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作物杂志,2023, 第3期: 246–253 doi: 10.16035/j.issn.1001-7283.2023.03.034

• 植物保护 • 上一篇    下一篇

稻瘟病菌类SRRM1转录因子的功能分析

邱凯华(), 方淑梅, 梁喜龙()   

  1. 黑龙江八一农垦大学，163319，黑龙江大庆
  • 收稿日期:2021-11-24 修回日期:2022-02-17 出版日期:2023-06-15 发布日期:2023-06-16
  • 通讯作者: 梁喜龙，主要从事植物逆境生理及化学调控与高产等方面的研究工作，E-mail：xilongliang@126.com
  • 作者简介:邱凯华，研究方向为植物抗逆生理与化学调控，E-mail：1546650929@qq.com
  • 基金资助:
    黑龙江省自然科学基金(C2016047);黑龙江八一农垦大学研究生创新科研项目(YJSCX2019-Y14);校内培育课题(XZR2014-01)

Functional Analysis of SRRM1-Like Transcription Factor of Magnaporthe grisea

Qiu Kaihua(), Fang Shumei, Liang Xilong()   

  1. Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2021-11-24 Revised:2022-02-17 Online:2023-06-15 Published:2023-06-16

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摘要：

转录因子在稻瘟病菌（Magnaporthe grisea）的生长发育、逆境适应以及致病性中发挥着重要作用。为明确类SRRM1转录因子调控的pre-mRNA剪接与稻瘟病菌生长发育和逆境适应的关系，采用以基因重组原理为基础的基因定点敲除技术获取类SRRM1基因缺失菌株，并分析营养生长及逆境适应。结果表明，本研究成功构建敲除载体并敲除了稻瘟病菌Y34菌株中的类SRRM1基因，获得基因缺失突变体。稻瘟病菌中类SRRM1基因缺失未明显影响其营养生长，但对刚果红、十二烷基硫酸钠、山梨醇、NaCl及杀菌剂吡唑醚菌酯逆境胁迫敏感，表现为抗逆性减弱。类SRRM1在稻瘟病菌逆境适应过程中发挥重要作用。

关键词: 稻瘟病菌, 剪接, PWI结构域, 生物信息学分析, 基因敲除

Abstract:

Transcription factors played an important role in the growth and development, adversity adaptation and pathogenicity of Magnaporthe grisea. To clarify the relationship between pre-mRNA splicing regulated by transcription factor SRRM1-like and the growth and adversity adaptation of M.grisea, the SRRM1-like gene- deficient strains were obtained by gene-specific knockout technology based on the principle of gene recombination, and analyzed for vegetative growth and adversity adaptation. The results showed that the knockout vector was successfully constructed and the SRRM1-like gene in the rice blast fungus Y34 strain was knocked out, and three gene deletion mutants were obtained. The deletion of the SRRM1-like gene did not significantly affect the biotrophic growth of M.grisea, but it was sensitive to stress by Congo-Red, SDS, sorbitol, NaCl and the fungicide pyraclostrobin, and showed weakened stress resistance. SRRM1-like played an important role in the adversity adaptation of M.grisea.

Key words: Magnaporthe grisea, Splicing, PWI domain, Bioinformatics analysis, Gene knockout

表1

敲除载体构建及突变体验证所用引物信息

引物名称
Primer name		 引物序列
Primer sequence (5′→3′)		 产物长度
Product length (bp)		 酶切位点
Restriction site
M-0-L-S GAATTCTATCCGAAACAAACCTGAG 1152 EcoR I
M-0-L-A GAGCTCGGAGATCGGGTTGTTGTAG Sac I
M-0-R-S GGATCCCTACAACATCCGCCCTTAC 1305 BamH I
M-0-R-A AAGCTTGTCTGAGCCTTCGGAACAT Hind III
M-0-G-S AGGCTGGATGTCACGCTAA 653 -
M-0-G-S ATTTCGACGATGCAGGAGA -
Hyg-S GCCCTTCCTCCCTTTATT 753 -
Hyg-A TGTTGGCGACCTCGTATT -
M-0-L-H-S TACATCAGCACCCAAGGC 2044 -
M-0-L-H-A GCTATTTACCCGCAGGAC -
M-O-H-R-S CCGTGGTTGGCTTGTATG 2193 -
M-O-H-R-A CGGGCTCTAACCTCCAGTA -

图1

类SRRM1蛋白的空间结构 (a) 类SRRM1蛋白二级结构；(b) 类SRRM1蛋白结构域；(c) 类SRRM1蛋白三级结构

图2

类SRRM1蛋白的进化关系与结构分析

图3

M-0基因左、右臂的PCR扩增

图4

pXEH20、P-L、P-LR质粒电泳图 P为敲除载体pXEH20；P-L为连接基因左臂的重组载体；P-LR为连接基因左、右臂的重组载体

图5

M-0基因敲除载体的构建示意图及验证 (a)：敲除载体pXEH20-LR的构建及重组示意图；(b)：敲除载体验证电泳图，泳道1、3为水对照，泳道2、4分别为M-0基因左、右臂

图6

M-0转化子目的基因验证电泳图 泳道1为野生型对照；泳道2为水对照；泳道3、4、5分别为突变体M9、M11、M17中Hyg片段；泳道6、7、8分别为突变体M9、M11、M17中Gene片段；泳道9为水对照；泳道10为野生型对照

图7

M-0转化子侧翼序列验证电泳图 泳道1为野生型对照；泳道2为水对照；泳道3、4、5分别为突变体M9、M11、M17中M-0-L-H片段；泳道6、7、8分别为突变体M9、M11、M17中M-0-H-R片段；泳道9为水对照；泳道10为野生型对照

图8

基因敲除对稻瘟病菌营养生长及逆境适应的影响 不同小写字母表示差异显著（P < 0.05）

[1] Will C L, Lührmann R. Spliceosome structure and function. Cold Spring Harbor Perspectives in Biology, 2011, 3(7):a003707.
[2] Kelly S M, Corbett A H. Messenger RNA export from the nucleus:a series of molecular wardrobe changes. Traffic, 2009, 10(9):1199-1208.
doi: 10.1111/j.1600-0854.2009.00944.x pmid: 19552647
[3] Nino C A, Herissant L, Babour A, et al. mRNA nuclear export in yeast. Chemical Reviews, 2013, 113(11):8523-8545.
doi: 10.1021/cr400002g pmid: 23731471
[4] Katahira J. Nuclear export of messenger RNA. Genes, 2015, 6(2):163-184.
doi: 10.3390/genes6020163 pmid: 25836925
[5] Burge C B, Tuschl T, Sharp P A. Splicing of precursors to mRNAs by the spliceosomes. Cold Spring Harbor Monograph Series, 1999, 37:525-560.
[6] Jurica M S, Moore M J. Pre-mRNA splicing:awash in a sea of proteins. Molecular Cell, 2003, 12(1):5-14.
doi: 10.1016/S1097-2765(03)00270-3
[7] Kastner B, Will C L, Stark H, et al. Structural insights into nuclear pre-mRNA splicing in higher eukaryotes. Cold Spring Harbor Perspectives in Biology, 2019, 11(11):a032417.
doi: 10.1101/cshperspect.a032417
[8] Moore M J, Query C C, Sharp P A. Splicing of precursors to mRNAs by the spliceosome. Cold Spring Harbor Monograph Series, 1993, 24:303.
[9] Cáceres J F, Kornblihtt A R. Alternative splicing:multiple control mechanisms and involvement in human disease. Trends in Genetics, 2002, 18(4):186-193.
doi: 10.1016/s0168-9525(01)02626-9 pmid: 11932019
[10] Fu X D. The superfamily of arginine/serine-rich splicing factors. RNA, 1995, 1(7):663-680.
pmid: 7585252
[11] Graveley B R. Sorting out the complexity of SR protein functions. RNA, 2000, 6(9):1197-1211.
pmid: 10999598
[12] Blencowe B J, Bowman J A L, McCracken S, et al. SR-related proteins and the processing of messenger RNA precursors. Biochemistry and Cell Biology, 1999, 77(4):277-291.
pmid: 10546891
[13] Wagner S, Chiosea S, Nickerson J A. The spatial targeting and nuclear matrix binding domains of SRm160. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(6):3269-3274.
[14] Szymczyna B R, Bowman J, McCracken S, et al. Structure and function of the PWI motif:a novel nucleic acid-binding domain that facilitates pre-mRNA processing. Genes and Development, 2003, 17(4):461-475.
pmid: 12600940
[15] Blencowe B J, Ouzounis C A. The PWI motif:a new protein domain in splicing factors. Trends in Biochemical Sciences, 1999, 24(5):179-180.
pmid: 10322432
[16] Wagner S, Chiosea S, Ivshina M, et al. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160. The Journal of Cell Biology, 2004, 164(6):843-850.
doi: 10.1083/jcb.200307002
[17] Kataoka N, Yong J, Kim V N, et al. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Molecular Cell, 2000, 6(3):673-682.
pmid: 11030346
[18] Kim V N, Kataoka N, Dreyfuss G. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. Science, 2001, 293(5536):1832-1836.
pmid: 11546873
[19] Le Hir H, Moore M J, Maquat L E. Pre-mRNA splicing alters mRNP composition:evidence for stable association of proteins at exon-exon junctions. Genes and Development, 2000, 14(9):1098-1108.
pmid: 10809668
[20] Le Hir H, Izaurralde E, Maquat L E, et al. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. The European Molecular Biology Organization Journal, 2000, 19(24):6860-6869.
doi: 10.1093/emboj/19.24.6860
[21] Lejeune F, Ishigaki Y, Li X, et al. The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells:dynamics of mRNP remodeling. The European Molecular Biology Organization Journal, 2002, 21(13):3536- 3545.
doi: 10.1093/emboj/cdf345
[22] Beckwith E J, Hernando C E, Polcowñuk S, et al. Rhythmic behavior is controlled by the SRm 160 splicing factor in Drosophila melanogaster. Genetics, 2017, 207(2):593-607.
doi: 10.1534/genetics.117.300139 pmid: 28801530
[23] McCracken S, Longman D, Johnstone I L, et al. An evolutionarily conserved role for SRm 160 in 3′-end processing that functions independently of exon junction complex formation. Biological Chemistry, 2003, 278(45):44153-44160.
[24] Long J C, Caceres J F. The SR protein family of splicing factors:master regulators of gene expression. Biochemical Journal, 2009, 417(1):15-27.
doi: 10.1042/BJ20081501
[25] Jeong S. SR proteins:binders,regulators, and connectors of RNA. Molecules and Cells, 2017, 40(1):1.
[26] Blencowe B J, Issner R, Nickerson J A, et al. A coactivator of pre-mRNA splicing. Genes and Development, 1998, 12(7):996-1009.
pmid: 9531537
[27] McCracken S, Longman D, Marcon E, et al. Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin. Biological Chemistry, 2005, 280(51):42227-42236.
[28] Cheng C, Sharp P A. Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Molecular Cell Biology, 2006, 26(1):362-370.
doi: 10.1128/MCB.26.1.362-370.2006
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