CN1051576C - 经引入编码草酸氧化酶的基因生产抗桑条菌核病核盘霉攻击的植物 - Google Patents

经引入编码草酸氧化酶的基因生产抗桑条菌核病核盘霉攻击的植物 Download PDF

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CN1051576C
CN1051576C CN92101989A CN92101989A CN1051576C CN 1051576 C CN1051576 C CN 1051576C CN 92101989 A CN92101989 A CN 92101989A CN 92101989 A CN92101989 A CN 92101989A CN 1051576 C CN1051576 C CN 1051576C
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乔治·弗雷西内
阿兰·萨扬
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Abstract

1)编码草酸氧化酶的DNA序列。
2)可用于使植物对抗由核盘霉真菌引起的病害。它可由嵌合基因和含有编码序列的载体提供。
3)它可使植物提高对核盘霉真菌引起之病害的抵抗力。

Description

经引入编码草酸氧化酶的基因生产抗桑条菌核病核盘霉攻击的植物
本发明涉及编码草酸氧化酶的基因、由该基因编码的蛋白质，包含该基因的嵌合基因、以及将它们用于植物转化以赋予植物抗真菌病抗性的方法。
菌核病是影响大量双子叶植物的主要真菌病。其病原因子桑条菌核病核盘霉(Sclerotinia Sclerotiorum)是表现有很少宿主特异性的多属主真菌。
该真菌可直接在植物的茎部攻击植物，也可在叶子上攻击然后播散到茎部，也可在花的头状花序水平上攻击植物。前两种情况，植物因营养供应中断而枯萎；后一种情况，则可导致花的萎缩进而损害收成。
该真菌可产生破坏被感染植物细胞壁的溶解酶，并促进其在植物中的发育繁殖。这些酶在致病性中起着重要但似乎并非足够的作用。这种真菌也产生草酸(Godov et al，1990)。这种草酸可降低被感染组织的pH值，进而促进溶解酶对细胞壁的水解作用。减少草酸的产生量或破坏之，将会延缓甚至抑制真菌的发育。
为产生″nos″抗性植物，可采用使草酸解毒的战略。破坏该酸将限制被攻击植物组织细胞内pH值的降低，从而使溶解酶只能在远离表现其活性和效力最适pH值之pH值条件下发挥作用。这样便可导致真菌致病性的降低。可使用催化下列反应的草酸氧化酶实现本发明的目的；
可从各种植物通常是单子叶植物中分离草酸氧化酶(Pieta etal，1984)，例如，可使用常规层析技术(Sephadex G-75过滤凝胶和MonoQ离子交换凝胶，Pharmacia)从大麦中纯化这种蛋白质，按照下述比色法(0bzansky和Richardson，1983)监测酶促活性：
MBTH＝3-甲基-2-苯并噻唑啉酮腙
DMA＝N，N-二甲基α-丙氨酸
经过上述处理后，有可能于变性条件下在丙烯酰胺凝胶上纯化分子量为26,000道尔顿的蛋白质。用一部分纯化的草酸氧化酶制取兔抗草酸氧化酶抗体；将蛋白质的其余部分用来进行天然蛋白质(N末端)的序列分析，或者在溴化氰裂解后，对某些内部肽段作序列分析。所得结果如下：
N末端：TDPDPLQDF-VADLDGKAVSVNGH
       S
内部肽No.2：HFQFNVGKTEAY
CDNA
将上述肽序列与得自蛋白质文库Swiss-Prot的资料相比较，就能够鉴定称为Germine并由Dratewka-kos等人于1989年公开的小麦蛋白质。完成实验，使我们能够确定作者公开的CDNA编码表现出草酸氧化酶活性的含201个氨基酸的蛋白质。涉及本专利申请中提到的其它实验描述部分，我们将使用作者在J.Biol，chem，264，4896-4900-文之图2中的核苷酸序列编号。
该CDNA的序列长度为1075个核苷酸，其中5′非转译区有85个残基，开放读码为672个核苷酸(从86-757位)，3′非转译区有318个残基。
根据此CDNA序列推断的蛋白质序列与测定天然蛋白质得知的序列相比较，表明CDNA不仅编码成熟草酸氧化酶，而且还编码N末端部分中23个氨基酸的信号肽。因此草酸氧化酶最初是以前蛋白质的形式合成的(即信号肽加上成熟肽)，后者须经过一个成熟过程，除去信号肽以释放出成熟的活性酶。
下文中，我们将使用编码前蛋白质的部分(核苷酸86到757)，或只使用编码成熟蛋白质的部分(从155位到757位)，在后一种情况下，AUG密码子(编码蛋氨酸)应位于ACC密码子(编码苏氨酸，即成熟蛋白质的第一个氨基酸)的前面。
桑条菌核病核盘霉对植物的攻击基本上是通过植物的茎，这样便有利于在叶绿体组织中或植物的其他不同组织中表达草酸氧化酶；在前一种情况下，可使用1，5-二磷酸核酮糖羧基酶小亚基(Helianthus annuus)(SSuHa，Waksman et al，1987)的启动子，对于后一种情况，我们将使用普遍存在的花椰菜花叶病毒(其一部分是复制的、并被称为″双(CaMV)″)的35SRNA(CaNV35S)的启动子。
例如可由下列元件来构造本发明的嵌合基因：
A、双CaMV启动子后面连接一部分编码前蛋白质(信号肽加上成熟肽)的草酸氧化酶cDNA及得自pTi37蓝曙红(nopaline)合成酶基因(Bevan et al，1983)的终止子″nos″。
B、双CaMV启动子后面接有部分只编码成熟蛋白质的草酸氧化酶cDNA，再后面是终止子″nos″。
C、基因与″A″相同，但以向日葵1，5-二磷酸核酮糖羧基酶小亚基(SSuHa)的启动子代替双CaMV启动子。
D、基因与″B″相同，但用SSuHa的启动子代替双CaMV启动子。
以使用土壤杆菌的系统或任何已知用于转化植物细胞的其他系统将各嵌合基因导入植物细胞中。由这些转化的细胞再生植物。这些植物均提高了对桑条菌核病核盘霉的抗性。
实施例1：制备两个编码序列
A、前蛋白质：它得自于用HindII消化(在位置66处)的上述cDNA。其粘性末端用klenow聚合酶处理成平头。然后用NheI消化(在位置811处)该DNA。
用SacI平行消化质检PVC19(Yanisoh-Perro et al.，1985)。
经用Klenow聚合酶处理将所得到粘性末端修成平头。然后用Xgal(与Nhel相配合)消化质粒。
连接如上制备的cDNA片段和质粒。如此得到的新质粒定名为pRPA-oxo-01，其酶切图示于图1中。
B、成熟蛋白质：它得自于用BstNI消化(在位置173处)的上述cDNA。连接所得片段和该序列的接头：
5′                  3′
ATGACCGACCCAGACCCTCTCC
TACTGGCTGGGTCTGGGAGAGGT
3′                 5′
如此便导致对跨越TDPDPLQ至MTDPDPLQ之成熟蛋白质N末端序列的修饰。
然后用NheI消化(在位置811处)该cDNA片段，使之能够与按上述方法制得的质粒PUC19相连接。如此形成的新质粒被称为pRPA-oxo-02，其酶切图示于图1中。
实施例2：制备嵌合基因
a、制备含有启动子和终止子nos的载体
以双CaMV为例：该载体得自于按下述方法制得的质粒PRPA-BL-410：
″玉米RuBisCo/AroA基因之SSU的转运肽″融合体：
玉米RuBisCO基因之SSU的转运肽衍生于192bp的EcoRI-SphI片段；它得自于Lebrun等人(1987)所述玉米RuBisco基因之SSu基因的cDNA，带有跨越转移起始密码子的Ncol位点和相当于转送肽之切割位点的SphI位点。
用T4噬菌体聚合酶处理SphI末端，并使其与用EcoRI再切割之pRPA-BL104中的AroA基因并经klenow聚合酶处理的NcoI末端连接，获得玉来转运肽和细菌EPSPS基因间的转移融合体。
″玉米RuBisCO之SSU的转运肽/玉米RuBisCO之SSU成熟部分的22氨基酸序列/AroA基因″融合体：
以相似方式，将玉米RuBisCO基因之SSU的228bp cDNA的EcoRI-Hind II片段与klenow聚合酶处理的PRPA-BL104中之AroA基因的NcoI末端相连接并用EcoRI再次切割。得到玉米RuBisCO之SSU的转运肽、玉米RuBisCO之SSU成熟部分的22氨基酸和细菌EPSPS基因间的转移融合体。
向日葵RuBisCO之SSU的转换肽：
该片段得自于由Waksman和Freyssinet(1987)分离的cDNA。按照Zoller和Smith(1984)的方法在转换肽的裂解位点产生SphI位点。如此制得的向日葵RuBisCO之SSu的转换肽即为171bp的EcoRI-SphI片段。
″向日葵RuBisCO之SSu的转换肽/玉米RuBisCO之SSU成熟部分22氨基酸序列/AroA基因″融合体：
用相当于所说的向日葵RuBisCO基因之SSU的转换肽的171bpEcoRI-SphI切割含有″玉米RuBisCO之SSU的转换肽/玉米基因成熟部分之玉米RuBisCO之SSU的22氨基酸序列″融合体的构建体。所得之构建体呈现有EcoRI-SphI片段的取代，并是″向日葵RuBisCO之SSU转运肽/玉米RuBisCO之SSU成熟部分的22氨基酸序列/AroA基因″融合体。
使EcoRI-SalI片段与含有T-DNA之3′nos序列和右侧端的SalI-SstI片段相连接。所得到的含有″向日葵RuBisCO之SSU的转换肽/玉米RuBisCO之SSU成熟部分的22氨基酸序列/AroA基因/3′nos/T-DNA右侧端″的EcoRI-SstI片段取代了含有双CaMV启动子质粒150Aα2的T-DNA右侧端的EcoRI-SstI片段。转移融合体″双CaNV/向日葵RuBisCO之SSU的转换肽/玉米RuBisCO之SSU成熟部分的22氨基酸序列/AroA基因/3′nos″在载体150α2中称为PRPA-BL294。
″向日葵RuBisCO之SSU的转换肽/玉米RuBisCO之SSU的22氨基酸序列/玉米RuBisCO之SSU的转换肽/AroA基因″融合体：
用NcoI-HindIII切割上述构建体以释放出AroA基因。然后将其与含有″玉米RuBisCO之SSU的转换肽/AroA基因″融合体的1.5kbpNcoI-HindIII片段相连接。所得构建体呈现NcoI-HindIII片段的取代并且是转运融合体″向日葵RuBisCO之SSU的转换肽/玉米基因成熟部分RuBisCO之SSU的22氨基酸序列/玉米RuBisCO之SSU的转运肽/AroA基因″。
a、使EcoRI-SalI片断与含有T-DNA之3′nos序列和右侧端的SalI-SstI片断连续。所得到的含有″向日葵RuBisCO之SSU的转运肽/玉米基因成熟部分RuBisCO之SSU 22氨基酸序列/玉米RuBisCO之SSU的转运肽/AroA基因/3′nos/T-DNA右侧端的EcoRI-SstI片断取代了含有包括双CaMV启动子之质粒150Aα2T-DNA右侧端的EcoRI-SstI片断。转录融合体″双CaMV/向日葵RuBisCO之SSU的转运肽/玉米基因成熟部分RuBisCO之SSU的22氨基酸序列/玉米RuBisCO之SSU的转运肽/3′nos″在载体150Aα2中称为PRPA-BL410。用EcoRI和SalI消化该质粒，以除去结构基因″最佳化转运肽一成熟EPSPS编码区″，即消除PRPA-BL-410(见图1)。
以SSUHa为例：该载体得自于质粒PRPA-BL-207(已在欧洲专利申请，0,337,899中述及)，用EcoRI和HindIII消化所说的质粒以除去腈水解酶编码区，即消除PRPA-BL-201(见图1)。
b、嵌合基因的构建：
PRPA-oxo-03：它是用EcoRI和SalI消化PRPA-oxo-01得到的。然后将所得到的编码前蛋白质的片段分别插入在双CaMV下游和终止子nos上游的EcoRI和SalI位点之间。
PRPA-oxo-04：它是用EcoRI和SalI消化PRPA-oxo-02得到的。然后将所得到的编码成熟蛋白质的片段分别插入在双CaMV下游和终止子nos上游的EcoRI和SalI之间。
PRPA-oxo-05：它是用EcoRI和HindIII消化PRPA-oxo-01得到的。然后将所得的编码前蛋白质的片段分别插入位于双SSUHa下游和终止子nos上游的EcoRI和HindIII位点之间。
PRPA-oxo-06：它是用EcoRI和HindIII消化PRPA-oxo-02得到的。然后将所得到的编码成熟蛋白质的片断分别插入位于SSUHa启动子下游和终止子nos上游的EcoRI和HindIII之间。
表I：四个嵌合基因的简略说明代号        启动子    草酸氧化    终止子
                  酶编码区PRPA-oxo-03  dCaMV    前蛋白质      nosPRPA-oxo-04  dCaMV    成熟蛋白质    nosPRPA-oxo-05  SSuHa    前蛋白质      nosPRPA-oxo-06  SSuHa    成熟蛋白质    nos
实施例3：生产转基因油菜
a、转化
将上述各载体导入携带装配型质粒pTVK291(Komari et al，1986)的非致瘤性瘤病土壤杆菌菌株EHA101(Hood et al，1987)中。
转化油菜(westar变种)的方法基本上与Boulter等人(1990)所述相同，其中使用的细菌浓度为2.5×109个/ml(OD600nm＝1)。
b、植株再生
再生方法基本上与Boulter等人(1990)所述相同。植物在DeBlock等人所介绍(1989)的培养基中生根。然后在温室内进入开花阶段。
实施例4：检测油菜对桑条菌核病核盘霉的抗性
体外实验：
一叶片：在含有激素、添加1mM草酸的Murashige和Skoog(MS)培养基上生长11天后，对三片叶片称重以检测抗真菌抗性。
在这些条件下，观察到：从使用嵌合基因PRPA-oxo-03、PRPA-oxo-04、PRPA-oxo-05和PRPA-oxo-06之一修饰的油菜得到的叶片之质量有明显增加，而从未经修饰的油菜得到的叶片质量则无变化甚至减少。
一根伸长：在添加5mM草酸的水上生长两天后检查根伸长，体外测定上述抗性。在这种情况下观察到用嵌合基因PRPA-oxo-03，PRPA-oxo-03，PRPA-oxo-04之一修饰之油菜植物的根能够生长并增加了长度，而未修饰的油菜的根则在这些条件下未见生长。
体内实验：
在第一个花出现时，立即在花瓣上涂敷桑条菌核病核盘霉孢子，从而在花落期间自然发生叶子的感染，或者直接将菌丝体系涂敷在叶子上或将浸渍了菌丝体的花瓣置于叶子上，以污染再生形成的油菜植物，然后在温室中体内检测抗性。用嵌合基因PRPA-oxo-03，PRPA-oxo-04、pRPA-oxo-05和pRPA-oxo-06之一修饰的植物使真菌不能发育，而且完全没有出现菌核病的特征性枯萎病症，而未修饰的植物则因桑条菌核病核盘霉的发育迅速枯萎。

Claims (7)

1.转化植物以使其抗核盘霉真菌所致病害的方法，其特征在于：将编码草酸氧化酶蛋白质或前蛋白质的DNA导入植物细胞。
2.如权利要求1所述的转化植物的方法，其中DNA编码序列包含在嵌合基因中。
3.如权利要求1所述的转化植物的方法，其中包含DNA编码序列的嵌合基因包含在载体中。
4.如权利要求1所述的转化植物的方法，其中被转化的植物细胞含有如权利要求3所述的载体。
5.如权利要求4所述的转化植物的方法，进一步包括从转化的植物细胞得到转化的植物。
6.如权利要求5所述的方法，其中转化的植物为双子叶植物。
7.如权利要求6所述的方法，其中双子叶植物为油菜。
CN92101989A 1991-03-05 1992-02-28 经引入编码草酸氧化酶的基因生产抗桑条菌核病核盘霉攻击的植物 Expired - Lifetime CN1051576C (zh)

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