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二氧化氯处理促进厚皮甜瓜果实的采后愈伤

2019-02-20郑晓渊王调兰张静荣姜红王斌毕阳

中国农业科学 2019年3期
关键词:厚皮丙烷类黄酮

郑晓渊,王调兰,张静荣,姜红,王斌,毕阳



二氧化氯处理促进厚皮甜瓜果实的采后愈伤

郑晓渊,王调兰,张静荣,姜红,王斌,毕阳

(甘肃农业大学食品科学与工程学院,兰州 730070)

【目的】研究二氧化氯(ClO2)处理对厚皮甜瓜果实采后愈伤的影响,为厚皮甜瓜的采后愈伤提供方法和理论依据。【方法】以‘玛瑙’厚皮甜瓜为试材,人工模拟损伤后,用25 mg·L-1的ClO2浸泡损伤果实10 min,于常温黑暗条件下进行愈伤。测定愈伤期间损伤果实的失重率以及损伤接种粉红单端孢果实的病情指数,通过甲苯胺蓝和间苯三酚—盐酸染色法观察聚酚软木脂、聚酯软木脂和木质素在伤口部位的积累,并用IS Capture图像软件对聚酚软木脂、聚酯软木脂和木质素积累量进行分析。测定伤口表面的色度值,分析伤口处组织愈伤期间苯丙烷代谢活性以及过氧化物酶和多酚氧化酶的活性变化。【结果】ClO2处理显著降低了损伤果实的失重率和损伤接种果实的病情指数,愈伤第7天时,处理比对照低10.3%。果实在损伤后不同时间段接种粉红单端孢,经1周培养观察,处理果实的病情指数显著低于对照,第7天时处理果实的病情指数比对照低56.9%。处理显著促进了果实伤口处聚酚软木脂、聚酯软木脂和木质素的积累,处理果实的积累量在愈伤的中后期显著高于对照,三者比对照分别高25.3%、77.7%和35.5%。愈伤期间,处理果实伤口处的值显著低于对照,值显著高于对照,在愈伤第5天时,处理果实的值比对照低6.1%,第3天时的值比对照高17.8%。处理明显提高了果实伤口处的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶活性,在愈伤第7天时,处理果实伤口处的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶活性分别高于对照34.3%、80.5%和15.7%。此外,处理果实伤口处的总酚、类黄酮和木质素含量也显著高于对照,第7天时,分别高于对照14.7%、16.8%和15.6%。【结论】ClO2处理可有效促进厚皮甜瓜果实的采后愈伤,ClO2对愈伤的促进作用与激活伤口处的苯丙烷代谢,提高POD和PPO活性,促进软木脂和木质素的积累密切相关。

ClO2;厚皮甜瓜;采后;愈伤

0 引言

【研究意义】厚皮甜瓜(L.)是我国西北地区特色水果,由于果实个体较大,在采收和采后过程中易受机械损伤[1],而机械损伤造成的表面伤口为病原物的侵染提供了通道,加剧了采后腐烂的发生[2]。因此,有效降低伤口性病原菌的侵染率是采后厚皮甜瓜亟待解决的问题。【前人研究进展】不同果实表面形成的伤口具有不同程度的愈合能力,通过在伤口部位积累软木脂和木质素等具有保护作用的天然聚合物[3],从而抑制伤口部位水分的大量蒸腾,阻止病原物经由伤口的侵入[4]。近期研究发现,某些化学药物还具有促进伤口愈合的作用。例如,苯丙噻重氮可以促进采后梨果实的愈伤[5],脱落酸能提高采后番茄[4]和猕猴桃[6]果实的愈伤能力。ClO2是国际公认的A1级安全高效消毒剂,可杀灭病原物,对果蔬风味和品质无明显影响[7-8]。有报道表明,ClO2处理可减轻龙眼[9-10]和番茄果实[11]的采后病害,延缓苹果成熟衰老,减轻采后腐烂[12],还可一定程度上抑制鲜切哈密瓜的后熟[13]。而ClO2在减轻采后病害中的作用与增强果实苯丙烷代谢和提高氧化酶活性密切相关[14]。【本研究切入点】虽然已有ClO2诱导采后果实抗病性的报道,但该化合物是否影响厚皮甜瓜果实采后愈伤尚未见报道。【拟解决的关键问题】本研究以‘玛瑙’厚皮甜瓜果实为试材,用ClO2处理人工损伤的果实后在常温条件下进行愈伤,测定愈伤期间损伤果实的失重率以及接种果实的病情指数,观察愈伤组织的色度以及聚酚软木脂、聚酯软木脂和木质素的积累变化。分析氧化酶和苯丙烷代谢关键酶活性及其代谢产物的含量。评价ClO2处理对厚皮甜瓜果实采后愈伤能力的影响,为ClO2处理在厚皮甜瓜的采后应用提供方法和理论依据。

1 材料与方法

1.1 材料与设备

供试‘玛瑙’甜瓜于2017年7月采自甘肃省民勤县收成乡露地大田,选取八成熟、外观整齐、大小一致、无病虫伤和机械伤的果实,单果套网套后装入瓦楞纸包装箱,于当天运抵实验室,在常温下(20—25℃,RH 70%—80%)贮藏待用。

粉红单端孢()为甘肃厚皮甜瓜产区最常见的采后病原真菌[15],由本实验室提供,于PDA培养基上保存待用。

ClO2购自天津张大科技有限公司,有效浓度120 mg·g-1,于4℃冰箱保存。

刮皮刀(HF036型,阳江市阳东区焦点刀具有限公司,中国);恒温培养箱(SPX-30085H-II型,上海新苗医疗器械制造有限公司,中国);超净工作台(SW-CJ-2FD型,苏净集团苏州安泰空气技术有限公司,中国);立式压力蒸汽灭菌锅(LDZX-30KBS 型,上海申安医疗器械厂,中国);正置万能显微镜(CX21FS1C型,OLYMPUS公司,日本);Ci6x分光光度仪(Ci6x型,日本爱色丽有限公司,日本);台式高速冷冻离心机(3K30型,Sigma公司,德国);紫外-可见光分光光度计(UV-2450 型,岛津,日本)。

1.2 方法

1.2.1 果实人工损伤及愈伤 参照姜红等[16]的方法并进行修改。果实先用清水冲洗,然后用1%的次氯酸钠浸泡1 min进行表面消毒,再用无菌水冲洗,晾干后用刮皮刀在果实的赤道部位分别刮出4条长30 mm、宽30 mm、深2 mm的伤口。在室温条件下暴露0.5 h后,将损伤的果实浸入25 mg∙L-1的ClO2浸泡10 min,取出晾干后分别装入打孔的聚乙烯保鲜袋(25 cm×40 cm,厚度0.02 mm),于常温、避光条件下进行愈伤,以清水处理作对照。每处理用果实120个,重复3次。

1.2.2 愈伤效果的评价

1.2.2.1 失重率及病情指数的测定 失重率的测定采用重量法[17]。每处理用果实9个,重复3次。

病情指数的测定参照姜红等[16]的方法并修改。在培养了1周的培养皿中加入一定量的无菌水,用涂布器刮下孢子用4层纱布过滤至锥形瓶中,在振荡器上振荡15 s,经过血球计数板计数配置成浓度为1×106个/mL的孢子悬浮液。分别在果实损伤后的第0、1、3、5、7天,用涂布器将20 μL配好的孢子悬浮液均匀涂于创口表面,晾干后装入打孔的聚乙烯保鲜袋中,常温培养7天后统计病情指数。每个处理用果实8个,重复3次。

式中,发病级别的标准为:4级,创口表面全部发病;3级,创口表面3/4的面积发病;2级,创口表面1/2面积发病;1级,创口表面1/4面积发病;0级,创口表面不发病。

1.2.2.2 聚酚软木脂、聚酯软木脂和木质素沉积的观察 聚酚软木脂(suberin poly phenolic,SPP)和聚酯软木脂(suberin poly aliphatic,SPA)的沉积观察参照Lulai[18-19]的方法并修改。用不锈钢刀片垂直伤口表面切成厚0.2—0.3 mm,长和宽各为1 cm左右的薄片。采用如下步骤进行染色:用0.1%小檗碱(0.05%甲苯胺蓝)染色45 min后,先吸去染料,再用蒸馏水和75%酒精洗2—3遍,最后用95%酒精洗1—2遍,即脱去染料,紧接着在0.25%甲苯胺蓝(1%中性红)中放置1—2 min进行复染,最后用蒸馏水和75%酒精洗去染料,SPP(SPA)即染为紫蓝色。将染好色的薄片置于载玻片上,在显微镜下荧光观察拍照。每个果实切片4处,重复3次。

木质素的沉积观察参照Alba等[20]的方法并修改。用不锈钢刀片垂直伤口表面切成厚0.2—0.3 mm,长和宽各为1 cm左右的薄片,滴加1%间苯三酚染色1.5 min后再加1—2滴浓盐酸,木质素即染为红色,置于显微镜下观察拍照。每个果实切片4处,重复3次。

愈伤组织的SPP、SPA和木质化细胞层的厚度根据文献[21]的方法通过IS Capture图像软件进行测量计算。

1.2.3 愈伤组织色度的测定 在愈伤的0、1、3、5和7 d用Ci6x分光光度仪垂直于愈伤组织表面进行色度的测定,依次测定、和值,每个处理测12处愈伤组织。

1.2.4 生化测定取样 参照Bi等[22]的方法。在愈伤的0、1、3、5和7 d,用不锈钢刀片垂直伤口表面下取2—3 mm深的伤口组织3 g,用锡箔纸包好后用液氮冷冻,在-80℃超低温冰箱中保存备用。

1.2.5 苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶的活性测定 苯丙氨酸解氨酶(phenylalnine ammonia- lyase,PAL)的测定参照Liu等[23]的方法并修改。取冷冻样品3 g,于5 mL硼酸-硼砂缓冲液(pH 8.8,含40 g∙L-1聚乙烯吡咯烷酮(polyvinyl pyrrolidone,PVP),2 mmol∙L-1乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)和5 mmol∙L-1β-巯基乙醇)中冰浴研磨成浆,在4℃、12 000×条件下离心30 min,上层酶液即为粗酶液。反应体系包括:0.1 mL粗酶液,3 mL硼酸-硼砂缓冲溶液(50 mmol∙L-1、pH 8.8)液,0.5 mL食物L-苯丙氨酸(20 mmol∙L-1),以蒸馏水为参比,测定反应体系混合10 s后在290 nm波长处的吸光值作为初始值(OD0),将混合液在37℃水浴锅中保温1 h后在290 nm波长处的吸光值作为终止值(OD1)。以每小时吸光值变化值增加0.01为一个酶活性单位(U),以U·g-1FW表示。

过氧化物酶(peroxidase,POD)和多酚氧化酶(polyphenol oxidase,PPO)的测定参照LI[24]的方法。取冷冻样品3 g,于5 mL乙酸-乙酸钠缓冲液(pH 5.5,含1 mmol∙L-1聚乙二醇(polyethylene glycol,PEG),4%交联聚乙烯吡咯烷酮(crosslinking polyvingypyrrolidone,PVPP)和1%聚乙二醇辛基苯基醚(Triton X-100))中研磨成浆,在4℃、12 000×条件下离心30 min,收集上层液用即为粗酶液。POD反应体系:3 mL 25 mmol∙L-1愈创木酚,0.1 mL酶提取液,0.2 mL H2O2(5 mmol∙L-1)。以蒸馏水为参比,在反应进行到15 s时测定混合液在470 nm波长处的吸光值2 min。以每分钟吸光值变化值增加1为一个酶活性单位(U),以U·g-1FW表示,重复3次。PPO反应体系:4 mL的乙酸-乙酸钠缓冲液(50 mmol∙L-1、pH 5.5),1 mL邻苯二酚溶液(50 mmol∙L-1),0.1 mL酶提取液。以蒸馏水为参比,在反应进行到15 s时测定混合液在420 nm波长处的吸光值2 min。以每分钟吸光值变化值增加1为一个酶活性单位(U),以U·mg-1FW表示。

1.2.6 总酚、类黄酮和木质素的含量测定 总酚和类黄酮的测定参照Pirie等[25]的方法并作修改。取冷冻样品3 g,于预冷的4 mL HCL-甲醇溶液中冰浴研磨成浆,在4℃避光条件提取20 min,期间摇动数次,过滤收集上层清液待用。以1% HCL-甲醇溶液做为参比,分别测定滤液在280 nm和325 nm波长处的吸光度值作为总酚和类黄酮的含量,分别以OD280·g-1FW和OD325·g-1FW表示。

木质素的含量测定参照YIn等[26]的方法进行测定。取冷冻样品3 g,于预冷5 mL 95%乙醇中研磨成浆,在4℃,14 000×条件下离心30 min,弃去上清液,将沉淀物依次用95%乙醇,乙醇(V)﹕正己烷(V)=1﹕2冲洗3次,将清洗后的沉淀物在60℃烘箱中干燥24 h后转移至离心管中,溶于1 mL 25%溴化乙酰冰醋酸溶液,70℃恒温水浴30 min后加入1 mL NaOH(2 mol∙L-1)终止反应。最后加入2 mL冰醋酸和0.1mL盐酸羟胺(7.5 mol∙L-1),在4℃、12 000×条件下离心30 min,取上清液0.5 mL并用冰醋酸定容至5 mL,在280 nm波长处测定吸光值,木质素含量以OD280·g-1FW表示。

1.3 数据统计

上述测定均重复3次。全部数据用Excel 2010计算平均值和标准误(±SE),用SPSS 19.0进行Duncan’s多重差异显著性分析及相关性分析(<0.05)。

2 结果

2.1 ClO2处理对愈伤期间果实失重率和病情指数的影响

愈伤期间,处理和对照果实的失重率均逐渐升高,但处理果实的失重率显著低于对照,第7天时,比对照低10.3%(<0.05)(图1-A)。处理和对照果实的病情指数均随愈伤时间的延长逐渐下降,处理果实显著低于对照,第7天时,仅如对照的43.1%(<0.05)(图1-B)。失重率和病情指数的结果表明,ClO2处理有效促进了厚皮甜瓜果实的采后愈伤。

2.2 ClO2处理对果实伤口处SPP、SPA和木质素积累的影响

愈伤期间,处理和对照果实伤口处的SPP和SPA积累量均逐渐增加,处理果实的积累量在愈伤的中后期均显著高于对照(图2-A、B)。SPP和SPA的积累差异分别始于第1天和第3天,第7天时SPP和SPA的积累厚度分别比对照高25.3%和77.7%(<0.05)(图3-A、B)。处理和对照果实伤口处的木质素积累始于愈伤中期,处理显著高于对照。在第7天时,处理果实木质素的积累厚度比对照高35.5%(<0.05)(图3-C)。SPP、SPA和木质素的积累结果表明,ClO2有效促进了厚皮甜瓜果实伤口处的木栓化。

*代表显著性差异(P<0.05)。下同 * indicate significant differences (P<0.05). The same as below

P:聚酚软木脂Suberin poly phenolic;A:聚酯软木脂 Suberin poly aliphatic;L:木质素 Lignin

图3 ClO2处理对伤口处SPP(A)、SPA(B)和木质化(C)细胞层厚度的影响

2.3 ClO2处理对果实伤口处色度的影响

愈伤期间,处理和对照果实伤口处的值均先上升后下降,在愈伤的后期显著低于对照,第5天和第7天时,分别比同期对照低6.1%和5.8%(<0.05)(图4-A)。处理和对照果实的值差异不显著(结果未显示)。两者值总体先降后升,在愈伤的前期和中期显著高于对照。第3天时,比对照高17.8%(<0.05)(图4-B)。

图4 ClO2处理对伤口处的L*值(A)和b*值(B)的影响

2.4 ClO2处理对伤口处PAL、POD和PPO活性以及总酚、类黄酮、木质素含量的影响

愈伤期间,处理和对照果实伤口处的PAL活性均逐渐升高,但处理果实的PAL活性显著高于对照,第7天时,比对照高34.3%(<0.05)(图5-A)。对照果实的POD和PPO活性随愈伤时间的延长逐渐升高,而处理果实的活性则先略有降低后显著升高,在愈伤的后期显著高于对照,第7天时,POD和PPO活性分别比对照高80.5%和15.7%(<0.05)(图5-B、C)。处理果实伤口处的总酚、类黄酮和木质素含量在愈伤期间均呈先降后升的趋势,在愈伤的后期显著高于对照。第7天时,分别比对照高14.7%、16.8%和15.6%(<0.05)(图5-D、E、F)。PAL、POD和PPO活性以及总酚、类黄酮和木质素含量的增加结果表明,ClO2激活了厚皮甜瓜果实伤口处的苯丙烷代谢以及氧化酶活性。

3 讨论

ClO2可通过增强苯丙烷代谢和提高氧化酶活性来诱导果实的采后抗病性[12]。本研究发现,ClO2处理可通过激活采后厚皮甜瓜果实伤口处的苯丙烷代谢及氧化酶活性,加速软木脂和木质素在伤口处的沉积,从而促进厚皮甜瓜果实的采后愈伤。

苯丙烷代谢可合成愈伤组织形成所需的多种次生代谢产物,在果实愈伤中具有积极的作用[27]。PAL是苯丙烷代谢的限速酶[4],可催化苯丙烷代谢的第一步反应,使L-苯丙氨酸脱氨生成反式肉桂酸[27],反式肉桂酸又会进一步转化生成多酚和类黄酮以及木质素等愈伤组织的基本成分[28-29]。PAL的活性高低与果实的愈伤能力显著相关[6],果实的愈伤能力越强,其PAL活性就越高。本研究发现,ClO2处理显著提高了厚皮甜瓜伤口处PAL活性,该结果与赵明惠等[12]采用ClO2处理苹果后观察到的结果类似。苯丙烷代谢产生的肉桂酸、富马酸和咖啡酸等酚类物质[29],在细胞质内合成后被运送至细胞壁,与肉桂酸羟化酶催化形成的羟基肉桂酸和羟基肉桂酰醇在POD和H2O2的作用下氧化交联并开始沉积,形成SPP[30]。此外,伤口处的脂肪代谢也发生了显著的变化[31]。细胞内一些新的脂肪酸单体开始合成,主要包括超长链脂肪酸、-链烷醇、-羟基脂肪酸和,-二酸等,这些脂肪酸单体经质膜上的ATP结合通道转运蛋白被运送至膜外,在质膜和细胞壁间聚合形成SPA[30-31]。但这些脂肪酸单体或低聚物如何和甘油组装形成SPA尚有待揭示。在番茄果实上的研究结果表明,SPP和SPA都对病原物具有抵抗作用,但两者之间存在着差异。其中,SPP对细菌性病害有防御作用,SPA对真菌性病害有抵抗作用[32]。至于SPP和SPA在甜瓜果实愈伤中的功能是否与番茄一致尚有待证实。本研究观察到,ClO2处理促进了SPP、SPA以及木质素的积累,对于SPP的促进作用要先于SPA和木质素。苯丙烷代谢形成的4-香豆酸、阿魏酸和芥子酸等是木质素的合成前体,这些酚酸首先在CAD的作用下还原,形成相应的醇后再通过POD的作用聚合为木质素[33]。木质素是苯丙烷代谢的终产物,是细胞壁次生壁的组成物质,木质素在保持水分和维持细胞结构稳定中发挥着重要作用[28]。木质素也是伤口周皮的主要成分,可使细胞壁更加坚固[34],从而在伤口表面形成一个有效的物理屏障,限制病原物从伤口处获取营养,提高果实对病原物侵染的抵抗能力[35]。黄酮类物质在伤口处的含量随PAL活性的提高而增加[6]。黄酮类物质作为抗氧化剂,具有较强的抗氧化和自由基清除能力,可直接抑制真菌的孢子萌发和菌丝生长[33]。本研究发现,ClO2处理在愈伤后期显著提高了POD活性,促进了总酚、类黄酮和木质素的积累。但在愈伤前期,ClO2处理的POD活性以及总酚、类黄酮和木质素含量均显著低于对照,这可能与ClO2抑制POD活性和苯丙烷代谢相关。

图5 ClO2处理对厚皮甜瓜果实伤口处PAL(A)、POD(B)和PPO(C)活性以及总酚(D)、类黄酮(E)和木质素(F)含量的影响

本研究发现,ClO2处理可显著提高厚皮甜瓜果实伤口处的PPO活性。处理果实伤口处值高于对照,值低于对照的结果表明,PPO参与了愈伤组织的形成,处理果实伤口处的值在0 d显著高于对照可能源于ClO2的漂白作用。在愈伤中由于细胞内膜被破坏,液泡中的酚类底物会和PPO发生反应,氧化为醌,醌再进一步聚合为黑色或褐色的物质[36]。这些聚合物不仅引起果实组织褐变,而且可直接抑制病原菌生长,钝化病原菌分泌的胞外酶[37],该结果与Wei[38]等在猕猴桃愈伤期间观察到的结果一致。

4 结论

ClO2采后处理可有效降低损伤果实的失重率和损伤接种果实的病情指数,促进厚皮甜瓜的采后愈伤。ClO2处理对采后厚皮甜瓜愈伤的促进作用与激活果实苯丙烷代谢,提高POD和PPO活性,促进SPP、SPA和木质素在伤口处的积累密切相关。

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Using Chlorine Dioxide Treatment to Promote Wound Healing of Postharvest Muskmelon Fruit

ZHENG XiaoYuan, WANG TiaoLan, ZHANG JingRong, JIANG Hong, WANG Bin, BI Yang

(College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070)

【Objective】The aims of this study were to investigate effect of chlorine dioxide (ClO2) treatment on the wound healing of harvested muskmelons and its mechanism, providing methods and theoretical basis for wound healing of postharvest muskmelon fruit.【Method】The muskmelon fruit ‘cv. Manao’ was used as material. After artificially wounded, fruits were dipped with ClO2at 25 mg∙L-1for 10 min, and the treated fruit and control were wound healed at ambient temperature in dark. The weight loss of fruit and the disease index ofinoculated fruit were measured during healing. The accumulation of suberin poly phenolic, suberin poly aliphatic and lignin at the wounded sites of fruit were observed by Toluidin blue O-neutral red staining and phloroglucinol-HCl staining method, and the amount of accumulation of the three compositions were measured by IS Capture image software. Moreover, the color values of wounded surface were measured. The enzyme activities of phenylpropanoid metabolism and changes of peroxidase and polyphenol oxidase enzyme activities were analyzed during the fruit wound healing stage. 【Result】The weight loss of wounded fruit and the disease index of inoculated fruit were significantly reduced by ClO2treatment. The weight loss of treated fruit was 10.3% lower than that of control after 7 days of healing. Wounded fruit were inoculated byat different wound healing periods. After one week cultivated, the disease index of the treated fruit was significantly lower than that of control, which was 56.9% lower at 7 days of healing. The ClO2treatment significantly promoted the accumulation of suberin poly phenolic, suberin poly aliphatic and lignin. The treated fruit were significantly higher than that of control at the mid and late stage of healing. After 7 days of healing, the thickness of suberin poly phenolic cell layers, suberin poly aliphatic and lignin cell layers of treated fruit was 25.3%, 77.7% and 35.5% higher than that of control, respectively. Compared with the control, thevalue of wounded surface in the treated fruit was significantly lower andvalue was significantly higher during healing. Thevalue of the treated fruit was 6.1% lower than that of control after 5 days of healing. And thevalue of the treated fruit was 17.8% higher than that of control after 3 days of healing. The ClO2treatments increased enzymes activities of phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase at wounded sites of fruit, which were 34.3%, 80.5% and 15.7% higher than that of control after 7 days of healing, respectively. Meanwhile, the treatment also improved the accumulation of total phenols, flavonoids and lignin at wounded sites, which were 14.7%, 16.8%, and 15.6% higher than that of control after 7 days of healing, respectively.【Conclusion】ClO2treatment effectively promoted wound healing of harvested muskmelons by eliciting the phenylpropanoid metabolism. In addition, it also increased the enzymes activities of peroxidase and polyphenol oxidase, and promoted accumulation of suberin and lignin at wounded sites.

chlorine dioxide; muskmelons; postharvest; wound healing

10.3864/j.issn.0578-1752.2019.03.011

2018-07-18;

2018-09-13

国家公益性行业(农业)科研专项(201303075)

郑晓渊,Tel:18894310260;E-mail:1427426541@qq.com。通信作者毕阳,Tel:13119421362;E-mail:biyang@gsau.edu.cn

(责任编辑 赵伶俐)

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