基于宿主诱导的基因沉默技术创制抗拟轮枝镰孢玉米自交系

doi:10.3864/j.issn.0578-1752.2021.09.002

摘要/Abstract

摘要：

【目的】拟轮枝镰孢（Fusarium verticillioide）是一种主要的病原真菌,侵染玉米可以导致穗粒腐病、茎腐病、苗期根腐病及引起种子腐烂。由拟轮枝镰孢引起的病害不仅影响玉米的产量和品质,而且其病原菌代谢过程中产生的伏马菌素等多种真菌毒素严重威胁了人畜安全。通过宿主诱导的基因沉默（host-induced gene silencing,HIGS）技术创制抗拟轮枝镰孢的玉米种质,为玉米抗病育种提供新的优异抗源。【方法】通过同源克隆方法克隆可能与拟轮枝镰孢生长发育相关的关键基因,并通过体外转录获得相应的dsRNA片段;将不同基因的dsRNA与拟轮枝镰孢的分生孢子悬浮液预混后,用于后续的体外RNA沉默试验;对感病玉米自交系西502的种子进行消毒与接种,在培养皿中28℃避光培养48 h,调查种子的发病程度;在混有dsRNA的孢子悬浮液中加入葡萄糖,25℃培养24 h后,在显微镜下观察孢子萌发与菌丝生长情况;将三叶期的西502幼苗转移至预混dsRNA的孢子悬浮液中进行培养,7 d后观察苗期根腐病发病状况;通过种子鉴定与苗期鉴定体系,逐步筛选具有显著抑制效果的沉默靶标基因;合成筛选出的重点靶标基因片段,构建沉默载体并转化感病玉米自交系西502;对转基因株系的种子接种鉴定,验证转化玉米株系的抗性;提取接种后转基因种子的总RNA,对拟轮枝镰孢的靶标基因进行荧光定量分析,确定HIGS株系的沉默效果。【结果】从拟轮枝镰孢中克隆出18个与其生长发育相关的候选基因;通过种子接种鉴定,发现11个候选基因被沉默后,种子的发病等级极显著降低;进一步筛选出6个沉默后影响拟轮枝镰孢的孢子萌发和菌丝生长的候选靶标基因deo、Ras2、Dpdc、Hsp90、Frp1和Atg15;通过苗期接种鉴定,最后筛选出3个在体外具有显著抑制效果的沉默靶标基因deo、Atg15和Frp1;进而将3个靶标基因的特异区段人工融合成一段序列并构建沉默载体,获得转基因植株;鉴定发现转基因植株的T₂代种子对拟轮枝镰孢的抗性显著增强,且3个靶标基因的表达量均显著下降。【结论】拟轮枝镰孢基因deo、Atg15、Frp1与其生长发育密切相关,且沉默后能够显著提升玉米对拟轮枝镰孢的抗性。

关键词: 玉米, 拟轮枝镰孢, 宿主诱导基因沉默, 转基因, 穗粒腐病

Abstract:

【Objective】Fusarium verticillioides (F. verticillioides) is a common pathogen, which can cause ear rot, stalk rot, seedling blight, and seed rot in maize. These diseases caused by Fusarium verticillioides not only affected the yield and quality of maize, but also seriously threatened to the safety of human and livestock by a variety of fungal toxins such as fumonisin which produced during the metabolic process of the pathogen. So far, there is no report about the major resistance gene cloned and utilized for Fusarium verticillioides in maize. Using host-induced gene silencing technology provides a new strategy for resistance breeding in maize. 【Method】Key genes associated with the Fusarium verticillioides development were cloned using homologous gene sequence method, and the dsRNA were produced by in-vitro transcription (IVT) assay. The dsRNA for different genes was premixed with suspension spores of Fusarium verticillioides used for RNA silencing experiment in vitro. For investigate the degree of disease, the seeds of the susceptible inbred line Xi502 were sterilized and inoculated, and then were cultured in a petri dish at 28℃ in the dark for 48 h. For investigate the incidence of the seeds after inoculation, glucose was added to the spore suspension mixed with dsRNA, then spore germination and mycelia growth were observed under the microscope after 25℃ culture for 24 h. The Xi502 seedlings of the trifoliate stage were transferred to the spore suspension with premixed dsRNA for culture, and the incidence of seedlings blight was observed after 7 days. In order to select the target gene for HIGS, combine the seed morphological observation result after inoculation and seedling inoculation result. Then, the silent vector about these key target genes were constructed and transferred into the susceptible inbred line Xi502. The transgenic seeds were evaluated by artificial inoculation. The total RNA of the transgenic seeds after inoculation was extracted, and the relative expression of target genes in F. verticillioide was analyzed by qRT-PCR to determine the silencing effect of HIGS line. 【Result】Eighteen candidate genes related to growth were cloned by homologous cloning method in Fusarium verticillioides. It was found that the disease level of seeds was significantly reduced after 11 candidate genes silencing by seed inoculation experiments. Furthermore, six of the 11 candidate target genes, deo, Ras2, Dpdc, Hsp90, Frp1, and Atg15, were found that response to the spore germination and mycelium growth after gene silencing. Finally, based on the results of seedling inoculation, 3 silencing target genes deo, Atg15 and Frp1 with significant inhibitory effect in vitro were selected. Then the silencing vector was constructed by combine three specific segments from the three target genes, transgenic plants were obtained. It was found that the resistance level was highly increased in T2-generation seeds compared to the none-transgenic plants. As well as the expression levels of all the three target genes were significantly decreased in Fusarium verticillioides. 【Conclusion】Three genes, deo, Atg15 and Frp1, are important for development of Fusarium verticillioides. By constructing transgenic HIGS plants for target gene deo, Atg15 and Frp1, the increase the resistance to Fusarium verticillioides in maize.

Key words: maize (Zea mays L.), Fusarium verticillioides, host-induced gene silencing, transgenic, ear rot

赫可伟,陈甲法,周子键,吴建宇. 基于宿主诱导的基因沉默技术创制抗拟轮枝镰孢玉米自交系[J]. 中国农业科学, 2021, 54(9): 1835-1845.

HE KeWei,CHEN JiaFa,ZHOU ZiJian,WU JianYu. Fusarium verticillioides Resistant Maize Inbred Line Development Using Host-Induced Gene Silencing Technology[J]. Scientia Agricultura Sinica, 2021, 54(9): 1835-1845.

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参考文献 46

[1]	王晓鸣, 晋齐鸣, 石洁, 王作英, 李晓. 玉米病害发生现状与推广品种抗性对未来病害发展的影响. 植物病理学报, 2006,36(1):1-11.
	WANG X M, JIN Q M, SHI J, WANG Z Y, LI X. The status of maize diseases and the possible effect of variety resistance on disease occurrence in the future. Acta Phytopathologica Sinica, 2006,36(1):1-11. (in Chinese)
[2]	KAMLE M, MAHATO D K, DEVI S, LEE K E, KANG S G, KUMAR P. Fumonisins: Impact on agriculture, food, and human health and their management strategies. Toxins, 2019,11(6):328.
[3]	KNUTSEN H K, ALEXANDER J, BARREGÅRD L, BIGNAMI M, BRÜSCHWEILER B, CECCATELLI S, COTTRILL B, DINOVI M, EDLER L, GRASL-KRAUPP B, HOGSTRAND C, HOOGENBOOM L, NEBBIA C S, PETERSEN A, ROSE M, ROUDOT A C, SCHWERDTLE T, VLEMINCKX C, VOLLMER G, WALLACE C, DALL'ASTA G S, TARANU I, ALTIERI A, ROLDÁN- TORRES R, OSWALD I P. Risks for animal health related to the presence of fumonisins, their modified forms and hidden forms in feed. EFSA Journal, 2018,16(5):5242.
[4]	ROSS P F, RICE L G, PLATTNER R D, OSWEILER G D, WILSON T M, OWENS D L, NELSON H A, RICHARD J L. Concentrations of fumonisin B1 in feeds associated with animal health problems. Mycopathologia, 1991,114(3):129-135.
[5]	MARASAS W F O. Fumonisins: Their implications for human and animal health. Natural Toxins, 1995,3(4):193-198.
[6]	GELDERBLOM W C A, JASKIEWICZ K, MARASAS W F O, THIEL P G, HORAK R M, VLEGGAAR R, KRIEK N P. Fumonisins: Novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme. Applied & Environmental Microbiology, 1988,54(7):1806-1811.
[7]	YOSHIZAWA T, YAMASHITA A, LUO Y. Fumonisin occurrence in corn form high-risk and low-risk areas for human esophageal cancer in China. Applied and Environmental Microbiology, 1994,60(5):1626-1629.
[8]	UENO Y, IIJIMA K, WANG S D, SUGIURA Y, SEKIJIMA M, TANAKA T, CHEN C, YU S Z. Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China, by HPLC and ELISA. Food and Chemical Toxicology, 1997,35(12):1143-1150.
[9]	ZUO W, CHAO Q, ZHANG N, YE J, TAN G, LI B, XING Y, ZHANG B, LIU H, FENGLER K A, ZHAO J, ZHAO X, CHEN Y, LAI J, YAN J, XU M. A maize wall-associated kinase confers quantitative resistance to head smut. Nature Genetics, 2015,47(2):151-157.
[10]	WANG C, YANG Q, WANG W, LI Y, GUO Y, ZHANG D, MA X, SONG W, ZHAO J, XU M. A transposon-directed epigenetic change in ZmCCT underlies quantitative resistance to Gibberella stalk rot in maize. The New Phytologist, 2017,215(4):1503-1515.
[11]	LUNSFORD J N, FUTRELL M C, SCOTT G E. Maternal influence on response of corn to Fusarium moniliforme. Phytopathology, 1974,65:223-225.
[12]	PÉREZ-BRITO S, JEFFERS D, GONZÀLEZ-DE-LEÓN D, KHAIRALLAH M, CORTÉS-CRUZ M, VELÀZQUEZ-CARDELAS G, AZPIROZ-RIVERO S, SRINIVASAN G. QTL mapping of Fusarium moniliforme ear rot resistance in highland maize, México. Agrociencia, 2001,35:181-196.
[13]	ROBERTSON-HOYT L A, JINES M P, BALINT-KURTI P J, KLEINSCHMIDT C E, WHITE D G, PAYNE G A, MARAGOS C M, MOLNÁR T L, HOLLAND J B. QTL mapping for Fusarium ear rot and fumonisin contamination resistance in two maize populations. Crop Science, 2006,46(4):1734-1745.
[14]	MASCHIETTO V, COLOMBI C, PIRONA R, PEA G, STROZZI F, MAROCCO A, ROSSINI L, LANUBILE A. QTL mapping and candidate genes for resistance to Fusarium ear rot and fumonisin contamination in maize. BMC Plant Biology, 2017,17(1):20.
[15]	SEPTIANI P, LANUBILE A, STAGNATI L, BUSCONI M, NELISSEN H, MARIO ENRICO P, DELL’ACQUA M, MAROCCO A. Unravelling the genetic basis of Fusarium seedling rot resistance in the MAGIC maize population: Novel targets for breeding. Scientific Reports, 2019,9(1):5665.
[16]	张帆, 万雪琴, 潘光堂. 玉米抗穗粒腐病QTL定位. 作物学报, 2007,33(3):491-496.
	ZHANG F, WAN X Q, PAN G T. Molecular mapping of QTL for resistance to maize ear rot caused by Fusarium moniliforme. Acta Agronomica Sinica, 2007,33(3):491-496. (in Chinese)
[17]	DING J Q, WANG X M, CHANDER S, YAN J B, LI J S. QTL mapping of resistance to Fusarium ear rot using a RIL population in maize. Molecular Breeding, 2008,22(3):395-403.
[18]	STAGNATI L, LANUBILE A, SAMAYOA L F, BRAGALANTI M, GIORNI P, BUSCONI M, HOLLAND J B, MAROCCO A. A genome wide association study reveals markers and genes associated with resistance to Fusarium verticillioides infection of seedlings in a maize diversity panel. Genes, Genomes, Genetics, 2019,9(2):571-579.
[19]	ZILA C T, OGUT F, ROMAY M C, GARDNER C A, BUCKLER E S, HOLLAND J B. Genome-wide association study of Fusarium ear rot disease in the U.S.A. maize inbred line collection. BMC Plant Biology, 2014,14:372.
[20]	LANUBILE A, FERRARINI A, MASCHIETTO V, DELLEDONNE M, MAROCCO A, BELLIN D. Functional genomic analysis of constitutive and inducible defense responses to Fusarium verticillioides infection in maize genotypes with contrasting ear rot resistance. BMC Genomics, 2014,15(1):710.
[21]	YAO L, LI Y, MA C, TONG L, DU F, XU M. Combined genome-wide association study and transcriptome analysis reveal candidate genes for resistance to Fusarium ear rot in maize. Journal of Integrative Plant Biology, 2020,62(10):1535-1551.
[22]	SUDARSHANA M R, ROY G, FALK B W. Methods for engineering resistance to plant viruses. Methods in Molecular Biology, 2007,354:183-195.
[23]	WATERHOUSE P M, FUSARO A F. Viruses face a double defense by plant small RNAs. Science, 2006,313(5783):54-55.
[24]	BAUM J A, BOGAERT T, CLINTON W, HECK G R, FELDMANN P, ILAGAN O, JOHNSON S, PLAETINCK G, MUNYIKWA T, PLEAU M, VAUGHN T, ROBERTS J. Control of coleopteran insect pests through RNA interference. Nature Biotechnology, 2007,25(11):1322-1326.
[25]	KHATRI M, RAJAM M V. Targeting polyamines of Aspergillus nidulansby siRNA specific to fungal ornithine decarboxylase gene. Medical Mycology, 2007,45(3):211-220.
[26]	TINOCO M, BÁRBARA D, DALL'ASTTA R, JOÃO P, ARAGÃO F. In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biology, 2010,8:27.
[27]	NOWARA D, GAY A, LACOMME C, SHAW J, RIDOUT C, DOUCHKOV D, HENSEL G, KUMLEHN J, SCHWEIZER P. HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. The Plant Cell, 2010,22(9):3130-3141.
[28]	KOCH A, KUMAR N, WEBER L, KELLER H, IMANI J, KOGEL K H. Host-induced gene silencing of cytochrome P450 lanosterol C14 -demethylase-encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences of the USA, 2013,110(48):19324-19329.
[29]	THAKARE D, ZHANG J, WING R A, COTTY P J, SCHMIDT M A. Aflatoxin-free transgenic maize using host-induced gene silencing. Science Advances, 2017,3(3):e1602382.
[30]	BLUHM B H, ZHAO X, FLAHERTY J E, XU J R, DUNKLE L D. RAS2 regulates growth and pathogenesis in Fusarium graminearum. Molecular Plant-Microbe Interactions, 2007,20(6):627-636.
[31]	JIANG J, LIU X, YIN Y, MA Z. Involvement of a velvet protein FgVea in the regulation of asexual development, lipid and secondary metabolisms and virulence in Fusarium graminearum. PLoS ONE, 2011,6(11):e28291.
[32]	ADÁM A L, KOHUT G, HORNOK L. Fphog1, a hog-type map kinase gene, is involved in multistress response in Fusarium proliferatum. Journal of Basic Microbiology, 2010,48(3):151-159.
[33]	COLABARDINI A C, BROWN N A, SAVOLDI M, GOLDMAN M H S, GOLDMAN G H. Functional characterization of Aspergillus nidulans ypka, a homologue of the mammalian kinase SGK. PLoS ONE, 2013,8(3):e57630.
[34]	CHOI Y E, SHIM W B. Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit. Microbiology, 2008,154(1):326-336.
[35]	EATON C J, CABRERA I E, SERVIN J A, WRIGHT S J, COX M P, BORKOVICH K A. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa. PLoS ONE, 2012,7(10):e48026.
[36]	VIJAI B, SABINE B, ALBERT V, GOPALAN S, WEI Y D. Alanine: glyoxylate aminotransferase 1 is required for mobilization and utilization of triglycerides during infection process of the rice blast pathogen, Magnaporthe oryzae. Plant Signaling & Behavior, 2012,7(9):1206-1208.
[37]	NGUYEN L N, JÖRG B, LE G T, STÄRKEL C, SCHÄFER W. Autophagy-related lipase FgATG15 of Fusarium graminearum is important for lipid turnover and plant infection. Fungal Genetics and Biology, 2011,48(3):217-224.
[38]	ZHANG H, GUO J, VOEGELE R T, ZHANG J, DUAN Y, LUO H, KANG Z. Functional characterization of calcineurin homologs pscna1/pscnb1 in Puccinia striiformis f sp tritici using a host-induced RNAi system. PLoS ONE, 2012,7(11):e49262.
[39]	DUYVESTEIJN R G E, WIJK R V, BOER Y, REP M, HARING M A. Frp1 is a Fusarium oxysporum f-box protein required for pathogenicity on tomato. Molecular Microbiology, 2005,57(4):1051-1063.
[40]	LAMOTH F, JUVVADI P R, FORTWENDEL J R, STEINBACH W J. Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus. Eukaryotic Cell, 2012,11(11):1324-1332.
[41]	ZHAO P B, REN A Z, XU H J, LI D C. The gene fpk1, encoding a camp-dependent protein kinase catalytic subunit homolog, is required for hyphal growth, spore germination, and plant infection in Fusarium verticillioides. Journal of Microbiology & Biotechnology, 2010,20(1):208.
[42]	WORIEDH M, HAUBER I, MARTINEZ-ROCHA A L, VOIGT C, MAIER F J, SCHRÖDER M, MEIER C, HAUBER J, SCHÄFER W. Preventing fusarium head blight of wheat and cob rot of maize by inhibition of fungal deoxyhypusine synthase. Molecular Plant-Microbe Interactions, 2011,24(5):619-627.
[43]	CHEN J, DING J, LI H, LI Z, SUN X, LI J, WANG R, DAI X, DONG H, SONG W, CHEN W, XIA Z, WU J. Detection and verification of quantitative trait loci for resistance to Fusarium ear rot in maize. Molecular Breeding, 2012,30(4):1649-1656.
[44]	MU C, GAO J, ZHOU Z, WANG Z, SUN X, ZHANG X, DONG H, HAN Y, LI X, WU Y, SONG Y, MA P, DONG C, CHEN J, WU J. Genetic analysis of cob resistance to F. verticillioides: Another step towards the protection of maize from ear rot. Theoretical and Applied Genetics, 2019,132(4):1049-1059.
[45]	DOLORES B R, FERNANDO C G, CARLOS L G A, VICTOR H A R, JOSE L C S. Responses of maize landrace seedlings to inoculations of Fusarium spp. Open Access Library Journal, 2017,4(6):1-14.
[46]	JU M, ZHOU Z, MU C, ZHANG X, GAO J, LIANG Y, CHEN J, WU Y, LI X, WANG S, WEN J, YANG L, WU J. Dissecting the genetic architecture of Fusarium verticillioides seed rot resistance in maize by combining QTL mapping and genome-wide association analysis. Scientific Reports, 2017,7:46446.

基因 Gene	编号 ID	注释 Description	参考文献 Reference
rasG	FVEG_09577	Ras GTP酶Ras GTPase putative expressed	[30]
vea	FVEG_09521	Velvet蛋白Velvet protein putative expressed	[31]
mapk	FVEG_05063	促分裂原活化蛋白Mitogen-activated protein kinase	[32]
pka1	FVEG_05331	cAMP依赖蛋白激酶2 cAMP-dependent protein kinase type 2	[33]
CPP1	FVEG_09543	丝苏氨酸蛋白磷酸酶PP1-1 Serinethreonine-protein phosphatase PP1-1	[34]
gna2	FVEG_02792	鸟嘌呤核苷酸结合蛋白alpha-2亚基Guanine nucleotide-binding protein alpha-2 subunit	[35]
aga1	FVEG_11573	丙氨酸乙醛酸盐氨基转移酶1 Alanine-glyoxylate aminotransferase 1	[36]
Atg15	FVEG_09194	自噬相关脂肪酶Autophagy-like lipase putative expressed	[37]
cnb1	FVEG_07853	磷酸酶B亚基Calcineurin subunit B	[38]
Frp1	FVEG_01458	F-box蛋白F-box protein putative expressed	[39]
adeny	FVEG_01363	腺苷酸环化酶Adenylate cyclase	[33]
hsp90	FVEG_07470	热激蛋白90 Heat shock protein 90	[40]
Dpdc	FVEG_07987	DNA聚合酶delta催化亚基DNA polymerase delta catalytic subunit	[41]
gna3	FVEG_04170	鸟嘌呤核苷酸结合蛋白alpha-3亚基Guanine nucleotide-binding protein alpha-3 subunit	[35]
cna1	FVEG_04738	丝苏氨酸蛋白磷酸酶2B催化亚基Serinethreonine-protein phosphatase 2B catalytic subunit	[38]
ras2	FVEG_02837	Ras GTP酶Ras GTPase putative expressed	[30]
deo	FVEG_00771	脱氧辅蛋白合成酶Deoxyhypusine synthase	[42]
gna1	FVEG_06962	鸟嘌呤核苷酸结合蛋白Guanine nucleotide-binding protein putative expressed	[35]