谷氧还蛋白(AbGrx-1)对水稻干尖线虫在氧化胁迫下的保护作用

doi:10.16819/j.1001-7216.2019.9016

摘要/Abstract

摘要：

目的谷氧还蛋白(glutaredoxin, Grx)作为一种抗氧化酶,在通过清除多余活性氧来维持生物细胞氧化还原平衡、降低细胞膜损伤过程中发挥重要作用。水稻干尖线虫(Aphelenchoides besseyi)能在高温、渗透及氧化胁迫等多种逆境压力中存活。本研究旨在探究Grx在水稻干尖线虫抗氧化胁迫中的作用。方法通过cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE),获得了一个水稻干尖线虫谷氧还蛋白基因AbGrx-1,进行了序列比对和遗传进化分析;通过qRT-PCR检测了AbGrx-1在线虫响应氧化和温度胁迫中的表达差异,通过原核表达获得了AbGrx-1的重组蛋白,并分析了AbGrx-1蛋白浸泡对水稻干尖线虫在氧化和高温胁迫下存活的影响。结果 AbGrx-1基因全长包括90 bp的5＇非翻译区(UTR)、321 bp的编码区和97 bp的3＇UTR,开发阅读框(横跨91至411位)编码106个氨基酸。AbGrx-1蛋白的第24至27位点具有谷氧还蛋白催化残基(CPYC),分别在第69至第72和第83至第86位点存在保守的谷胱甘肽结合位点RSVP和GGDD,归为Ⅰ类谷氧还蛋白。遗传进化树显示AbGrx-1与燕麦真滑刃线虫(Aphelenchus avenae)Grx亲缘关系最近,位于同一进化分支。AbGrx-1在H₂O₂处理和12℃下时显著上调表达,但在0℃, 4℃, 37℃和45℃极端温度中下调表达。高浓度H₂O₂和高温导致水稻干尖线虫死亡率增加,AbGrx-1重组蛋白能显著提高暴露于高浓度H₂O₂中线虫的存活率,但不影响高温下线虫的存活率。结论 AbGrx-1参与调控水稻干尖线虫的抗氧化免疫反应,在抵抗氧化损伤、维持线虫生存方面具有重要功能。

关键词: 水稻干尖线虫, 谷氧还蛋白, 基因克隆, 重组蛋白, 抗氧化

Abstract:

【Objective】 The antioxidant glutaredoxin (Grx) plays a crucial role in regulating intracellular redox homeostasis via scavenging of excess reactive oxygen species. The white-tip nematode Aphelenchoides besseyi can survive in adverse environments including high temperature, osmosis and oxidative stresses. To reveal the antioxidant function of Grx in A. besseyi, 【Method】 herein the full-length cDNA of glutaredoxin (named AbGrx-1) from A. besseyi was cloned by using rapid amplification of cDNA ends (RACE), and AbGrx-1 protein and evolutionary relationship were characterized; differential gene expression level was detected in the nematodes under oxidant and temperature stresses by using quantitative real-time PCR; the effect of the recombinant AbGrx-1 protein on A.besseyi survival was also tested.【Result】 The full-length AbGrx-1 cDNA contains a 5' UTR of 90, an ORF of 321 bp encoding a polypeptide of 106 amino acids and a 3' UTR of 97 bp. The deduced amino acid sequence of AbGrx-1 shares a high similarity with other nematodes’ Grxs, and the catalytic residue (CPYC) and glutathione binding sites (RSVP and GGDD) indicate that AbGrx-1 is categorized into Class I Grx. The phylogenetic tree showed AbGrx-1 is located in the same clade with the plant parasitic nematode Aphelenchus avenae. AbGrx-1 mRNA is highly induced in A. besseyi exposed to H₂O₂ solution and 12℃, but is suppressed in the nematodes at 0, 4, 37 and 45℃. The high concentration H₂O₂ solutions and high temperature are adverse to survival of A. besseyi, but the survival rate increases with the nematodes pre-soaked in AbGrx-1 recombinant protein solution. 【Conclusion】 AbGrx-1 is required for A. besseyi antioxidative immunity, and plays an essential role in overcoming oxidative damage and nematode survival.

Key words: Aphelenchoides besseyi, glutaredoxin, gene cloning, recombinant protein, antioxidation

中图分类号:

S435.111.4⁺8

冯辉, 范亚磊, 张金凤, 朱红利, 魏利辉. 谷氧还蛋白(AbGrx-1)对水稻干尖线虫在氧化胁迫下的保护作用[J]. 中国水稻科学, 2019, 33(6): 565-574.

Hui FENG, Yalei FAN, Jinfeng ZHANG, Hongli ZHU, Lihui WEI. Protective Effect of Glutaredoxin (AbGrx-1) on Aphelenchoides besseyi Under Oxidative Stress[J]. Chinese Journal OF Rice Science, 2019, 33(6): 565-574.

图/表 7

表1 本研究引物列表

Table 1 Primers used in this study.

引物名 Primer name	序列 Sequences (5′→3′)
degGrx-F	TGTCCYTACTGCRASAARGCSAAG
degGrx-R	GTGTCGTCGTCGTCRCCTCCRCCKAWGAA
AbGrx-F1	ATCCTTCAATCTAAAGCCCGGAG
AbGrx-R1	TGGCACCAACGTCTTTCAACT
AbGrx-R2	TATCATCACCTCCTCCTAAGAA
AbGrx-fullF	GGGAAAGTAAACACCGAAATCG
AbGrx-fullR	TCCCAACACTGATTATTTGCC
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT (long);CTAATACGACTCACTATAGGGC (short)
NUP	AAGCAGTGGTATCAACGCAGAGT
Ab-18S-F	TGCGGTAATTCTGGAGCCAA
Ab-18S-R	CCCGTTAGAACCATGGCAGT
AbGrx-28aF	CATGCATATGTCCGTCAAGAGTTTTGTCGA
AbGrx-28aR	CATGCTCGAGTTAGATGGCACCAACGTCTTTCAA

图1 AbGrx-1 完整cDNA和推测的氨基酸序列5＇和3＇非翻译区用小写字母表示,开放阅读框用大写字母表示;谷氧还蛋白催化残基用方框表示;用于扩增全长序列的基因特异引物用下划线标注;终止子（TAA）用星号表示。

Fig. 1. Complete nucleotide and deduced amino acid sequences of the AbGrx-1 cDNA (GenBank Accession No. KP190142). The 5＇- and 3＇-UTR are shown in lowercase letters and the open reading frame (ORF) is shown in uppercase letters. The predicted catalytic residues of glutaredoxin is boxed, and the gene-speciﬁc primers used for full sequence amplification of AbGrx-1 are underlined. The stop codon (TAA) is marked with an asterisk.

图2 水稻干尖线虫和其他线虫谷氧还蛋白序列比对方框表示谷胱甘肽结合位点,向下箭头表示谷氧还蛋白的催化残基;一致和相似的序列分别用星号和圆点表示。

Fig. 2. Sequences alignment of A. besseyi and other nematode glutaredoxin proteins. Boxed amino acids are glutathione (GSH) binding sites, and arrows indicate catalytic residues of glutaredoxin. Consensus and similar sequences similarity are separately shaded with dark (asterisks) and gray (dots) shadows.

图3 线虫谷氧还蛋白的遗传发育树

Fig. 3. Phylogenetic relationship among different glutaredoxin proteins in the known nematodes species.

图4 氧化胁迫(A, H2O2处理)和温度胁迫(B)下水稻干尖线虫的存活率

Fig. 4. Cumulative survival rates of A. besseyi exposed to H2O2 (A) and temperature (B) stress.

图5 暴露于不同浓度H2O2和不同温度12 h AbGrx-1的相对表达水平柱条表示标准误;*,**分别表示与对照(0mml/L或25℃)差异达0.05, 0.01显著水平。

Fig. 5. Relative expression level of AbGrx-1 in A. besseyi exposed to H2O2 and temperature stress for 12 hours. Bars are stardand error. *, ** indicate significant difference between the treatment and the CK(0mml/L or 25℃)at 0.05 and 0.01 levels. The same as in figures below.

图6 AbGrx-1蛋白预处理对水稻干尖线虫在氧化和高温逆境下存活率的影响 A,AbGrx-1蛋白的表达和纯化;M,标记;1,纯化的蛋白;2,IPTG诱导表达的蛋白;3,未诱导蛋白;4,空载;B,不同浓度AbGrx1溶液浸泡对线虫存活率的影响;C~D,AbGrx-1蛋白预处理的线虫在氧化（C）和高温逆境（D）处理中的存活率。**表示处理与对照间差异达0.01显著水平。

Fig. 6. Role of AbGrx-1 protein in the response of A. besseyi to oxidization and temperature stresses. A, Prokaryotic expression and purification of AbGrx1 protein; M, Marker; Lanes 1 to 5, Purified protein, induced protein with IPTG, non-induced protein and empty vector; B, Cumulative survival rate of A. besseyi soaked in AbGrx-1 solution; C-D, Cumulative survival rate of A. besseyi presoaked in AbGrx-1 solution and then exposed to H2O2 (C) and high temperature stresses (D). **, Significant difference between the treatment and the CK at 0.01 level.

参考文献 30

[1]	Kalinina E V, Chernov N N, Novichkova M D.Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes.Biochemistry (Moscow), 2014, 79(13): 1562-1583.
[2]	Lillig C H, Berndt C, Holmgren A.Glutaredoxin systems.BBA-Gen Subjects, 2008, 1780(11): 1304-1317.
[3]	Morita S, Yamashita Y, Fujiki M, Todaka R, Nishikawa Y, Hosoki A, Yabe C, Nakamura J I, Kawamura K, Suwastika I N, Sato M H, Masumura T, Ogihara Y, Tanaka K, Satoh S.Expression of a rice glutaredoxin in aleurone layers of developing and mature seeds: Subcellular localization and possible functions in antioxidant defense.Planta, 2015, 242(5): 1195-1206.
[4]	Morgan K L, Estevez A O, Mueller C L, Cacho-Valadez B, Miranda-Vizuete A, Szewczyk N J, Estevez M.The glutaredoxin GLRX-21 functions to prevent selenium- induced oxidative stress inCaenorhabditis elegans. Toxicol Sci, 2010, 118(2): 530-543.
[5]	Vanina E. Marquez, Diego G. Arias, Claudia V. Piattoni, Carlos Robello, Alberto A. Iglesias, Guerrero A S A. Cloning, expression, and characterization of a dithiol glutaredoxin fromTrypanosoma cruzi. Antioxid Redox Sign, 2010, 12(6): 787-791.
[6]	Fu Z, Agudelo P, Wells C E.Induction of glutaredoxin expression in response to desiccation stress in the foliar nematodeAphelenchoides fragariae. J Nematol, 2012, 44(4): 370-367.
[7]	Shigeru H, Katsumi T.Effect of water-soaking and air-drying on survival of Aphelenchoides besseyi in Oryza sativa seeds. J Nematol, 2000, 32(3): 303-308
[8]	Tenente R V V, Wetzel M M V S, Manso E S B G, Marques A S A. Survival of Aphelenchoides besseyi in infested rice seeds stored under controlled conditions. Nematol Bras, 1994, 18: 85-92.
[9]	Feng H, Wei L, Chen H, Zhou Y.Calreticulin is required for responding to stress, foraging, and fertility in the white-tip nematode,Aphelenchoides besseyi. Exp Parasitol, 2015, 155: 58-67.
[10]	冯辉, 陈曦, 束兆林, 姚克兵, 魏利辉. 水稻干尖线虫Hsp90基因克隆及在不同逆境、侵染早期和取食过程的表达差异. 农业生物技术学报, 2016, 24(11): 1741-1753.
	Feng H, Chen X, Shu Z L, Yao K B, Wei L H.Characterization of the Hsp90 Gene in the white tip nematode (Aphelenchoides besseyi) and its expression in response to environmental stresses, early infection and feeding. Chin J Agric Biotechol, 2016, 24(11): 1741-1753. (in Chinese with English abstract)
[11]	陈曦, 冯辉, 束兆林, 姚克兵, 魏利辉. 水稻干尖线虫海藻糖酶Ab-tre-1基因克隆与逆境条件下的表达分析. 核农学报, 2016, 30(12): 2304-2311.
	Chen X, Feng H, Shu Z L,Yao K B, Wei L H.Isolation and expression analysis of a trehalase gene from white tip nematode.J Nucl Agric Sci, 2016, 30(12): 2304-2311. (in Chinese with English abstract)
[12]	陈俏丽, 王峰, 李丹蕾, 零雅茗, 张瑞芝. 水稻干尖线虫Ab-lea 基因在高渗透压下的表达. 中国水稻科学, 2017, 31(6): 652-657.
	Chen Q L, Wang F, Li D L, Ling Y M, Zhang R Z.Expression under hypertonic osmotic stress of Ab-lea from Aphelenchoides besseyi. Chin J Rice Sci, 2017, 31(6): 652-657. (in Chinese with English abstract)
[13]	Staheli J P, Boyce R, Kovarik D, Rose T M.CODEHOP PCR and CODEHOP PCR Primer Design. In: PARK D J, PCR Protocols. Totowa, NJ: Humana Press 2011: 57-73.
[14]	Schmittgen T D, Livak K J.Analyzing real-time PCR data by the comparative CT method.Nat Protoc, 2008, 3(6): 1101-1108.
[15]	An S, Zhang Y, Wang T, Luo M, Li C.Molecular characterization of glutaredoxin 2 from Ostrinia furnacalis. Integr Zool, 2013, 8(Suppl 1): 30-38.
[16]	Yao P, Chen X, Yan Y, Liu F, Zhang Y, Guo X, Xu B.Glutaredoxin 1, glutaredoxin 2, thioredoxin 1, and thioredoxin peroxidase 3 play important roles in antioxidant defense in Apis cerana cerana.Free Radical Bio Med, 2014, 68: 335-346.
[17]	Zhang S D, Shen Z J, Liu X M, Li Z, Zhang Q W, Liu X X.Molecular identification of three novel glutaredoxin genes that play important roles in antioxidant defense in Helicoverpa armigera. Insect Biochem Mol Biol, 2016, 75: 107-116.
[18]	Olahova M, Taylor S R, Khazaipoul S, Wang J, Morgan B A, Matsumoto K, Blackwell T K, Veal E A.A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance.Proc Natl Acad Sci U S A, 2008, 105(50): 19839-19844.
[19]	Isermann K, Liebau E, Roeder T, Bruchhaus I.A peroxiredoxin specifically expressed in two types of pharyngeal neurons is required for normal growth and egg production in Caenorhabditis elegans. J Mol Biol, 2004, 338(4): 745-755.
[20]	Dubreuil G, Deleury E, Magliano M, Jaouannet M, Abad P, Rosso M N.Peroxiredoxins from the plant parasitic root-knot nematode, Meloidogyne incognita, are required for successful development within the host. Int J Parasitol, 2011, 41(3-4): 385-396.
[21]	Zhou T, Gao C, Du L, Feng H, Wang L, Lan Y, Sun F, Wei L, Fan Y, Shen W, Zhou Y.Genetic analysis and QTL detection for resistance to white tip disease in rice.PLoS One, 2014, 9(8): e106099.
[22]	Sun M J, Liu W H, Lin M S.Effects of temperature, humidity and different rice growth stages on vertical migration of Aphelenchoides besseyi. Rice Sci, 2009, 16(4): 301-306.
[23]	Kung S-P, Gaugler R, Kaya H K.Effects of soil temperature, moisture, and relative humidity on entomopathogenic nematode persistence. J Invertebr Pathol, 1991, 57(2): 242-249.
[24]	Heald C M, Inserra R N.Effect of Temperature on Infection and Survival of Rotylenchulus reniformis.J Nematol, 1988, 20(3): 356-361.
[25]	Browne J A, Dolan K M, Tyson T, Goyal K, Tunnacliffe A, Burnell A M.Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae. Eukaryot Cell, 2004, 3(4): 966-975.
[26]	Stringham E G, Candido E P M. Transgenic hsp 16-Lacz strains of the soil nematode caenorhabditis elegans as biological monitors of environmental stress.Environ Toxicol Chem, 1994, 13(8): 1211-1220.
[27]	Li F, Ma X, Cui X, Li J, Wang Z.Recombinant buckwheat glutaredoxin intake increases lifespan and stress resistance via hsf-1 upregulation in Caenorhabditis elegans. Exp Gerontol, 2018, 104: 86-97.
[28]	Henkle-Duhrsen K, Kampkotter A.Antioxidant enzyme families in parasitic nematodes.Mol Biochem Parasitol, 2001, 114: 129-142.
[29]	Zhen L, Xiaoxia L, Yanna C, Yan W, Qingwen Z, Xuguo Z.Cloning and characterization of a 2-Cys peroxiredoxin in the pine wood nematode,Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation. Int J Biol Sci, 2011, 7(6): 823-836.
[30]	Lin B, Zhuo K, Chen S, Hu L, Sun L, Wang X, Zhang L H, Liao J.A novel nematode effector suppresses plant immunity by activating host reactive oxygen species-scavenging system.New Phytol, 2016, 209(3): 1159-1173.