CN113957079B - MtBGLU17基因在调控植物类黄酮合成中的应用 - Google Patents
MtBGLU17基因在调控植物类黄酮合成中的应用 Download PDFInfo
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Abstract
本发明提供了MtBGLU17基因在调控植物类黄酮合成中的应用。所述MtBGLU17基因的表达产物能够催化水解类黄酮糖苷底物。本发明首次研究MtBGLU17基因在调控植物类黄酮合成的作用,MtBGLU17基因的表达产物具有糖苷水解酶的活性,能够催化水解多种类黄酮化合物。MtBGLU17能够增加植物体内黄酮苷元的含量并且增强植物对非生物胁迫的抗性。
Description
技术领域
本发明涉及MtBGLU17基因在调控植物类黄酮合成中的应用,属于基因工程应用技术领域。
背景技术
类黄酮是广泛存在于植物中的一种重要的次生代谢产物,在植物生理中发挥着重要的作用,具有抗氧化、清除自由基等作用。类黄酮不仅可作为细胞信号途径的调节因子,还参与抵抗生物和非生物胁迫。此外,类黄酮可以提高牧草品质,增加反刍动物的营养,减少青贮中蛋白的降解。对人类而言,类黄酮化合物具有较高的药物活性;还可以通过诱导人体的保护酶系统对心血管疾病、癌症及一些慢性疾病具有防御作用。
类黄酮化合物的水解过程通常是由β-糖苷水解酶(β-glucosidase,BGLU)催化水解的。糖基化有利于类黄酮化合物的储存,而植物处于逆境时,则激活了BGLU水解储存形式的糖苷化合物生成活性的类黄酮苷元抵御逆境。因此,糖基化和去糖基化过程是植物适应环境、调控体内代谢化合物平衡的重要机制,阐明类黄酮途径的水解过程对植物的生长发育及抗逆具有重要的意义。
发明内容
本发明的目的是提供MtBGLU17基因在调控植物类黄酮合成中的应用,MtBGLU17能够增加植物体内类黄酮苷元的含量并且增强植物对非生物胁迫的抗性。
根据本申请的一个方面,提供了MtBGLU17基因在调控植物类黄酮合成中的应用。
在一些具体实施方案中,所述MtBGLU17基因的表达产物能够催化水解类黄酮糖苷底物。
在一些具体实施方案中,所述类黄酮糖苷底物选自黄酮醇、黄酮、异黄酮和黄烷酮中的至少一种。
在一些优选实施方案中,所述类黄酮糖苷底物选自类黄酮7位连接的糖苷。
在一些具体实施方案中,所述MtBGLU17基因的表达产物定位在叶绿体中。
根据本申请的另一个方面,还提供了MtBGLU17基因在调控植物对非生物胁迫响应中的应用。
在一些具体实施方案中,所述MtBGLU17基因能够增强植物对非生物胁迫的抗性,所述植物对非生物胁迫的抗性选自抗旱性或耐盐性。
根据本申请的另一个方面,还提供了MtBGLU17基因在调控植物的抗氧化活性中的应用。
在一些具体实施方案中,MtBGLU17基因在提高植物自由基清除能力中的应用。
在一些具体实施方案中,所述抗氧化活性选自超氧化物歧化酶、丙二醛和过氧化氢酶活性中的至少一种。
本发明的有益效果为:
1、本发明首次研究MtBGLU17基因在调控植物类黄酮合成的作用,MtBGLU17基因的表达产物具有糖苷水解酶的活性,能够催化水解多种类黄酮糖苷化合物的水解,
2、本发明通过对MtBGLU17过表达及突变体材料进行非生物胁迫处理,发现MtBGLU17基因过表达植株比野生型及突变体植株对干旱及NaCl处理耐性更强。进一步研究抗氧化酶活性发现,干旱和NaCl处理后,MtBGLU17基因过表达植株的DPPH,CAT,SOD活性显著高于野生型及突变体植株,且MDA含量比野生型及突变体植株低。因此,MtBGLU17能够增加植物体内黄酮苷元的含量并且增强植物对非生物胁迫的抗性。
附图说明
图1为融合蛋白的SDS-PAGE胶检测结果;
图2为MtBGLU17重组蛋白对不同种类类黄酮糖苷为底物的HPLC图谱。图a、b、c、d、e、f、g、h分别代表MtBGLU17重组蛋白催化的槲皮素3-O-葡萄糖苷、槲皮素7-O-葡萄糖苷、山奈酚3-O-葡萄糖苷、山奈酚7-O-葡萄糖苷、芹菜素7-O-葡萄糖苷、木犀草苷、染料木苷和樱桃苷作为底物的HPLC图。每个图的上排表示MtBGLU17重组蛋白添加相应的底物所催化的酶活反应;而下排表示仅添加相应的类黄酮糖苷底物作为阴性对照;
图3为MtBGLU17重组蛋白对相同浓度(10mM)不同类黄酮糖苷底物的转化率;
图4为蒺藜苜蓿不同组织中MtBGLU17基因的表达水平;
图5为在烟草叶片表皮细胞中瞬时转化MtBGLU17-RFP蛋白检测亚细胞定位的结果;
图6为蒺藜苜蓿MtBGLU17转基因植株鉴定。a、MtBGLU17转基因植株的PCR鉴定,每一列代表一个转基因株系。b、蒺藜苜蓿MtBGLU17转基因的qPCR分析。c、MtBGLU17转基因植株的表型;
图7为蒺藜苜蓿MtBGLU17的Tnt1插入突变体的鉴定结果;a、突变体NF14206,NF17318在MtBGLU17基因中的插入位置示意图;b、突变体NF14206,NF17318中Tnt1插入位点的PCR鉴定;c、突变体NF14206,NF17318的花中MtBGLU17基因表达水平的半定量分析(28个循环);d、突变体NF14206,NF17318植株的图片,左边R1代突变体图片,右边是筛选的纯合体材料;
图8为MtBGLU17过表达、突变体叶片中类黄酮含量的HPLC分析;由上往下依次代表:野生型植株R108(对照);过表达植株OE28;突变体植株NF14206;Luteolin标品;Lutiolin-7-O-glucoside标品;
图9为非生物胁迫处理对MtBGLU17基因过表达和突变体植株的影响;
图10为非生物胁迫处理对MtBGLU17基因过表达和突变体植株抗氧化活性的影响。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本研究首先对蒺藜苜蓿基因组中BGLU家族的成员进行分析,共鉴定51个MtBGLU基因,并通过系统进化分析把它们分成了五个簇。氨基酸序列比对结果表明这些MtBGLU蛋白中有多个保守的、具有特征性的基序。在MtBGLU基因的启动子中发现了许多可能与非生物胁迫和植物激素有关的顺式调控元件。此外,对基因芯片数据分析表明,这些MtBGLU基因在不同的组织中表现出不同的表达模式,并对不同的非生物胁迫和激素处理产生应答。
其次,本研究对蒺藜苜蓿芯片数据库中不同组织的表达数据进行加权基因共表达网络分析(Weighted Gene Co-Expression Network Analysis,WGCNA),鉴定到了5个类黄酮特异的表达模块,筛选到了模块中特异表达的MtBGLUs基因。接着,本研究对如上候选的MtBGLUs基因在非生物胁迫及激素处理下进行qPCR验证,发现MtBGLU17、MtBGLU21、MtBGLU22、MtBGLU28这四个基因的表达水平受多种非生物胁迫和激素胁迫的显著诱导。
实施例2
MtBGLU基因的克隆及重组酶的纯化
依据MtBGLU17、MtBGLU21、MtBGLU22和MtBGLU28基因的核苷酸序列,设计了基因特异性引物(表1),并以蒺藜苜蓿的根、茎、叶、花、果荚和种子的混合cDNA为模板,根据东洋纺(上海)生物科技有限公司提供的高保真酶KOD说明书进行PCR反应。
结果获得了4个MtBGLU的目标片段,接着将目标基因构建入pMAL-C2X载体并转入大肠杆菌进行原核蛋白表达。pMAL-C2X原核表达载体的构建的方法参照宝生物工程(大连)有限公司T4DNA连接酶说明书,将载体和DNA稍加离心后,取一个灭菌的Eppendorf管,建立连接反应,其反应体系如表2,轻轻混匀,25℃反应1h以上。
表1基因克隆所用引物列表
表2T4 DNA连接酶体系
最终,通过麦芽糖融合蛋白纯化系统获得纯化的目标重组蛋白结果如图1所示。其中,原核表达样品制备纯化及活性分析的方法包括:
1、原核表达实验样品制备步骤:
1)取20μL负80度冰箱保存的pMAL-C2X-BGLUs单克隆菌液于2mL LB(含Carb100mg/L)液体培养基中,37℃过夜振荡(200rpm)培养;
2)取1.5mL过夜培养的菌液于300mL新鲜的LB液体培养基中(含Carb 100mg/L,过膜灭菌的0.4%(w/v)葡萄糖)中,37℃摇床培养至OD600nm值为0.5-0.6时,取1mL菌液作为诱导前对照;
3)在300mL菌液中加入终浓度为1/3mM IPTG(异丙基-β-D-硫代半乳苷),于16℃振荡(150rpm)培养24h;
4)4℃,8,000g离心3mins,收集菌体,并放负20度冻存。
2、原核表达蛋白纯化步骤:
根据pMAL-C2x融合蛋白纯化系统手册(New England BioLab Inc.)对重组BGLUs蛋白进行纯化,所有步骤都在0-4℃环境下进行。
1)提前半天把冻存在负20度的菌液粗蛋白拿出来放在冰上解冻,同时清洗纯化柱(含麦芽糖的colum|buffer清洗2遍,再用colum|buffer清洗4遍,流速控制在3-4s/1滴);
2)将解冻的粗蛋白进行超声破碎处理,每5ml菌液粗蛋白超声5min;
3)9,000g离心30min,上清液加入约30ml colum|buffer;
4)将稀释的蛋白提取上清液加入纯化柱中,吸附于膜上,先用column buffer冲洗2遍,在用含有麦芽糖的column buffer冲洗约20ml,并收集洗脱蛋白;
5)将洗脱下来的粗蛋白利用Millipore(30KDa)浓缩蛋白液,并4,000g离心15min最终置换成酶活反应缓冲液;
6)纯化蛋白在SDS-PAGE电泳后,经考马斯亮蓝染液进行染色,确认重组蛋白的大小。
实施例3
对目的融合蛋白进行活性分析,检测目的融合蛋白体外类黄酮β葡萄糖苷水解酶的功能。具体包括以下步骤:
1、酶活反应体系为50μL,如表3。30℃,反应30min后,用等体积甲醇中止反应,12,000rpm离心10mins。取5μL上样。
2、酶动力学分析
类黄酮糖苷准品浓度在0-1000μM范围内,设置6-7个浓度梯度进行检测。每个反应体系为50μL,加入5μg重组蛋白,30℃反应30分钟,然后加入50μL甲醇终止反应。每个反应重复三次。
3、酶活产物分析与鉴定
1)HPLC条件
流动相:A相:0.1%甲酸水溶液;B相:乙腈。
洗脱梯度:0-10mins,95%B相线性梯度降至70%B相,平衡2mins。
DAD检测波长:254nm(黄酮、异黄酮和黄烷酮)、280nm(原花色素单体)、353nm(黄酮醇)和530nm(花青素)。
表3重组蛋白酶活反应体系
本研究分别以5类(黄酮醇类、黄酮类、异黄酮类、花青素类和黄烷醇类)共11种类黄酮葡萄糖苷为底物(蒺藜苜蓿体内类黄酮主要以葡萄糖苷的形式存在),对这4个融合蛋白进行体外酶活检测(表4)。
体外酶活实验结果显示,这个四个候选的MtBGLU蛋白只有MtBGLU17对多种类黄酮底物具有活性。其中,黄酮醇类的四种底物(槲皮素-3-O-葡萄糖苷,山奈酚-3-O-葡萄糖苷,槲皮素-7-O-葡萄糖苷,山奈酚-7-O-葡萄糖苷)都可以被MtBGLU17催化水解,两种花青苷都不能被MtBGLU17催化,两种黄酮(芹菜素-7-O-吡喃葡萄糖苷,木犀草苷)都可以被MtBGLU17催化,一种异黄酮(染料木苷)和黄烷酮(樱桃甙)也可以被MtBGLU17催化生成相应产物。
表4MtBGLU17酶活实验所测试的底物及其活性列表
注:“+”代表相应的蛋白对该底物有可检测到的活性;“-”代表相应的蛋白对该底物无可检测到的活性。
MtBGLU17蛋白催化的类黄酮糖苷底物的液相色谱图如图2所示,由图2可以看出:MtBGLU17对7位连接的黄酮醇(槲皮素-7-O-葡萄糖苷,山奈酚-7-O-葡萄糖苷)及黄酮(芹菜素-7-O-吡喃葡萄糖苷,木犀草苷)具有很高的活性;对3位连接的黄酮醇(槲皮素-3-O-葡萄糖苷,山奈酚-3-O-葡萄糖苷),异黄酮(染料木苷)和黄烷酮(樱桃甙)有较低的活性。
等量的MtBGLU17纯化蛋白(10μg)加入同样浓度的不同类黄酮糖苷底物进行酶活反应,结果如图3所示,由图3可以看出:MtBGLU17对黄酮及黄酮醇-7-O-葡萄糖苷活性最高。
为探究MtBGLU17在体外的最适底物,本研究以不同的类黄酮糖苷进行酶动力学试验。结果如表5所示,由表5的结果表明:MtBGLU17对Gen7G具有最低的Km(米氏常数)值54.50μM;其次Qu7G为110.09μM;Lu7G、Ka7G和Ap7G的Km值依次为:166.47μM,166.90μM和380.43μM;根据Km值越低,酶对该底物的亲和力越强的理论,表明MtBGLU17对Gen7G具有最高的亲和力,而对Qu7G、Lu7G和Ka7G等具有中等的亲和力,然而对于Ap7G的亲和力最低。MtBGLU17对这些底物的最大反应速率大约维持在10-2-10-1nmol·s-1的范围,Kcat一般在10-1s-1左右。MtBGLU17对不同底物的转化率(Kcat/Km)结果显示对这五种底物转化率差异不大,在0.97s-1·mM-1和2.28s-1·mM-1之间,对Lu7G的转化率最低为0.97s-1·mM-1,而其对Ap7G的转化率最高为2.28s-1·mM-1.
表5MtBGLU17对不同类黄酮糖苷底物的动力学参数列表
实施例4
为了探究MtBGLU17基因在植物体内的生物学功能,本研究对MtBGLU17基因在蒺藜苜蓿共14种不同组织中,包括根,茎,叶,花,果实,种子及发育过程中的花和果荚(花芽,花,花后3天,6天,9天,12天,16天,20天,24天,36天果荚)的表达量进行分析,提取不同组织中的RNA,根据生产商的说明,使用Eastep Super总RNA提取试剂盒(Promega,Shanghai,China)。使用2×RealStar Green快速混合液(GeneStar,中国上海)和ABI 7500实时检测系统(美国Applied Biosystems)进行荧光定量PCR。PCR产物胶回收按照广州飞扬生物工程有限公司E.Z.N.A.胶回收试剂盒说明书进行操作。
结果如图4所示,由图4的结果表明MtBGLU17基因在花,花芽和幼果荚中表达量较高,特别是花后3天的果荚,而在其余组织中的表达水平很低。
为了探究MtBGLU17亚细胞定位的情况,MtBGLU17-RFP融合蛋白被瞬时转化到烟草的叶片中,并通过共聚焦荧光显微镜观察红色荧光信号。烟草亚细胞定位的具体步骤包括:将含有阳性质粒的农杆菌GV3101菌株(保存在甘油中,-80℃)用灭菌的枪头接种于含有利福平和卡那霉素的液体培养基中(1mL),于28℃200rpm过夜培养,活化菌液。第二天,再将活化的菌液接种于含有利福平和卡那霉素的液体培养基中(25mL),于28℃200rpm过夜培养。室温下5,000rpm离心10mins,收集菌体,再用同体积的蒸馏水重悬菌液,室温下5000rpm离心10mins,弃上清。用1mL重悬液(0.5%D-葡萄糖,50mM MES,2mM Na3PO4·12H2O,0.1mM乙酰丁香酮)重悬菌体。用重悬液将农杆菌稀释至OD600=0.8,用于注射。注射前一天将烟草浇水,使气孔张开以利于注射渗透。在烟草叶片背面主脉两侧各选1-2个注射点,用5mL的无针头的注射器缓慢将菌液注入烟草叶片,尽可能将菌液注射满烟草叶片,注射完成后等待48h,培养条件为每天光照14h(28℃)、黑暗10h(28℃)。48h内使用激光共聚焦显微镜观察RFP荧光(参数:激发波长543nm,发射波长560-660nm)。
由图5的结果所示:MtBGLU17-RFP在烟草叶片中出现很多明显的红色荧光信号,并且这些信号都与叶绿体自发荧光(绿色)信号重叠,呈橘黄色。结果表明MtBGLU17定位在叶绿体中。
实施例5
MtBGLU17在体外可水解多种类黄酮糖苷,为了探究MtBGLU17基因的体内功能,本研究构建了植物表达载体MtBGLU17-PB2GW7,通过农杆菌介导的遗传转化方法将其在蒺藜苜蓿中过表达。蒺藜苜蓿外植体农杆菌侵染转化及转基因植株再生的方法包括以下步骤:
1)需提前准备并灭菌的材料:无抗生素的SH3a固体培养基、液体培养基、含有相应抗生素的SH3a固体培养基、离心管50mL(2个)、培养皿(2个)、剪好的滤纸、三角瓶、2瓶ddH2O;
2)转化前一天早上,取负80℃冰箱保存的农杆菌单克隆菌液20μL,接种于1mL LB液体培养基(含rif抗生素+对应载体抗生素)中,28度摇床震荡(200转)12h,前一天晚上取200μL活化菌液加入100mL LB液体培养基(含rif抗生素+对应载体抗生素),28度摇床震荡(200转)过夜培养;
3)转化当天,用灭菌的100mL三角瓶取50片R108蒺藜苜蓿生长良好的叶片,先用自来水清洗3-5min,然后用蒸馏水洗两遍,把水倒掉后把含有叶片的三角瓶放进超净工作台,加入75%的乙醇灭菌2min,然后用0.1%的生汞灭菌6-8min(期间操作步骤4),蒸馏水洗三遍,把叶片放入灭过菌的培养皿中,加入少量灭过菌的水,叶片待切;
4)当菌液OD600达到0.6-0.8左右,将菌液分别倒入2个灭过菌的50mL离心管中,常温下6,000rpm离心6min;
5)菌液离心后,加入适量重悬液(100mL)。用1mL枪头重悬,然后转移到干净的三角瓶,待侵染;
6)叶片用刀片切成横条,再将侵染液放入超净台,切好的叶片放入侵染液静置15min,并包上锡箔纸放在超净台内,15min后叶片用大约4片滤纸吸干后,正面朝下,放入SH3a无抗生素的共培培养基;
7)离体的R108叶片在经过两天共培养后,第三天开始转入SH3a抗生素的诱导培养基,并每10天换一次诱导培养基;
8)待大约2个月左右,当愈伤组织再分化出不定苗时,将不定苗从愈伤组织上切下,叶片向上插入含有相应抗生素的1/2SH9筛选固体培养基上继续培养;
9)当再生苗生长到较为健壮时,转入蛭石:营养土=1:1的生长基质中,覆膜后在24℃、光照16h/黑暗8h的条件下生长;
10)待植株成活后揭膜继续生长;
11)T1代阳性苗(PCR鉴定)收的种子播到含600mg/L PPT抗性的MS固体培养基中,筛选阳性后代(植株根能正常分化侧根且地上部分生长正常),进一步将T2代阳性苗种下,按此方法接着筛T3代纯合转基因植株。
过表达植株通过PCR鉴定,结果如图6a所示,阳性转基因植株达到80%-90%左右。最终,本研究共获得了大约80株MtBGLU17转基因株系。接下来,通过RT-PCR进行筛选,本研究选取了其中三个表达量较高的株系OE28、OE75和OE143,通过R2代和R3代筛选,最终获得这三个转基因纯合株系,结果如图6b所示。通过图6c可以看出,OE28、OE75和OE143对蒺藜苜蓿的生长发育没有显著的影响。
实施例6
为了筛选MtBGLU17基因的突变体,本研究从蒺藜苜蓿Tnt1突变体数据库中查找并进一步通过序列比对分析,筛选到了两个插入到MtBGLU17基因的外显子区域的突变体NF14206(Tnt1反向插入在第8个外显子)和NF17318(Tnt1反向正向插入在第11个外显子),结果如图7a所示。
接着,通过对两个突变体材料进行PCR鉴定,在两代突变体繁殖之后最终筛选得到了纯合突变体材料,结果如图7b,d所示。接着,本研究分别以相同浓度(100ng/μL)的R108、NF14206、NF17318的花的cDNA为模板,用MtBGLU17的定量引物进行PCR,结果发现只有野生型材料能扩增出相应的基因产物,而突变体没有条带,表明,突变NF14206和NF17318的花中检测不到MtBGLU17基因的表达,结果如图7c所示。
为了研究MtBGLU17基因在蒺藜苜蓿体内的功能,本研究测定了两个月、生长条件一致的野生型R108、MtBGLU17基因过表达及突变体材料的叶片中的类黄酮含量。
从-80度冰箱取出蒺藜苜蓿鲜样粉末,称取100mg左右,加入500μL的80%甲醇,超声破碎30min,然后放4度冰箱过夜,12,000rpm离心20min,重复三次,然后取100μL上清液加入液相色谱瓶,待测。液相色谱条件同上酶活分析。结果如图8所示,由图8可以看出:过表达MtBGLU17(OE28)在蒺藜苜蓿中具有催化木犀草素7-O-葡萄糖苷(Lutiolin-7-O-glucoside)水解的作用,含量明显低于野生型(R108)和突变体材料(NF14206);而对应的产物木犀草素(Lutiolin)在OE28叶片中较R108和NF14206显著增加。另外,由于R108和NF14206叶片中MtBGLU17基因的表达量都很低,因此突变体较野生型而言,木犀草素7-O-葡萄糖苷及木犀草素的含量没有收到明显影响。这些结果证明MtBGLU17在蒺藜苜蓿体内特异催化黄酮糖苷(木犀草素7-O-葡萄糖苷)的水解生成黄酮苷元(木犀草素)。
实施例7
为了探究MtBGLU17基因是否影响植物参与非生物胁迫响应过程,本研究对MtBGLU17基因过表达株系、突变体及野生型R108材料进行自然干旱及NaCl处理。将蒺藜苜蓿MtBGLU17基因过表达纯合体、突变纯合体、野生型材料的种子均匀发芽并转移到冰箱春化三天后,放于人工气候箱生长3-5天后移入营养土中在16h光照/8h黑暗的生长室中继续生长。所有材料种植条件(土壤,光照及浇水)一致。其中,蒺藜苜蓿生长2个月时,从每一株系中选择成熟且大小相似的植株(每个试验有6个生物学重复)。一部分材料取叶、花放-80℃冰箱备用,用于提RNA和类黄酮测定。另一部分进行干旱和NaCl处理,在干旱处理中,所有植物在水中完全浸泡3小时,然后停止浇水1周取样待测。NaCl处理时,用1.5%NaCl溶液浸泡24小时,5天后取样待测。这些实验在所有测量中至少重复三次。
结果如图9所示,由图9的结果发现:在连续自然干旱7d后,野生型及突变体(NF14206,NF17318)材料相对于过表达材料(OE28,OE143)而言,植株叶片开始变黄,变干。在NaCl处理5d后,MtBGLU17突变体及野生型较过表达植株而言,叶片变黄,变干,叶片开始脱落。整体而言,在蒺藜苜蓿中过表达MtBGLU17基因能显著增加植株的抗旱性及耐盐性,但突变体和野生型R108之间没有明显差异。
为了进一步探究MtBGLU17基因过表达植株对于抵抗非生物胁迫的潜在机制,本研究对不同材料的抗氧化活性分别使用不同的检测方法(DPPH,CAT,SOD,MDA)进行测定。
从-80℃冰箱取出研磨好的蒺藜苜蓿新鲜叶片粉末,根据索莱宝DPPH,CAT,SOD活性及MDA含量检测试剂盒提供的说明书进行测定,结果如图10所示,由图10的结果可以看出:在干旱和NaCl处理后过表达植株(OE28,OE143)的叶片中的DPPH,CAT,SOD活性相对高于野生型R108和突变体植株(NF14206和NF17318),其中过表达植株的SOD活性显著高于野生型及突变体植株4倍以上。另外,干旱和NaCl处理后过表达材料MDA的含量较野生型相比也明显减少,而突变体植株MDA含量比野生型有所升高。整体而言,在蒺藜苜蓿中过表达MtBGLU17基因显著增加了植株的抗氧化活性,从而有助于抵抗非生物胁迫。
以上对本发明所提供的MtBGLU17基因在调控植物类黄酮合成中的应用进行了详细介绍。本文中应用了具体个例对本发明的原理及实施方式进行了阐述,以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求的保护范围内。
Claims (5)
1.MtBGLU17基因在植物对非生物胁迫响应中的应用,其特征在于,所述植物为苜蓿,所述非生物胁迫选自干旱或高盐。
2.根据权利要求1所述的应用,其特征在于,所述MtBGLU17基因能够增强苜蓿对非生物胁迫的抗性,所述苜蓿对非生物胁迫的抗性选自抗旱性或耐盐性。
3.MtBGLU17基因在调控苜蓿的抗氧化活性中的应用。
4.MtBGLU17基因在提高苜蓿自由基清除能力中的应用。
5.根据权利要求3所述的应用,其特征在于,所述抗氧化活性选自超氧化物歧化酶、丙二醛和过氧化氢酶活性中的至少一种。
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