CN113957085A - 圆锥铁线莲异戊烯基转移酶pt1基因的应用及其过表达拟南芥株系和构建方法 - Google Patents
圆锥铁线莲异戊烯基转移酶pt1基因的应用及其过表达拟南芥株系和构建方法 Download PDFInfo
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Abstract
本发明公开了一种圆锥铁线莲异戊烯基转移酶PT1基因的应用及其过表达拟南芥株系和构建方法。其中,圆锥铁线莲异戊烯基转移酶PT1基因其核苷酸序列如SEQ ID NO.1所示。本发明采用模式植物拟南芥,通过构建植物PT1基因过表达载体及转基因操作,获得拟南芥转PT1基因的阳性株系。本发明证实转基因拟南芥株系的植物形态具有叶片表面多腺毛、叶片颜色深的特点。在受到紫外胁迫后,转基因型拟南芥叶片中O2 ‑生成速率和H2O2含量增加均显著低于野生型拟南芥,叶片中MDA含量增加显著低于野生型拟南芥叶片,叶片电导率的增加显著低于野生型拟南芥叶片。在受到极限紫外胁迫后,转基因型拟南芥的存活率显著高于野生型拟南芥。
Description
技术领域
本发明属于植物基因工程领域,提供一种圆锥铁线莲异戊烯基转移酶PT1(Prenyltransferase 1,PT1)基因的应用及其过表达拟南芥株系和构建方法。
背景技术
圆锥铁线莲(Clematis terniflora DC.)为毛茛科铁线莲属(Clematis)植物,作为中药及民间草药已有很长的应用历史,临床和民间常用做利尿通淋或祛风止痛类药物,全草用于治疗慢性咽炎和慢性前列腺炎等,具有较高的药用价值。
植物或者微生物体内的异戊烯基化转移酶,能够将生物体内甲羟戊酸或者甲基赤藓醇磷酸途径产生的异戊烯基取代基,连接到不同小分子化合物或化合物不同的取代位置。异戊烯基作为一种活性基团,还可以在修饰酶的作用下进一步发生氧化、环合、脱水以及重排等反应,促进了异戊烯基化合物结构多样性。芳香类异戊烯基化合物种类众多、结构更加变化多样,一方面在植物的正常生长发育中扮演着不可替代的作用,另一方面也与植物防御逆境有着很大的联系,而且,异戊烯基类芳香类化合物往往具有更强的药理活性,常常被作为创新药物筛选的先导化合物或者前体物。
异戊烯基转移酶广泛分布在各类植物或微生物中,参与超过6万种异戊烯基化合物的形成或修饰。鉴于异戊烯基转移酶的重要性和特异性,对异戊烯基转移酶分子水平的研究越来越受到关注。最早的腺苷酸异戊烯基转移酶是在各类细菌中发现的;2008年,Sasaki等人第一次克隆表达出苦参中类黄酮的异戊烯基转移酶(naringenin 8-dimethylallytransferase,SDN8DT1)。在此之后,大豆、白色羽扇豆、紫花苜蓿、洋芹欧洲防风草等植物中的芳香族异戊烯基转移酶也陆续得到了克隆表达。但是,对异戊烯基转移酶功能挖掘以及对其参与的生理生化反应的研究还非常欠缺,亟待深入。
紫外光作为一种非生物逆境胁迫对植物的根、茎、叶的生长状态有一定影响,尤其是短波紫外线UV-B会对植物造成一定程度的生理性损伤。一般的,植物会通过改变形态、调节生理指标和光合速率、促进某些次生代谢产物含量的增加以及加强抗氧化防御系统来应对低剂量紫外胁迫的伤害。但是,应对高剂量的紫外胁迫,仅靠植物自身的调节已经不能完全修复带来的损伤。采用人为的技术手段加强植物对紫外胁迫的抗性是行之有效的方法。目前,采用基因技术增强植物抗紫外胁迫能力的研究尚不够深入。
因此,亟需提供一种能增强野生型拟南芥植物的植物形态特征和/或提高植物抗紫外胁迫的基因及其方法。
发明内容
本发明的目的在于克服现有技术中的缺陷,并提供一种圆锥铁线莲异戊烯基转移酶PT1(Prenyltransferase 1,PT1)基因的应用及其过表达拟南芥株系和构建方法。本发明在长期实验研究的基础上,克隆表达出圆锥铁线莲的异戊烯基转移酶PT1基因;通过转基因拟南芥的表型分析及功能验证,证实了过表达PT1 基因的拟南芥具有更强的抗紫外胁迫能力。
本发明所采用的具体技术方案如下:
第一方面,本发明提供了一种圆锥铁线莲异戊烯基转移酶PT1基因的应用,所述圆锥铁线莲异戊烯基转移酶PT1基因的核苷酸序列如SEQ ID NO.1所示,通过将该基因的表达载体转化到野生型拟南芥植物中进行过表达,从而改变植物形态特征和/或提高植物抗紫外胁迫。
作为优选,所述改变植物形态特征的具体表现为:过表达PT1基因能增加叶片表面腺毛且能加深叶片颜色。
作为优选,所述提高植物抗紫外胁迫的具体表现为:在植物受到紫外胁迫后,过表达PT1基因能降低叶片中的O2 -生成速率、H2O2增加量、丙二醛增加量和电导率的增加。
作为优选,所述提高植物抗紫外胁迫的具体表现为:在植物受到极限紫外胁迫后,过表达PT1基因能增加植物的存活率。
作为优选,所述圆锥铁线莲异戊烯基转移酶PT1基因能作为培育强抗紫外胁迫植物的基因资源。
第二方面,本发明提供了一种基于圆锥铁线莲异戊烯基转移酶PT1基因的过表达拟南芥株系的构建方法,具体如下:
S1:利用Trizol法提取圆锥铁线莲RNA,反转录获得cDNA,利用高保真酶从cDNA模板中扩增如SEQ ID NO.1所示的PT1基因序列,以获得圆锥铁线莲异戊烯基转移酶PT1基因CDS片段;扩增PT1基因序列时采用的正向引物和反向引物为:
正向引物:5’-ATGATTTCTGCTTTAGCTTCTCCA-3’
反向引物:5’-TCAACAAACAAAGGGAATAAGCAAA-3’;
S2:构建含有圆锥铁线莲异戊烯基转移酶PT1基因编码区的表达载体,具体步骤如下:
1)利用DNA凝胶回收试剂盒对已连接同源臂的圆锥铁线莲异戊烯基转移酶PT1基因进行胶回收,获得纯化PCR产物;
2)利用限制性内切酶对所述PCR纯化产物和质粒进行酶切;
3)使用同源重组法将目的基因与载体pCAMBIA2302连接,通过热激法导入大肠杆菌中,经菌液PCR扩增、电泳和测序后获得含有目的基因的重组质粒,实现表达载体的构建;
S3:将所述表达载体通过农杆菌介导法转化到野生型拟南芥植株中,具体步骤如下:
1)使用冻融法将重组质粒导入农杆菌中,平板筛选获得阳性克隆菌株;
2)取阳性克隆的农杆菌菌液加入到液体LB培养基中连续扩繁两次,然后将菌块重悬浮并加入表面活性剂后搅拌均匀,将拟南芥花蕾浸入菌液浸花后,黑暗保湿放置,然后置于光照培养箱培养,待其生长成熟时收种;
3)培养浸花转化获得的拟南芥种子至幼苗生长出两片真叶,喷施Basta工作液,培养5~15天后,将抗性单株移栽到新的基质中培养,待植株长出5~8片真叶时,分别提取野生型植株与转基因型植株RNA,反转录获得cDNA后进行 PCR扩增,将PCR产物进行琼脂糖凝胶电泳检测,以筛选获得过表达拟南芥T1 代种子,进而得到过表达拟南芥株系。
作为优选,所述步骤S3中,将拟南芥花蕾浸入菌液浸花10~20s后,黑暗保湿放置24~48h。
作为优选,所述步骤S3中,Basta工作液的浓度为0.001%~0.005%。
作为优选,所述步骤S3中,利用Primer 5软件设计一对特异性引物:
正向引物PT1yz1Fp:5’-GGCGGCAGCAACTATAACCA-3’
反向引物PT1yz1Rp:5’-CTCCCTCAAACTTGACTTCAGCAC-3’
进行过表达拟南芥T1代筛选,其中引物PT1yz1Fp在目标片段序列中设计,而PT1yz1Rp在目标载体序列上设计,使该特异引物能特异的检测出转化成功的拟南芥T1代单株,最终筛选出过表达拟南芥株系。
第三方面,本发明提供了一种利用如第二方面任一所述构建方法得到的基于圆锥铁线莲异戊烯基转移酶PT1基因的过表达拟南芥株系。
本发明相对于现有技术而言,具有以下有益效果:
本发明采用模式植物拟南芥,通过构建植物PT1基因过表达载体及转基因操作,获得拟南芥转PT1基因的阳性株系。本发明证实转基因拟南芥株系的植物形态具有叶片表面多腺毛、叶片颜色深的特点。在受到紫外胁迫后,转基因型拟南芥叶片中O2 -生成速率和H2O2含量增加均显著低于野生型拟南芥,叶片中 MDA含量增加显著低于野生型拟南芥叶片,叶片电导率的增加显著低于野生型拟南芥叶片。在受到极限紫外胁迫后,转基因型拟南芥的存活率显著高于野生型拟南芥。由此可以看出,本发明提供的转基因型拟南芥较野生型拟南芥对紫外胁迫表现出更强的抗性,该圆锥铁线莲异戊烯基转移酶基因PT1可以作为培育抗紫外胁迫植物的基因资源。本发明对植物中同源基因功能的阐述以及通过基因工程选育抗紫外胁迫品种具有重要的意义。
附图说明
图1为采用RT-PCR试验检测PT1的基因的表达,其中,1-6号为转基因型拟南芥植物,7号为ddH2O阴性对照,8号为重组质粒-阳性对照。
具体实施方式
下面结合附图和具体实施方式对本发明做进一步阐述和说明。本发明中各个实施方式的技术特征在没有相互冲突的前提下,均可进行相应组合。
本发明提供了一种圆锥铁线莲异戊烯基转移酶PT1基因的应用,其中,圆锥铁线莲异戊烯基转移酶PT1基因的核苷酸序列如SEQ ID NO.1所示,通过将该基因的表达载体转化到野生型拟南芥植物中进行过表达,从而改变植物形态特征和/或提高植物抗紫外胁迫。
本发明将所构建的表达载体转化到野生型拟南芥植物,得到转基因拟南芥植物,并进一步对转基因拟南芥植物表型、生理参数进行了研究。这里的转基因拟南芥植物指的是将该基因的表达载体转化到野生型拟南芥植物中进行过表达所得植物,下面为了方便表述,转基因拟南芥植物、转基因型拟南芥植物和过表达拟南芥株系均为相同指代含义。
本发明对比研究了转基因拟南芥植物叶片与野生型拟南芥植物叶片的表面特征,发现转基因植株具有叶片表面多腺毛、叶片颜色深的特点。
同时,本发明研究对比发现,在受到紫外胁迫后,转基因型拟南芥叶片中 O2 -生成速率和H2O2含量增加均显著低于野生型拟南芥;转基因型拟南芥叶片中丙二醛(MDA)含量增加显著低于野生型拟南芥叶片;转基因型拟南芥叶片电导率的增加显著低于野生型拟南芥叶片。在受到极限紫外胁迫后,转基因型拟南芥的存活率显著高于野生型拟南芥。
此外,本发明研究证实,转基因型拟南芥较野生型拟南芥对紫外胁迫表现出更强的抗性。圆锥铁线莲异戊烯基转移酶PT1基因能作为培育强抗紫外胁迫植物的基因资源。
本发明提供了一种基于圆锥铁线莲异戊烯基转移酶PT1基因的过表达拟南芥株系的构建方法的优选方式,具体如下:
S1:获得PT1基因CDS片段:利用Trizol法提取圆锥铁线莲RNA,反转录获得cDNA,利用高保真酶从cDNA模板中扩增PT1基因序列(SEQ ID NO.1)。
正向引物:5’-ATGATTTCTGCTTTAGCTTCTCCA-3’
反向引物:5’-TCAACAAACAAAGGGAATAAGCAAA-3’
所述圆锥铁线莲异戊烯基转移酶PT1基因的核苷酸序列如SEQ ID NO.1所示;
S2:构建含有PT1基因编码区的表达载体,具体的该方法包括以下步骤:
I.纯化PCR产物
利用SteadyPure DNA凝胶回收试剂盒(AG公司)对已连接同源臂的PT1 基因进行胶回收。
II.质粒-双酶切
利用内切酶NcoI和Bgl II对PCR纯化产物和pCAMBIA2302质粒进行酶。
III.重组质粒
使用同源重组法将目的基因与载体pCAMBIA2302连接,热激法导入大肠杆菌中,经菌液PCR扩增、电泳、测序后获得含有目的基因的重组质粒。
S3:采用农杆菌介导植物转化。具体包括以下步骤:
I.重组载体导入农杆菌
使用冻融法将重组质粒导入农杆菌GV3101中,平板筛选获得阳性克隆菌株。
II.浸花法侵染拟南芥
取阳性克隆的农杆菌菌液加入到液体LB培养基中连续扩繁两次,然后将菌块重悬浮后加入Silwet L-77,搅拌均匀,将拟南芥花蕾浸入菌液浸花10~20S 后,黑暗保湿放置24-48h,然后置于光照培养箱正常培养,待其生长成熟时收种。
III.筛选获得过表达拟南芥T1代种子
培养浸花转化获得的拟南芥种子至幼苗生长出两片真叶,喷施0.001%-0.005%的Basta工作液,培养5-15天后,将抗性单株移栽到新的基质中培养,待植株长出5-8片真叶时,分别提取野生型植株与转基因型植株RNA,反转录获得cDNA 后进行PCR扩增,将PCR产物进行琼脂糖凝胶电泳检测。转基因拟南芥基因组 DNA中通过扩增得到目标条带PT1,野生型拟南芥基因组没有目标条带(如图1 所示)。作为优选,具体操作如下:
利用Primer 5软件设计一对特异性引物:
正向引物PT1yz1Fp:5’-GGCGGCAGCAACTATAACCA-3’
反向引物PT1yz1Rp:5’-CTCCCTCAAACTTGACTTCAGCAC-3’
进行过表达拟南芥T1代筛选,其中引物PT1yz1Fp在目标片段序列中设计而PT1yz1Rp在目标载体序列上设计,因此该特异引物能特异的检测出转化成功的拟南芥T1代单株。
实施例1
本实施例进行了圆锥铁线莲异戊烯基转移酶PT1基因的克隆与表达,具体方法如下:
利用Trizol法提取圆锥铁线莲RNA,反转录获得cDNA,利用高保真酶从 cDNA模板中扩增PT1基因序列(SEQ ID NO.1)。
正向引物:5’-ATGATTTCTGCTTTAGCTTCTCCA-3’
反向引物:5’-TCAACAAACAAAGGGAATAAGCAAA-3’
PCR扩增体系为:模板1μL,2×PCR缓冲液10μL,上、下游引物各0.8μL, dNTP 0.4μL,Polymerase 0.4μL,补充ddH2O至50μL。95℃变性3min;95℃15 S,52℃30S,72℃1min,35个循环;72℃延伸5min,4℃保存。PCR产物经电泳、回收、测序后-20℃保存备用。
利用SteadyPure DNA凝胶回收试剂盒(AG公司)对已连接同源臂的PT1 基因进行胶回收。利用内切酶NcoI和Bgl II对PCR纯化产物和pCAMBIA2302 质粒进行酶切,酶切体系为:10×缓冲液2μL,pCAMBIA2302质粒≤1μg,NcoI 和Bgl II各1μL,补充ddH2O至20μL。37℃2h;65℃20min,4℃保存。
使用同源重组法将目的基因与载体pCAMBIA2302连接,热激法导入大肠杆菌中,经菌液PCR扩增、电泳、测序后获得含有目的基因的重组质粒。
使用冻融法将重组质粒导入农杆菌GV3101感受态细胞中,平板筛选获得阳性克隆菌株。利用基因特异引物进行PCR鉴定,并将该质粒转化到E.coli DH5α感受态细胞中,筛选阳性克隆,进一步测序验证。利用经过测序验证的阳性克隆的农杆菌菌液,进行浸花法转化哥伦比亚野生型拟南芥(Col.0)。具体操作如下:
将收获的拟南芥种子点播于育苗块表面,置于温度21±2℃的光照培养箱中生长,光照周期为16h光照/8h黑暗,光照强度为8000Lx。待主花薹长至10cm 左右高时,剪去花薹,然后待长至盛花期时,进行浸花转化。
取20μL阳性克隆的农杆菌菌液加入到15mL液体LB培养基(加Spc、Rif、 Str)中28℃下摇菌24h进行扩繁,然后取5mL新摇的菌液加入到500mL液体LB培养基(加Spc、Rif、Str)中28℃下摇菌36h,将菌液分装至100mL大离心管中常温下8000rpm离心10min。弃上清后,加入5%的蔗糖对菌块进行重悬浮至OD值为1.5-2.0,按照7μL/mL的比例加入Silwet L-77,搅拌均匀后,将拟南芥花蕾浸入菌液浸花10s后,黑暗保湿放置24h,然后置于光照培养箱正常培养,待其生长成熟时收种。
培养浸花转化获得的拟南芥种子,待大部分幼苗生长出两片真叶时,喷施0.0015%的Basta工作液(100mL水,15μL Basta、20μL Silvet)1次,再培养1 周后,将抗性单株株移栽到新的基质中培养,待植株长出5片真叶时,分别提取野生型植株与转基因型植株RNA,反转录获得cDNA后进行PCR扩增,将PCR 产物进行琼脂糖凝胶电泳检测,转基因拟南芥基因组DNA中通过扩增得到目标条带PT1,而未转化的植株没有得到目标条带。具体操作如下:
利用Primer 5软件设计一对特异性引物:
正向引物PT1yz1Fp:5’-GGCGGCAGCAACTATAACCA-3’
反向引物PT1yz1Rp:5’-CTCCCTCAAACTTGACTTCAGCAC-3’
进行过表达拟南芥T1代筛选,其中引物PT1yz1Fp在目标片段序列中设计而PT1yz1Rp在目标载体序列上设计,因此该特异引物能特异的检测出转化成功的拟南芥T1代单株。经过上述筛选过程,本实施例得到6个过表达拟南芥株系。
实施例2
本实施例利用实施例1所得过表达拟南芥株系进行了筛选,得到四个转基因拟南芥植株,并将四个转基因拟南芥植株与原始的野生型拟南芥植株进行了PT1 基因在改变植株表型方面的研究,具体如下:
分别播种6个过表达拟南芥株系以及原始的野生型拟南芥各12株,具体操作及培养条件为:将拟南芥种子点播于育苗块表面,置于温度21±2℃的光照培养箱中生长,光照周期为16h光照/8h黑暗,光照强度为8000Lx,约15天后进行移苗,将拟南芥从育苗块转移至6cm×6cm方形花盆中,培养条件保持不变。
对各转基因拟南芥进行引物验证实验,如图1所示的凝胶电泳结果表明,1、 3、4、6号泳道目的条带清晰且明亮,分别对应PT1-8、PT1-10、PT1-11、PT1-13 四个过表达拟南芥株系,表明这四个拟南芥株系可作为后续实验材料。为了便于后续描述,将这四个拟南芥株系命名为转基因拟南芥植株。
在植株长出7片真叶时进行形态观察:与野生型拟南芥相比,转基因拟南芥叶片明显增厚,叶片颜色加深,腺毛密集且粗,推测这些性状与转基因拟南芥抗逆性有关。
实施例3
本实施例利用实施例2所得转基因拟南芥与原始的野生型拟南芥植株,进行了转基因植株在提高植物抗紫外胁迫方面的功能验证,具体如下:
(1)紫外照射处理
分别给予转基因拟南芥植株与野生型拟南芥植株同等条件紫外照射。照射条件如下:照射时间72h,照射强度约为14000Lx。
(2)紫外处理后表型分析
可以看出,在紫外处理72h后,转基因拟南芥的生长状态明显优于对照。野生型拟南芥叶片严重失水,出现干枯萎蔫状态,而转基因拟南芥叶片生长状态良好,仅少数叶片出现萎蔫,多数叶片呈现绿色健康生长状态。
(3)紫外处理后生理参数检测
1)对叶片活性氧生成的影响
采用超氧阴离子自由基(O2 -)试剂盒、过氧化氢(H2O2)试剂盒进行测定。
表1转基因型拟南芥和野生型拟南芥在紫外胁迫72h后叶片中O2 -生成速率的影响
表2转基因型拟南芥和野生型拟南芥在紫外胁迫72h后叶片中H2O2含量变化
由表1-2可以看出,野生型拟南芥在受到紫外胁迫后,其叶片中O2 -生成速率和H2O2含量显著增加,而转基因型拟南芥叶片变化不大。
2)丙二醛MDA的测定
采用硫代巴比妥酸法测定MDA含量。
分别称取转基因型拟南芥和野生型拟南芥新鲜叶片0.1g,放于液氮预冷的研钵之中,加入5mL10%三氯乙酸(TCA)研磨至匀浆,常温下4000r/min离心10min,取得上清液。吸取上清液2mL置于试管中,再向试管中加入2m L 0.6%硫代巴比妥酸TBA,摇匀后置沸水浴15min,取出试管冷却至室温后4000r/min 离心10min,2mL上清液加10%TCA为对照,测定在波长450nm、532nm、 600nm处的吸光值,计算各处理组的丙二醛含量。
表3转基因型拟南芥和野生型拟南芥在紫外胁迫72h后叶片中MDA含量
由表3可以看出,野生型拟南芥在受到紫外胁迫后,其叶片中MDA含量显著增加,而转基因型拟南芥叶片变化不大。
3)电导率的测定
分别称取转基因型拟南芥和野生型拟南芥新鲜叶片0.2g,用蒸馏水洗去表面污垢后用滤纸擦干,剪碎放入25mL试管,加入20mL去离子水,测定一次去离子水电导率,摇匀浸泡24h后用DDS-307电导仪测定初电导率,再将各处理试管加盖放到沸水水浴锅中煮30min后取出冷却,到室温后测定终电导率,计算叶片相对电导率。
表4转基因型拟南芥和野生型拟南芥在紫外胁迫72h后叶片相对电导率的变化
由表4可以看出,野生型拟南芥在受到紫外胁迫后,其叶片中电导率均显著增加,但转基因型拟南芥增幅显著低于野生型拟南芥。
由此可以看出,本发明提供的转基因型拟南芥较野生型拟南芥对紫外胁迫表现出更强的抗性,该圆锥铁线莲异戊烯基转移酶基因PT1可以作为培育抗紫外胁迫植物的基因资源。
以上所述的实施例只是本发明的一种较佳的方案,然其并非用以限制本发明。有关技术领域的普通技术人员,在不脱离本发明的精神和范围的情况下,还可以做出各种变化和变型。因此凡采取等同替换或等效变换的方式所获得的技术方案,均落在本发明的保护范围内。
序列表
<110> 浙江理工大学
<120> 圆锥铁线莲异戊烯基转移酶PT1基因的应用及其过表达拟南芥株系和构建方法
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 1203
<212> DNA
<213> 圆锥铁线莲(Clematis terniflora DC.)
<400> 1
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actaggcccc tggatggttt cagtctacgg tctcatgagt ccagaaatct gacttccagc 120
aagaacagaa gatgtagtcg aacagtttct tcgtctttgc cgcgcaaatt tgaaaatatt 180
gattcgcaaa tctcaagcag agagaaccca tcgcccaagt attctgtctc cccaccattt 240
gatagcgtgg atggaattat tcaagaaaag gaattttcca tttactcgtg gactaatatt 300
gccaagaaat tgggtgcatg ttatcagttc attcgtcctt tccaaatacg ctcagcaatt 360
ctggaaataa tagtgatctc actttttcct gtgcaagcaa tatctgatct ttccccgaca 420
ttcttcgttc gactcttgaa gacgattata cctgtggttt tcgtttatgt gtacgcgaat 480
ggcctgaatg aaataactga ccttgaaatc gataagatta acaagccgtt tcttcccctt 540
gcttcagggg atttatctct taccgaagcc acagcaataa taagcatttc tagtgttata 600
atctttctca taacggccac gctaaactcg gtaccgatgt cgattgccat aatgagcatc 660
tttttagcct ttagtgcata ttcaattgat cttcctcttc tcaggtggaa aaaaaatgca 720
gtgtcggcgg cagcaactat aaccactata caaggaattg tgacgccact atgtttcttt 780
gcccatgccc agacgtacat tcttggtaaa cccttagtgt ttacaaaaac agtattcttt 840
actatggcca tcatgactag ctttggtgtt gtcatctcaa tattcaagga tatatctgat 900
ttggaaggag ataaagcaca tggagttcga tcaacaagtg ccatttacgg ggtagaaaaa 960
gttttttggt cctctattta tatattgttg gcaacatatg gaggtgctgc tatttttggc 1020
gctacttcac ctaatctcct tacaaagatt attgctgttt tggggcatgg aattcctgct 1080
attatacttt ggactcggac aaagtctact aatatctcag acccaacaac cacatttcct 1140
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tga 1203
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<212> PRT
<213> 圆锥铁线莲(Clematis terniflora DC.)
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Claims (10)
1.一种圆锥铁线莲异戊烯基转移酶PT1基因的应用,其特征在于,所述圆锥铁线莲异戊烯基转移酶PT1基因的核苷酸序列如SEQ ID NO.1所示,通过将该基因的表达载体转化到野生型拟南芥植物中进行过表达,从而改变植物形态特征和/或提高植物抗紫外胁迫。
2.根据权利要求1所述的应用,其特征在于,所述改变植物形态特征的具体表现为:过表达PT1基因能增加叶片表面腺毛且能加深叶片颜色。
3.根据权利要求1所述的应用,其特征在于,所述提高植物抗紫外胁迫的具体表现为:在植物受到紫外胁迫后,过表达PT1基因能降低叶片中的O2 -生成速率、H2O2增加量、丙二醛增加量和电导率的增加。
4.根据权利要求1所述的应用,其特征在于,所述提高植物抗紫外胁迫的具体表现为:,在植物受到极限紫外胁迫后,过表达PT1基因能增加植物的存活率。
5.根据权利要求1所述的应用,其特征在于,所述圆锥铁线莲异戊烯基转移酶PT1基因能作为培育强抗紫外胁迫植物的基因资源。
6.一种基于圆锥铁线莲异戊烯基转移酶PT1基因的过表达拟南芥株系的构建方法,其特征在于,具体如下:
S1:利用Trizol法提取圆锥铁线莲RNA,反转录获得cDNA,利用高保真酶从cDNA模板中扩增如SEQ ID NO.1所示的PT1基因序列,以获得圆锥铁线莲异戊烯基转移酶PT1基因CDS片段;扩增PT1基因序列时采用的正向引物和反向引物为:
正向引物:5’-ATGATTTCTGCTTTAGCTTCTCCA-3’
反向引物:5’-TCAACAAACAAAGGGAATAAGCAAA-3’;
S2:构建含有圆锥铁线莲异戊烯基转移酶PT1基因编码区的表达载体,具体步骤如下:
1)利用DNA凝胶回收试剂盒对已连接同源臂的圆锥铁线莲异戊烯基转移酶PT1基因进行胶回收,获得纯化PCR产物;
2)利用限制性内切酶对所述PCR纯化产物和质粒进行酶切;
3)使用同源重组法将目的基因与载体pCAMBIA2302连接,通过热激法导入大肠杆菌中,经菌液PCR扩增、电泳和测序后获得含有目的基因的重组质粒,实现表达载体的构建;
S3:将所述表达载体通过农杆菌介导法转化到野生型拟南芥植株中,具体步骤如下:
1)使用冻融法将重组质粒导入农杆菌中,平板筛选获得阳性克隆菌株;
2)取阳性克隆的农杆菌菌液加入到液体LB培养基中连续扩繁两次,然后将菌块重悬浮并加入表面活性剂后搅拌均匀,将拟南芥花蕾浸入菌液浸花后,黑暗保湿放置,然后置于光照培养箱培养,待其生长成熟时收种;
3)培养浸花转化获得的拟南芥种子至幼苗生长出两片真叶,喷施Basta工作液,培养5~15天后,将抗性单株移栽到新的基质中培养,待植株长出5~8片真叶时,分别提取野生型植株与转基因型植株RNA,反转录获得cDNA后进行PCR扩增,将PCR产物进行琼脂糖凝胶电泳检测,以筛选获得过表达拟南芥T1代种子,进而得到过表达拟南芥株系。
7.根据权利要求6所述的构建方法,其特征在于,所述步骤S3中,将拟南芥花蕾浸入菌液浸花10~20s后,黑暗保湿放置24~48h。
8.根据权利要求6所述的构建方法,其特征在于,所述步骤S3中,Basta工作液的浓度为0.001%~0.005%。
9.根据权利要求6所述的构建方法,其特征在于,所述步骤S3中,利用Primer 5软件设计一对特异性引物:
正向引物PT1yz1Fp:5’-GGCGGCAGCAACTATAACCA-3’
反向引物PT1yz1Rp:5’-CTCCCTCAAACTTGACTTCAGCAC-3’
进行过表达拟南芥T1代筛选,其中引物PT1yz1Fp在目标片段序列中设计,而PT1yz1Rp在目标载体序列上设计,使该特异引物能特异的检测出转化成功的拟南芥T1代单株,最终筛选出过表达拟南芥株系。
10.一种利用权利要求6~9任一所述构建方法得到的基于圆锥铁线莲异戊烯基转移酶PT1基因的过表达拟南芥株系。
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