, Fujian Provincial Key Laboratory of Crop Breeding by Design, Plant Immunity Center, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Institute of Rice, Fujian Academy of Agricultural Sciences, Fuzhou 350018, Fujian, China
  • Received:2021-04-02 Accepted:2021-09-09 Published:2022-05-12 Published online:2021-10-18
  • Contact: LI Sheng-Ping,TANG Ding-Zhong
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    Special Fund for Agro-scientific Research in the Public Interest of Fujian Province(2020R11010016-3);Natural Science Foundation of Fujian Province(2019J01424);Major Science and Technology Projects of Fujian Province(2020NZ08016)

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摘要：

稻瘟病是水稻最重要的病害之一, 对农业生产造成巨大的经济损失。研究表明, 水稻中S-腺苷-L-甲硫氨酸合成酶OsSAMS1参与了水稻衰老相关的进程, 我们实验室前期利用转录组测序分析发现, OsSAMS1基因的表达水平受稻瘟病菌诱导后明显提高。然而, OsSAMS1是否参与水稻的免疫反应, 尚未明确。基于此, 本研究选取野生型ZH11为背景材料, 通过构建OsSAMS1基因敲除突变体来探究该基因在水稻抗病中的功能。结果表明, OsSAMS1主要在水稻叶片中表达; 且其表达明显受稻瘟病菌侵染所诱导。亚细胞定位结果显示, OsSAMS1在细胞膜、细胞质和细胞核内均有表达。通过接种稻瘟病菌发现, 与对照相比, 2个等位敲除突变体ossams1-1和ossams1-2均表现为更加感病, 且体内病程相关基因的表达也明显更低, 同时突变体中乙烯合成相关基因的表达也受到明显抑制。综上所述, OsSAMS1参与了水稻的免疫反应, 且正调控水稻稻瘟病的抗性。本研究为深入揭示OsSAMS1在稻瘟病免疫反应的分子机理奠定了基础, 并为稻瘟病抗病育种研究提供了基因资源。

关键词: 水稻, 稻瘟病, OsSAMS1基因, 乙烯, 功能研究

Abstract:

Rice blast is one of the most devastating diseases in rice, which causes great economic losses to agricultural production. It has been reported that S-Adenosyl-l-Mmethionine Synthetase 1 (OsSAMS1) is involved in the process of senescence in rice. Transcriptome sequencing analysis showed that the relative expression level of OsSAMS1 was significantly increased after inoculation with Magnaporthe oryzae (M. oryzae). However, it remains unclear whether OsSAMS1 is involved in rice immunity. To verify this, we constructed the knock-out mutants of OsSAMS1 in the wild type variety ZH11. The results showed that OsSAMS1 was mainly expressed in rice leaves, and its expression was significantly induced by M. oryzae inoculation. Subcellular localization revealed that OsSAMS1 was distributed in the plasma membrane, cytoplasm, and nucleus. Compared to the wild type, the two knockout mutants, ossams1-1 and ossams1-2, displayed enhanced susceptibility upon M. oryzae infection, and the expression of pathogenesis-related (PR) genes was significantly inhibited. In addition, ethylene synthesis-related genes were also dramatically decreased in both two mutants. These results suggested that OsSAMS1 was involved in rice immune response and positively regulated rice blast resistance, which lays a foundation for further revealing the molecular mechanism of OsSAMS1 in plant immunity and provides genetic resources for rice breeding of blast resistance.

Key words: rice, rice blast disease, OsSAMS1, ethylene, function research

表1

研究OsSAMS1所用引物信息"

引物名称
Primer name		 前引物
Forward sequence (5′-3′)		 后引物
Reverse sequence (5′-3′)
PR1a CGTGTCGGCGTGGGTGT GGCGAGTAGTTGCAGGTGATG
PR5 CAACAGCAACTACCAAGTCGTCTT CAAGGTGTCGTTTTATTCATCAACTTT
PR6 CAACAGCAACTACCAAGTCGTCTT CAAGGTGTCGTTTTATTCATCAACTTT
PR10 CCCTGCCGAATACGCCTAA CTCAAACGCCACGAGAATTTG
OsACS1 ACCAAGATGTCCAGCTTCGG GAGGAGGTACTGCGTCTGGG
OsACS2 GGAATAAAGCTGCTGCCGAT TGAGCCTGAAGTCGTTGAAGC
OsACS4 GATGTTGCGCTGGAGAGGATA TTCCCAATTGTTGCTTTGCA
OsACS6 ACAATCAGGCAAAGAAGCGAG TTGGATATGAGAACCCCACGA
SAMS1-GFP ATTTGGAGAGGACAGGGTACCATGGCCGCACTTGATACCTTC AGTGTCGACTCTAGAGGATCCGGCAGAAGGCTTCTCCCACT
SAMS1-qRT-PCR TTCTCTGGCAAGGACCCAAC GGACACCGATGGCGTATGAT
Ubiquitin AACCAGCTGAGGCCCAAGA ACGATTGATTTAACCAGTCCATGA
gRNAs-sams1-1 GTGAGACCTGCACCAAGACA GAGATGAGGACGGTGTGGAC
gRNAs-sams1-2 GTGAGACCTGCACCAAGACA GAGATGAGGACGGTGTGGAC

图1

稻瘟病菌Guy11侵染后OsSAMS1的表达变化 A: 对稻瘟病菌侵染前和侵染后12、24和36 h后的样品进行转录组测序分析, 发现OsSAMS1明显受稻瘟病菌侵染而诱导表达; B: 对稻瘟病菌侵染前和侵染后12、24、48和72 h后的样品进行qRT-PCR分析, 发现与水处理对照相比, 稻瘟病菌侵染后OsSAMS1的表达水平明显升高, 侵染24 h后达到最高。"

图2

OsSAMS1的时空表达模式 利用qRT-PCR对生长2周、4周和6周水稻的根、茎、叶, 0.5~1.0 cm、1~3 cm、3~5 cm和5~10 cm的小穗, 萌发和成熟的种子及愈伤组织中OsSAMS1的表达水平进行分析。误差线表示从3个独立的样本获得数值的标准偏差。"

图3

ossams1-1 (A)和ossams1-2 (B)敲除突变体的鉴定"

图4

ZH11、ossams1-1和ossams1-2的生长发育表型 对正常生长灌浆期的水稻进行表型观察, 标尺为10 cm。"

表2

野生型与2个敲除系在株高、穗长和千粒重等主要农艺性状比较"

性状Trait ZH11 ossams1-1 ossams1-2
株高 Plant height (cm) 99.82 ± 1.86 85.94 ± 1.76** 86.12 ± 1.92**
穗长 Panicle length (cm) 23.15 ± 1.32 18.85 ± 1.41* 18.96 ± 1.31*
有效穗数 Number of effective panicle 9.20 ± 1.04 7.84 ± 1.02* 8.11 ± 1.01*
每穗颖花数 Spikelets per panicle 144.46 ± 4.26 122.26 ± 5.16* 120.16 ± 5.06*
结实率 Seed setting rate (%) 95.32 ± 1.16 96.12 ± 1.06 96.02 ± 1.32
千粒重 Thousand-grain weight (g) 26.62 ± 0.46 26.22 ± 0.52 26.82 ± 0.51
粒长 Grain length (mm) 7.92 ± 0.11 7.89 ± 0.12 7.88 ± 0.11
粒宽 Grain width (mm) 3.62 ± 0.08 3.68 ± 0.09 3.60 ± 0.06

图5

ossams1-1和ossams1-2与对照ZH11相比更感稻瘟病菌 A: 喷雾接种Guy11后, ossams1-1和ossams1-2植株比ZH11植株产生更多的病斑; B: 发病叶片中的真菌生物量分析, 星号代表显著差异(*: P < 0.05, **: P < 0.01, 采用t检验)。"

图6

ossams1-1、ossams1-2和ZH11中病程相关基因的表达分析 图中A、B、C、D分别表示利用qRT-PCR检测接种稻瘟病菌Guy11后, ossams1-1、ossams1-2和ZH11中病程基因PR1a、PR5、PR6和PR10转录本的积累情况(*: P < 0.05; **: P < 0.01)."

图7

接菌前后ossams1突变体与野生型ZH11中乙烯合成相关基因的表达分析 图中A、B、C、D分别代表OsACS1、OsACS2、OsACS4和OsACS6在稻瘟病菌诱导前后ossams1突变体与野生型ZH11中的表达变化情况(*: P < 0.05, **: P < 0.01, 采用t检验)。"

图8

OsSAMS1的亚细胞定位 利用激光共聚焦显微镜观察OsSAMS1-GFP在烟草细胞中的表达部位, 表明OsSAMS1-GFP在细胞核、细胞质和细胞膜内均有表达, 标尺为20 μm。"

图9

OsSAMS1在植物中的进化树分析 利用BLAST分析在NCBI、RGAP和TAIR蛋白数据库中对OsSAMS1进行同源蛋白搜索, 获得获得LOC_Os01g18860和LOC_Os01g22010基因编码的2个同源蛋白, 拟南芥中2个同源蛋白AtSAM1和AtSAM2, 以及玉米、高粱、黍稷和粟中的SAMS1蛋白, 然后利用MEGA7.0软件进行进化树分析。"

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