烟草半胱氨酸蛋白酶家族和相应miRNAs的鉴定及其对PVY的响应

doi:10.13560/j.cnki.biotech.bull.1985.2023-0297

摘要/Abstract

摘要：

植物半胱氨酸蛋白酶（cysteine proteases, CPs）广泛影响着植物的生理过程及抗病调控。为研究烟草半胱氨酸蛋白酶在调控马铃薯Y病毒（potato virus Y, PVY）中的功能机理，本研究在全基因组水平上鉴定栽培烟草CPs基因家族，使用生物信息学方法分析CPs基因家族的进化关系、Motif基序及启动子调控元件；筛选CPs对应的miRNAs，分析CPs基因及其对应的miRNAs在PVY感染后的表达特性。结果表明，栽培烟草中共鉴定到70个CPs基因家族成员，根据其系统发育特征和结构可分为5个亚家族，其中C1A 39个、C2A 2个、C12 5个，C13 8个，C14A 16个；Motif分析表明，同一亚家族CPs具有相似的motif分布；启动子顺式作用元件分析表明，CPs家族基因含有光应答元件、激素响应元件以及低温、干旱、高温、盐胁迫等响应元件。基于转录组和small RNA测序结果发现，PVY感染后有38个CPs基因上调表达，18个CPs基因下调表达。有28个CPs基因受51个miRNAs调控，PVY感染后，38个miRNAs表达上调，13个miRNAs表达下调，其中7个在PVY感染后与miRNAs负相关。RT-qPCR结果证明PVY感染后，有15个CPs基因表达量显著上调。研究结果有助于深入理解烟草CPs基因家族功能，及其与相应miRNAs通过互作调控烟草对PVY的响应，为研究茄科作物抗PVY分子机制提供理论依据。

关键词: 烟草, 半胱氨酸蛋白酶, 基因家族, miRNA, 互作

Abstract:

Plant cysteine proteases（CPs）widely affect plant physiological processes and disease resistance regulation. To study the functional mechanism of tobacco cysteine proteases in regulating potato virus Y（PVY）, the CPs family genes of Nicotiana tabacum L. at the genome-wide level were identifed. The evolutionary relationships, motifs and promoter cis-acting elements of CPs family genes were analyzed by bioinformatics. Then the miRNAs corresponding to CPs were screend, the expression characteristics of CPs genes and their corresponding miRNAs after PVY infection were analyzed. The results showed that a total of 70 CPs genes were identified in Nicotiana tabacum L. genome, which were divided into five subfamilies, of which C1A contained 39 CPs, C2A contained 2 CPs, C12 contained 5 CPs, C13 contained 8 CPs and C14A contained 16 CPs. Motif analyses showed that the CPs of subfamily had similar motif distribution. Promoter cis-acting elements analysis showed that several CPs family gene promoters contained light response elements, hormone response elements and low temperature, drought, high temperature, salt stress response elements. Based on the sequencing results of transcriptome and small RNA, it was found that 38 CPs were up-regulated while 18 CPs were down-regulated after PVY infection.Total 51 miRNAs were found to be targeted to 28 members of cysteine protease gene family, 38 miRNAs were up-regulated while 13 were down-regulated after PVY infection,7 of them were negatively correlated with miRNAs.The results of RT-qPCR showed that 15 CPs genes were significantly up-regulated after PVY infection. This study is conducive to better understanding the functions of CPs genes and their corresponding miRNAs in regulating tobacco PVY responses, which may provide evidence for the molecular mechanism of anti-PVY in Solanaceae crops.

Key words: tobacco, cysteine protease, gene family, miRNA, interaction

尹国英, 刘畅, 常永春, 羽王洁, 王兵, 张盼, 郭玉双. 烟草半胱氨酸蛋白酶家族和相应miRNAs的鉴定及其对PVY的响应[J]. 生物技术通报, 2023, 39(10): 184-196.

YIN Guo-ying, LIU Chang, CHANG Yong-chun, YU Wang-jie, WANG Bing, ZHANG Pan, GUO Yu-shuang. Identification of the Cysteine Protease Family and Corresponding miRNAs in Nicotiana tabacum L. and Their Responses to PVY[J]. Biotechnology Bulletin, 2023, 39(10): 184-196.

图/表 7

图1 烟草半胱氨酸蛋白酶家族的系统发育进化树

Fig. 1 Phylogenetic tree of cysteine protease family in tobacco

图2 烟草半胱氨酸蛋白的保守结构域模式图

Fig. 2 Conserved domain pattern of cysteine protein in tobacco

图3 烟草半胱氨酸蛋白酶顺式作用元件模式图

Fig. 3 Cis-acting element mode diagram of cysteine protein in tobacco

图4 烟草半胱氨酸蛋白酶家族基因及其对应 miRNAs 调控的网络图黄色代表靶基因，蓝色代表miRNAs

Fig. 4 Networks of corresponding cysteine protease family genes and their relatived miRNAs in tobacco Yellow represents target gene and blue represents miRNAs

图5 烟草半胱氨酸蛋白酶家族基因及对应miRNAs在PVY感染后的表达 A、B分别为半光氨酸蛋白酶家族基因和miRNAs表达热图；C、D分别为半光氨酸蛋白酶家族基因和miRNAs散点图；PVY指感染PVY，CK指对照

Fig. 5 Expressions of cysteine protease family genes and miRNAs infected PVY in tobacco A, B indicate the expression heatmap of cysteine protease family genes and miRNAs; C, D indicate the scatter plot of cysteine protease family genes and miRNAs, respectively; PVY refers to infection with PVY, while CK refers to control

图6 烟草半胱氨酸蛋白酶家族基因及对应miRNAs在PVY感染后表达量负相关调控图

Fig. 6 Negative correlation network of expressions of cysteine protease family genes and corresponding miRNAs after PVY infection in tobacco

图7 PVY感染后烟草半胱氨酸蛋白酶基因RT-qPCR分析 “CK”和“PVY”分别指对照和感染PVY；*与**分别表示基因表达量在感染PVY前后在P<0.05与P<0.01水平上差异显著

Fig. 7 RT-qPCR analysis of the cysteine protease genes in PVY-infected tobacco “CK” and “PVY” indicate the control and infected PVY, respectively; * and * * respectively indicate significant differences in gene expression levels at the P<0.05 and P<0.01 levels before and after infection with PVY

参考文献 38

[1]	Grudkowska M, Zagdańska B. Multifunctional role of plant cysteine proteinases[J]. Acta Biochim Pol, 2004, 51(3): 609-624. pmid: 15448724
[2]	吴丹丹, 陈永坤, 杨宇, 等. 小桐子半胱氨酸蛋白酶家族和相应miRNAs的鉴定及其对低温锻炼的响应[J]. 植物学报, 2021, 56(5): 544-558. doi: 10.11983/CBB21014
	Wu DD, Chen YK, Yang Y, et al. Identification of the cysteine protease family and corresponding miRNAs in Jatropha curcas and their response to chill-hardening[J]. Chin Bull Bot, 2021, 56(5): 544-558.
[3]	Li YK, Cui W, Wang R, et al. microRNA858-mediated regulation of anthocyanin biosynthesis in kiwifruit(Actinidia arguta)based on small RNA sequencing[J]. PLoS One, 2019, 14(5): e0217480. doi: 10.1371/journal.pone.0217480 URL
[4]	Karrer KM, Peiffer SL, DiTomas ME. Two distinct gene subfamilies within the family of cysteine protease genes[J]. Proc Natl Acad Sci USA, 1993, 90(7): 3063-3067. pmid: 8464925
[5]	Rawlings ND, Morton FR, Kok CY, et al. MEROPS: the peptidase database[J]. Nucleic Acids Res, 2008, 36(suppl_1): D320-D325. doi: 10.1093/nar/gkm954 URL
[6]	Wilkinson KD, Laleli-Sahin E, Urbauer J, et al. The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3[J]. J Mol Biol, 1999, 291(5): 1067-1077. pmid: 10518943
[7]	Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database[J]. Nucleic Acids Res, 2010, 38(Database issue): D227-D233. doi: 10.1093/nar/gkp971 URL
[8]	Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis[J]. Annu Rev Biochem, 1999, 68: 383-424. pmid: 10872455
[9]	Dando PM, Fortunato M, Strand GB, et al. Pyroglutamyl-peptidase I: cloning, sequencing, and characterization of the recombinant human enzyme[J]. Protein Expr Purif, 2003, 28(1): 111-119. doi: 10.1016/S1046-5928(02)00632-0 URL
[10]	Vorster BJ, Cullis CA, Kunert KJ. Plant vacuolar processing enzymes[J]. Front Plant Sci, 2019, 10: 479. doi: 10.3389/fpls.2019.00479 pmid: 31031794
[11]	丁修恒. 南方根结线虫效应蛋白MiV98抑制植物免疫及与番茄半胱氨酸蛋白酶Rcr3^pim互作研究[D]. 南京: 南京农业大学, 2019.
	Ding XH. Inhibition of plant immunity and interaction with tomato cysteine protease Rcr3^pim by the Meloidogyne incognita effector MiV98[D]. Nanjing: Nanjing Agricultural University, 2019.
[12]	Bernoux M, Timmers T, Jauneau A, et al. RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector[J]. Plant Cell, 2008, 20(8): 2252-2264. doi: 10.1105/tpc.108.058685 pmid: 18708476
[13]	Bozkurt TO, Schornack S, Win J, et al. Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface[J]. PNAS, 2011, 108(51): 20832-20837. doi: 10.1073/pnas.1112708109 pmid: 22143776
[14]	Zhao P, Zhou XM, Zhang LY, et al. A bipartite molecular module controls cell death activation in the Basal cell lineage of plant embryos[J]. PLoS Biol, 2013, 11(9): e1001655. doi: 10.1371/journal.pbio.1001655 URL
[15]	Yang XL, Li YZ, Wang AM. Research advances in potyviruses: from the laboratory bench to the field[J]. Annu Rev Phytopathol, 2021, 59: 1-29. doi: 10.1146/annurev-phyto-020620-114550 pmid: 33891829
[16]	Glais L, Tribodet M, Kerlan C. Genomic variability in potato potyvirus Y(PVY): evidence that PVY(N)W and PVY(NTN)variants are single to multiple recombinants between PVY(O)and PVY(N)isolates[J]. Arch Virol, 2002, 147(2): 363-378. pmid: 11890528
[17]	Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene Lin-4 encodes small RNAs with antisense complementarity to Lin-14[J]. Cell, 1993, 75(5): 843-854. doi: 10.1016/0092-8674(93)90529-y pmid: 8252621
[18]	Bazzini AA, Hopp HE, Beachy RN, et al. Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development[J]. PNAS, 2007, 104(29): 12157-12162. doi: 10.1073/pnas.0705114104 pmid: 17615233
[19]	Tang JY, Chu CC. microRNAs in crop improvement: fine-tuners for complex traits[J]. Nat Plants, 2017, 3: 17077. doi: 10.1038/nplants.2017.77 pmid: 28665396
[20]	Li F, Pignatta D, Bendix C, et al. microRNA regulation of plant innate immune receptors[J]. PNAS, 2012, 109(5): 1790-1795. doi: 10.1073/pnas.1118282109 pmid: 22307647
[21]	Park JH, Shin C. The role of plant small RNAs in NB-LRR regulation[J]. Brief Funct Genomics, 2015, 14(4): 268-274. doi: 10.1093/bfgp/elv006 pmid: 25777149
[22]	Iqbal MS, Hafeez MN, Wattoo JI, et al. Prediction of host-derived miRNAs with the potential to target PVY in potato plants[J]. Front Genet, 2016, 7: 159. doi: 10.3389/fgene.2016.00159 pmid: 27683585
[23]	詹琳琳. 烟草抗马铃薯Y病毒miRNA的筛选及相关miRNA的功能分析[D]. 杭州: 浙江农林大学, 2015.
	Zhan LL. Screening for miRNA of tobacco resistance to potato virus Y and function analysis of relevant miRNA[D]. Hangzhou: Zhejiang A & F University, 2015.
[24]	Bao ML, Bian HW, Zha YL, et al. miR396a-Mediated basic helix-loop-helix transcription factor bHLH74 repression acts as a regulator for root growth in Arabidopsis seedlings[J]. Plant Cell Physiol, 2014, 55(7): 1343-1353. doi: 10.1093/pcp/pcu058 URL
[25]	Guo YS, Jia MG, Yang YM, et al. Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provides insight into tobacco-PVY interactions[J]. Sci Rep, 2017, 7(1): 4895. doi: 10.1038/s41598-017-05155-w
[26]	Niño MC, Kim MS, Kang KK, et al. Genome-wide identification and molecular characterization of cysteine protease genes in rice[J]. Plant Biotechnol Rep, 2020, 14(1): 69-87. doi: 10.1007/s11816-019-00583-8
[27]	Clark K, Franco JY, Schwizer S, et al. An effector from the huanglongbing-associated pathogen targets citrus proteases[J]. Nat Commun, 2018, 9(1): 1718. doi: 10.1038/s41467-018-04140-9 pmid: 29712915
[28]	Hao L, Hsiang T, Goodwin PH. Role of two cysteine proteinases in the susceptible response of Nicotiana benthamiana to Colletotri-chum destructivum and the hypersensitive response to Pseudomonas syringae pv. tomato[J]. Plant Sci, 2006, 170(5): 1001-1009. doi: 10.1016/j.plantsci.2006.01.011 URL
[29]	Mueller AN, Ziemann S, Treitschke S, et al. Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2[J]. PLoS Pathog, 2013, 9(2): e1003177. doi: 10.1371/journal.ppat.1003177 URL
[30]	van der Linde K, Mueller AN, Hemetsberger C, et al. The maize cystatin CC9 interacts with apoplastic cysteine proteases[J]. Plant Signal Behav, 2012, 7(11): 1397-1401. doi: 10.4161/psb.21902 pmid: 22960758
[31]	Bar-Ziv A, Levy Y, Hak H, et al. The tomato yellow leaf curl virus(TYLCV)V2 protein interacts with the host papain-like cysteine protease CYP1[J]. Plant Signal Behav, 2012, 7(8): 983-989. doi: 10.4161/psb.20935 pmid: 22827939
[32]	Ueda T, Seo S, Ohashi Y, et al. Circadian and senescence-enhanced expression of a tobacco cysteine protease gene[J]. Plant Mol Biol, 2000, 44(5): 649-657. pmid: 11198425
[33]	Zang QW, Wang CX, Li XY, et al. Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat[J]. J Biosci, 2010, 35(3): 379-388. doi: 10.1007/s12038-010-0043-1 URL
[34]	马岩岩, 张军, 陈娇, 等. 柑橘半胱氨酸蛋白酶基因CsCysP的分离、亚细胞定位及表达分析[J]. 园艺学报, 2014, 41(4): 621-630.
	Ma YY, Zhang J, Chen J, et al. Isolation, subcellular localization and expression analysis of a Citrus cysteine protease gene, Cs-CysP[J]. Acta Hortic Sin, 2014, 41(4): 621-630.
[35]	闫龙凤, 杨青川, 韩建国, 等. 植物半胱氨酸蛋白酶研究进展[J]. 草业学报, 2005, 14(5): 11-19.
	Yan LF, Yang QC, Han JG, et al. Summary of cysteine protease in plants[J]. Acta Pratacultural Sci, 2005, 14(5): 11-19.
[36]	Uno Y, Furihata T, Abe H, et al. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions[J]. PNAS, 2000, 97(21): 11632-11637. doi: 10.1073/pnas.190309197 pmid: 11005831
[37]	罗健达. 马铃薯Y病毒侵染对烟草叶绿体蛋白降解的调控机理研究[D]. 北京: 中国农业科学院, 2021.
	Luo JD. The research of the regulation mechanism of chloroplast protein degradation induced by potato virus Y in tobacco[D]. Beijing: Chinese Academy of Agricultural Sciences, 2021.
[38]	裴悦宏, 李凤巍, 刘维娜, 等. 本氏烟半胱氨酸蛋白酶基因家族特征及其在TMV侵染中的功能[J]. 中国农业科学, 2022, 55(21): 4196-4210. doi: 10.3864/j.issn.0578-1752.2022.21.008
	Pei YH, Li FW, Liu WN, et al. Characteristics of cysteine proteinase gene family in Nicotiana benthamiana and its function during TMV infection[J]. Sci Agric Sin, 2022, 55(21): 4196-4210.