低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响

低温胁迫下嫁接对黄瓜叶片S OD 和CAT基因表达与活性变化的影响*高俊杰1,2秦爱国1于贤昌1**(1山东农业大学园艺科学与工程学院作物生物学国家重点实验室,山东泰安271018;2泰安市农业局,山东泰安271000)摘 要 研究了低温胁迫下嫁接和自根黄瓜叶片M n -SOD 、Cu /Zn -SOD 和C AT mRNA 基因表达和酶活性变化及其与抗冷性的关系.结果表明:低温胁迫下,嫁接与自根黄瓜叶片Cu /Zn -SOD 、M n -SOD mRNA 基因相对表达量变化分别与其Cu /Zn -SOD 、M n -SOD 活性变化相吻合,而CATmRNA 相对表达量变化与其C AT 活性变化并不一致;嫁接黄瓜叶片Cu /Zn -SOD 和M n -SOD mRNA 相对表达量及SOD 、Cu /Zn -SOD 和M n -SOD 活性均高于自根黄瓜,MDA 含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜较高的SOD 基因表达量调控的较高SOD 活性是其抗冷性强于自根黄瓜的主要因素;嫁接黄瓜的功能叶CAT mRNA 相对表达量略高于自根黄瓜,而幼叶C AT mRNA 相对表达量低于后者,但两者CAT 活性差异不大,说明低温胁迫对嫁接黄瓜叶片C AT mRNA 相对表达量及CAT 活性的影响不大.关键词 嫁接黄瓜 低温胁迫 SOD C AT MDA 文章编号 1001-9332(2009)01-0213-05 中图分类号 S642.2 文献标识码 AEffects of grafting on cucu m ber leaf S OD and CAT gene expression and activities under lowte m perat ure stress .GAO Jun -jie 1,2,Q IN A -i guo 1,YU X ian -chang 1(1State K ey Laborator y of C rop B iology ,Colle g e of H orticult u re Science and Eng i n eering,Shandong Agricult u ral Universit y ,Tai .an 271018,Shandong,Ch i n a;2Tai .an M unici p al Bureau o f Agriculture ,Tai .an 271000,Shandong,China ).-Chin.J.App l .E col .,2009,20(1):213-217.A bstract :This paper stud i e d the re lative expressi o n ofM n-SOD,Cu /Zn-SOD and C AT mRNA s and t h e changes o f SOD,M n -SOD,Cu /Zn-SOD and CAT activ ities i n g rafted and o w n -r ooted c u -c um ber leaves under lo w te m perat u re stress ,and the ir relations w ith the cold resistance of cucum -ber .For bo t h grafted and o w n -rooted cucumber leaves ,the relati v e expressi o n o f M n -SOD and Cu /Zn -SOD mRNAs under lo w te m perat u re stress w as respecti v ely accor dance w it h t h e changes o f M n -SOD and Cu /Zn -SOD activ iti e s ,w h ile the expressi o n o f CAT mRNA w as not accordance w ith the change of CAT activ ity .The relative expressi o n o fM n -SOD and Cu /Zn -SOD mRNA s and the activ ities o f SOD,M n -SOD and Cu /Zn -SOD i n grafted cucu m ber leaves w ere higher than those i n o w n -r ooted cucumber leaves ,w hile the MDA content and e lectrolytic l e akage w ere in adverse .The h i g her SOD activ ity regulated by t h e h i g her SOD mRNA s expression i n grafted cucu m ber leaves m i g ht be the key factor o f grafted cucum ber hav i n g a higher cold resistance to l o w te m perat u re stress than o wn-roo ted cucum ber .The relati v e expression o f C AT mRNA w as slightly h i g her i n functi o na l leaves but l o w er i n young leaves of g rafted cucu m ber ,wh ile less difference w as observed i n CAT ac -tiv ity ,co m pari n g w ith own -rooted cucum ber ,w hich ill u strated t h at l o w te m perature stress had lesser effects on the relati v e expression ofCAT mRNA and the activ ity of CAT i n grafted cucum ber leaves .K ey words :grafted cucum ber ;lo w te m perature stress ;SOD ;C AT ;MDA.*国家自然科学基金项目(30571271)和山东省自然科学基金资助项目(Y2005D05).**通讯作者.E-m ai:l xcyu@s d au .edu .c n 2008-06-23收稿,2008-10-29接受.低温胁迫下,植物体产生的大量活性氧可破坏叶片叶绿体、细胞膜、蛋白质及核酸等[1-4],而超氧化物歧化酶(SOD)和过氧化氢酶(CAT)等抗氧化酶对防御活性氧毒害具有重要作用[5-6].研究表明,与自根黄瓜相比,以黑籽南瓜为砧木的嫁接苗的抗冷性可增强约3e[7-8],且嫁接苗抗氧化酶活性的增应用生态学报 2009年1月 第20卷 第1期 Ch i nese Journa l of A pp lied E co logy ,Jan .2009,20(1):213-217强是其抗冷性增强的主要原因之一[9-10],但嫁接增强黄瓜抗冷性的分子机制研究尚未见报道.为此,本试验从基因转录和酶活性水平,研究了低温胁迫下黄瓜嫁接苗和自根苗叶片Cu/Zn-SOD、M n-SOD和CAT基因表达和酶活性变化及其与抗冷性的关系,以探讨嫁接增强黄瓜抗冷性的分子机制.1材料与方法111试验设计试验于2007年3)5月在山东农业大学作物生物学国家重点实验室进行.供试黄瓜品种为-津绿3号.(Cucum is sativus L.cv.Jun lv No.3),砧木为黑籽南瓜(Cucurbita f icifolia Bouche),黄瓜和南瓜种子均在25e催芽后,取芽长一致的种子播于10c m@ 10c m(直径@高度)的营养钵中.营养钵育苗基质为草炭B蛭石B鸡粪=3B2B1(体积比).当黄瓜和黑籽南瓜心叶初显时,以黄瓜为接穗,分别以黑子南瓜和黄瓜为砧木进行嫁接.当黄瓜/黑籽南瓜嫁接苗(嫁接黄瓜)和黄瓜/黄瓜嫁接苗(自根黄瓜)长至7叶1心时,同时移入人工气候室进行处理.移入人工气候室的第1天至第2天为15e/ 10e(昼/夜)预处理,第3天至第5天为5e低温胁迫处理,处理期间的光照时间为11h,光照强度为55163L m o l#m-2#s-1.低温胁迫0、24和48h,分别选取长势一致的嫁接和自根黄瓜幼叶(上数第1叶和第2叶)和功能叶(上数第4叶和第5叶)测定各项指标.嫁接黄瓜的幼叶和功能叶分别标记为GY和GG,自根黄瓜的幼叶和功能叶分别标记为OY和OG.试验各处理均3次重复.样品经液氮冷冻后,于-80e超低温保存.112测定项目与方法11211RNA提取取011g黄瓜叶片于液氮中研磨成粉末状,以Tr izo l试剂(Sangon公司)提取黄瓜叶片总RNA(上海生工T rizo l试剂盒说明),在-80e 保存.11212荧光定量PCR(rea-l ti m e quan titati v e RT-PCR)采用SYBR Pri m eScri p t TM RT-PCR K it(Ta K a Ra)试剂盒,利用The L i g h t C ycler115仪器(Roche公司)测定Cu/Zn-SOD、M n-SOD和C AT基因表达量,采用的内参为a l p ha-tubulin.荧光定量PCR引物由Ta Ka R a 公司合成,分别为:CuF:5.-GACTGGGCC A-C ATTTCAACC-3.,CuR:5.-GCCTTGCCATC TTC AC-C AA-3.(EF121763),M nF:5.-CAATGGCGGAGGTC A-C ATTA-3.,M nR:5.-AGAGCAAGCCACACCCATC-3.(EF203086),CF:5.-AATGGCCGGAGGATGTGA-3., CR:5.-CC AACGACATAGAGAAAGCC AAC-3.(EF468-517),检测al p ha-tubuli n的引物为aF:5.-C ACTA CACCGTTGGAAAGGAAA-3.,a R:5.-C AAAAGGAG-GGAGCCGAGA-3..11213SOD及其同工酶活性测定参照Prochazkova 等[11]的方法:取叶片015g,加入115m l磷酸缓冲液(0105m o l#L-1,pH=718),冰浴研磨后,再加115m l磷酸缓冲液,倒入离心管中,再用2m l缓冲液洗钵,低温(0e~4e)4000r pm离心20m i n,上清液即为酶粗提液.SOD活性用NB T还原法[10]测定:3m l反应液(内含217m l144mmo l#L-1的蛋氨酸,011m l2215L m ol#L-1氮蓝四唑,011m l3L m o l #L-1的EDTA-N a2,011m l60L m ol#L-1的核黄素)加入20L l酶提取液,72L m ol#m-2#s-1光照下反应30m i n,于560n m比色,以抑制NBT光化还原的50%为一个酶活性单位;Cu/Zn-SOD和M n-SOD活性的测定方法为:在提取液中加入5mm o l#L-1 KCN,由于Cu/Zn-SOD对KC N敏感,故只能检测出M n-SOD和Fe-SOD,在提取液中加入5mm o l#L-1 KCN和10mmo l#L-1H2O2,由于Cu/Zn-SOD和M n-SOD对其敏感,故只能检测出Fe-SOD.用SOD 活性减去加入5mm ol#L-1KCN后测得的SOD活性即得出Cu/Zn-SOD活性,用加入5mm o l#L-1 KCN后SOD活性减去加入5mm o l#L-1KCN和10mm ol#L-1H2O2后测得的SOD活性即得出M n-SOD活性.11214C AT活性、MDA含量和电解质渗漏率测定CAT活性采用比色法[12]测定,MDA含量采用巴比妥酸显色法[13]测定,电解质渗漏率采用OR I W TDS 电导仪[13]测定.113数据处理采用M icrosoft Exce l软件处理数据和作图,采用DPS统计软件中的Duncan新复极差法对数据进行统计分析.2结果与分析211低温胁迫下嫁接黄瓜叶片SOD基因的相对表达与SOD活性变化21111SOD活性由图1可知,与自根黄瓜相比,低温胁迫下嫁接黄瓜幼叶和功能叶的SOD活性明显增强,且以功能叶的增强尤为明显,胁迫48h时,嫁接黄瓜幼叶和功能叶SOD活性分别较自根黄瓜增加了3013%和8111%.低温胁迫下,嫁接黄瓜幼叶214应用生态学报20卷图1低温胁迫下嫁接和自根黄瓜叶片SOD活性的变化F i g.1Chang es of SOD acti v iti es i n g ra fted and own-rooted cu-cu m be r leaves under l ow temperature stress.GY:嫁接黄瓜幼叶Young leaf of grafted cu cu m ber;GG:嫁接黄瓜功能叶Fun cti on al leaf of grafted cucumb er;OY:自根黄瓜幼叶Young l eaf of o w n-rooted cucumb er;OG:自根黄瓜功能叶Functi onal leaf of o w n-rooted cucumb er.下同The s a m e b el o w.和功能叶SOD活性均呈明显增强趋势,而自根黄瓜幼叶和功能叶SOD活性增强不明显.21112M n-SOD mRNA表达与M n-SOD活性由图2可知,在低温胁迫24h时,除嫁接黄瓜功能叶M n-SOD mRNA相对表达量有所增加外,其他叶片与胁迫前相比变化不大;在低温胁迫48h时,嫁接和自根黄瓜幼叶和功能叶M n-SOD mRNA相对表达量均明显增加,但前者的增幅明显大于后者,且以功能叶的表现更为明显.在低温胁迫下,嫁接和自根黄瓜幼叶和功能叶M n-SOD活性的变化趋势与M n-SOD mRNA相对表达量的变化趋势基本一致.21113Cu/Zn-SOD mRNA表达与Cu/Zn-SOD活性低温胁迫下,嫁接和自根黄瓜幼叶及功能叶Cu/ Zn-SOD mRNA相对表达量均呈先增加后下降趋势,但前者的变幅明显高于后者,且以功能叶的表现更为明显,胁迫24h时分别提高6115%和1113%, 48h时分别提高3914%和7913%.低温胁迫下,嫁接和自根黄瓜幼叶和功能叶Cu/Zn-SOD活性的变化趋势与Cu/Zn-SOD mRNA相对表达量的变化趋势基本一致(图2).212低温胁迫下嫁接黄瓜叶片C AT mRNA基因的相对表达与CAT活性变化由图3可知,低温胁迫下,嫁接和自根黄瓜幼叶及功能叶CAT mRNA相对表达量均明显增加,前者功能叶的CAT mRNA相对表达量明显高于后者,胁迫24h和48h,分别提高6512%和4718%;而幼叶CAT mRNA相对表达量则明显低于后者,胁迫24h 和48h,分别降低1414%和716%.低温胁迫下,嫁图2低温胁迫下嫁接和自根黄瓜叶片M n-SOD、Cu/Zn-SOD mRNA相对表达量和M n-S OD、Cu/Zn-SOD活性变化Fig.2Chang es o f M n-S OD mRNA,Cu/Zn-SOD mRNA re l a-tive expressi on and M n-S OD,Cu/Zn-SOD acti v ities in g ra fted and o w n-roo ted cucu m be r leaves under l ow temperature stress.接和自根黄瓜幼叶及功能叶C AT活性均无明显变化,且两者差异不大.213低温胁迫下嫁接黄瓜叶片MDA含量和电解质渗漏率的变化由图4可知,低温胁迫下,除嫁接黄瓜功能叶MDA含量的升高趋势不明显外,嫁接和自根黄瓜叶片MDA含量均呈升高趋势,且后者较前者更为明显.胁迫48h,自根黄瓜幼叶和功能叶MDA含量分别提高5512%和5618%,而嫁接黄瓜幼叶和功能叶仅分别提高3714%和1317%.低温胁迫下,嫁接黄瓜幼叶和功能叶电解质渗漏率均分别低于自根黄瓜,胁迫24h时分别降低1710%和1718%,48h2151期高俊杰等:低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响时分别降低2312%和2813%.3讨论有研究表明,部分抗氧化酶基因表达量的增加可提高植物的抗冷性[14].M c K ersie等[15]研究认为,在转基因的紫花苜蓿中,过量表达烟草M n-SOD基因的植株的抗冷性明显提高;Gupta等[16]也报道,在烟草中Cu/Zn-SOD基因的过量表达可提高其对环境胁迫的忍受力.在逆境胁迫条件下,植物通常提高一种或多种抗氧化酶的活性,这些酶活性水平的提高与植物对胁迫条件的忍受力密切相关[17-19].在植物体中,SOD主要清除O2-#,生成O2和H2O2,后者被C AT进一步分解为H2O和O2,在叶绿体中这步反应被APX催化[20-21].因此,植物体内SOD活性增强,可以有效清除O2-#等活性氧自由基,减少细胞膜受损伤及细胞内物质的渗漏程度.本试验中,嫁接黄瓜幼叶及功能叶Cu/Zn-SOD、M n-SOD mRNA相对表达量及SOD、Cu/Zn-SOD和M n-SOD活性均分别高于自根黄瓜,其幼叶和功能叶M DA含量和电解质渗漏率均分别低于自根黄瓜,说明低温胁迫下嫁接黄瓜较高的Cu/Zn-SOD和M n-SOD相对表达量调控的较高SOD活性是其抗冷性强于自根黄瓜的主要因素之一.嫁接黄瓜功能叶C AT mRNA相对表达量明显高于自根黄瓜,而幼叶C AT mRNA相对表达量低于自根黄瓜,两者CAT活性差异不大,说明嫁接对低温胁迫下黄瓜叶片C AT mRNA表达及C AT活性的影响较小.但低温胁迫下,嫁接黄瓜幼叶和功能叶MDA含量和电解质渗漏率均分别低于自根黄瓜,表现出膜脂过氧化程度与C AT活性不一致甚至相反的现象,这是因为在低温胁迫下,植物体内导致细胞膜损伤及细胞内物质渗漏的H2O2并不完全由CAT 清除,其他清除机制如APX催化等可能起着重要作用.CAT不足时,叶绿体中H2O2主要依赖于APX及相应的AsA-GSH循环系统来清除[22-23].低温胁迫下黄瓜嫁接苗的A s A含量明显高于自根苗[10],西瓜嫁接后耐冷性的提高与其体内活性氧清除系统中抗氧化剂含量和抗氧化酶活性的提高有关[17],均说明了这一点.与自根黄瓜相比,尽管低温胁迫下嫁接黄瓜幼叶和功能叶SOD和C AT的基因表达及活性明显增加,MDA含量和电解质渗漏率明显降低,但其幼叶的增加或降低幅度与功能叶相比均较小.说明嫁接增强黄瓜幼叶抗冷性的作用较小,这可能与其生理发育尚未完成,细胞器结构与功能尚不完善有关.参考文献[1]E lstner EF,O ss w a l d W.M echanis m s o f oxygen acti va-ti on duri ng plan t stress.Procee d ings of the Royal Societ y216应用生态学报20卷of Edinburgh,1994,102:131-154[2]P rasad TK,A nderson M D,M a rti n B A.Ev i dence forchilli ng i nduce ox ida ti ve stress i n m a i ze seedli ng s andregu latory role for hydrogen perox i de.P lant Cell,1994,6:65-74[3]Q ueiroz CGS,A l onso A,M ares-Gu i a M.Ch illi ng-i n-duce changes i n m embrane fl u i d it y and anti ox i dant en-zym e ac tiv iti es i n Coffea arabiga L.roots.B iolog iaP lantarum,1998,41:403-413[4]Scanda lios J G.O xyg en stress and superox i de d i s m uta-ses.P lant Phy 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sdaugao@责任编辑张凤丽2171期高俊杰等:低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响。