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作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1466-1479.doi: 10.3724/SP.J.1006.2023.22039

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

泛素受体蛋白OsDSK2b负向调控水稻叶瘟和渗透胁迫抗性

丁杰荣1,**(), 马雅美1,**(), 潘发枝2, 江立群1, 黄文洁3, 孙炳蕊1, 张静1, 吕树伟1, 毛兴学1, 于航1, 李晨1,*(), 刘清1,*()   

  1. 1广东省农业科学院水稻研究所/广东省水稻育种新技术重点实验室/广东省水稻工程实验室, 广东广州 510640
    2华南农业大学, 广东广州 510642
    3广东省农业科学院农业生物基因研究中心/广东省农作物种质资源保护与利用重点实验室, 广东广州 510640
  • 收稿日期:2022-06-22 接受日期:2022-11-25 出版日期:2023-06-12 网络出版日期:2022-12-07
  • 通讯作者: *刘清, E-mail: liuqing198504@126.com;李晨, E-mail: 2369538973@qq.com
  • 作者简介:丁杰荣, E-mail: jierongdjr@163.com;
    马雅美, E-mail: mayamei@gdaas.cn第一联系人：**同等贡献
  • 基金资助:
    广东省发展和改革委、农业农村厅投资项目(粤发改农经[2021]272号);科技创新战略专项资金: 广东省农业科学院高水平人才建设项目(R2020PY-JX001);广东省重点实验室运行费专项(2020B1212060047)

Ubiquitin receptor protein OsDSK2b plays a negative role in rice leaf blast resistance and osmotic stress tolerance

DING Jie-Rong1,**(), MA Ya-Mei1,**(), PAN Fa-Zhi2, JIANG Li-Qun1, HUANG Wen-Jie3, SUN Bing-Rui1, ZHANG Jing1, LYU Shu-Wei1, MAO Xing-Xue1, YU Hang1, LI Chen1,*(), LIU Qing1,*()   

  1. 1Rice Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of New Technology in Rice Breeding/Guangdong Rice Engineering Laboratory, Guangzhou 510640, Guangdong, China
    2South China Agricultural University, Guangzhou 510642, Guangdong, China
    3Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory for Crop Germplasm Resources Preservation and Utilization, Guangzhou 510640, Guangdong, China
  • Received:2022-06-22 Accepted:2022-11-25 Published:2023-06-12 Published online:2022-12-07
  • Contact: *E-mail: liuqing198504@126.com;E-mail: 2369538973@qq.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    Investment Project of Department of Agriculture and Rural Affairs, Development and Reform Commission, Guangdong province(Yue Fa Gai Nong Jing [2021]272);Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science(R2020PY-JX001);Guangdong Key Laboratory of New Technology in Rice Breeding(2020B1212060047)

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摘要：

泛素受体蛋白DSK2 (dominant suppressor of KAR2)在植物生长发育和逆境胁迫中发挥重要作用, 但其在水稻抗病和渗透胁迫中的作用尚未见报道。本研究发现OsDSK2b受多种逆境的调控, 该基因的表达水平在稻瘟病菌侵染和20% PEG-6000处理后显著降低。时空表达分析表明OsDSK2b基因在三叶期幼苗中的表达水平最高, 亚细胞定位结果显示该蛋白定位于细胞质。接种稻瘟病菌(GD08-T13和Guy11)后, 敲除植株的病斑面积约为0.05 cm2和0.10~0.13 cm2, 远小于野生型植株的病斑面积(0.24 cm2和0.31 cm2)。敲除OsDSK2b显著增强水稻的叶瘟抗性。而且在病原菌侵染后, 敲除植株中病程相关蛋白(PR)基因的表达受到明显诱导。敲除OsDSK2b也显著增强水稻的渗透胁迫抗性, 20% PEG-6000模拟渗透胁迫处理后, OsDSK2b敲除植株的存活率为58.3%~74.0%, 显著高于野生型植株的存活率(17.0%)。OsDSK2b敲除植株的离子渗透率和植株失水率则显著低于野生型植株。扫描显微镜的结果表明, 无论在20% PEG-6000处理前后, 敲除OsDSK2b能够促进气孔的闭合, 且在20% PEG-6000处理后, 这种促进作用会更强。此外, qRT-PCR结果表明, 在20% PEG-6000处理后, OsDSK2b敲除植株中DREB基因以及脱落酸(ABA)合成或通路相关基因的表达水平显著高于野生型植株, 且敲除植株ko6和ko14的内源ABA含量分别为314.2 ng g-1和344.4 ng g-1, 显著高于野生型植株的内源ABA含量(206.8 ng g-1), 揭示OsDSK2b参与调控渗透胁迫的过程同时涉及ABA依赖和ABA非依赖途径。本研究通过解析OsDSK2b基因在水稻应对生物与非生物胁迫时所发挥的调控作用, 为水稻抗性品种的选育提供了新的候选基因。

关键词: 水稻(Oryza sativa L.), 稻瘟病, 渗透胁迫, PR, DREB, ABA

Abstract:

The ubiquitin receptor protein DSK2 (dominant suppressor of KAR2) plays an important role in the growth, development, and stress tolerance in plant, but its role in rice disease resistance and osmotic stress has not been reported yet. In this study, we identified that OsDSK2b was regulated by various stresses, and the relative expression level of this gene decreased significantly after Magnaporthe oryzae infection and 20% PEG-6000 treatment. The spatio-temporal expression analysis showed that the relative expression level of OsDSK2b was highest at three-leaf seedling stage. The subcellular localization analysis demonstrated that OsDSK2b was localized in the cytoplasm in rice protoplast. The lesion area of OsDSK2b knockout plants was about 0.05 cm2 and 0.10-0.13 cm2, which was much smaller than wild-type plants (0.24 cm2 and 0.31 cm2) after inoculation with Magnaporthe oryzae (GD08-T13 and Guy11). Compared with wild-type plants, knockout of OsDSK2b significantly enhanced the leaf blast resistance in rice, and the relative expression levels of pathogenesis-related protein (PR) genes were induced significantly in OsDSK2b knockout plants after Magnaporthe oryzae (Guy11) infection. The knockout of OsDSK2b also significantly enhanced the osmotic stress tolerance in rice. OsDSK2b knockout plants had higher survival rate (58.3%-74.0%) than wild-type plants (17.0%) after 20% PEG-6000 treatment. Meanwhile, compared with wild-type plants, OsDSK2b knockout plants had lower ion permeability and water loss rate after 20% PEG-6000 treatment. Scanning microscopy revealed that knockout of OsDSK2b could promote the stomatal closure both before and after 20% PEG-6000 treatment, and the promotion effect was stronger after osmotic stress treatment. In addition, qRT-PCR results showed that the relative expression level of DREB genes and abscisic acid (ABA) synthesis or pathway-related genes were significantly higher in OsDSK2b knockout plants than wild-type plants after osmotic stress treatment. The endogenous ABA contents of ko6 and ko14 knockout plants were 314.2 ng g-1 and 344.4 ng g-1, respectively, which were significantly higher than wild-type plants (206.8 ng g-1). These results indicated that OsDSK2b could regulate rice osmotic stress through both the ABA-dependent and ABA-independent pathways. This study provides a new candidate gene for the breeding of rice resistant varieties by analyzing the regulatory role of OsDSK2b in rice coping with biotic and abiotic stresses.

Key words: rice (Oryza sativa L.), rice blast, osmitic stress, PR, DREB, ABA

表1

本研究所使用的引物信息表"

引物名称
Primer name		 引物序列
Primer sequence (5°−3°)
OsDSK2b-6a-F/R TTGGTTGGTTAGAACCCTGACGGCAGCCAAGCCAGCA/TCAGGGTTCTAACCAACCAAGTTTTAGAGCTAGAAAT
OsDSK2b-6b-F/R CTGCTAGGAGCAGCTGAAGCAACACAAGCGGCAGC/CTTCAGCTGCTCCTAGCAGGTTTTAGAGCTAGAAAT
Pps-GGL/Pgs-GG2 TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG/AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Pps-GG2/Pgs-GGR TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG/AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC
U-F/gR-R CTCCGTTTTACCTGTGGAATCG/CGGAGGAAAATTCCATCCAC
SP1/SP2 CCCGACATAGATGCAATAACTTC/GCGCGGTGTCATCTATGTTACT
OsDSK2bJC-F/R TCTTTTCTTGCTTCTCACACTCC/TTGCCAGTAGAACCTGCCCG
OsDSK2b-F/R TGGTAGCCAAGGAGGCAATG/ATTGCCAAGAAGACGCTCCA
PR1a-F/R CGTCTTCATCACCTGCAACTACTC/CATGCATAAACACGTAGCATAGCA
PR2-F/R CCTTCACCAAGTATCTGCGA/GTCTAGCGCATTCTGCAAAC
PR5-F/R ACGAAATACTCCAAGTTCTTCAAGG/GTAATTTGTTCCGGCAGGGC
PR8-F/R TCTACGACGTGCAGAACAACTTCAG/TCCAACTCAACCACTGTGCAAGTAA
PR10a-F/R TGTGAGCCACGACAAATCCA/TGATCTCCCAGCAGCAAACA
PR10b-F/R GAGAGGCTGTGGAAGGTCTG/CCTTTAGCACGTGAGTTGCG
DREB1A-F/R TGGAGCTACTAGAGCTCAATCAACTG/TGGCATCGGAAGCCAGAA
DREB1B-F/R ACAGAGTAGGCAATGAGACTGAGGAT/TTACAGGAATTCATTGACTGCACAT
DREB2A-F/R GGAATCTCCTCCTTTCATCGTG/TTCCGCTCCTGACAAACACG
DREB2B-F/R TTGTGCTTATGACGAGGCAGC/GCTAGTGCAACCTGAACGGAAG
引物名称
Primer name		 引物序列
Primer sequence (5°−3°)
LEA3-F/R CGGCAGCGTCCTCCAAC/CGGTCATCCCCAGCGTG
LIP9-F/R CCTCTTCGACAACCTCCTTGGCA/CTTCCTCATCACTCGACGAGCT
Rab16A-F/R CACACCACAGCAAGAGCTAAGTG/TGGTGCTCCATCCTGCTTAAG
ABI2-F/R TTGTGGAGACTCACGGGCAGTG/CGAGACATTCGTCATCCTTTGC
NCED3-F/R ACGTGATCAAGAAGCCGTACCT/GCTGGTCGAGCGGGATCT
NCED4-F/R GCCGAGACACGCATTGG/GTGAAGGTGGCGACAGCAA
EF1α-F/R TTTCACTCTTGGTGTGAAGCAGAT/GACTTCCTTCACGATTTCATCGTAA

图1

OsDSK2b的表达受多种胁迫影响以及OsDSK2b在日本晴中的时空表达模式 A: OsDSK2b在稻瘟病分离株Guy11、20% PEG-6000、150 mmol L-1 NaCl和100 μmol L-1ABA处理前后的相对表达量; B: OsDSK2b在日本晴不同组织(2周龄幼苗、幼根、茎节、倒二叶、旗叶、孕穗期根、幼穗和抽穗期穗)中的相对表达量; C: OsDSK2b蛋白在水稻原生质体中的亚细胞定位(标尺为10 μm)。数据均为3个生物学重复的平均值(±标准差) (Dunnett’s test, *: P < 0.05, **: P < 0.01)"

图2

敲除OsDSK2b增强水稻对稻瘟病的抗性 A: OsDSK2b的结构和各敲除株系的靶序列突变情况。外显子和内含子分别用黑色矩形和黑线表示, 红色字体表示CRISPR载体构建的靶序列, 蓝色字体表示插入的碱基, 省略号表示缺失的碱基; B: 打孔接种GD08-T13菌株7 d后, 野生型日本晴和OsDSK2b敲除株系的表型图; C: 打孔接种GD08-T13菌株7 d后, OsDSK2b敲除株系和野生型日本晴的病斑面积统计图; D: 离体接种Guy11菌株7 d后, 野生型日本晴和OsDSK2b敲除株系的表型图; E: 接种Guy11菌株7 d后, OsDSK2b敲除株系和野生型日本晴的病斑面积统计图。 数据为3个生物学重复的平均值(±标准差) (Dunnett’s测验, *: P < 0.05, **: P < 0.01)。"

图3

OsDSK2b调控病程相关蛋白PR基因的表达 稻瘟病菌Guy11接种前(0 h)和接种后(2 d和4 d), 野生型日本晴和OsDSK2b敲除植株叶片中的PR基因的相对表达量。数据均为3个生物学重复的平均值(±标准差) (Dunnett’s测验, *: P < 0.05; **: P < 0.01)。"

图4

敲除OsDSK2b增强水稻对渗透胁迫的抗性 A: 20% PEG-6000处理前与处理3?d再复水生长5?d后, 野生型日本晴和OsDSK2b敲除株系的表型图; B: 20% PEG-6000处理3?d再复水生长5 d后, 野生型日本晴和OsDSK2b株高统计图; C: 20% PEG-6000处理前与处理5?d再复水生长5 d后, 野生型日本晴和OsDSK2b敲除株系的表型图; D: 20% PEG-6000处理5 d再复水生长5 d后, 野生型日本晴和OsDSK2b的存活率统计图; E: 20% PEG-6000处理前后, 野生型日本晴和OsDSK2b的离子渗透率统计图; F: 野生型日本晴与OsDSK2b敲除株系地上部在0、30、60、90、120、180、240和300 min的离体失水率统计图。数据均为3个生物学重复的平均值(±标准差) (Dunnett’s测验, *: P < 0.05; **: P < 0.01)。"

图5

敲除OsDSK2b降低气孔开度 A: 不同开放程度的气孔图。标尺为10 μm; B: 正常条件下和20% PEG-6000处理4 h后, 野生型日本晴和OsDSK2b敲除株系中3种不同开放程度的气孔占比统计图。结果以3个生物学重复的平均值(±标准差)表示, 每个重复至少统计300个以上气孔; C: 野生型日本晴和OsDSK2b敲除株系中气孔密度统计图; D: 野生型日本晴和OsDSK2b敲除株系中气孔大小统计图。结果以3个生物学重复的平均值(±标准差)表示, 每个重复至少统计100个气孔。"

图6

OsDSK2b调控抗逆相关基因的表达 A: 20% PEG-6000处理前(0 h)和处理后(4 h)野生型日本晴和OsDSK2b敲除株系中DREB基因的相对表达量; B: 20% PEG-6000处理前(0 h)和处理后(4 h)野生型日本晴和OsDSK2b敲除株系中ABA信号通路相关基因的相对表达量。数据均为3个生物学重复的平均值(±标准差) (Dunnett’s测验, *: P < 0.05; **: P < 0.01)。"

图7

OsDSK2b影响20% PEG-6000处理下水稻内源ABA的积累 数据均为6个生物学重复的平均值(±标准差) (Dunnett’s测验, *: P < 0.05; **: P < 0.01)。"

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