马铃薯腐烂茎线虫Dd-mel-26基因的克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0599

摘要/Abstract

摘要：

为更深入地了解马铃薯腐烂茎线虫并寻找具有RNAi应用价值的靶标基因资源,本研究从马铃薯腐烂茎线虫基因库中选取了一个潜在致死基因底物特异性适配体基因Dd-mel-26。利用RACE技术对该基因进行了克隆,并通过生物信息学、荧光定量PCR技术以及dsRNA体外浸泡诱导线虫靶基因沉默的方法对该基因进行了功能分析。结果显示,Dd-mel-26基因的cDNA全长为1 594 bp,包含一个1 158 bp的开放阅读框,预测编码一个含385个氨基酸的蛋白质,分子量为43.52 kD,等电点为5.4。基因组结构中包含10个外显子和9个内含子序列。Dd-MEL-26蛋白属于MATH-BTB蛋白家族,不具有信号肽,预测其在细胞核中与相关的蛋白互作从而行使其功能。系统进化分析结果显示Dd-MEL-26蛋白与植物寄生线虫聚为一组,且与象耳豆根结线虫（Meloidogyne enterolobii）的亲缘关系最近。通过dsRNA体外浸泡法将该基因沉默后显著地增加了线虫的繁殖量和垂直迁移力。因此,Dd-mel-26基因在一定条件下可以正调控马铃薯腐烂茎线虫的繁殖和垂直移动,但将该基因做为RNAi靶标从而实现对该线虫的防治可能需要更加深入的研究。

关键词: 马铃薯腐烂茎线虫, 甘薯茎线虫病, Dd-mel-26基因, MATH-BTB基因家族

Abstract:

To further understand the nematode Ditylenchus destructor and seek the target gene resources with RNAi application value,a potential lethal gene substrate specific adaptor gene,Dd-mel-26,was selected from the gene pool of D. destructor. The gene was cloned by RACE,and its function was analyzed by bioinformatics,real-time PCR and dsRNA induced in vitro RNAi of nematode target gene. The results showed that the full-length cDNA of Dd-mel-26 gene was 1 594 bp and contained an open reading frame of 1 158 bp. It was predicted to encode a protein containing 385 amino acids with a molecular weight of 43.52 kD and an isoelectric point of 5.4. Its genome structure consisted of 10 exons and 9 intron sequences. Dd-MEL-26 belonged to the MATH-BTB protein family and did not possess signal peptides. It is predicted that Dd-MEL-26 interacted with related proteins in the nucleus to perform its function. Phylogenetic analysis demonstrated that Dd-MEL-26 was grouped into plant parasitic nematodes,and was closely related to Meloidogyne enterolobii. Silencing the gene via dsRNA significantly increased the nematode fertility and vertical migration. Therefore,the Dd-mel-26 gene can positively regulate the propagation and vertical migration of D. destructor under certain conditions. However,deeper study may be needed if this gene is used as RNAi target for achieving the control of this nematode.

Key words: Ditylenchus destructor, sweet potato stem nematode disease, Dd-mel-26 gene, MATH-BTB gene family

高波, 马娟, 李秀花, 李焦生, 王容燕, 陈书龙. 马铃薯腐烂茎线虫Dd-mel-26基因的克隆与功能分析[J]. 生物技术通报, 2021, 37(7): 107-117.

GAO Bo, MA Juan, LI Xiu-hua, LI Jiao-sheng, WANG Rong-yan, CHEN Shu-long. Cloning and Functional Analysis of Dd-mel-26 Gene from Ditylenchus destructor[J]. Biotechnology Bulletin, 2021, 37(7): 107-117.

图/表 10

表1 本研究所用引物信息

Table 1 Primer information used in this study

引物Primer	序列信息Sequence（5'-3'）	备注Remarks
mel-26F	GATGCCTTCTGAATCTGTTG	cDNA中间区段扩增
mel-26R	GCAGTAAGAACCAGGAACAG	cDNA中间区段扩增
mel26-5GSP1	GTCGGAATGTGACGGGTTTG	5'RACE引物
mel26-5GSP2	TTTCCGTTTGTGAGGCGTTG	5'RACE引物
mel26-3GSP1	ATCGGGCAGATTTGTTACGC	3'RACE引物
mel26-3GSP2	CCCGCAGAGCTTTCTTCGTC	3'RACE引物
Q_T	CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT	RACE加帽引物
Q_O	CCAGTGAGCAGAGTGACG
Q_I	GAGGACTCGAGCTCAAGC
mel-26RT	CACAAGAACGGTGATGCTAT	5'RACE反转录引物
mel-26GF	GATGCCTTCTGAATCTGTTG	基因组序列扩增
mel-26GR	ACATCACAGACATAGCCAGA	基因组序列扩增
26T7aF	TAATACGACTCACTATAGGGTGCCTTCTGAATCTGTTGCC	dsRNA合成模板扩增
26T7aR	TAATACGACTCACTATAGGGGAATGTGACGGGTTTGGATG
26T7bF	TAATACGACTCACTATAGGGTCCAAACCCGTCACATTCCG
26T7bR	TAATACGACTCACTATAGGGACGAGCTGCGTCCGCAATAA
26T7cF	TAATACGACTCACTATAGGGCGTCCGAAATCAACGAACTT
26T7cR	TAATACGACTCACTATAGGGCGGAGATGGAACTTAGAGGG
GFP_L1	TAATACGACTCACTATAGGGGACTTCTTCAAGTCCGCCAT	阴性对照dsRNA模板合成引物
GFP_R1	TAATACGACTCACTATAGGGAAGTTCACCTTGATGCCGTT	阴性对照dsRNA模板合成引物
mel26q_L	TTGAAATGCGAATTCATCCA	qPCR扩增引物
mel26q_R	TCATAATTGCAAACGCCAAG
actin_L	AGGACTTGTACGCCAACACC
actin_R	ACATTTGCTGGAAGGTGGAG
mel26-probe-F	TCCAAACCCGTCACATTCCG	Southern blotting探针引物
mel26-probe-R	ACGAGCTGCGTCCGCAATAA	Southern blotting探针引物

图1 马铃薯腐烂线虫Dd-mel-26 cDNA及其基因组序列扩增结果 A：3'RACE;B：5'RACE;C：cDNA中间区段;D：Dd-mel-26基因组序列

Fig. 1 Amplification results of the cDNA and genomic sequence of Dd-mel-26 A：3' RACE;B：5' RACE;C：intermediate region of the cDNA;D：Dd-mel-26 genomic sequence

图2 Dd-mel-26基因组结构图

Fig. 2 Genome structure of Dd-mel-26

图3 Dd-MEL-26蛋白的二级结构预测图

Fig. 3 Predicted secondary structure of Dd-MEL-26 protein

图4 Dd-MEL-26蛋白的三级结构图橘黄色单体为26_A,紫罗兰色单体为26_B,红色的两个α螺旋结构为3-box结构域,结构的上半部分富含β折叠股的蝶状结构为MATH功能结构域部分（MATH_A和MATH_B）,下半部分富含α螺旋的碟状结构为BTB功能结构域部分（BTB_A和BTB_B）

Fig. 4 Tertiary structure of Dd-MEL-26 protein The orange monomer is 26_ A,the violet monomer is 26_ B,the two red α-helix structures are 3-box domains,and the butterfly-like structure rich in β-strand in the upper part of the structure is the MATH domain（MATH_ A and MATH_B）,and the butterfly-like structure rich in α-helix in the lower part is BTB domain（BTB_ A and BTB_B）

图5 基于Dd-mel-26基因的氨基酸序列利用邻接法构建的系统发育树只列出进化树中大于70%的Bootstrap值

Fig. 5 Neighbor-Joining tree calculated from the sequences of Dd-mel-26 amino acid sequence Only bootstrap values more than 70% are listed at the branches points

图6 Dd-mel-26基因的Southern杂交结果图

Fig. 6 Southern blotting results of Dd-mel-26 gene

图7 马铃薯腐烂茎线虫不同发育时期Dd-mel-26基因的相对表达量

Fig. 7 Relative expressions of Dd-mel-26 gene at different developmental stages of D. destructor

图8 受Dd-mel-26的a、b和c片段dsRNA处理的线虫Dd-mel-26的相对表达量

Fig. 8 Relative expressions of Dd-mel-26 after treatment with dsRNA of fragments a,b and c of Dd-mel-26

图9 受Dd-mel-26 dsRNA处理线虫靶基因的相对表达量（A）、沙柱平均通过率（B）及其平均繁殖量（C）

Fig. 9 Relative expressions of target gene（A）,average passing rate in sand column（B）,and average reproduction（C）in the Dd-mel-26 dsRNA-treated nematodes

参考文献 46

[1]	丁再福, 林茂松. 甘薯、马铃薯和薄荷上的茎线虫的鉴定[J]. 植物保护学报, 1982, 9(3):169-172, 219.
	Ding ZF, Lin MS. Identification of the stem nematodes from the sweet-potatoes, potatoes, and mints in China[J]. J Plant Prot, 1982, 9(3):169-172, 219.
[2]	刘维志, 刘清利, 尼秀媚. 马铃薯茎线虫Ditylenchus destructor Thorne, 1945的描述[J]. 莱阳农学院学报, 2003, 20(1):1-3.
	Liu WZ, Liu QL, Ni XM. Description of stem nematodes:Ditylenchus destructor thorne, 1945[J]. J Laiyang Agric Coll, 2003, 20(1):1-3.
[3]	刘先宝, 葛建军, 谭志琼, 等. 马铃薯腐烂茎线虫在国内危害马铃薯的首次报道[J]. 植物保护, 2006, 32(6):157-158.
	Liu XB, Ge JJ, Tan ZQ, et al. First report of Ditylenchus destructor infesting potato in China[J]. Plant Prot, 2006, 32(6):157-158.
[4]	周忠, 马代夫. 甘薯茎线虫病的研究现状和展望[J]. 杂粮作物, 2003, 23(5):288-290.
	Zhou Z, Ma DF. Research status and prospect of sweet potato stem nematode disease[J]. Rain Fed Crops, 2003, 23(5):288-290.
[5]	王欣, 李强, 曹清河, 等. 中国甘薯产业和种业发展现状与未来展望[J]. 中国农业科学, 2021, 54(3):483-492.
	Wang X, Li Q, Cao QH, et al. Current status and future prospective of sweetpotato production and seed industry in China[J]. Sci Agric Sin, 2021, 54(3):483-492.
[6]	王治文, 高翔, 马德君, 等. 核酸农药——极具潜力的新型植物保护产品[J]. 农药学学报, 2019, 21(Z1):681-691.
	Wang ZW, Gao X, Ma DJ, et al. Nucleic acid pesticides—the new plant protection products with great potential[J]. Chin J Pestic Sci, 2019, 21(Z1):681-691.
[7]	李宇, 谢辉, 徐春玲, 等. Cathepsin B基因的沉默对香蕉穿孔线虫繁殖力的影响[J]. 中国农业科学, 2010, 43(8):1608-1616.
	Li Y, Xie H, Xu CL, et al. RNAi effect of Cathepsin B gene on reproduction of Radopholus similis[J]. Sci Agric Sin, 2010, 43(8):1608-1616.
[8]	彭焕. 马铃薯腐烂茎线虫内切葡聚糖酶新基因的克隆及基于RNAi的功能初步研究[D]. 北京:中国农业科学院, 2009.
	Peng H. Cloning two new genes of β-1, 4-endoglucanase from Ditylenchus destructor and functional analysis with RNA interference[D]. Beijing:Chinese Academy of Agricultural Sciences, 2009.
[9]	Dalzell JJ, Warnock ND, Stevenson MA, et al. Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality[J]. Int J Parasitol, 2010, 40(11):1303-1310. doi: 10.1016/j.ijpara.2010.03.010 URL
[10]	Fan WJ, Wei ZR, Zhang M, et al. Resistance to Ditylenchus destructor infection in sweet potato by the expression of small interfering RNAs targeting unc-15, a movement-related gene[J]. Phytopathology, 2015, 105(11):1458-1465. doi: 10.1094/PHYTO-04-15-0087-R URL
[11]	Niu JH, Jian H, Xu JM, et al. RNAi silencing of the Meloidogyne incognita Rpn7 gene reduces nematode parasitic success[J]. Eur J Plant Pathol, 2012, 134(1):131-144. doi: 10.1007/s10658-012-9971-y URL
[12]	Joshi I, Kumar A, Kohli D, et al. Conferring root-knot nematode resistance via host-delivered RNAi-mediated silencing of four Mi-msp genes in Arabidopsis[J]. Plant Sci, 2020, 298:110592. doi: 10.1016/j.plantsci.2020.110592 pmid: 32771150
[13]	Klink VP, Kim KH, Martins V, et al. A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female Heterodera glycines cyst formation during infection of Glycine max[J]. Planta, 2009, 230(1):53-71. doi: 10.1007/s00425-009-0926-2 URL
[14]	梅眉, 黄永红, 茆振川, 等. 南方根结线虫乳酸脱氢酶基因克隆及其沉默效应分析[J]. 中国农业科学, 2013, 46(16):3384-3391.
	Mei M, Huang YH, Mao ZC, et al. Molecular clone and its RNA interference effect analysis of lactate dehydrogenase gene in Meloidogyne incognita[J]. Sci Agric Sin, 2013, 46(16):3384-3391.
[15]	Huang YH, Mei M, Mao ZC, et al. Molecular cloning and virus-induced gene silencing of MiASB in the southern root-knot nematode, Meloidogyne incognita[J]. Eur J Plant Pathol, 2014, 138(1):181-193. doi: 10.1007/s10658-013-0321-5 URL
[16]	Banerjee S, Banerjee A, Gill SS, et al. RNA interference:a novel source of resistance to combat plant parasitic nematodes[J]. Front Plant Sci, 2017, 8:834. doi: 10.3389/fpls.2017.00834 pmid: 28580003
[17]	Kim YH, Yang JW. Recent research on enhanced resistance to parasitic nematodes in sweetpotato[J]. Plant Biotechnol Rep, 2019, 13(6):559-566. doi: 10.1007/s11816-019-00557-w URL
[18]	Pintard L, Willis JH, Willems A, et al. The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase[J]. Nature, 2003, 425(6955):311-316. doi: 10.1038/nature01959 URL
[19]	Luke-Glaser S, Pintard L, Lu CG, et al. The BTB protein MEL-26 promotes cytokinesis in C. elegans by a CUL-3-independent mechanism[J]. Curr Biol, 2005, 15(18):1605-1615. pmid: 16169482
[20]	Wilson KJ, Qadota H, Mains PE, et al. UNC-89(obscurin)binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1(katanin)in striated muscle of Caenorhabditis elegans[J]. Mol Biol Cell, 2012, 23(14):2623-2634.
[21]	Quintin S, Mains PE, Zinke A, et al. The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo[J]. EMBO Rep, 2003, 4(12):1175-1181. pmid: 14634695
[22]	Simmer F, Moorman C, van der Linden AM, et al. Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions[J]. PLoS Biol, 2003, 1(1):e12. DOI: 10.1371/journal.pbio.0000012. doi: 10.1371/journal.pbio.0000012 URL
[23]	Fraser AG, Kamath RS, Zipperlen P, et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference[J]. Nature, 2000, 408(6810):325-330. doi: 10.1038/35042517 URL
[24]	Sönnichsen B, Koski LB, Walsh A, et al. Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans[J]. Nature, 2005, 434(7032):462-469. doi: 10.1038/nature03353 URL
[25]	Lu CG, Mains PE. The C. elegans anaphase promoting complex and MBK-2/DYRK kinase act redundantly with CUL-3/MEL-26 ubiquitin ligase to degrade MEI-1 microtubule-severing activity after meiosis[J]. Dev Biol, 2007, 302(2):438-447. doi: 10.1016/j.ydbio.2006.09.053 URL
[26]	Johnson JL, Lu C, Raharjo E, et al. Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis[J]. Dev Biol, 2009, 330(2):349-357. doi: 10.1016/j.ydbio.2009.04.004 URL
[27]	Elvin M, Snoek LB, Frejno M, et al. A fitness assay for comparing RNAi effects across multiple C. elegans genotypes[J]. BMC Genomics, 2011, 12:510. doi: 10.1186/1471-2164-12-510 pmid: 22004469
[28]	Young HK, Seung HO. In vitro culture and factors affecting population changes of Ditylenchus destructor of ginseng[J]. Korean J Plant Pathol, 1995, 11(1):39-46.
[29]	Scotto-Lavino E, Du G, Frohman MA. 3’ end cDNA amplification using classic RACE[J]. Nat Protoc, 2006, 1(6):2742-2745. pmid: 17406530
[30]	Scotto-Lavino E, Du G, Frohman MA. 5’ end cDNA amplification using classic RACE[J]. Nat Protoc, 2006, 1(6):2555-2562. pmid: 17406509
[31]	丁中, 彭德良, 何旭峰, 等. 不同地理种群甘薯茎线虫对不同类型杀线剂的敏感性[J]. 农药, 2007, 46(12):851-853.
	Ding Z, Peng DL, He XF, et al. Susceptibility of different populations of Ditylenchus destructor to different type of nematicides in Hebei Province of China[J]. Agrochemicals, 2007, 46(12):851-853.
[32]	Perry R, Warrior P, Twomey U, et al. Effects of the biological nematicide, DiTera®, on movement and sensory responses of second stage juveniles of Globodera rostochiensis, and stylet activity of G. rostochiensis and fourth stage juveniles of Ditylenchus dipsaci[J]. Nematology, 2002, 4(8):909-915. doi: 10.1163/156854102321122520 URL
[33]	Hennessy J, Clarke AJ. Movement of Globodera rostochiensis(wollenweber)juveniles stimulated by potato-root exudate[J]. Nematologica, 1984, 30(2):206-212. doi: 10.1163/187529284X00103 URL
[34]	Blumenthal T, Steward K. RNA processing and gene structure[M]//Riddle DL, Blumenthal T, Meyer BJ, et al. C elegans II. Cold Spring Harbor(NY);Cold Spring Harbor Laboratory Press, 1997:117-45.
[35]	Conrad R, Fen Liou R, Blumenthal T. Functional analysis of a C. elegans trans-splice acceptor[J]. Nucl Acids Res, 1993, 21(4):913-919. doi: 10.1093/nar/21.4.913 URL
[36]	Zhang H, Blumenthal T. Functional analysis of an intron 3’ splice site in Caenorhabditis elegans[J]. RNA, 1996, 2(4):380-388. pmid: 8634918
[37]	Ogg SC, Anderson P, Wickens MP. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract[J]. Nucl Acids Res, 1990, 18(1):143-149. doi: 10.1093/nar/18.1.143 URL
[38]	Gorlova O, Fedorov A, Logothetis C, et al. Genes with a large intronic burden show greater evolutionary conservation on the protein level[J]. BMC Evol Biol, 2014, 14(1):50. doi: 10.1186/1471-2148-14-50 pmid: 24629165
[39]	Dow MR, Mains PE. Genetic and molecular characterization of the Caenorhabditis elegans gene, mel-26, a postmeiotic negative regulator of Mei-1, a meiotic-specific spindle component[J]. Genetics, 1998, 150(1):119-128. pmid: 9725834
[40]	Thomas JH. Adaptive evolution in two large families of ubiquitin-ligase adapters in nematodes and plants[J]. Genome Res, 2006, 16(8):1017-1030. pmid: 16825662
[41]	Xu L, Wei Y, Reboul J, et al. BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3[J]. Nature, 2003, 425(6955):316-321. doi: 10.1038/nature01985 URL
[42]	Weber H, Bernhardt A, Dieterle M, et al. Arabidopsis AtCUL3a and AtCUL3b form complexes with members of the BTB/POZ-MATH protein family[J]. Plant Physiol, 2005, 137(1):83-93. doi: 10.1104/pp.104.052654 URL
[43]	Zhuang M, Calabrese MF, Liu J, et al. Structures of SPOP-substrate complexes:insights into molecular architectures of BTB-Cul3 ubiquitin ligases[J]. Mol Cell, 2009, 36(1):39-50. doi: 10.1016/j.molcel.2009.09.022 pmid: 19818708
[44]	Chen J, Ou Y, Yang Y, et al. KLHL22 activates amino-acid-dependent mTORC1 signalling to promote tumorigenesis and ageing[J]. Nature, 2018, 557(7706):585-589. doi: 10.1038/s41586-018-0128-9 URL
[45]	Lilley CJ, Davies LJ, Urwin PE. RNA interference in plant parasitic nematodes:a summary of the current status[J]. Parasitology, 2012, 139(5):630-640. doi: 10.1017/S0031182011002071 pmid: 22217302
[46]	Li WS, Gao BX, Lee SM, et al. RLE-1, an E3 ubiquitin ligase, regulates C. elegans aging by catalyzing DAF-16 polyubiquitination[J]. Dev Cell, 2007, 12(2):235-246. doi: 10.1016/j.devcel.2006.12.002 URL