水杨酸诱导黄瓜PCD的鉴定及相关基因的表达分析

doi:10.7668/hbnxb.2018.06.008

摘要/Abstract

摘要： 为揭示叶绿体在水杨酸（SA）诱导黄瓜PCD过程中的作用，选择黄瓜幼苗四叶期时，叶片滴加10 mmol/L SA，在滴加SA的位置取材，首先进行ROS原位检测、Trypan blue染色、PI和FDA染色、TUNEL检测，验证SA可诱导黄瓜PCD过程；继而根据高通量测序结果，从1 759个高通量测序得到的差异表达基因中，筛选出15个注释含chloroplast的基因，RT-PCR验证这些基因在SA诱导的黄瓜叶片PCD中有表达，初步认定这些基因参与了SA诱导的黄瓜P CD过程。对筛选得到的基因进行qRT-PCR表达分析，这15个基因在SA诱导的黄瓜PCD中起正调控或负调控的作用，上调表达的5个基因分别是：类囊体加工肽酶1基因、6-磷酸葡萄糖酸脱氢酶基因、果糖1，6-二磷酸醛缩酶3基因、α-葡聚糖-水二激酶1基因和丙二烯氧化物环化酶4基因；下调表达的10个基因分别是：甜菜碱脱氢酶1基因、钙敏感受体基因、脱镁叶绿酸a单加氧酶基因、叶绿素b还原酶1基因、σ因子结合蛋白1基因、NADPH-原叶绿酸酯还原酶基因、2-琥珀苯甲酸-辅酶A连接酶基因、ATP依赖的锌金属蛋白酶基因、烟酰胺腺嘌呤二核苷酸转运子基因和胱硫醚β合成酶（CBS）蛋白基因。为进一步揭示叶绿体在SA诱导黄瓜PCD中的作用奠定了基础。

关键词: 黄瓜, 水杨酸, 细胞程序性死亡, 基因表达

Abstract: To reveal the role of chloroplast in the PCD of cucumber induced by salicylic acid (SA),10 mmol/L salicylic acid was added to the leaves of cucumber seedlings at the four-leaf period,and the samples were collected at the location where SA was added. ROS in situ detection,Trypan blue staining,PI and FDA staining,and TUNEL detection were performed. These results showed that SA could induce PCD process in cucumber. According to the results of high-throughput sequencing,15 genes annotated with chloroplast were screened out among 1 759 differentially expressed genes. These genes were expressed in cucumber leaves treated with SA was confirmed by RT-PCR. It was preliminarily identified that these genes were involved in the PCD process induced by SA in cucumber. According to the expression of qRT-PCR,the 15 genes played a role of positive regulation or negative regulation in PCD induced SA in cucumber,upon regulating characteristics five kinds of genes were raised:Probable thylakoidal processing peptidase 1 gene, 6-phosphogluconate dehydrogenase gene, Probable fructose-bisphosphate aldolase 3 gene, Alpha-glucan water dikinase 1 gene and Allene oxide cyclase 4 gene, down-regulated expression of 10 genes were:Betaine aldehyde dehydrogenase 1 gene, Calcium sensing receptor gene, Pheophorbide a oxygenase gene, Probable chlorophyll(ide) b reductase1 gene, Sigma factor binding protein 1 gene, NADPH-protochlorophyllide oxidoreductase gene, 2-succinylbenzoate-CoA ligase gene, ATP-dependent zinc metallopro-tease FtsH1 gene, Nicotinamide adenine dinucleotide transporter 1 gene and CBS domain-containing protein1 gene. It lays a foundation for further revealing the role of chloroplast in cucumber PCD induced by SA.

Key words: Cucumber, Salicylic acid, Programmed cell death, Gene expression

中图分类号:

Q78
S642.03

陈冲, 刘双, 王丹丹, 池春玉, 朱宏, 金晓霞, 丁国华. 水杨酸诱导黄瓜PCD的鉴定及相关基因的表达分析[J]. 华北农学报, 2018, 33(6): 56-63. doi: 10.7668/hbnxb.2018.06.008.

CHEN Chong, LIU Shuang, WANG Dandan, CHI Chunyu, ZHU Hong, JIN Xiaoxia, DING Guohua. Identification of PCD Induced by Salicylic Acid and Expression Analysis of Genes Related with the PCD in Cucumber[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(6): 56-63. doi: 10.7668/hbnxb.2018.06.008.

http://www.hbnxb.net/CN/Y2018/V33/I6/56

参考文献

[1] 孙军利,赵宝龙,郁松林. 外源水杨酸(SA)对高温胁迫下葡萄幼苗耐热性诱导研究[J]. 水土保持学报,2014,28(3):290-294,299.
[2] 孟雪娇,邸昆,丁国华. 水杨酸在植物体内的生理作用研究进展[J]. 中国农学通报,2010,26(15):207-214.
[3] 王楠,高静,黄文静,等. 旱、盐胁迫下黄芪种子萌发及其对水杨酸的响应[J]. 草业科学,2018,35(1):106-114.
[4] 符冠富,张彩霞,杨雪芹,等. 水杨酸减轻高温抑制水稻颖花分化的作用机理研究[J]. 中国水稻科学,2015,29(6):637-647.
[5] 徐晓昀,郁继华,颉建明,等. 水杨酸和油菜素内酯对低温胁迫下黄瓜幼苗光合作用的影响[J]. 应用生态学报,2016,27(9):3009-3015.
[6] 单长卷,赵新亮,汤菊香. 水杨酸对干旱胁迫下小麦幼苗抗氧化特性的影响[J]. 麦类作物学报,2014,34(1):91-95.
[7] 吴华,苏倩,陈金慧,等. 水杨酸对紫外线胁迫下杉木组培苗的影响[J]. 江苏农业科学,2016,44(9):207-210.
[8] Khan M,Fatma M,Per T S,et al. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants[J]. Frontiers in Plant Science,2015,6:462.
[9] Anil K,Das S N,Podile A R. Induced defense in plants:a short overview[J]. Proceedings of the National Academy of Sciences India,2014,84(3):669-679.
[10] Wani A B,Chadar H,Wani A H,et al. Salicylic acid to decrease plant stress[J]. Environmental Chemistry Letters,2017,15(1):101-123.
[11] Malamy J,Carr J P,Klessig D F,et al. Salicylic acid:a likely endogenous signal in the resistance response of tobacco to viral infection[J]. Science,1990,250(4983):1002-1004.
[12] Slaymaker D H,Navarre D A,Clark D,et al. The tobacco salicylic acid-binding protein 3(SABP3) is the chloroplast carbonic anhydrase,which exhibits antioxidant activity and plays a role in the hypersensitive defense response[J]. Proceedings of the National Academy of Sciences of the United States of America,2002,99(18):11640-11645.
[13] Van Wees S C,Glazebrook J. Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid[J]. The Plant Journal:for Cell and Molecular Biology,2003,33(4):733-742.
[14] Lee B,Park Y S,Yi H S,et al. Systemic induction of the small antibacterial compound in the leaf exudate during benzothiadiazole-elicited systemic acquired resistance in pepper[J]. The Plant Pathology Journal,2013,29(3):350-355.
[15] Heath M C. Hypersensitive response-related death[J]. Plant Molecular Biology,2000,44(3):321-334.
[16] Greenberg J T,Guo A,Klessig D F,et al. Programmed cell death in plants:a pathogen-triggered response activated coordinately with multiple defense functions[J]. Cell,1994,77(4):551-563.
[17] Greenberg J T,Yao N. The role and regulation of programmed cell death in plant-pathogen interactions[J]. Cellular Microbiology,2004,6(3):201-211.
[18] Zhao Y,Luo L,Xu J,et al. Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana[J]. Cell Research,2018,28(4):448-461.
[19] Green D R,Reed J C. Mitochondria and apoptosis[J]. Science,1998,281(5381):1309-1312.
[20] Reape T J,Kacprzyk J,Brogan N,et al. Mitochondrial markers of programmed cell death in Arabidopsis thaliana[J]. Methods in Molecular Biology,2015,1305(10):211-221.
[21] 王艳杰,邓雯,张鹏飞. 细胞色素C与细胞凋亡研究进展[J]. 动物医学进展,2012,33(7):89-92.
[22] Balk J,Leaver C J,Mccabe P F. Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants[J]. FEBS Letters,1999,463(1/2):151-154.
[23] Ambastha V,Tripathy B C,Tiwari B S. Programmed cell death in plants:A chloroplastic connection[J]. Plant Signaling & Behavior,2015,10(2):e989752.
[24] Van Aken O,Van Breusegem F. Licensed to kill:mitochondria,chloroplasts,and cell death[J]. Trends in Plant Science,2015,20(11):754-766.
[25] Samuilov V D,Lagunova E M,Kiselevsky D B,et al. Participation of chloroplasts in plant apoptosis[J]. Bioscience Reports,2003,23(2/3):103-117.
[26] Doyle S M,Diamond M,Mccabe P F. Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures[J]. Journal of Experimental Botany,2010,61(2):473-482.
[27] Lee K P,Kim C,Landgraf F,et al. EXECUTER1-and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America,2007,104(24):10270-10275.
[28] Kim C,Meskauskiene R,Zhang S,et al. Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway[J]. The Plant Cell,2012,24(7):3026-3039.
[29] Peters J S,Chin C. Evidence for cytochrome f involvement in eggplant cell death induced by palmitoleic acid[J]. Cell Death and Differentiation,2005,12(4):405-407.
[30] Zuppini A,Gerotto C,Moscatiello R,et al. Chlorella saccharophila cytochrome f and its involvement in the heat shock response[J]. Journal of Experimental Botany,2009,60(14):4189-4200.
[31] Wang H,Zhu X,Li H,et al. Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system[J]. Scientific Reports,2014,4:5989.
[32] Yao N,Greenberg J T. Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death[J]. The Plant Cell,2006,18(2):397-411.
[33] Zhao Y,Luo L,Xu J,et al. Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana[J]. Cell Research,2018,28(4):448-461.
[34] Adam Z,Rudella A,Van Wijk K J. Recent advances in the study of Clp,FtsH and other proteases located in chloroplasts[J]. Current Opinion in Plant Biology,2006,9(3):234-240.
[35] Carrión C A,Costa M L,Martínez D E,et al. In vivo inhibition of cysteine proteases provides evidence for the involvement of ‘senescence-associated vacuoles’ in chloroplast protein degradation during dark-induced senescence of tobacco leaves[J]. Journal of Experimental Botany,2013,64(16):4967-4980.
[36] Luis C O,Armando B G,Julio M,et al. AtMCP1b,a chloroplast-localised metacaspase,is induced in vascular tissue after wounding or pathogen infection[J]. Functional Plant Biology,2007,34(12):1061-1071.
[37] Yang Y,Jin H,Chen Y,et al. A chloroplast envelope membrane protein containing a putative LrgB domain related to the control of bacterial death and lysis is required for chloroplast development in Arabidopsis thaliana[J]. The New Phytologist,2012,193(1):81-95.
[38] 刘双,乔禹,王芳,等. 基于数字基因表达谱筛选黄瓜中水杨酸诱导基因[J]. 核农学报,2015,29(5):874-884.
[39] Ramel F,Sulmon C,Gouesbet G,et al. Natural variation reveals relationships between pre-stress carbohydrate nutritional status and subsequent responses to xenobiotic and oxidative stress in Arabidopsis thaliana[J]. Annals of Botany,2009,104(7):1323-1337.
[40] Stone J M,Heard J E,Asai T,et al. Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants[J]. The Plant Cell,2000,12(10):1811-1822.
[41] Bowling S A, Clarke J D, Liu Y, et al. The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[J]. Plant Cell, 1997, 9:1573-1584.
[42] 仝贝. 植物叶绿体电子传递蛋白在细胞程序性死亡中的功能[D]. 西安:西北大学,2017.
[43] Petrov V,Hille J,Mueller-Roeber B,et al. ROS-mediated abiotic stress-induced programmed cell death in plants[J]. Frontiers in Plant Science,2015,6:69.
[44] Pétriacq P,De Bont L,Tcherkez G,et al. NAD:not just a pawn on the board of plant-pathogen interactions[J]. Plant Signaling & Behavior,2013,8(1):e22477.
[45] Yun L J,Chen W L. SA and ROS are involved in methyl salicylate-induced programmed cell death in Arabidopsis thaliana[J]. Plant Cell Reports,2011,30(7):1231-1239.
[46] 刘双. 诱导的黄瓜超敏反应相关基因的筛选和表达分析[D]. 哈尔滨:哈尔滨师范大学,2016.
[47] Narusaka M,Kawai K,Izawa N,et al. Gene coding for SigA-binding protein from Arabidopsis appears to be transcriptionally up-regulated by salicylic acid and NPR1-dependent mechanisms[J]. Journal of General Plant Pathology,2008,74(5):345-354.
[48] Xie Y D,Li W,Guo D,et al. The arabidopsis gene SIGMA FACTOR-BINDING PROTEIN 1 plays a role in the salicylate-and jasmonate-mediated defence responses[J]. Plant,Cell & Environment,2010,33(5):828-839.
[49] Kaurilind E,Brosché M. Stress marker signatures in lesion mimic single and double mutants identify a crucial leaf Age-Dependent salicylic acid related defense signal[J]. PLoS One,2017,12(1):e0170532.
[50] Rodrigues R A,Silva-Filho M C,Cline K. FtsH2 and FtsH5:two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex[J]. The Plant Journal:for Cell and Molecular Biology,2011,65(4):600-609.
[51] Malnoë A,Wang F,Girard-Bascou J,et al. Thylakoid FtsH protease contributes to photosystem Ⅱ and cytochrome b6f remodeling in Chlamydomonas reinhardtii under stress conditions[J]. The Plant Cell,2014,26(1):373-390.
[52] Soleimani M J. Possible effects of pathogen inoculation and salicylic acid pre-treatment on the biochemical changes and proline accumulation in Green bean[J]. Archives of Phytopathology and Plant Protection,2015,48(3):212-222.
[53] Mur L A,Aubry S,Mondhe M,et al. Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis[J]. The New Phytologist,2010,188(1):161-174.
[54] Christ B,Egert A,Süssenbacher I,et al. Water deficit induces chlorophyll degradation via the ‘PAO/phyllobilin’ pathway in leaves of homoio-(Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants[J]. Plant,Cell & Environment,2014,37(11):2521-2531.
[55] Horie Y, Ito H, Kusaba M, et al. Participation of Chlorophyll b reductase in the initial step of the degradation of light-harvesting Chlorophyll a/b-protein complexes in Arabidopsis[J]. The Journal of Bioloical Chemistry, 2009, 284(26):17449-17456.
[56] 曹晏彬,柏素花,戴洪义. 苹果丙二烯氧化物环化酶基因MdAOC1的克隆与表达分析[J]. 林业科学,2013,49(12):73-80.

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