CN112430602A - 毛果杨hda909基因在提高植物抗真菌能力中的应用 - Google Patents
毛果杨hda909基因在提高植物抗真菌能力中的应用 Download PDFInfo
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Abstract
本发明公开了毛果杨HDA909基因在提高植物抗真菌能力中的应用,涉及基因工程技术领域。本发明根据毛果杨HDA909基因序列,构建毛果杨HDA909过表达载体,采用农杆菌介导法导入受体植物,使得转基因植物中的HDA909基因过量表达,获得抗真菌植物。利用链格孢菌侵染植物,发现转基因植株的菌斑数量和菌斑面积显著降低,所受链格孢菌侵染损伤的程度较轻。本发明证明了毛果杨HDA909基因的过量表达能够提高植物抗真菌能力,为未来进一步创建抗真菌的转基因植株提供了新的思路。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及一种毛果杨HDA909基因在提高植物抗真菌能力中的应用。
背景技术
随着社会经济发展,环境污染日趋严重,原有的森林树种将会遇到与自然选择下有重大不同的环境胁迫压力。为保障人类生存环境和生活质量不再劣化,人类必须学会协助林木去更好更快地适应所遇到的各种随人类社会现代化而加剧和新出现的环境胁迫因素。由于林木生长周期长,遗传杂合性高,利用常规育种手段往往难以满足不同目的定向培育树木新品种的要求。近年来生物技术的发展以及分子育种研究的兴起,为利用基因工程手段改良林木、加速林木新品种的选育奠定了基础。
病原菌侵染是造成植物生产力大面积损失的重要因素。链格孢菌有299种,它不仅是人类对花粉和哮喘病的过敏源,更是主要的植物病原体。链格孢菌的孢子在植物表面生长繁殖时会产生大量菌丝,破坏植物细胞组织,致使植物受到侵害。
发明内容
本发明的目的是提供一种毛果杨HDA909基因在提高植物抗真菌能力中的应用,以解决上述现有技术存在的问题。
为实现上述目的,本发明提供了如下方案:
本发明提供毛果杨HDA909基因在提高植物抗真菌能力中的应用,所述的毛果杨HDA909基因序列如SEQ ID NO.1所示,所述毛果杨HDA909基因编码的氨基酸序列如SEQ IDNO.2所示。
本发明还提供一种抗真菌植物的创制方法,根据权利要求1所述的毛果杨HDA909基因序列,构建毛果杨HDA909过表达载体,采用农杆菌介导法导入受体植物,使得转基因植物中的HDA909基因过量表达,获得抗真菌能力。
优选的,所述的受体植物是毛果杨。
优选的,所述的真菌为链格孢菌。
优选的,毛果杨HDA909过表达载体的构建方法,包括以下步骤:
1)以野生型毛果杨的叶片cDNA为模板,HDA909-Primer-F和HDA909-Primer-R为引物,获得毛果杨HDA909基因目的片段;
2)利用Plasminal Mini KitⅠ将所得毛果杨HDA909基因目的片段和pMD18-TVector载体连接,连接产物为pMD18-HDA909;连接产物转化到大肠杆菌感受态细胞,得到pMD18-HDA909重组质粒;
3)用BamHⅠ和KpnⅠ分别对pMD18-HDA909重组质粒和pROKⅡ质粒进行双酶切,双酶切后得到的HDA909基因片段和pROKⅡ载体线性片段用Gel Extraction Kit进行回收,回收产物利用T4连接酶连接,得到、pROKⅡ-HDA909质粒,即为毛果杨HDA909过表达载体。
优选的,HDA909-Primer-F引物序列如SEQ ID NO.3所示,HDA909-Primer-R引物序列如SEQ ID NO.4所示。
本发明公开了以下技术效果:
本发明利用农杆菌介导法将毛果杨HDA909基因遗传转化到毛果杨中,从而使其在转基因植株中过量表达。选用HDA909转基因和非转基因植株第3、4节点的叶片进行离体真菌接种实验。在接种链格孢菌1周后,与非转基因植株相比,转基因植株的菌斑数量和菌斑面积显著降低。通过二氨基联苯胺法对染病叶片进行染色,结果显示,野生型植株病变叶片产生棕红色斑点数量明显多于转基因植物染病叶片,且病斑颜色较深,说明野生型植株叶片细胞中积累的H2O2较多,转基因植株叶片细胞中积累的H2O2较少;野生型植株叶片所受链格孢菌侵染产生的损伤较为严重,转基因植株叶片所受链格孢菌侵染损伤的程度较轻。以上结果表明,毛果杨中HDA909基因的过量表达可以显著提高转基因植株叶片对链格孢菌的抗性。本发明证明了毛果杨HDA909基因的过量表达能够提高植物抗真菌能力,为下一步构建抗真菌的植物品种提供了工具。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为HDA909基因片段的PCR扩增结果,图中M为DL 2000Marker;1为HDA909编码区片段扩增结果;
图2为pROKⅡ-HDA909重组质粒的酶切鉴定结果,图中M为DL 2000Marker;1为毛果杨HDA909编码区片段;2为pROKⅡ-HDA909重组质粒酶切产物;3为pROKⅡ质粒酶切产物;
图3为pROKⅡ-HDA909重组质粒PCR电泳结果,图中M为DL 10000Marker;1-3为检测的质粒;4为阴性对照(ddH2O);
图4为HDA909基因遗传转化毛果杨的生长图,图中A为侵染的茎段在毛果杨分化培养基中生长情况,B为转基因毛果杨在含Kan的生根培养基中生长,C为转基因植株在含Kan的生根培养基上生根情况;
图5为转基因植株的RT-PCR检测结果,图中1为非转基因毛果杨;2为HDA909转基因植株Tr-43;3为HDA909转基因植株Tr-10;4为HDA909转基因植株Tr-46;5为阴性对照(ddH2O);
图6为接种真菌后毛果杨叶片染病情况,图中A为叶片感染链格孢菌;B为毛果杨叶片接种链格孢菌的染病率;**为P<0.01,****为P<0.0001;
图7为转基因毛果杨叶片产生的病斑的情况,图中A为毛果杨病变叶片的病斑情况;B为毛果杨叶片接种链格孢菌的病斑面积;****为P<0.0001;
图8为转基因毛果杨病变叶片DAB染色情况。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明实施例所使用的材料与试剂均可由商业途径获得。
实施例1
毛果杨HDA909基因的克隆
1、实验材料
植物材料为野生型毛果杨(Populus trichocarpa)无菌组培苗,菌种与载体见表1,主要试剂见表2。
表1菌种与载体
表2主要试剂
2、溶液配制
(1)0.1%DEPC水溶液:将超纯水与DEPC原液按9:1的比例充分混合,搅拌2h,灭菌20min。
(2)5M NaCl溶液:取0.1%DEPC水45mL溶解14.625g的NaCl,定容至50mL,37℃孵育12h,灭菌20min。
(3)75%乙醇溶液:将经高温灭菌并冷却至室温的0.1%DEPC水与无水乙醇按照1:3的比例混合均匀,置于-20℃条件下保存。
3、实验方法
3.1、毛果杨总RNA的提取
(1)在预冷的研钵中,将毛果杨叶片在液氮中研磨为粉末。
(2)将约0.1g样品粉末与4℃预冷的pBIOZOL试剂充分混合,然后放置5min。
(3)12000×g离心2min,取400μL上清液加到新的RNase-free的离心管中。
(4)加入100μL 5M NaCl溶液,颠倒混匀,加入300μL氯仿混匀,于4℃、12000×g离心10min。
(5)取上清液加到一个新的RNase-free的离心管中,加入等体积异丙醇,混合均匀,25℃静置10min,于4℃、12000×g离心10min。
(6)弃掉上清液(不要失去白色沉淀),向沉淀中加入800μL 75%乙醇,于4℃、12000×g离心8min,弃掉上清液,风干残留液体。
(7)向RNA沉淀中加入30μL无菌的0.1%DEPC水,冰浴0.5h,于-80℃保存。
3.2、毛果杨cDNA的合成
利用RT reagent Kit试剂盒将毛果杨叶片总RNA合成cDNA,反应体系及条件见表3、表4。
表3去除基因组DNA反应
反应条件:42℃,10min。
表4反转录反应
反应条件:42℃,60min;85℃,5min,4℃保存。
3.3、毛果杨HDA909基因的克隆
3.3.1、PCR扩增目的片段
以毛果杨叶片的cDNA为模板,利用HDA909基因的特异性引物扩增出HDA909基因的全长编码序列,反应体系和反应条件见表5、表6。
表5PCR扩增反应体系
表6PCR扩增反应程序
3.3.2、回收并纯化目的片段
PCR产物电泳,用胶回收试剂盒将基因片段纯化回收。
3.3.3、HDA909基因片段与pMD18-T Vector载体连接
将HDA909基因片段与pMD18-T Vector载体连接,连接体系见表7。
表7pMD18-T Vector载体连接体系
连接条件:16℃恒温过夜。连接产物为pMD18-HDA909。
3.3.4、连接产物转化感受态细胞及鉴定
把连接产物转化到DH5α中,将其均匀涂抹在含有Amp(25mg/L)的固体LB培养基上,于37℃倒置培养16h。将白色单菌落接种在含有Amp(25mg/L)的液体LB培养基中,在37℃,200rpm/min的条件下进行扩大培养。以菌液为模板进行PCR鉴定(反应体系、条件见表8、表9),将鉴定结果正确的菌液送至北京六合华大基因科技股份有限公司进行测序。
表8菌液PCR反应体系
表9菌液PCR反应程序
4、实验结果
本发明利用HDA909特异性引物,从毛果杨中克隆出HDA909基因的编码区片段,经测序其核苷酸序列长度为1323bp。PCR结果如图1所示,毛果杨HDA909基因的ORF序列长度为1323bp,如SEQ ID NO.1所示;HDA909基因编码的蛋白质由440个氨基酸构成,如SEQ IDNO.2所示。
实施例2
HDA909基因遗传转化毛果杨及其功能分析
1、实验材料
野生型毛果杨组培苗。链格孢菌(Alternaria alternata)属半知菌亚门,其分生孢子梗为褐色或青褐色倒棒形,多为单生或数根束生,分生孢子多为3~6个串生有纵膈膜1~2个、横膈膜3~4个。购自中国林科院森林生态环境与保护研究所。pROKⅡ载体为商业途径购买。主要试剂如表10所示。
表10主要试剂
毛果杨生根培养基:WPM培养基2.41g/L,蔗糖20g/L,琼脂粉5.8g/L,pH=5.75,IBA0.1mg/L。
毛果杨分化培养基:WPM培养基2.41g/L,蔗糖20g/L,琼脂粉5.8g/L,pH=5.75,6-BA 0.3mg/L,IBA 0.02mg/L和TDZ 0.0008mg/L。
2、实验方法
2.1、植物过量表达载体的构建
2.1.1、pMD18-HDA909与pROKⅡ载体的酶切与连接
利用Plasminal Mini Kit Ⅰ制备pMD18-HDA909重组质粒和pROKⅡ质粒。用BamH Ⅰ和Kpn Ⅰ分别对以上两种质粒进行双酶切,反应体系见表11。
表11双酶切反应体系
反应条件:37℃恒温酶切2.5h。
用干净的刀片切下双酶切后得到的HDA909基因片段和pROKⅡ载体线性片段,用Gel Extraction Kit进行回收,回收产物利用T4连接酶连接,连接体系见表12。
表12连接反应体系
连接条件:16℃恒温金属浴过夜。
2.1.2、连接产物转化大肠杆菌及鉴定
将pROKⅡ-HDA909的连接产物转化到DH5α中,在选择培养基(含Kan 50mg/L)上培养16h,任意挑取几个抗性单菌落分别放置于5mL LB液体培养基(含Kan 50mg/L)中进行扩大摇菌。以菌液为模板进行PCR扩增。
2.1.3、pROKⅡ-HDA909质粒提取与酶切鉴定
利用Plasmid Mini Kit Ⅰ试剂盒进行重组质粒的制备。利用限制性核酸内切酶BamH Ⅰ和Kpn Ⅰ对pROKⅡ-HDA909质粒进行双酶切鉴定,反应体系见表13。
表13双酶切反应体系
反应条件:37℃恒温酶切2.5h。
2.1.4、pROKⅡ-HDA909质粒转化农杆菌及鉴定
将pROKⅡ-HDA909质粒转化到农杆菌EHA105中,在选择培养基上培养2d。任意挑取抗性单菌落,放置到LB液体培养基中(含Kan 50mg/L),在28℃、180rpm/min的条件下过夜培养。以菌液为模板进行PCR验证,反应体系和反应条件见表14、表15。利用质粒提取试剂盒,从农杆菌菌液中提取pROKⅡ-HDA909质粒。将pROKⅡ-HDA909质粒转化到DH5α,并通过BamHⅠ和Kpn Ⅰ的双酶切进行鉴定,反应体系见表16。鉴定得到的阳性农杆菌菌液与40%的甘油等比例混合,于-80℃条件下保存,用于毛果杨的遗传转化实验。
表14菌液PCR反应体系
表15菌液PCR反应程序
表16双酶切反应体系
反应条件:37℃恒温酶切2.5h。
2.2、毛果杨的培养
在超净工作台中,切取无菌的三周龄毛果杨鲜嫩的不带芽点的茎段,茎段约为0.8cm长,放置在毛果杨分化培养基上,在25℃光周期为16h光照/8h黑暗的环境中进行茎段分化。待茎段分化出绿色幼芽,生长至约0.8~1cm高时,切取幼芽放置于毛果杨生根培养基中,待幼芽生长约为1.5cm时,切取单株幼芽***到毛果杨生根培养基中进行培养,3周后长出的植株的茎用于毛果杨的遗传转化。
2.3、毛果杨的遗传转化
取新鲜嫩绿状态良好的3周龄左右的毛果杨组培苗的茎段,利用农杆菌介导法遗传转化毛果杨。
(1)在无菌条件下,将携带HDA909基因的农杆菌菌液用经酒精灯外焰灼烧后并冷却至室温的接种环蘸取,采用三区划线法在含有Kan(50mg/L)和Rif(250mg/L)的固体LB培养基上倒置培养2d,培养条件为恒温28℃。
(2)在5mL液体LB培养基(含50mg/L Kan和250mg/L Rif)中接种在线上单一生长且直径约为1-2mm的白色圆形菌落,在220rpm/min、28℃的恒温条件下培养约16h。
(3)在30mL液体LB培养基中加入5mL培养好的菌液,在恒温28℃、220rpm/min的条件下培养约4h,此时菌液OD600值为0.6~0.8最为适宜。
(4)于4℃、4000×rpm的离心机中离心10min,倒掉上清液,用无菌水将剩余菌体重悬稀释至OD600值为0.4,重悬液用作侵染液。
(5)取新鲜嫩绿状态良好的3周龄左右的毛果杨组培苗,用经高温灭菌器灭菌并冷却至室温的剪刀将植株的叶片剪掉,保留约0.8cm叶柄与叶片连接的部分;再将植株的茎剪掉,用镊子将植株的茎放置在培养皿中,用手术刀片将植株的茎切成约为0.8cm长的茎段(不含腋芽部分)。
(6)用镊子将带一小部分叶片的叶柄和茎段放入事先准备好的侵染液中侵染10min,在这个过程中需要不时轻柔摇晃侵染液,使它与茎段伤口得到更充分的接触。用镊子将在侵染液中浸泡过的茎段取出,然后放置于事先准备好的经过高温灭菌的滤纸上,用于吸除植物茎段表面多余的菌液,不过要保持茎段的湿润,然后在分化培养基上利用无菌的镊子将茎段摆好,置于25℃的环境中暗培养2d。
(7)暗培养后,在无菌环境中,用镊子将侵染后的茎段放置到毛果杨分化筛选培养基上,在25℃光周期为16h光照/8h黑暗的环境中继续培养。
2.4、转基因毛果杨的抗性筛选
将在毛果杨分化培养基(含Kan 40mg/L)上生长出的带有鲜嫩抗性芽的茎段放入毛果杨生根培养基(含Kan 40mg/L)中继续筛选,然后将生长的新鲜嫩绿且单独的幼苗用无菌的刀切下,用无菌的镊子将其***到含有Kan的毛果杨生根筛选培养基中进行生根。待植株长到约为5cm时,在无菌环境中剪取植株叶片并快速冷冻,在-80℃条件下保存,作为后续分子检测的实验材料。
2.5、转基因毛果杨的分子检测
参照实施例1的实验方法提取卡那抗性转基因植株叶片的RNA并合成cDNA。以上述cDNA为模板进行定量RT-PCR,使用HDA909基因特异性引物来进行检测,反应体系和反应条件见表17、18。
表17菌液PCR反应体系
表18菌液PCR反应程序
2.6、转基因毛果杨真菌抗性分析
分析HDA909转基因毛果杨对链格孢菌的抗性。在接种链格孢菌的实验中,选取形状、大小及生长状态一致的HDA909转基因与非转基因的2.5月龄毛果杨大苗第3节点或第4节点的叶片。处理前用灭菌后的超纯水对叶片的正反面进行3次喷洒清洗。无菌环境中,用移液器吸取10mL无菌水将双层无菌滤纸打湿。在培养好的链格孢菌的平板内加入20mL无菌水并用无菌的玻璃棒多次反复刮擦,使大量菌丝和孢子溶于无菌水中。在无菌漏斗中从上至下依次铺入300目、400目及500目的过滤网进行过滤。当使用血球计数板计算孢子悬浮液中孢子的浓度达到2×107个/mL时,即可用于侵染。将清水清洗3次并晾干表面水分的叶片放入培养皿中,叶片下方是铺垫好的湿润的滤纸,在主叶脉两侧各滴2滴10μL孢子悬浮液,对照组则在主叶脉两侧各滴2滴无菌水。将侵染后的叶片放置在25℃,16h光照/8h黑暗的环境中培养。
2.7、二氨基联苯胺(DAB)染色
取接种链格孢菌7d后的毛果杨叶片,在1mg/mL(pH3.8)的DAB溶液中过夜染色,第二天使用乙醇进行脱色,在10%的水合氯醛溶液中,使叶片透化,光学显微镜下观察叶片。
3、实验结果
3.1HDA909过量表达载体的构建
利用毛果杨HDA909基因的特异性引物,通过PCR方法从毛果杨获得HDA909基因的编码区片段。并将pMD18-T载体与PCR产物连接,并进行测序,进而获得pMD18-HDA909中间载体。用BamH Ⅰ和Kpn Ⅰ分别对pMD18-HDA909中间载体和pROKⅡ载体进行双酶切,连接这两个酶切产物,从而获得pROKⅡ-HDA909的过量表达载体。将连接产物转化到大肠杆菌中并提取重组质粒,利用BamH Ⅰ和Kpn Ⅰ对重组质粒进行双酶切鉴定。酶切结果显示,酶切片段与HDA909基因片段大小一致,证明成功构建了pROKⅡ-HDA909的过量表达载体(图2)。
把构建的pROKⅡ-HDA909重组质粒转化到EHA105中,并制备该质粒。利用HDA909基因的特异性引物,以转化菌液作为PCR的模板来进行鉴定。电泳结果显示,在1323bp处有清晰的扩增条带(图3),表明pROKⅡ-HDA909成功转入到EHA105中,可用于植物的遗传转化。
3.2、转基因毛果杨的获得及分子鉴定
通过农杆菌介导法将HDA909基因遗传转化到毛果杨中。用携带HDA909基因的农杆菌侵染毛果杨茎段,然后将毛果杨茎段置于分化选择培养基(含Kan)中,茎段两端的伤口约在3周时可以分化出嫩绿的幼芽(图4A)。将幼芽移入生根选择培养基(含Kan)中继续培养,待幼芽生长至株高约为3cm的单独幼苗时,在新的生根培养基(含Kan)中***幼苗继续培养。HDA909转基因植株在毛果杨生根培养基(含Kan)上生长状态良好可以正常生根(图4B,4C),而非转基因植株则不能正常生根。
提取野生型和转基因毛果杨叶片的总RNA,反转录为cDNA,作为RT-PCR的模板;利用HDA909特异性引物进行RT-PCR鉴定。结果显示,HDA909基因能够在转基因植株中转录(图5)。
3.3、HDA909基因在真菌胁迫应答反应中的功能
叶枯病严重威胁着杨树的正常生长,作为一种常见的病害,其病原体为链格孢菌。本论文研究了毛果杨HDA909基因在链格孢菌侵染叶片中的作用。选取形状、大小及生长状态一致的HDA909转基因与非转基因2.5月龄毛果杨大苗第3节点或第4节点的叶片进行链格孢菌接种实验,分析HDA909转基因植株对链格孢菌的抗性。接种链格孢菌1周时,转基因植株的叶片与非转基因植株的叶片都受到了不同程度上的侵害,然而HDA909转基因植株对链格孢菌的抗性明显高于野生型植株(图6A)。在接种链格孢菌7天时,野生型植株叶片产生病斑的百分率为72.5%,然而转基因株系Tr-43、Tr-10、Tr-46叶片产生病斑的百分率分别为0%,25%和17.5%(图6B)。非转基因植株叶片上的病斑面积大于转基因植株叶片上的病斑面积(图7A),分别是转基因株系Tr-43、Tr-10、Tr-46叶片病斑面积的69.6倍、6.5倍和9.5倍(图7B)。
过氧化氢广泛地影响着许多的生命活动,是一种重要的活性氧。病原体侵染可以使细胞内积累大量的H2O2。DAB可以在产生H2O2的地方与H2O2发生反应,生成棕红的斑点。因此,利用DAB染色方法,可以检测植株受真菌侵染的损伤程度,植株受损程度越大,其产生的红棕色斑点越多,颜色越深。在4倍光学显微镜下观察病变叶片的DAB染色情况,转基因植株染病叶片产生的棕红色斑点与野生型植株染病叶片相比数量明显较少、颜色较浅(图8)。因此,相对非转基因植株,HDA909转基因植株的叶片不易感染链格孢菌,感染的叶片受损程度也相对较轻。综上所述,HDA909基因在毛果杨中的过量表达显著提高了转基因植株对真菌胁迫的抗性。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 东北林业大学
<120> 毛果杨HDA909基因在提高植物抗真菌能力中的应用
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Ala His Asn Leu Ile Ile Asn Tyr Ala Leu His Arg Arg Met Glu Ile
50 55 60
Asn Arg Pro Phe Pro Ala Gly Pro Glu Asp Ile Gly Trp Phe His Ser
65 70 75 80
Asp Asp Tyr Val Glu Phe Leu Ser Ser Val Ser Pro Gln Ser Ala Ser
85 90 95
Asp Pro Ala His Gly Arg Gln Leu Lys Arg Phe Asn Ile Gly Glu Asp
100 105 110
Cys Pro Val Phe Tyr Gly Leu Phe Glu Phe Cys Gln Ala Ser Ala Gly
115 120 125
Gly Ser Ile Gly Cys Ala Val Lys Leu Asn Arg Gly Asp Ala Asp Ile
130 135 140
Ala Leu Asn Trp Ala Gly Gly Leu His His Ala Lys Arg Ser Glu Ala
145 150 155 160
Ser Gly Phe Cys Tyr Val Asn Asp Ile Val Leu Gly Ile Leu Glu Leu
165 170 175
Leu Lys Val His Lys Arg Val Leu Tyr Val Asp Ile Asp Val His His
180 185 190
Gly Asp Gly Val Glu Glu Ala Phe Tyr Thr Thr Asp Arg Val Met Thr
195 200 205
Val Ser Phe His Lys Tyr Gly Asp Phe Phe Pro Gly Thr Gly His Ile
210 215 220
Lys Asp Ile Gly Val Gly Lys Gly Lys Asn Tyr Ala Leu Asn Ile Pro
225 230 235 240
Leu Lys Asp Gly Met Asp Asp Glu Cys Phe Arg Ala Leu Phe Arg Pro
245 250 255
Leu Ile Gln Lys Val Met Glu Val Tyr Gln Pro Asp Ala Val Val Leu
260 265 270
Gln Cys Gly Ala Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn
275 280 285
Leu Ser Val Lys Gly His Ala Asp Cys Leu Arg Phe Ile Arg Ser Phe
290 295 300
Asn Val Pro Leu Met Ile Leu Gly Gly Gly Gly Tyr Thr Ile Arg Asn
305 310 315 320
Val Ala Arg Cys Trp Cys Tyr Glu Thr Ala Val Ala Val Gly Val Glu
325 330 335
Pro Asp Asn Lys Leu Pro Tyr Asn Glu Tyr Tyr Glu Tyr Phe Gly Pro
340 345 350
Glu Tyr Thr Leu His Ala Asp Pro Ser Asn Met Glu Asn Leu Asn Thr
355 360 365
Pro Lys Asp Met Glu Arg Ile Arg Asn Ile Leu Leu Glu Gln Leu Ser
370 375 380
Arg Leu Pro Asn Ala Pro Ser Val Pro Phe Gln Thr Thr Pro Ala Thr
385 390 395 400
Thr Glu Val Pro Glu Glu Asp Glu Glu Asn Met Asp Gln Arg Pro Lys
405 410 415
Arg His Val Trp Asn Gly Val Asp Tyr Glu Ser Asp His Asp Glu Asp
420 425 430
Glu Lys Pro Glu Pro Gly Phe Phe
435 440
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgggatccat gctggacact aatggagg 28
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggggtacctc agaaaaatcc gggttccggt 30
Claims (6)
1.毛果杨HDA909基因在提高植物抗真菌能力中的应用,其特征在于,所述的毛果杨HDA909基因序列如SEQ ID NO.1所示,所述毛果杨HDA909基因编码的氨基酸序列如SEQ IDNO.2所示。
2.一种抗真菌植物的创制方法,其特征在于,根据权利要求1所述的毛果杨HDA909基因序列,构建毛果杨HDA909过表达载体,采用农杆菌介导法导入受体植物,使得转基因植物中的HDA909基因过量表达,获得抗真菌能力。
3.根据权利要求2所述的一种抗真菌植物的创制方法,其特征在于,所述的受体植物是毛果杨。
4.根据权利要求2所述的一种抗真菌植物的创制方法,其特征在于,所述的真菌为链格孢菌。
5.根据权利要求2所述的一种抗真菌植物的创制方法,其特征在于,毛果杨HDA909过表达载体的构建方法,包括以下步骤:
1)以野生型毛果杨的叶片cDNA为模板,HDA909-Primer-F和HDA909-Primer-R为引物,获得毛果杨HDA909基因目的片段;
2)利用Plasminal Mini KitⅠ将所得毛果杨HDA909基因目的片段和pMD18-T Vector载体连接,连接产物为pMD18-HDA909;连接产物转化到大肠杆菌感受态细胞,得到pMD18-HDA909重组质粒;
3)用BamHⅠ和KpnⅠ分别对pMD18-HDA909重组质粒和pROKⅡ质粒进行双酶切,双酶切后得到的HDA909基因片段和pROKⅡ载体线性片段用Gel Extraction Kit进行回收,回收产物利用T4连接酶连接,得到pROKⅡ-HDA909质粒,即为毛果杨HDA909过表达载体。
6.根据权利要求5所述的一种抗真菌植物的创制方法,其特征在于,HDA909-Primer-F引物序列如SEQ ID NO.3所示,HDA909-Primer-R引物序列如SEQ ID NO.4所示。
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