甲基磺酸乙酯诱变的棘豆链格孢菌菌株苦马豆素合成基因簇相关基因表达模式分析

doi:10.11843/j.issn.0366-6964.2022.04.024

摘要/Abstract

摘要： 旨在对棘豆链格孢菌SWN基因簇各基因表达模式与苦马豆素合成的相关性进行分析。本研究应用甲基磺酸乙酯(EMS)对棘豆链格孢菌UA003诱变处理，筛选苦马豆素产率显著变化的诱变菌株。对棘豆链格孢菌UA003、EMS诱变菌株及甘肃链格孢菌EA菌株SWN基因簇AATL、swnR、swnN、swnH1、swnH2、swnK(A、KS、SDRe1、SDR)等基因的表达水平和swnK基因的突变位点进行分析。结果表明：E02、E23和E25等诱变菌株苦马豆素产率显著增加，其AATL、swnR、swnN、swnH1及swnH2等基因表达显著下调(P<0.05或P<0.01)，A、KS、SDR和SDRe1基因表达显著上调(P<0.05或P<0.01)。突变菌株E02 swnK基因13个核苷酸位点发生突变，其氨基酸序列6个位点发生突变。与UA003相比，EA菌株163个核苷酸位点不同，氨基酸序列67个位点不同，其编码产物起始段缺失一段由MLTPAVSLKNLTKPK组成的氨基酸序列，在AT基因编码产物的1 135与1 143处插入了一段由TEVDGVP组成的氨基酸序列。综上所述，EMS诱变处理能显著改变棘豆链格孢菌的苦马豆素合成能力及SWN基因簇各基因的表达水平。其SWN基因簇中AATL、swnR、swnN、swnH1、swnH2等基因的表达模式与苦马豆素合成呈现负相关，A、KS、SDR和SDRe1等基因的表达模与苦马豆素合成呈现正相关，EMS突变菌株苦马豆素合成的变化可能与SWN基因簇相关基因结构和功能的变化有关。

关键词: 疯草内生真菌, 甲基磺酸乙酯诱变, 苦马豆素, 苦马豆素合成基因簇, 基因表达

Abstract: The objective of this study is to investigate the correlation between the expression pattern of genes in swainsonine biosynthesis gene cluster (SWN) and swainsonine production in Alternaria oxytropis, fungal endophyte from locoweed. A. oxytropis UA003 was treated with Ethyl methanesulfonate (EMS). The SW content of each mutated strain was determined to screen strains with significant changes in SW yield. The expression levels of AATL, swnR, swnN, swnH1, swnH2, swnK (A, KS, SDRe1, SDR) in SWN gene cluster of some strains including A. oxytropis UA003, EMS mutagenesis strains and A. gansuense were analyzed. The swnK gene sequences of the above-mentioned strains were further analyzed to mutation sites. The results showed as follows: the yield of SW in E02, E23 and E25 strains induced by EMS was significantly increased, and the gene expressions of AATL, swnR, swnN, swnH1, swnH2 were significantly down-regulated (P<0.05 or P<0.01). The gene expressions of A, KS, SDR and SDRe1 were significantly up-regulated (P<0.05 or P<0.01 in all strains). E02 strain mutated by EMS had 13 nucleotide mutation sites of swnK gene and 6 amino mutation acid sites, respectively. Compared with UA003, EA strain had 163 different nucleotide sites and 67 different amino acid sites. The amino acid sequence MLTPAVSLKNLTKPK deletion was absent in the initial segment of the transcription product, and an amino acid sequence of TEVDGVP was inserted in the position 1135 and 1143 of the AT gene transcription product. In conclusion, EMS mutagenesis could significantly change the biosynthesis of swainsonine and gene expression of SWN gene cluster in A. oxytropis. The expression patterns of AATL, swnR, swnN, swnH1 and swnH2 genes in the SWN gene cluster were negatively correlated with the biosynthesis of swainsonine in E02, E23 and E25 strains. However, the expression patterns of A, KS, SDR and SDRe1 were positively correlated with the biosynthesis of swainsonine in above-mentioned strains. The biosynthesis changes of SW in endophytic fungi may be related to the structural and functional changes of related genes in SWN gene cluster.

Key words: fungal endophyte from locoweed, ethyl methanesulfonate mutagenesis, swainsonine, swainsonine biosynthesis gene cluster, gene expression

中图分类号:

Q933

余永涛, 毛彦妮, 赵清梅, 李金荣, 白晓南, 薛佳祺, 张浩东. 甲基磺酸乙酯诱变的棘豆链格孢菌菌株苦马豆素合成基因簇相关基因表达模式分析[J]. 畜牧兽医学报, 2022, 53(4): 1241-1251.

YU Yongtao, MAO Yanni, ZHAO Qingmei, LI Jinrong, BAI Xiaonan, XUE Jiaqi, ZHANG Haodong. Expression Pattern Analysis of Related Genes in SWN Gene Cluster of Alternaria oxytropis Mutated by Ethyl Methylate[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1241-1251.

参考文献 31

[1]	MOLYNEUX R J, JAMES L F. Loco intoxication indolizidine alkaloids of spotted locoweed (Astragalus lentiginosus )[J]. Science, 1982, 216(4542):190-191.
[2]	WU C C, WANG W L, LIU X X, et al. Pathogenesis and preventive treatment for animal disease due to locoweed poisoning[J]. Environ Toxicol Pharmacol, 2014, 37(1):336-347.
[3]	GARDNER D R, MOLYNEUX R J, RALPHS M H. Analysis of swainsonine:extraction methods, detection, and measurement in populations of locoweeds (Oxytropis spp.)[J]. J Agric Food Chem, 2001, 49(10):4573-4580.
[4]	COOK D, RALPHS M H, WELCH K D, et al. Locoweed poisoning in livestock[J]. Rangelands, 2009, 31(1):16-21.
[5]	BRAUN K, ROMERO J, LIDDELL C, et al. Production of swainsonine by fungal endophytes of locoweed[J]. Mycol Res, 2003, 107(8):980-988.
[6]	PRYOR B M, CREAMER R, SHOEMAKER R A, et al. Undifilum, a new genus for endophytic Embellisia oxytropis and parasitic Helminthosporium bornmuelleri on legumes[J]. Botany, 2009, 87(2):178-194.
[7]	COOK D, GARDNER D R, GRUM D, et al. Swainsonine and endophyte relationships in Astragalus mollissimus and Astragalus lentiginosus [J]. J Agric Food Chem, 2011, 59(4):1281-1287.
[8]	MUKHERJEE S, DAWE A L, CREAMER R. Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis [J]. J Microbiol Methods, 2010, 81(2):160-165.
[9]	LI X, LU P. Transcriptome profiles of Alternaria oxytropis provides insights into swainsonine biosynthesis[J]. Sci Rep, 2019, 9(1):6021.
[10]	LU H, QUAN H Y, REN Z H, et al. The genome of Undifilum oxytropis provides insights into swainsonine biosynthesis and locoism[J]. Sci Rep, 2016, 6:30760.
[11]	COOK D, DONZELLI B G G, CREAMER R, et al. Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi[J]. G3 (Bethesda), 2017, 7(6):1791-1797.
[12]	TAN X M, CHEN A J, WU B, et al. Advance of swainsonine biosynthesis[J]. Chin Chem Lett, 2018, 29(3):417-422.
[13]	LUO F F, HONG S, CHEN B, et al. Unveiling of swainsonine biosynthesis via a multibranched pathway in fungi[J]. ACS Chem Biol, 2020, 15(9):2476-2484.
[14]	YU Y T, ZHAO Q M, WANG J N, et al. Swainsonine-producing fungal endophytes from major locoweed species in China[J]. Toxicon, 2010, 56(3):330-338.
[15]	COOK D, GRUM D S, GARDNER D R, et al. Influence of endophyte genotype on swainsonine concentrations in Oxytropis sericea [J]. Toxicon, 2013, 61:105-111.
[16]	GRUM D, COOK D, GARDNER D R, et al. Influence of seed endophyte amounts on swainsonine concentrations in Astragalus and Oxytropis locoweeds[J]. J Agric Food Chem, 2011, 60(33):8083-8089.
[17]	LI Y Z, NAN Z B. A new species, Embellisia astragali sp. nov.， causing standing milk-vetch disease in China[J]. Mycologia, 2007, 99(3):406-411.
[18]	NOOR A I, NEYAZ M, COOK D, et al. Molecular characterization of a fungal ketide synthase gene among swainsonine-producing Alternaria species in the USA[J]. Curr Microbiol, 2020, 77(9):2554-2563.
[19]	余永涛, 李金荣, 赵清梅, 等. 直立黄芪中产苦马豆素真菌的分离与鉴定[J]. 畜牧兽医学报, 2018, 49(8):1770-1780.YU Y T, LI J R, ZHAO Q M, et al. Isolation and identification of swainsonine-producing fungi from Astragalus adsurgens pall[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(8):1770-1780. (in Chinese)
[20]	白晓南, 余永涛, 赵清梅, 等. 变异黄芪中产苦马豆素内生真菌的分离与鉴定[J]. 畜牧与兽医, 2017, 49(10):95-102.BAI X N, YU Y T, ZHAO Q M, et al. Isolation and identification of swainsonine-producing fungal endophyte from Astragalus variabills bunge[J]. Animal Husbandry & Veterinary Medicine, 2017, 49(10):95-102. (in Chinese)
[21]	张蕾蕾, 余永涛, 何生虎, 等. 不同因素对疯草内生真菌合成苦马豆素的影响[J]. 畜牧兽医学报, 2015, 46(1):163-173.ZHANG L L, YU Y T, HE S H, et al. Influence of different factors on swainsonine production in fungal endophyte from locoweed[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(1):163-173. (in Chinese)
[22]	BELFON R, CREAMER R. RAPD-PCR analysis of fungal endophytes of locoweed[J]. Phytopathology, 2003, 93:S7.
[23]	OLDRUP E, MCLAIN-ROMERO J, PADILLA A, et al. Localization of endophytic Undifilum fungi in locoweed seed and influence of environmental parameters on a locoweed in vitro culture system[J]. Botany, 2010, 88(5):512-521.
[24]	张雨, 朱燕丽, 李博, 等. 金龟子绿僵菌发酵液中苦马豆素含量及催化酶基因mRNA表达分析[J]. 畜牧兽医学报, 2015, 51(4):881-887.ZHANG Y, ZHU Y L, LI B, et al. mRNA expression analysis of catalytic enzyme gene and content of swainsonine in the fermentation broth of Metarhizium anisopliae [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4):881-887. (in Chinese)
[25]	SHAFIQUE S, BAJWA R, SHAFIQUE S. Mutagenesis and genetic characterisation of amylolytic Aspergillus niger [J]. Nat Prod Res, 2010, 24(12):1104-1114.
[26]	KUNZ B A, GABRIEL M, KANG X L, et al. DNA repair modifies the site and strand specificity of ethyl methanesulfonate mutagenesis in yeast[J]. Mutagenesis, 1992, 7(6):461-469.
[27]	LI Y X, WANG M N, SEE D R, et al. Ethyl-methanesulfonate mutagenesis generated diverse isolates of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen[J]. World J Microbiol Biotechnol, 2019, 35:28.
[28]	SHAHID M G, NADEEM M, GULZAR A, et al. Novel ergot alkaloids production from Penicillium citrinum employing response surface methodology technique[J]. Toxins (Basel), 2020, 12(7):427.
[29]	SEGA G A. A review of the genetic effects of ethyl methanesulfonate[J]. Mutat Res, 1984, 134(2-3):113-142.
[30]	WU V W, THIEME N, HUBERMAN L B, et al. The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus[J]. Proc Natl Acad Sci U S A, 2020, 117(11):6003-6013.
[31]	MOJZITA D, RANTASALO A, JÄNTTI J. Gene expression engineering in fungi[J]. Curr Opin Biotechnol, 2019, 59:141-149.