Selection and validation of appropriate reference genes and expression analysis of jasmonic acid-related genes responding to Marssonina rosae in Rosa species and cultivars
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摘要:目的 为研究蔷薇属植物响应黑斑病侵染的稳定表达的最适内参基因，解析茉莉酸在蔷薇属植物响应蔷薇盘二孢侵染过程中的作用。方法 以黑斑病病原菌蔷薇盘二孢侵染的6个蔷薇属种/品种不同时间的离体叶片为材料，利用qRT-PCR技术及geNorm、NormFinder、BestKeeper软件对9个候选内参基因（ACT、GAPDH、PP2A、Rcl2、SAND、TIP、TUA、TUB、UBC）的表达量进行测定和分析，利用筛选出的内参基因，对蔷薇属植物茉莉酸（JA）抗病途径相关基因（COI1、OPR3、MYC2、JAR1）的表达水平进行定量分析。结果 （1）UBC可作为6种蔷薇属植物共同适用的内参基因，可用于后续分析JA抗病途径相关基因的表达水平。（2）内源JA含量在6种蔷薇属植物响应蔷薇盘二孢侵染的过程中存在差异。在黑斑病高抗植物受侵染0 ~ 4 d间JA含量下调，4 ~ 8 d间上调。在黑斑病易感植物受侵染0 ~ 8 d间，内源JA含量呈下调趋势。（3）JA合成相关基因OPR3及JAR1表达量在受侵染初期表达量趋势存在差异。在除荷花蔷薇外的其余5种植物中，OPR3在侵染初期（0 ~ 0.5 d）表达下调，JAR1在侵染初期表达上调。OPR3和JAR1在侵染后期均表达上调，黑斑病易感材料的上调程度高于高抗材料。（4）JA信号传导相关基因COI1及MYC2在受侵染初期表达量趋势同样存在差异。COI1在黑斑病高抗材料受侵染初期上调表达，在黑斑病易感材料下调表达，MYC2在6种植物受侵染0 ~ 2 d中均下调表达。COI1及MYC2表达量在受侵染2 d后均上调表达，且在黑斑病易感植物中的上调程度大于黑斑病高抗材料。结论 与JA信号传导相关的MYC2、COI1在蔷薇属植物抵御黑斑病病菌入侵过程中发挥负调控作用，且由JA通路介导的抵御死体营养型病原菌的侵染在后期发挥了作用。Abstract:Objective This paper aims to select the appropriate reference gene and analyze the role of jasmonic acid in Rosa species and cultivars responding to Marssonina rosae.Method R. multiflora f. carnea, R. xanthina f. spontanea, R. glauca, R. rugosa, R. hybrida ‘Porcelina’ and R12-26 (hybrid of R. rugosa and R. hybrida ‘Porcelina’) were used as materials, qRT-PCR technology, geNorm, NormFinder, BestKeeper were used to evaluate the expression stability of nine candidate reference genes (ACT, GAPDH, PP2A, RCl2, SAND, TIP, TUA, TUB and UBC). The expression levels of jasmonic acid resistance pathway relatedgenes (COI1, OPR3, MYC2 and JAR1) in six Rosa species and cultivars were quantitatively analyzed.Result (1) UBC was the common reference gene of six Rosa species and cultivars responding to M. rosae, and can be used for analyzing the related gene expression levels to JA resistance pathway. (2) There were differences in JA content among the six plants responding to M. rosae. In disease-resistant plants, JA concentration decreased at 0−4 d and increased at 4−8 d. In disease-susceptible plants, JA concentration decreased at 0−8 d and increased significantly at 8−10 d. (3) The expression levels of JA synthesis-related genes OPR3 and JAR1 were different at the early stage of infection. The expression level of OPR3 was down-regulated and JAR1 up-regulated in five plants except for R. multiflora f. carnea. OPR3 and JAR1 were all up-regulated in the late stage of infection, however the up-regulation degree in black-spot susceptible species was significantly higher than that of resistant species. (4) There were also differences in the expression levels of JA signaling related genes COI1 and MYC2 at the early stage of infection. The expression of COI1 was up-regulated in disease-resistant plants and down-regulated in disease-susceptible plants at the early stage of infection, MYC2 was down-regulated in 6 plants infected 0−2 d. COI1 and MYC2 were up-regulated after 2 d of infection, and the up-regulation degree in black-spot susceptible species was significantly higher than that of resistant species.Conclusion MYC2 and COI1, which are related to JA signaling, play a negative regulatory role in the resistance of Rosa plants to the invasion of black spot pathogens, and the JA pathway mediated resistance to the invasion of dead vegetative pathogens plays a role in the later stage of infection.
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Keywords:
- Rosa /
- Marssonina rosae /
- reference gene selection /
- jasmonic acid /
- expression analysis
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植物寄生线虫对农林业发展构成严重的威胁[1-2]，为了防治线虫病害，并避免传统化学防治带来的环境污染[3]，开发利用杀线虫生防制剂已成为当今的研究热点[4-5]。真菌次生代谢产物具有药效高、环境相容性好、不容易造成二次污染等优点已被越来越多的人所关注[6-7]。丝状真菌Sr18(Syncephalastrum racemosum)(简称Sr18真菌)，是一株以松材线虫(Bursaphelenchus xylophilus)为靶标，自主分离获得的具有广谱杀线虫活性的丝状真菌，其代谢物对大豆孢囊线虫、甘薯茎线虫、南方根结线虫等都具有良好的防治作用，用1/2浓度的Sr18发酵液处理松材线虫、南方根结线虫、大豆孢囊线虫等不同线虫24 h，校正死亡率均能达到100%[8-9]。前期研究通过实验已证实发酵液中杀线虫活性组分主要为小分子物质[10]，其小分子活性组分具有水溶性好且耐热的优势。进一步的机理研究表明：小分子活性组分对线虫体内的超氧化物岐化酶(Superoxide dismutase, SOD)、过氧化物酶(Peroxidase, POD)、过氧化氢酶(Catalase, CAT)等抗氧化保护酶系，神经递质相关的总胆碱酯酶(Total Cholinesterase, TChE)，以及解毒酶谷胱甘肽转硫酶(Glutathione S-transferases, GST)均有明显的抑制作用[11]。同时还发现小分子活性组分对线虫卵的孵化也有很强的抑制作用，原始浓度发酵液处理南方根结线虫的虫卵48 h，孵化抑制率能达到85%以上[9]。在此基础上，实验借助场发射扫描电镜和高分辨透射电镜的手段，对1/2浓度Sr18发酵液小分子活性组分处理后的松材线虫进行了体表和内部结构观察[12-13]，旨在为进一步揭示Sr18发酵液小分子活性组分毒杀线虫的机制提供依据。
1. 材料与方法
1.1 实验材料
1.1.1 供试线虫
松材线虫，中国农业科学院植物保护研究所提供。
1.1.2 菌种
Sr18真菌，由本研究室筛选及保藏。
灰葡萄孢霉(Batrytis cinerea)(简称BC菌)，由天津师范大学提供。
1.2 实验方法
1.2.1 发酵液的小分子活性组分的制备
在工厂用5t发酵罐采取优化放大工艺[14](发酵条件：罐压0.1 mPa；排气1；温度27 ℃；转速120 r/min，变频器324.7；发酵时间40 h)制得Sr18发酵液[10]。
取原浓度的Sr18发酵液，加入等量无菌水稀释成1/2浓度的Sr18发酵液，经微孔滤膜(0.22 μm)过滤除菌后，采用截流分子量为6 000 da的UEOS-503型中空纤维膜组件对发酵液进行超滤分级[15]，除去大分子物质后所得到的小分子收集物即为实验所用的1/2浓度的Sr18发酵液小分子活性组分。
1.2.2 松材线虫培养与收集
在无菌操作条件下，将灰葡萄孢霉接种于察氏培养基上，21 ℃下避光培养7 d[16]，待菌丝长满培养皿后，将松材线虫接种于培养皿进行培养繁殖，26 ℃下避光培养5~7 d。
培养好的线虫采用贝尔曼(Baermann)漏斗法[17]进行收集，用无菌水离心洗涤3次(2 000 r/min，3 min)，制备成约15 000条/mL的悬液，供试。
1.2.3 松材线虫的处理
参照日本Kawazu等[18]处理线虫的实验方法(即1 mL杀线虫药剂+1 500头松材线虫)，取24孔板，每孔加200 μL线虫悬液，实验组加入2 mL 1/2浓度的Sr18发酵液小分子活性组分，对照组则加入等量的无菌水，于26 ℃培养箱中处理1~3 d，每24 h取样观察1次。
1.2.4 扫描电镜(SEM)制样
将处理的线虫按照预定时间(24、48、72 h)取样，离心后用无菌水洗4次，加入用磷酸缓冲液配制的2.5%戊二醛，4 ℃过夜后，再用0.1 M PBS漂洗3次，每次15 min；然后用磷酸缓冲液配制的1%锇酸室温下固定2 h，PBS漂洗3次，每次15 min；分别用30%、50%、70%、80%、90%、100%I、100%Ⅱ梯度乙醇溶液脱水，每次10 min；六甲基二硅胺烷法干燥；离子溅射镀膜仪镀膜[19]；场发射扫描电镜(型号：Nova NanoSEM 230)观察，拍照记录，每24 h取样观察1次。
1.2.5 透射电镜(TEM)制样
按照电镜样品制备的常规方法[20]，用PBS反复清洗包含有线虫的小琼脂块，2.5%戊二醛和1%锇酸双重固定(37 ℃，12 h；45 ℃，12 h；60 ℃，24 h)，分别用30%、50%、70%、80%、90%、100%I、100%Ⅱ梯度乙醇溶液脱水，每次10 min；环氧树脂梯度浸透，包埋，LEICA EMUC6超薄切片机进行超薄切片(70~80 nm)，铜网收集，醋酸双氧铀-柠檬酸铅双重染色[21]，Hitachi H-600透射电子显微镜观察并拍照，每24 h取样观察1次。
2. 结果与分析
2.1 松材线虫虫体及体壁的SEM观察
正常线虫及小分子活性组分处理后的线虫SEM观察结果如图 1所示。观察发现，对照组线虫虫体丰满，呈自然弯曲状态(图 1A)，线虫头部与虫体分界明显，体壁环纹清晰，尾部表面光滑，雄虫可见交合刺外凸。
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图 1 线虫虫体低倍观察结果A.对照(Bar=100 μm)；B.处理3天后的线虫(Bar=200 μm)。Figure 1. Ultrastructure observation of Bursaphelenchus xylophilus at low magnificationA, normal Bursaphelenchus xylophilus(Bar = 100 μm); B, 3 days treated Bursaphelenchus xylophilus by Sr18(Bar = 200 μm).与对照组相比，小分子活性组分处理后的线虫虫体皱缩并扭曲(图 1B)，体壁环纹模糊，表面凹凸不平，附着的细菌消失，表皮呈片状脱落，体壁出现明显的局部凹陷，放大后可见体表内陷形成的孔洞(图 2A、2B)，并可见内容物从体壁破损的孔洞溢出(图 2C)。
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图 2 处理后线虫体表SEM观察结果A.处理1天后线虫体表出现孔洞(Bar=4 μm)；B.放大后的孔洞(Bar=1 μm)；C.处理3天后内溶物由孔洞溢出(←)(Bar=1 μm)。Figure 2. Ultrastructure observation of the Bursaphelenchus xylophilu's body surface at high magnificationA, holes on body surface of nematode treated 1 day by Sr18(Bar=4 μm); B, magnifying hole on body surface(Bar=1 μm); C, dissolved substance inside the cell flew out from the holes after 3 days treated(Bar=1 μm).2.2 松材线虫头部表观结构的SEM观察
由图 3A和图 3B可以看出，正常线虫的头部与虫体界限分明, 连接处环形平台清晰，头部六片唇瓣之间有凹陷间隔(图 3B)，并可观察到唇片上存在乳状突起，唇片上角质环纹明晰, 6个唇片在口周形成角质口环, 环中可见口针外凸。此外，还发现线虫体表局部有携带细菌[22]的存在(图 3A)。与之相对应，小分子组分处理过的线虫头部和虫体皱缩严重，连接处环形平台缢缩；唇瓣之间有凹陷间隔消失，难以分辨出6片唇，角质口环和横纹消失、口针完全被破坏(图 3C)。
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图 3 线虫头部SEM观察结果A.对照线虫头部及体壁的细菌(Bar=20 μm)；B.对照线虫头部(Bar=3 μm)；C.处理3天后萎缩的线虫头部(Bar=1 μm)。Figure 3. Ultrastructure observation of the Bursaphelenchus xylophilu's head at high magnificationA, head and bacteria on body wall of normal nematode(Bar=20 μm); B, head of normal nematode(Bar=3 μm); C, shrinking head of nematode treated 3 days by Sr18(Bar=1 μm).2.3 松材线虫尾部的SEM观察
松材线虫的尾部为生殖孔所在部位，其结构的完好与否与繁殖密切相关。观察发现，正常线虫尾部因雌雄而异，雄虫侧面观察呈尖形(图 1A)，可以观察到交合刺，尾尖片状翼膜清晰(图 4A)；小分子活性组分处理后，尾部与虫体变化相似，表面凹凸不平，严重皱缩，交合刺消失，但尾尖片状翼膜变化不大(图 4B、4C)。
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图 4 线虫尾部SEM观察结果A.对照线虫尾部及交合刺(Bar=20 μm)；B.处理2天后线虫尾部缢缩，肿胀物形成(Bar=20 μm)；C.肿胀物(Bar=5 μm)。Figure 4. Ultrastructure observation of the Bursaphelenchus xylophilu's tail at high magnificationA, tail and spicule of normal nematode(Bar=20 μm); B, shrinking tail and swellings of nematode treated 2 days by Sr18(Bar=20 μm); C, magnifying swelling on body surface(Bar=5 μm).2.4 松材线虫内部结构的TEM超微观察
实验对小分子活性组分处理后引起的的松材线虫内部结构变化进行了TEM超微观察。观察发现，对照组线虫表皮、体壁等结构完整，其3层结构(角质层、下皮层、肌肉层)完整；体腔内的细胞界限清晰，细胞核核膜完整，核糖体丰富，分布均匀；线粒体形态完好、且多聚集于肌纤维[20]附近，内质网等内膜结构清楚(图 5A、5B)。
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图 5 线虫内部结构透射电镜(TEM)观察结果A.对照线虫肌纤维(M)及线粒体(MI)(Bar=500 nm)；B.正常的细胞核(n)(Bar=500 nm)；C.处理1天后的细胞核(n)及内质网(ER)(Bar=500 nm)；D.处理2天后的体壁(↘)及细胞损伤(Bar=500 nm)；E.处理3天后体壁分离(Bar=2 μm)；F.处理3天后体壁分离后组织细胞结构消失(Bar=2 μm)。Figure 5. Ultrastructure observation of the Bursaphelenchus xylophilu's internal structure at high magnificationA, muscle fiber and mitochondria of normal nematode(Bar=500 nm); B, cell nucleus of normal nematode(Bar=500 nm); C, damaged cell nucleus and endoplasmic reticulum of nematode treated 1 day by Sr18(Bar=500 nm); D, swellings on body wall and cellular damage of nematode treated 2 days by Sr18(Bar=500 nm); E, separation of cuticle and coelom of nematode treated 3 days by Sr18 (Bar=2 μm); F, disappearance of tissue and cell structure after separation of nematode treated 3 days by Sr18 (Bar=2 μm).经小分子活性组分处理后，体腔内部分细胞受损明显，细胞核形状不规则，核膜受损，染色质分散，内质网出现断裂，核糖体集聚，线粒体明显固缩(图 5C、5D)，局部体壁和体腔分离，肌纤维严重受损，已看不清体壁层次，并形成空腔，体腔内细胞间界限勉强可见；最终体内细胞界限消失，失去细胞功能(图 5E、5F)。
3. 讨论与结论
扫描电镜及透射电镜的超微结构观察结果显示，Sr18发酵液分离得到的小分子活性组分对松材线虫的杀线作用是“内外兼顾”的，全面影响线虫的结构。就超微观察结果而言，观察到体壁受损，层次消失，肌细胞溶解，线粒体、内质网等细胞器受损。核糖体聚集通常也是细胞损伤的重要指征，从而导致线虫死亡，表明其作用机理应为毒杀，而非触杀[23]。前期酶学研究结果也显示，Sr18发酵液的小分子活性组分对ATP、TChE、GST、以及SOD、POD、CAT等酶均有抑制作用[10]。这与超微观察的结果相对应。失去了这些酶的保护，细胞器和细胞的损伤在所难免，而细胞器或细胞的损伤也必然加剧对酶的影响[24]。
目前，对真菌代谢物对线虫抑制或致死作用相关研究主要集中在这些代谢产物的实验室或大田的时间-剂量效应研究上以及代谢物对J2的致死性，对虫卵孵化的抑制力以及抑制线虫侵染寄主的能力[25]；Vos等[26]研究发现丛枝菌根真菌可通过改变其寄主根系分泌物来减少根结线虫的侵染，而且其根系分泌物可在抑制线虫运动方面发挥一定的作用。另有一些则对线虫寄生真菌和食线虫真菌侵染性胞外酶的蛋白分子结构、酶动力学以及其作用于线虫的组织部位进行了相关的分析与研究[27]；现在许多微生物的杀线虫代谢产物的真正的作用机制还不清楚，代谢产物可通过不同的作用靶点破坏线虫的正常生活，已有的对微生物杀线代谢产物作用机制的研究多从代谢物对线虫的神经毒作用、体壁破坏作用、与物质代谢及能量代谢相关的酶的变化以及分泌植物生长激素等方面进行[28-33]，较深入系统的研究还很缺乏。由于这方面的理论问题没有真正搞清楚，因此大大限制了高效生物杀线虫农药的开发应用，这直接影响对它们更好的利用[29]。
近年来随着系统生物学中代谢组学技术的迅猛发展为在更深层次上揭示活性代谢物的杀线虫机理提供了强有力的手段和工具[34]。未来课题组将在前期研究的基础上，以对农林作物造成严重危害的植物寄生线虫为作用对象，将高分辨电镜、酶学及生理生化等研究手段与系统生物学中新兴的代谢组学的新技术方法相结合，充分利用学科交叉的优势，通过体内、外实验系统深入地研究探讨Sr18生物杀线剂防治植物根结线虫病害的作用机理，寻找生物标记物，确定其作用方式，为今后建立植物寄生线虫代谢组学研究平台及开发对黄瓜、番茄、辣椒等根结线虫病害具有防效高、增产作用明显[29]等特点的线虫生防菌株Sr18新型高效生物杀线剂做有益的探索。
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图 1 6种蔷薇属植物离体叶片侵染蔷薇盘二孢的发病情况
红色圆圈表示病斑开始出现。黑色圆圈表示病斑蔓延至整个叶片。Red circle indicates the disease onset with the appearance brown necrotic lesions. Black circles indicate the spread of the brown necrotic lesions throughout the whole leaf.
Figure 1. Incidence of in vitro leaves infected by M. rosae of 6 Rosa species and cultivars
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图 2 9个候选内参基因响应蔷薇盘二孢侵染时的Ct值
Figure 2. Ct value of 9 candidate reference genes in response to M. rosae
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图 3 利用geNorm软件计算的候选内参基因的表达稳定性及配对变异值
M代表各候选内参基因的表达稳定度。下同。M represents the average expression stability of each candidate reference gene. The same below.
Figure 3. Average expression stability values and variation values of candidate reference genes calculated by geNorm
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图 4 Normfinder软件计算获得的候选内参基因的表达稳定性
Figure 4. Expression stability of candidate reference genes and by pairwise variation using Normfinder
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图 5 BestKeeper软件计算获得的候选内参基因的表达稳定性
SD代表各候选内参基因Ct值的标准偏差。SD represents the standard deviation of Ct value of each candidate reference gene.
Figure 5. Expression stability of candidate reference genes calculated by BestKeeper
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图 6 内源茉莉酸在蔷薇属植物响应蔷薇盘二孢侵染的含量
Figure 6. Content of JA in Rosa species and cultivars responding to M. rosae
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图 7 茉莉酸相关基因在蔷薇盘二孢侵染不同时间的相对表达量
Figure 7. Relative expression level of JA-related genes at different time of M. rosae infection
表 1 基因引物基本信息
Table 1 Basic information of gene primers
基因
Gene引物序列（5′—3′）
Primer sequence (5′-3′)退火温度
Annealing temperature (Tm)/℃扩增效率
Amplification efficiency (E)/%r ACT F: TCAAGGATTGGTGGACTTCAGT
R: ACCAGAGAACAAGAATGCAAGC55 96.0 0.970 GAPDH F: TATGACCAGATCAAGGCTGCT
R: ACCAATGAAGTCGGTTGACAC55 102.5 0.999 PP2A F: TGTCACTGCATCAAAGGACAG
R: GACGAATTGTCTTCTCCACCA55 102.0 0.982 Rcl2 F: ATGGGAAATGCCCTACCT
R: CACTTGTCCGACTGTTGC55 95.7 0.969 SAND F: BGTGTTGAGGAGTTGCCTCTTG
R: AACCTGTCGGGAGAATCTGTT55 98.5 0.989 TIP F: GAATCCACGGCTGGGAAA
R: CAGTTCGTGGGTGGAGGAGTT55 104.2 0.998 TUA F: CATTGAGCGTCCCACCTA
R: CACATCCACATTCAGAGCC55 103.2 0.987 TUB F: GTACATGGCCTGCTGTTTGAT
R: ATGGTACGCTTGGTCTTGATG55 96.1 0.972 UBC F: GCCAGAGATTGCCCATATGTA
R: TCACAGAGTCCTAGCAGCACA55 103.5 0.996 COI1 F: AATGAGGGGCTGTTGCTTCA
R: GATCCCCTGTACCCTTGCAC56 102.2 0.984 MYC2 F: CGGCAGCAGCGTCAAGAAT
R: GAGGTCGGAGTGGTGGGAAT57 101.0 0.957 OPR3 F: CATCAGCGAAGGCACTTTGG
R: GGCGTCGACTACCTTCTTCC55 103.6 0.951 JAR1 F: TTGGGTGCTACTTCTTTCTCAG
R: AATTGGAGGAAGGAGGGTG57 104.4 0.952 
下载: 导出CSV
表 2 9个候选内参基因的表达稳定性排序
Table 2 Expression stability ranking of 9 candidate internal reference genes
种/品种
Species/cultivar方法 Method 1 2 3 4 5 6 7 8 9 荷花蔷薇
R. multiflora f. carneageNorm UBC TIP PP2A GAPDH TUB ACT SAND TUA Rcl2 Normfinder UBC TIP PP2A GAPDH TUB ACT TUA SAND Rcl2 Bestkeeper TIP ACT UBC TUB SAND PP2A TUA GAPDH Rcl2 RefFinder UBC TIP PP2A TUB ACT GAPDH SAND TUA Rcl2 单瓣黄刺玫
R. xanthina f. spontaneageNorm UBC PP2A SAND TIP GAPDH ACT Rcl2 TUB TUA Normfinder PP2A UBC TIP SAND GAPDH ACT Rcl2 TUB TUA Bestkeeper PP2A TIP UBC ACT GAPDH SAND Rcl2 TUB TUA RefFinder PP2A UBC TIP SAND GAPDH ACT Rcl2 TUB TUA 玫瑰
R. rugosageNorm UBC TIP GAPDH TUB TUA PP2A SAND Rcl2 ACT Normfinder UBC TIP PP2A GAPDH TUB SAND TUA Rcl2 ACT Bestkeeper UBC TIP TUA TUB GAPDH SAND ACT PP2A Rcl2 RefFinder UBC TIP GAPDH TUB TUA PP2A SAND ACT Rcl2 R12-26 geNorm PP2A TIP UBC SAND GAPDH TUB TUA ACT Rcl2 Normfinder TIP GAPDH UBC PP2A SAND TUB TUA ACT Rcl2 Bestkeeper UBC ACT GAPDH TIP PP2A SAND Rcl2 TUB TUA RefFinder UBC TIP PP2A GAPDH SAND ACT TUB TUA Rcl2 红叶蔷薇
R. glaucageNorm UBC TIP SAND PP2A TUA TUB GAPDH Rcl2 ACT Normfinder UBC TIP TUA SAND PP2A TUB GAPDH Rcl2 ACT Bestkeeper SAND TIP PP2A UBC ACT TUA TUB GAPDH Rcl2 RefFinder UBC TIP SAND PP2A TUA TUB GAPDH ACT Rcl2 ‘波塞妮娜’
R. hybrida ‘Porcelina’geNorm SAND TIP UBC GAPDH PP2A ACT TUB Rcl2 TUA Normfinder GAPDH TIP UBC SAND TUB PP2A ACT Rcl2 TUA Bestkeeper PP2A UBC SAND TIP GAPDH ACT TUB Rcl2 TUA RefFinder UBC TIP SAND GAPDH PP2A TUB ACT Rcl2 TUA
下载: 导出CSV
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