盐胁迫下四个水稻类受体蛋白激酶的功能分析

doi:10.16819/j.1001-7216.2021.0905

摘要/Abstract

摘要：

【目的】盐胁迫是制约水稻生长和产量主要逆境之一,研究盐胁迫响应基因对于了解植物耐盐机理和培育耐盐水稻品种具有重要意义。类受体蛋白激酶RLK（receptor-like protein kinases）广泛参与调控植物细胞信号转导和对逆境胁迫的响应过程。本研究的目的是分析盐胁迫下四个RLK基因的表达模式和生物学功能。【方法】通过荧光定量PCR检测4个RLK基因在NaCl处理下的表达变化以及在不同组织器官中的表达情况,同时利用CRISPR/Cas9对4个RLK基因分别进行编辑。【结果】4个RLK基因的转录均受NaCl诱导或抑制,其中Os04g0275100基因和Os07g0541900基因主要在根中表达;Os09g0353200基因主要在叶片中表达;Os01g0852100基因在根、茎、叶、叶鞘中均有表达。通过测序分别筛选到4个基因的功能缺失突变体,耐盐性实验结果表明四个基因的突变体对NaCl的敏感程度与野生型一致。【结论】鉴定的4个RLK基因的转录受NaCl调控且表达具有组织特异性,突变单个RLK基因不影响水稻的耐盐性。为进一步揭示盐胁迫下RLK基因的功能和作用机制奠定了基础。

关键词: 类受体蛋白激酶, 盐胁迫, 基因编辑, 粳稻

Abstract:

【Objective】Salinity stress is one of the main harsh environmental factor that greatly hinders rice growth and production, and it is meaningful to study salt-responsive genes for understanding the mechanisms behind plant response to salt stress and breeding salt tolerance variety. The receptor-like protein kinases are involved in regulation of plant cell signaling and responding to stress. In this study, we analyzed the expression pattern and function of four RLKs under salt stress. 【Method】 Quantitative real-time PCR was employed to detect the expression level of the four RLKs under NaCl treatment and its tissue specificity. 【Results】The transcription of Os04g0275100 was induced by NaCl, which was expressed mainly in roots. The transcription of Os07g0541900 was inhibited by NaCl, which was expressed mainly in roots. The transcription of Os09g0353200 was inhibited by NaCl, which was expressed mainly in leaves. The transcription of Os01g0852100 was inhibited by NaCl, which was expressed in roots, shoots, leaves and sheath. Loss-of-function mutants of the four RLKs were obtained by sequencing, which exhibited the identical response to NaCl as compared with the wild-type. 【Conclusion】The expression of the four RLKs identified in this study were regulated by NaCl and were tissue-specified. Mutation of one of the four RLKs has no effects on rice salt tolerance. This study lays a basis for revealing the biological function and mechanisms of RLKs under salt stress.

Key words: receptor-like protein kinase, salt stress, CRISPR/Cas9, japonica rice

路凯, 陈涛, 姚姝, 梁文化, 魏晓东, 张亚东, 王才林. 盐胁迫下四个水稻类受体蛋白激酶的功能分析[J]. 中国水稻科学, 2021, 35(2): 103-111.

Kai LU, Tao CHEN, Shu YAO, Wenhua LIANG, Xiaodong WEI, Yadong ZHANG, Cailin WANG. Functional Analysis on Four Receptor-like Protein Kinases Under Salt Stress in Rice[J]. Chinese Journal OF Rice Science, 2021, 35(2): 103-111.

图/表 7

表1 本研究所用引物序列

Table 1 Primers used in this study.

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
Os04g0275100-Target-1F Os04g0275100-Target-1R Os04g0275100-Target-2F Os04g0275100-Target-2R Os09g0353200-Target-1F Os09g0353200-Target-1R Os09g0353200-Target-2F Os09g0353200-Target-2R Os07g0541900-Target-1F Os07g0541900-Target-1R Os07g0541900-Target-2F Os07g0541900-Target-2R Os01g0852100-Target-1F Os01g0852100-Target-1R Os01g0852100-Target-2F Os01g0852100-Target-2R	GGCACGGCTCTGCTATGCCTCCT AAACAGGAGGCATAGCAGAGCCG GCCGCTACATCGGCGGCGACAACG AAACCGTTGTCGCCGCCGATGTAG GGCAGCCTCACTATCCGAAGCAG AAACCTGCTTCGGATAGTGAGGC GCCGCTGCGGTTCGATCTGCATCT AAACAGATGCAGATCGAACCGCAG GGCACCTCATCAGCGCCCTCCAG AAACCTGGAGGGCGCTGATGAGG GCCGCCTCCTGAACGCCACCGTG AAACCACGGTGGCGTTCAGGAGG GCCGTATAAGGGGAAGCTCAGGGA AAACTCCCTGAGCTTCCCCTTATA GCCGTGGCCGATTTCGGCTTCGCA AAACTGCGAAGCCGAAATCGGCCA
Os04g0275100-T1T2-F Os04g0275100-T1T2-R Os09g0353200-T1T2-F Os09g0353200-T1T2-R Os07g0541900-T1T2-F Os07g0541900-T1T2-R Os01g0852100-T1-F Os01g0852100-T1-R Os01g0852100-T2-F Os01g0852100-T2-R U-F	GTGGGACGGATGAAGTAGTATATCTTCG AATCAAGCAGCAAGGGGACCT ACGTGCAAGGATCTATTCAAAACT GGAGAGAACACATACCGGGTAAG ACATCCAAAATGATACGCATTCCA AGCTCCGACCTCAGGTTACACC GGTACTCGCACCAGGAATTTAGAG CATCATGCTTAGGGGTTCAACA TTGTCCTGAATAAACCAACAGTAGCACT CAACAACTTGGGAAACGTTAAACG CTCCGTTTTACCTGTGGAATCG
U-R	CGGAGGAAAATTCCATCCAC
Uctcg-B1’	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
gRctga-B2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Uctga-B2’	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
gRcggt-BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
Cas9-F	GATCCTTACTTTCCGTATTCCTTACTACG
Cas9-R OsActin1-qPCR-F OsActin1-qPCR-R	ATACCCTCCTCAATCCTCTTCATG GATGACCCAGATCATGTTTG GGGCGATGTAGGAAAGC

图1 NaCl、ABA、甘露醇对RLK基因表达的影响荧光定量检测RLK基因的相对表达水平。**代表处理间差异达显著水平（P<0.01）。

Fig. 1. Relative expression level of RLKs under NaCl, ABA and mannitol treatment. qPCR was conducted to detect the relative expression of RLK. ** represent significant difference at P<0.01.

图2 RLK基因在不同组织器官中的相对表达水平 qPCR检测RLK基因在灌浆期水稻的根、茎、叶、剑叶、叶鞘中的相对表达水平。

Fig. 2. Relative expression level of RLKs in different tissues and organs. qPCR was conducted to detect the relative RLK expression in root, shoot, leaf, flag leaf and sheath at the filling stage.

图3 RLK基因靶位点的选择和pYLCRISPR/Cas9PUbi-RLKs-sgRNA载体 A-RLK基因靶位点; B-pYLCRISPR/Cas9PUbi-RLKs-sgRNA载体。

Fig. 3. Target site of RLKs and diagram of pYLCRISPR/Cas9PUbi-RLKs-sgRNA vector. A, Target site of RLKs; B, Diagram of pYLCRISPR/Cas9PUbi-RLKs-sgRNA vector.

图4 PCR扩增Cas9基因以鉴定转基因植株 A–鉴定Os04g0275100基因编辑植株; B–鉴定Os07g0541900基因编辑植株; C–鉴定Os09g0353200基因编辑植株; D–鉴定Os01g0852100基因编辑植株。

Fig. 4. Identification of the transgenic plants by amplification of Cas9. A, Identification of the Os04g0275100-edited plant; B, Identification of the Os07g0541900-edited plant; C, Identification of the Os09g0353200-edited plant; D, Identification of the Os01g0852100- edited plant.

图5 RLK基因功能缺失突变体的鉴定 A-Os04g0275100基因缺失突变体的突变类型;B-Os09g0353200基因缺失突变体的突变类型;C-Os07g0541900基因缺失突变体的突变类型;D-Os01g0852100基因缺失突变体的突变类型。

Fig. 5. Identification of the loss-of-function mutants of RLKs. A, Identification of the mutation type of Os04g0275100; B, Identification of the mutation type of Os09g0353200; C, Identification of the mutation type of Os07g0541900; D, Identification of the mutation type of Os01g0852100.

图6 RLK基因功能缺失突变体的耐盐性分析 A-突变体和野生型的耐盐表型分析;B-突变体和野生型的存活率比较。

Fig. 6. Salt tolerance analysis of the loss-of-function mutants of RLKs. A, Phenotypic analysis on salt tolerance of mutants and WT; B, Comparison of survival rate between mutants and WT.

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