CN105755002A - 烟草西柏三烯一醇合成酶基因启动子及其应用 - Google Patents
烟草西柏三烯一醇合成酶基因启动子及其应用 Download PDFInfo
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Abstract
本发明涉及一种烟草西柏三烯一醇合成酶基因启动子,其核苷酸序列为:(1)SEQ ID NO.1所示的核苷酸序列;或(2)SEQ ID NO.1所示的核苷酸序列经重复、缺失、替换或移位等常规技术手段改造而形成的具有同等功能的核苷酸序列。本发明还涉及一种嵌合基因,以及包含该嵌合基因的植物转化载体、转基因植物细胞和转基因植物组织。利用本发明烟草西柏三烯一醇合成酶基因启动子对温度敏感响应的特性,可将其应用于烟草西柏三烯一醇合成调控过程,以改善烟草的品质,并提高烟草抵抗蚜虫和烟草花叶病的抗性。
Description
技术领域
本发明属于基因工程领域,具体涉及一种烟草西柏三烯一醇合成酶基因启动子及其应用。
背景技术
腺毛是烟草叶面分泌物的分泌器官,主要分泌西柏烷类化合物,占叶面化学成分的60%以上。在西柏烷类化合物中,西柏三烯二醇含量远远高于西柏三烯一醇,β-西柏三烯二醇含量稍高于α-西柏三烯二醇。在烟叶烘烤调制和陈化过程中,西柏烷类化合物降解产生茄酮及其衍生物等香味成分,是构成烟叶品质的重要组成成分。此外,西柏烷类化合物不仅对改善烟草的品质有极其重要的作用,对抵抗蚜虫和烟草花叶病的侵袭也有良好的抗性。
在烟草腺毛中,类萜代谢前体物有甲羟戊酸(MVA)途径和丙酮酸/3-磷酸甘油醛代谢(DXP)产生异戊烯焦磷酸(IPP)。IPP是合成萜类化合物的前体,MEP途径存在于质体中,为单萜、双萜、四萜生物合成提供IPP。IPP被IPP异构酶(IPI)和二价金属离子转化为具有活性的异构体DMAPP。IPP和DMAPP在牻牛儿基焦磷酸合成酶(GPPS)的作用下缩合成牻牛儿基焦磷酸(GPP),然后GPP再与2个IPP在牻牛儿基焦磷酸合成酶(GGPPS)的作用下缩合成牻牛儿基焦磷酸(GGPP)。GGPP在二萜合成酶的作用下生成二萜,分为两步:第一步,GGPP为二萜的形成提供了C20骨架结构,在西柏三烯一醇合成酶CBTS的作用下,GGPP发生环化反应,生成α-和β西柏三烯一醇(CBT-ol);第二步,在P450羟化酶CYP71D16的作用下,西柏三烯一醇发生羟化反应生成α-和β西柏三烯二醇(CBT-diol)。WangE等最先克隆了腺毛特异P450羟化酶基因CYP71D16,并对其功能进行了研究,认为该基因编码的是CBT-ol羟化酶,能将西柏三烯一醇转化为西柏三烯二醇。Hanane等以普通烟草腺毛特异性基因西柏三烯一醇合成酶(CYC)设计的引物从烟草中克隆出CBT基因,对其调控位点进行研究,认为CBT的产物能将GGPP催化成西柏三烯一醇,同时发现CBT和CYP71D16只能在烟草腺毛中特异表达。西柏三烯一醇合成酶可以调控西柏三烯一醇的合成。而编码该酶的基因CYC的启动子可以在转录水平对该基因的表达进行调控,因此,克隆和分离CYC基因的启动子并找到对应的调节元件是实现西柏三烯烷类合成的重要前提。
发明内容
为解决以上问题,本发明提供一种烟草西柏三烯一醇合成酶基因启动子,其驱动的GUS基因只在腺毛上特异表达,且在不同温度下的活性不同,在28℃条件下具有较高活性,可实现GUS基因的高水平表达。
为解决以上技术问题,本发明通过以下技术方案实现:
烟草品种8306是杨铁钊等培育出的高香气烤烟新品系,具有典型的烤烟香味,香气突出,显著高于其它烤烟栽培品种;且经过连续8代的自交选择,遗传性状表达稳定。本发明从8306中克隆出烟草西柏三烯一醇合成酶(CYC)基因启动子,构建pC-NtCYC-pro-GUS载体,转化烟草K326,利用报告基因GUS对NtCYC基因启动子的特异表达进行研究,并深入研究了该NtCYC基因启动子在对环境的应答机制,建立了烟草腺毛基因过表达体系,进一步为在腺毛中研究基因功能奠定了基础。
研究发现了一种烟草西柏三烯一醇合成酶基因启动子,其核苷酸序列为:
(1)SEQIDNO.1所示的核苷酸序列;
(2)SEQIDNO.1所示的核苷酸序列经重复、缺失、替换或移位等常规技术手段改造而形成的具有同等功能的核苷酸序列。
设计一种嵌合基因,其中包含上述烟草西柏三烯一醇合成酶基因启动子以及与之可操作连接的、编码目的基因的序列。
一种植物转化载体,其中包含上所述嵌合基因。
一种转基因植物细胞,其中包含上述嵌合基因。
一种转基因植物组织,其中包含上述嵌合基因。
利用上述烟草西柏三烯一醇合成酶基因启动子对温度敏感响应的特性,可将其应用于烟草西柏三烯一醇合成调控过程,以改善烟草的品质,并提高烟草抵抗蚜虫和烟草花叶病的抗性。
本发明具有以下积极有益技术效果:
发现并克隆出了一种对温度敏感响应的烟草西柏三烯一醇合成酶基因启动子,其驱动的GUS基因只在腺毛上特异表达,且在不同温度下的活性不同,在28℃条件下具有较高活性,可实现GUS基因的高水平表达,为灵活调控西柏三烯一醇的合成找到了有效的实现途径。
附图说明
图1.“8306”NtCYC启动子的PCR扩增电泳图;
孔道M为Marker,孔道1~2为“8306”NtCYC启动子。
图2.“8306”NtCYC启动子的菌落PCR扩增电泳图;
孔道M为Marker,孔道1~4为单菌落。
图3.重组阳性质粒pC-NtCYC-pro-GUS电泳图;
孔道M为Marker,孔道1~8为pC-NtCYC-pro-GUS质粒。
图4.NheⅠ酶切验证图;
孔道M为Marker,孔道1~8为pC-NtCYC-pro-GUS质粒。
图5.NtCYC启动子转化烟草图;
A为阳性对照:未转化叶片在共培养基上分化,B为阴性对照:未转化叶片培养基上不分化,C为转化叶片在筛选培养基上分化,D为阳性植株。
图6.植物组织染色对比图;
A为转基因植株叶片,B为转基因植株茎,C为转基因植株根(箭头指向根毛),D为K326叶片,E为K326茎,F为K326根。
图7.不同温度下的GUS活性。
具体实施方式
以下结合具体实施例进一步阐述本发明。下述实施例中所涉及的试验方法或分析方法,如无特别说明,均为常规方法,所用试剂如无特别说明,均为市售。
实施例一、pC-NtCYC-pro-GUS质粒的构建方法,包括以下步骤:
(1)烟草叶片DNA的提取:采用BioFlux植物DNA提取试剂盒(Cat.NO.DE-06111)提取烟草8306的叶片DNA。
(2)烟草NtCYC基因启动子的PCR扩增:以提取的烟草8306叶片DNA为模板,采用引物NtCYC-Pro-F:5'-AACTATGAAAATTTTTTAACAACTAATTTAT-3'和NtCYC-Pro-R:5'-CTCGTTTGGCGAGAGAAAGAGAGAG-3'进行PCR扩增。PCR扩增程序为:95℃3min,95℃15sec,55℃15sec,72℃1min,72℃5min,共40个循环。
(3)烟草pC-NtCYC-Pro-GUS重组载体的连接:将获得的PCR产物(电泳验证见图1)进行纯化后与目的载体pC-NPT-GUS进行同源重组反应。按照下表体系37℃反应30分钟后进行质粒的转化。
(4)烟草pC-NtCYC-Pro-GUS重组载体的转化:取200μL感受态宿主菌(DH-5α),放置冰上融化后加入1μL步骤(3)中的连接液,轻轻混匀后置于冰上30min;42℃水浴热休克90sec,迅速放入冰上2min。加入300μLSOC培养基(不含抗生素),置于37℃摇床,转速200rpm,复苏45min。菌液涂布于15cm培养皿上(Apr-IPTG/x-galLB固体培养基),37℃过夜。
(5)烟草pC-NtCYC-Pro-GUS重组载体的鉴定:挑取单克隆作为PCR模板。采用(2)的方法和体系进行菌落PCR验证(图2),对鉴定正确的克隆测序。
实施例二、转基因植株的制备
(1)无菌苗的培养:取烟草品种K326种子2g,在超净工作台中放入无菌培养皿中,先用75%酒精消毒40s;快速倒掉酒精,加入10%次氯酸钠浸泡种子8min;倒掉次氯酸钠,用无菌水冲洗烟草种子5~7次,防止残留次氯酸钠影响种子正常生长;将冲洗干净的种子放置在无菌滤纸上,吸干种子表面水分;将种子小心均匀地散布在MS固体培养基上,28℃光照培养,30天左右得到烟草无菌苗,作为转化材料待用。
(2)农杆菌感受态的制备:取出-80℃保存的农杆菌EHA105菌液在冰上解冻,划线接菌于YEB固体培养基(含Rif50mg/L)中,28℃恒温培养2~3天;在超净台中挑取农杆菌EHA105单克隆,接种于10mLYEB液体培养基(含Rif50mg/L)中,于恒温摇床中28℃、200rpm/min培养1~2天;取2mL菌液加入50mLYEB液体培养基(含Rif50mg/L)28℃、200rpm/min中继续培养,至OD600为0.5;取3mL菌液于1.5mL离心管,4℃、5000rpm/min、离心5min,弃去上清液;用10μL预冷的0.15mol/LCaCl2悬浮菌液,4℃、5000rpm/min、离心5min,弃去上清液;再用10μL预冷的0.15mol/LCaCl2悬浮菌液;冰浴30min、4℃、5000rpm/min,离心5min,弃去上清液;用1mL预冷的CaCl2(20mmol/L)悬浮,分装成100μL/管,每管中加入100μL预冷的40%甘油,即为农杆菌感受态细胞,液氮中速冻后至-80℃保存。
(3)转化农杆菌:取-80℃保存的农杆菌感受态细胞,置于冰上融化;加入2μL质粒pC-NtCYC-pro-GUS,用微量加样器抽打混匀;冰浴30min,立即置于液氮中,冰冻5min;迅速置于37℃水浴锅中,水浴5min;加入650μLYEB液体培养基(含Rif50mg/L)28℃、200rpm/min培养2~3天;于超净工作台上将菌液涂布于YEB固体培养基(含Rif50mg/L、Kana50mg/L);28℃于恒温箱中暗培养2~3天;挑选单菌落,提取质粒DNA,琼脂糖凝胶检测,得到了大小大于10000bp的条带,见图3;进一步采用NheⅠ酶切提取的农杆菌质粒,结果如图4所示,农杆菌来源的pC-NtCYC-pro-GUS质粒经过NheⅠ酶切均产生了三条带并且大小与预测大小一致,表明pC-NtCYC-pro-GUS质粒成功转入农杆菌EHA105中;检测出的重组阳性质粒,挑选单菌落,接种于YEB液体培养基(含Rif50mg/L,Kana30mg/L)中,28℃、200rpm/min震荡培养至OD600为0.5,取出菌液500μL,加入200μL的80%的无菌甘油,得重组农杆菌菌液,于-80℃保存。
(4)烟草无菌苗成苗后按照下列步骤用重组农杆菌菌液转化烟草:取烟草无菌组培苗,在超净工作台中将其剪成1cm2左右大小的叶盘,放在MS培养基上,28℃光照预培养2~3天;取-80℃保存的含有重组质粒的农杆菌,接种于3mlYEB液体培养基(含Rif50mg/L,Kana50mg/L)中,28℃、200rpm/min震荡培养过夜;取1mL菌液转至30mLYEB液体培养基(含Rif50mg/L,Kana30mg/L)中,28℃、200rpm/min震荡培养过夜;待菌液培养至OD600为0.5左右,在无菌条件下将培养好的烟草叶片侵染5~8min;将侵染后的烟草叶盘置于无菌滤纸上吸干烟草表面的菌液;将吸干菌液的烟草叶盘正面朝上放在MS固体培养基上,放置在恒温培养箱中28℃暗培养2天;培养2天后,将烟草叶盘转移至分化培养基中,伤口与培养基充分接触。28℃光照培养;出现分化芽后,将分化芽转移至筛选培养基中,约20天左右,部分分化芽出现黄化现象,分化芽继续生长至1cm左右时,修整分化芽转入生根培养基中诱导生根,生长为抗性苗。待根系生长发达后,将植株移栽到土壤中培养即可,生长为转基因植株。如图5所示,阳性对照K326叶片在共培养基上正常分化,阴性对照中在含kana50mg/mL的培养基上K326叶片发白不分化,农杆菌侵染后的烟草叶片在筛选培养基上分化后移至生根培养基生根,获得阳性植株。
实施例三、组织检验
1.化学组织染色检验:将转基因植株的根、茎、叶分别切成小快浸没在装有1mLGUS染液的1.5mL的离心管中,37℃过夜染色,染色后的组织经75%的乙醇脱色,待组织脱色为无色后,在OLYMPUSSZX16体视显微镜下观察并拍照;
GUS染液的配制:磷酸钠缓冲液(pH7.0)100mmol/L、Na2EDTA10mmol/L、K3[Fe(CN)6]1mmol/L、K4[Fe(CN)6]1mmol/L、TritonX-1000.5%(v/v)、甲醇20%(v/v)、X-GIuc0.5mg/mL,余量为ddH2O;
2.温度处理
取组培瓶中长势大小一致的转基因植株作为供试材料。设置两个温度作为处理,分别为4℃和42℃,处理12个小时,0小时为对照,分别在1小时、6小时、12小时取样,用液氮急速冷冻,将材料置于-80℃冰箱保存。
3.植物β-葡萄糖苷酸酶(GUS)酶联免疫分析
(1)配制GUS提取液:0.1mol/L磷酸缓冲液(pH7.0)50mL,10%SDS1mL;0.5mol/LEDTA(PH8.0)取2mL;TritonX-100取100μL;β-巯基乙醇100μL;用水定容至100mL;
(2)取样品约0.01g,加入到2mLEppendorf管中,用全自动样品快速研磨仪进行研磨,将研磨破碎的烟叶组织转移到1.5mLEp管中,并立即加入1mL的GUS提取缓冲液,充分混匀。
(3)12,000rpm,4℃离心5min,将上清液转移至另一洁净的Eppendorf管,冰上放置待用。
(4)稀释标准品:150μL的原倍标准品加入150μL标准品稀释液为5号标准品,150μL的5标准品加入150μL标准品稀释液为4号标准品,150μL的4号标准品加入150μL标准品稀释液为3号标准品,150μL的3号标准品加入150μL标准品稀释液为2号标准品,150μL的2号标准品加入150μL标准品稀释液为1号标准品。
(5)加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μL,待测样品孔中先加样品稀释液40μL,然后再加待测样品10μL(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。
(6)温育:用封板膜封板后置37℃温育30分钟。
(7)配液:将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
(8)洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30秒后弃去,如此重复5次,拍干。
(9)加酶:每孔加入酶标试剂50μL,空白孔除外。
(10)温育:操作同(3)。
(11)洗涤:操作同(5)。
(12)显色:每孔先加入显色剂A50μL,再加入显色剂B50μL,轻轻震荡混匀,37℃避光显色15分钟。
(13)终止:每孔加终止液50μL,终止反应(此时蓝色立转黄色)。
(14)测定:以空白空调零,450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15分钟以内进行。
4.NtCYC基因启动子表达活性
以NtCYC转基因植株根、茎和叶等部位为材料,经过GUS染色、酒精脱色处理后,在显微镜下观察拍照,结果如图6所示,转基因植株根、茎和叶的腺毛染上蓝色,而对照K326均没有染上颜色,说明NtCYC基因启动子驱动的GUS基因只在腺毛上特异表达;而对照K326的根、茎和叶无GUS表达活性。
5.温度对NtCYC启动子活性的影响
以28℃为对照,4℃、42℃分别处理后,测定的GUS活性浓度均小于转基因植株在28℃条件下的酶活性浓度,见图7,说明NtCYC启动子在烟草中能启动GUS报告基因表达,但启动子在不同温度下的活性不同,在4℃、42℃下该NtCYC启动子的活性显著低于28℃的活性。
综上,在类西柏烷二萜化合物的生物合成过程中,NtCYC基因的表达受到NtCYC基因启动子顺式作用元件的调控,研究表明,本发明NtCYC基因启动子只在烟草腺毛中特异表达,叶面分泌物只由烟草腺毛腺头细胞分泌可能与此有关;不同的温度条件对该NtCYC基因启动子的活性有显著影响。因此,通过本发明启动子转基因方式实现了目的基因高水平表达,提高作物的香味品质和天然抗性。
SEQUENCELISTING
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Claims (6)
1.一种烟草西柏三烯一醇合成酶基因启动子,其核苷酸序列为:
(1)SEQIDNO.1所示的核苷酸序列;或
(2)SEQIDNO.1所示的核苷酸序列经重复、缺失、替换或移位等常规技术手段改造而形成的具有同等功能的核苷酸序列。
2.一种嵌合基因,其中包含权利要求1所述烟草西柏三烯一醇合成酶基因启动子以及与之可操作连接的、编码目的基因的序列。
3.一种植物转化载体,其中包含权利要求2所述嵌合基因。
4.一种转基因植物细胞,其中包含权利要求2所述嵌合基因。
5.一种转基因植物组织,其中包含权利要求2所述嵌合基因。
6.权利要求1所述烟草西柏三烯一醇合成酶基因启动子在烟草西柏三烯一醇合成调控中的应用。
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