CN116286853A - 桂花OfWRKY139基因在增强香气物质合成中的应用 - Google Patents
桂花OfWRKY139基因在增强香气物质合成中的应用 Download PDFInfo
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- CN116286853A CN116286853A CN202211620457.3A CN202211620457A CN116286853A CN 116286853 A CN116286853 A CN 116286853A CN 202211620457 A CN202211620457 A CN 202211620457A CN 116286853 A CN116286853 A CN 116286853A
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- osmanthus fragrans
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Abstract
本发明公开了桂花OfWRKY139基因在增强香气物质合成中的应用,属于植物分子生物学领域。本发明公开了桂花OfWRKY139基因在增强香气物质合成中的应用,OfWRKY139基因的核苷酸序列如SEQ ID NO.1所示。本发明以日香桂盛花期花朵为材料,通过克隆得到桂花OfWRKY139全长基因,在此基础上构建其过量表达载体pBI121‑OfWRKY139,转入桂花花瓣中,得到转基因植株,基因功能鉴定结果表明OfWRKY139基因是增强桂花香气物质合成过程中重要转录因子,可用于桂花基因工程中花香观赏性状的改良及遗传品质等繁育工作。
Description
技术领域
本发明属于植物分子生物学领域,更具体地说,涉及桂花OfWRKY139基因在增强香气物质合成中的应用。
背景技术
桂花(Osmanthusfragrans)属木犀科(Oleaceae)木犀属(Osmanthus),是我国十大传统名花之一,也是目前最重要的园林植物之一。桂花素以树形优美、香气宜人为人民所喜爱。桂花是中国获得栽培品种国际登录权威的少数几种花卉之一,这也更凸显了桂花在中国花卉中的重要地位。
WRKY转录因子是植物十大转录因子家族之一,广泛参与植物对生物、非生物和激素胁迫的响应,以及植物生长发育和生理过程。目前WRKY转录因子对植物次生代谢物合成的调控研究主要集中在酚类、萜烯类和生物碱类。萜烯类化合物是指存在于自然界中,分子式为异戊二烯单位的倍数的烃类及其含氧衍生物,按照异戊二烯单位的多少,可将常见萜类化合物分为单萜、倍半萜、二萜、二倍半萜、三萜、四萜和多萜,例如芳樟醇、月桂烯、反式-β-罗勒烯、金合欢烯和橙花醇等,都是构成花香的重要组分。
WRKY转录因子可独立亦也可通过与其它转录因子相互作用而发挥调控植物次生代谢的作用。不同的WRKY转录因子能够结合次生代谢途径中不同的基因,也能对同一种次生代谢产物有不同的调控方式。因此,亟需发掘桂花基因组中调控花香物质合成的重要相关基因。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供桂花OfWRKY139基因在增强香气物质合成中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
桂花OfWRKY139基因在增强植物香气物质合成中的应用,所述桂花OfWRKY139基因的核苷酸序列如SEQ ID NO.1所示。
进一步地,所述的应用包括以下步骤:
(1)构建OfWRKY139基因的载体;
(2)将所构建的OfWRKY139基因的载体转化到植物或植物组织中;
(3)培育筛选得到香气物质合成得到增强的转基因植物或植物组织。
进一步地,所述的桂花香气物质为(S)-氧化芳樟醇和E-氧化芳樟醇。
进一步地,所述的植物为桂花或大花烟草。
进一步地,所述的植物组织为桂花花瓣或大花烟草叶片。
进一步地,所述的转化是通过农杆菌瞬时转化。
进一步地,所述的载体是植物表达载体。
进一步地,所述的植物表达载体是pBI121-OfWRKY139。
相比于现有技术,本发明的有益效果为:
本发明以日香桂盛花期花朵为材料,通过克隆得到桂花OfWRKY139全长基因。同时,在此基础上构建其过量表达载体pBI121-OfWRKY139,转入桂花花瓣中,OfWRKY139在桂花中高效表达。转基因桂花花瓣和未转基因桂花花瓣避光培养48h后,未转基因桂花花瓣中萜类香气物质的含量并没有明显提升。转基因桂花花瓣中的(S)-氧化芳樟醇和E-氧化芳樟醇这两个芳樟醇氧化物的含量在OfWRKY139瞬时侵染桂花花瓣后显著高于对照。转基因大花烟草花瓣中α/β-石竹烯的含量显著低于野生型,同时γ-焦烯、西柏烯和2,6-二甲基-2-辛烯的含量却高于对照。表明OfWRKY139基因是增强桂花萜类香气物质合成过程中重要转录因子,可用于桂花基因工程中花香观赏性状的改良及遗传品质等繁育工作。
附图说明
图1为目的基因扩增产物琼脂糖电泳图;
图2为转化农杆菌GV3101后的菌检琼脂糖电泳图;
图3为OfWRKY139瞬时侵染桂花花瓣后阳性检测图,注:A为35S::OfWRKY139融合蛋白的GUS染色情况图(CK:野生型、NC:空载)、B为OfWRKY139瞬时侵染桂花花瓣后OfWRKY139的表达情况图、C为OfWRKY139瞬时侵染桂花花瓣后在培养基上的生长情况图;
图4为OfWRKY139基因瞬时侵染桂花花瓣后物质代谢图,注:A为OfWRKY139转基因植株和野生型花朵挥发性物质代谢物的PCA-X散点图、B为OPLS-DA散点图、C为载荷分析散点图:红色和黄色箭头表示VIP>1的物质成分、D为图C中箭头标出的物质的相对含量分析图;
图5为过表达OfWRKY139的大花烟草的生长和再生过程图,注:A为侵染的烟草叶片的抗性愈伤;B-C为筛选培养基上抗性芽、抗性芽生根;D-E为抗性苗移栽到基质中生长;
图6为OfWRKY139转基因烟草花朵挥发性物质代谢图,注:A为OfWRKY139转基因大花烟草植株和野生型花朵挥发性物质代谢物的PCA-X散点图、B为OPLS-DA散点图、C为载荷分析散点图:红色和黄色箭头表示VIP>1的物质成分、D为图C中箭头标出的物质的相对含量分析图;
图7为OfWRKY139转基因植株叶片挥发性物质代谢图,注:A为OfWRKY139转基因植株和野生型叶片挥发性物质代谢物的PCA-X散点图、B为OPLS-DA散点图、C为载荷分析散点图:红色和黄色箭头表示VIP>1的物质成分、D为图C中箭头标出的物质的相对含量分析图;
图8为萜类合成途径关键基因表达分析图,注:数值用平均值±SE表示,小写字母代表基于单因素随机区组方差分析的显著差异(P<0.05)。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。以下实施例中,未详细叙述的操作均为常规生物学实验操作,可参照分子生物学实验手册以及现有公开的期刊文献等进行,或者按照试剂盒和产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:日香桂OfWRKY139基因的克隆、超表达载体构建
1、总RNA提取与cDNA合成
本申请所用材料为日香桂盛花期花朵,瞬时转化所用的桂花花瓣为生长在南京林业大学校园内的金桂品种‘波叶金桂’。
采用TIANGEN植物RNA提取试剂盒(DP432)提取植物总RNA。
采用TaKaRaPrimeScriptTMRTMasterMix(PerfectRealTime)反转录试剂盒将所提取到的RNA反转录成cDNA,最后得到的cDNA加水稀释10倍后于-20℃冰箱保存。
2、获取目的基因片段
根据前期课题组已发表的桂花全基因组数据库(Xiulian Yang,Yuanzheng Yue,et al.The chromosome-level quality genome provides insights into theevolution ofthe biosynthesis genes for aroma compounds ofOsmanthusfragrans.),筛选得到1个基因序列,与模式植物拟南芥的序列进行比对,判断该基因属于WRKY基因家族,根据该基因家族成员在染色体上的位置命名为OfWRKY139。
利用BioXM软件对该基因的核苷酸全长序列进行酶切位点分析,选择XbaI和BamHI作为限制性内切酶酶切位点。利用CE design软件设计引物。按要求进行相关信息的填写,具体包括载体上的酶切位点附近的序列、目的基因全长、按照5′端和3′端的顺序填写酶切位点,即可得到扩增引物。设计好的序列由捷瑞生物公司合成。引物序列如下:
pBI121-OfWRKY139-F:
5′-GAGAACACGGGGGACTCTAGAATGATGGCCTTTGGTAACATGAA-3′,
pBI121-OfWRKY139-R:
5′-GGACTGACCACCCGGGGATCCAAATACGCACCTCTTCTTGCAAT-3'。
以稀释10倍的日香桂OfWRKY139cDNA为模板,进行PCR扩增,反应体系(20μL)为:正向引物pBI121-OfWRKY139-F1μL;反向引物pBI121-OfWRKY139-R1μL;cDNA1μL;PrimeSTAR10μL(高保真PCR酶购买于宝日医生物技术(北京)有限公司高速高保真PCR酶R045A);ddH2O7μL。PCR反应程序为:98℃变性10s;58℃退火15s;72℃延伸2min,35个循环;72℃总延伸10min;16℃终止反应。
将扩增产物进行琼脂糖电泳检测(图1),获得目的基因片段,然后利用试剂盒对目的基因片段进行切胶回收。
3、目的基因片段与载体连接
1)提前将切pBI121载体从-80℃超低温冰箱中取出进行活化和摇菌,按照质粒提取试剂盒(北京天根生化科技有限公司)提取pBI121载体质粒,随后进行双酶切实验,反应体系(20μL)为:限制性内切酶BamHI1μL;限制性内切酶XbaI1μL;Buffer2μL(Buffer为Takara限制性内切酶附带);载体质粒XμL;ddH2O补足至20μL。
其中X(μL)=1000ng/载体质粒浓度(ng/μL)。将离心管微微震荡,使其混匀,瞬时离心6s,置于37℃的水浴锅内培养1h。将双酶切后的载体进行琼脂糖电泳,然后利用试剂盒进行切胶回收(湖南艾科瑞生物工程有限公司),下同。
2)连接体系(20μL)如下:目的基因回收产物XμL;pBI121载体双酶切回收产物YμL;连接酶2μL;Buffer1μL;ddH2O补足至20μL(连接酶和Buffer源于南京诺唯赞生物科技有限公司同源重组试剂c112)。
其中X(μL)=200ng/目的基因回收产物浓度(ng/μL),Y(μL)=200ng/质粒双酶切回收产物浓度(ng/μL)。将离心管微微震荡,使其混匀,瞬时离心6s,置于37℃的水浴锅内培养30min,冰上2min。
3)转化:超净工作台内,用移液枪取5μL的连接产物于50μL的TreliefTM5α感受态细胞,轻弹混匀,冰浴5min,42℃水浴60s,再冰浴2min,加250μL液体LB(不含Kana),37℃,200rppm的摇床内孵育30min。
4)涂板:取200μL孵育后的菌液,用灭菌的玻璃棒均匀涂布在LB固体培养基上(内含50mg/L的Kana)并晾干,用封口膜后倒置在37℃恒温培养箱内培养12-14h。
4、重组质粒筛选
当培养基上长出菌后,于超净工作台内进行单菌落检测。每个基因挑取8个饱满的单菌落,依序依次在含Kana抗性的LB固体培养基上进行备份,并用无菌牙签将相应的单菌落沾取至以下20μL体系中进行菌检:pBI121-OfWRKY139-F 1μL,pBI121-OfWRKY139-R 1μL,Green Mix 10μL(Green Mix购于南京诺唯赞生物科技有限公司),ddH2O 8μL。
PCR反应条件为:94℃预变性3min;94℃变性30s;58℃退火30s;72℃延伸1min,35个循环;72℃总延伸10min;16℃终止反应。将获得的扩增产物进行琼脂糖电泳,挑取3个长度正确的阳性单菌落送测,测得OfWRKY139基因的核苷酸序列如SEQ ID NO.1所示,片段长度为714bp,编码237个氨基酸的蛋白质,该蛋白氨基酸序列如SEQ ID NO.2所示。选取碱基错配率最低的且测序有重复的阳性单菌落提取质粒进行后续实验。
实施例2:OfWRKY139基因的转化及功能鉴定
1、重组质粒转化农杆菌
1)将保存在-80℃超低温冰箱内的GV3101感受态取出放在冰上融化。每33μL感受态加入1μL质粒,吸打混匀后依次冰浴20min、液氮速冻5min、37℃水浴5min、冰浴5min;
2)加500μL无抗性的LB液体培养基,28℃,200rppm摇床上培养1h;培养完成后,将菌液6000r,离心1min,弃去部分上清液,留100μL均匀涂布于LB固体培养基上(内含50mg/LKana),封口膜密封,倒置于28℃培养箱中培养40-48h;
3)菌检和备份:结果如图2所示,菌检中的目的条带正确且亮度一致,则将备份的板子中相对应的菌落挑入LB液体培养基(内含50mg/L Kana)中摇菌,再将菌液和50%甘油按3:7的体积比保菌,液氮中速冻后保存在-80℃超低温冰箱内。
2、农杆菌介导转化‘波叶金桂’桂花花瓣
1)摇菌:将pBI121空载、P19辅助表达载体和已转入农杆菌的GUS::pBI121-OfWRKY139目标基因融合表达载体的菌液于-80℃取出常温融化至冰水混合状态后插入冰中融化,按照300μL菌液加入30mL LB液体培养基中(含Kana 10μg·mL-1),28℃、200rpm避光振荡培养,直至菌液OD600=0.6-0.8之间;
2)准备混合菌液:称取0.0196g的乙酰丁香酮(AS)粉末,使用二甲基亚砜助溶(在通风橱操作),再加入适量无菌水定容到100mL,获得母液,取母液15mL加入85mL的无菌水,从而获得150μmol·L-1的AS;称取0.2035g的MgCl2和0.2135g的2-(N-吗啉)乙磺酸一水物(MES)加入到配好的150μmol·L-1的AS溶液中;将菌液时进行离心(4℃、5000rpm、10min),弃上清,收集菌体,使用当天配制的缓冲液进行重悬,最后按照最佳比例混合(P19辅助载体:目的基因(V:V)=5:7),充分震荡混匀后活化3h;
3)侵染:选取生长状态良好的桂花花瓣,使用真空泵将混合菌液注入桂花花瓣;
4)培养:将注射过目的基因混合菌液的瞬时转化植物和注射过空载体菌液的瞬时转化植物,避光培养48h,OfWRKY139瞬时侵染桂花花瓣后在培养基上的生长情况如图3C所示。
3、瞬时转化桂花花瓣的GUS染色
将上述的野生型桂花花瓣(CK)、侵染空载的桂花花瓣(NC)以及侵染浸GUS::pBI121-OfWRKY139的桂花花瓣泡于新鲜配置的GUS染液中,避光,37℃,染色1-2d后,使用75%的酒精进行脱色,更换酒精3-5次,再使用95%的无水乙醇脱色,至植物完全脱色后拍照。
结果如图3A所示,GUS染色情况表明pBI121载体与35S::OfWRKY139融合表达载体侵染桂花花瓣后,经GUS染色后花瓣均呈现明显的蓝色,而对照却不变色,说明OfWRKY139基因成功转入桂花花瓣中。
4、荧光定量分析:采用TIANGEN植物RNA提取试剂盒(DP432)提取植物总RNA。采用TaKaRa PrimeScriptTMRT Master Mix(Perfect Real Time)反转录试剂盒将所提取到的RNA反转录成cDNA,最后得到的cDNA加水稀释10倍后于取1μL作为模板,加入F 0.4μL,R 0.4μL,TBGreen Mix 5μL,Rox染液0.2μL,ddH2O 3μL,配成10μL的体系进行荧光定量试验,荧光定量试剂盒为TaKaRa TB Green Premix Ex Taq(RR420A),实验中使用的热循环条件如下:95℃持续30s,然后在95℃持续5s,60℃持续30s,95℃持续15s,60℃持续1分钟的40个循环后,在95℃下持续15s。相对表达水平的计算使用的2-△△CT法。
结果如图3B所示,再通过qRT-PCR的方法确定目标基因的超表达情况,OfWRKY139约为对照的3倍。
5、瞬时转化桂花花瓣的香气物质测定
室温下(25±2℃)用镊子夹取0.4g桂花花瓣,记录叶片重量后放置于密封的1mL萃取瓶中,加入内标溶液(1/10000葵酸乙酯:1微的葵酸乙酯标样加入到10mL的甲醇)后摇晃混匀后平衡15min;在温度45℃的水浴锅中,密封后用65μmDB/5MS的萃取头插入萃取瓶中部,萃取30min后将萃取头插入色谱柱(TRACE TR-5MS C30m×0.25mm×0.25gym),载气为高纯度氦气(He),氦气流速为1mL/min,不分流进样,进样口温度250℃,解吸附时间为3min;温度程序为:首先温度60℃,保持2min,第一个升温程序为60℃上升到150℃,速度为5℃/min,第二个升温程序为150℃升至250℃速度为10℃/min,再保持1min;质谱条件为离子源温度250℃,电离方式EI,电子能量70eV;
结果如图4所示,将数据中的硅氧化物杂质去除后,计算出各种物质的相对含量,将物质的相对含量导入SIMCA5.0中,根据OPLS-DA聚类,依据VIP值得大小,挑选VIP>1的物质进行LOADING分析,选择VIP大于1的物质做具体的含量分析,VIP大于1的物质包含β-石竹烯,倍半萜、α-石竹烯,倍半萜、γ-焦烯,单萜、西柏烯,双萜和2,6-二甲基-2-辛烯,结果表明转化了OfWRKY139转基因株系中α/β-石竹烯的含量显著低于野生型,同时γ-焦烯、西柏烯和2,6-二甲基-2-辛烯的含量却高于对照,表明OfWRKY139对差异性p<0.05的香气物质具有重要的调控作用。
实施例3:稳定转化大花烟草
1、大花烟草的稳定遗传转化
(1)侵染菌液的制备
提前一天晚上将pCAMBIA2300::OfWRKYs融合表达载体的菌液从超低温冰箱取出;取200μL加入到20mL含Kana的LB培养液中,于28℃摇箱中200rpm避光振荡培养约12-16h,摇至菌液的浓度为OD600=0.4-0.5。
(2)外植体消毒
外植体选择未开花且无病虫害的健壮烟草叶片,首先将叶片清洗干净,依次进行流水冲洗20min、75%乙醇消毒30s,ddH2O清洗2-3次、0.1%升汞消毒8-10min,ddH2O清洗3-5次。用锋利刀片切掉叶片边缘和主脉,切成1cm×1cm大小方块。
(3)农杆菌的转化
将切好的叶片放入准备好的菌液侵染10min左右,期间每隔2min轻轻地摇晃菌液。
(4)烟草的遗传转化过程
1)共培养
将侵染过后的叶盘置于无菌滤纸上,吸去多余的菌液,于共生培养基上暗培3d。
2)筛选培养
将共培后的材料转接到选择培养基上,25℃光照培养,每两周更换一次培养基。
3)壮芽培养
当叶盘周围的愈伤组织分化出抗性芽后,转接到壮芽培养基上生长,每两周更换一次培养基。
4)生根培养
当不定芽约2cm时,轻轻的切下,转接到生根培养基。
5)炼苗与移栽
根系发达时取出幼苗,清洗掉根部的培养基,于室内放置2d,后移栽到温室培养(图5)。
2、总RNA提取、反转录获得cDNA及目的基因扩增
为了确定OfWRKY139基因在转基因烟草中的表达状况,使用北京艾德莱(EASYspinPlus Plant RNAkit)试剂盒,采用液氮研磨法提取野生型和转基因大花烟草叶片的总RNA,步骤入下:向无RNA酶的1.5mL离心管中加入600μL裂解液RLT和60μL PLANTaid,轻轻吸打混匀,备用;研钵用液氮预冷,充分研磨一定量的植物材料,取约100mg粉末加入第1步中备用的离心管中,立即用手剧烈上下振荡,再使用振荡仪振荡30s,保证植物材料充分裂解;将上一步的裂解物13000r/min离心8min;取一个新离心管,并加入275μL的无水乙醇,吸取上一步中550μL的上清液于无水乙醇中,并快速吸打混匀;将混合物转移至收集管的吸附柱中,13000r/min离心2min,弃废液;往吸附柱中加入700μL去蛋白液RW1,室温放置4min后13000r/min离心1min,弃废液;往吸附柱中加入漂洗液RW(按照要求加入无水乙醇),室温放置5min后13000r/min离心1min,弃废液,重复一次;13000r/min离心2min;将吸附柱置于新的无RNA酶的离心管中,在膜的中央加入30μL 90℃预热的无RNA酶的无菌水,室温放置2min后12000r/min离心1min;取2μL用于TBE胶检测提取的RNA质量;取1μLRNA用于检测浓度。
将提取的总RNA置于冰上,马上用于反转录。使用北京全式金公司的One-Step试剂盒(AT311-03)合成各样品的第一链cDNA,具体步骤如下:
(1)加入TotalRNA5μg、AnchoredOligo(dt)18Primer(0.5μg/μL)1μL、2×TSReactionMix10μL、TransScriptRT/RIEnzymeMix1μL、gDNARemover1μL,并充分的吸打混匀,于PCR仪上42℃反应30min;
(2)85℃加热5s,置于冰上备用;
(3)反应液稀释20×后,取1μL为模板进行半定量PCR检测,反应体系为20μL,反应程序为:94℃4min;94℃30s,60℃30s,72℃45s,共30个循环;72℃10min;16℃保存。
(4)吸取5μLPCR反应液和5μLMarker跑胶(2%)。
以此为模板进行PCR扩增,OfWRKY139基因的半定量引物在转基因烟草中扩增出特异条带,证明OfWRKY139基因已经转入到大花烟草植株的基因组中,从而确定9株超量表达的转基因株系。不同株系之间在表达量上有一定差异,通过半定量的条带亮度挑选超表达强度最高的3个株系进行后续实验并收种。
3、GC-MS检测转基因系的花香成分
利用GC-MS检测转基因系的花香成分,采取转基因植株以及野生型烟草盛花初期的花朵,即花朵开放后尚未散粉且柱头湿润的时期,设置3个重复,每个重复挑2个大小一致的花朵,保持花朵的重量一致。顶空固相微萃取方法萃取烟草花香物质的条件为:室温下(25±2℃),将要检测的烟草花朵,记录花朵重量后放置于密封的20ml萃取瓶中平衡15min,并加入内标溶液(1/2000的癸酸乙酯)。其他条件同瞬时转化桂花花瓣的香气物质测定部分。
结果如图6所示,VIP大于1的物质包含β-Caryophyllene(β-石竹烯,倍半萜)、α-Caryophyllene(α-石竹烯,倍半萜)、γ-Pyronene(γ-焦烯,单萜)、(+)-Cembrene(西柏烯,双萜)和2-Octene,2,6-dimethyl(2,6-二甲基-2-辛烯),结果表明转化了OfWRKY139转基因株系中α/β-石竹烯的含量显著低于野生型,同时γ-焦烯、西柏烯和2,6-二甲基-2-辛烯的含量却高于对照。
4、GC-MS检测转基因系的叶片成分
转基因株系叶片挥发性物质GC-MS测定与分析,为了探究OfWRKY139基因是否能在转基因叶片中也有抑制倍半萜物质合成的作用,根据T0代阳性转基因烟草的表达,挑选表达量最好的三个转基因株系与野生型大花烟草种植,经阳性检测后获得T1代转基因植株,在大花烟草开花前,选取野生型与转基因植株的相同部位的叶片,去除主脉和较大的侧脉,将叶片剪成2mm左右的长条,称取0.3g进行顶空固相微萃取,萃取条件为:室温下,条状叶片置于密封的10ml萃取瓶中平衡15min,并加入内标溶液(1/10000的癸酸乙酯),使用65μmDB/5MS的萃取头,60℃萃取35min。气相色谱条件与质谱条件同上。
结果如图7所示,结果显示,1-Hexadecanol(棕榈醇)、trans-Farnesol(反式-金合欢醇)、Hexadecanoicacid,methylester(棕榈酸甲酯)和Hexadecane(十六烷)在两者存在差异,其中反式-金合欢醇为倍半萜类物质。
5、转基因株系中MEP-MVA代谢途径关键酶基因的表达分析,我们选取OfWRKY139转基因株系中高表达的3个株系T1代叶片,每个株系3个重复提取RNA,经过反转录获得cDNA,通过qRT-PCR分析萜类合成途径中关键酶基因的表达,具体程序同瞬时侵染桂花花瓣的荧光定量实验部分。
结果如图8所示,OfWRKY139基因在转基因株系均有超表达。MVA途径上游关键基因NtHMGS表达无变化,但NtHMGR1、NtHMGR2和NtIPPI基因在转基因株系中下调表达,MEP途径上游关键基因NtDXS、NtDXR也呈下调表达的趋势。在MVA途径倍半萜途径中,两个NtEAS基因在转基因植株中均下调表,但是MEP途径单萜和双萜途径的基因表达均无明显差异。
Claims (8)
1.桂花OfWRKY139基因在增强植物香气物质合成中的应用,所述桂花OfWRKY139基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,包括以下步骤:
(1)构建OfWRKY139基因的载体;
(2)将所构建的OfWRKY139基因的载体转化到植物或植物组织中;
(3)培育筛选得到香气物质合成得到增强的转基因植物或植物组织。
3.根据权利要求1或2所述的应用,其特征在于,所述的桂花香气物质为(S)-氧化芳樟醇和E-氧化芳樟醇。
4.根据权利要求1或2所述的应用,其特征在于,所述的植物为桂花或大花烟草。
5.根据权利要求2所述的应用,其特征在于,所述的植物组织为桂花花瓣或大花烟草叶片。
6.根据权利要求2所述的应用,其特征在于,所述的转化是通过农杆菌瞬时转化。
7.根据权利要求2所述的应用,其特征在于,所述的载体是植物表达载体。
8.根据权利要求7所述的应用,其特征在于,所述的植物表达载体是pBI121-OfWRKY139。
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