CN103045628A - Method for reducing toxicity of Vip3 protein to transgenic plant through gene fusion - Google Patents

Abstract

Translated fromChinese

本发明公开了一种通过基因融合降低Vip3蛋白对转基因植物毒性的方法，1）、将一个多肽（polypeptide）基因的C端与基因vip3的N端直接相连；中间加以4个氨基酸组成的连接肽连接；2）、融合基因在植物细胞表达的融合蛋白质不集中在植物的细胞膜上。Vip3蛋白为以下任意一种：为Bt营养期杀虫蛋白Vip3类蛋白的任意一种；为SEQ ID NO：1所示的氨基酸序列；为与SEQ ID NO： 1具有至少70%相似性的氨基酸序列。多肽（polypeptide）为绿色荧光蛋白GFP；连接肽为Gly-Asp-Pro-Thr。将本发明所得的融合基因转入植物中，从而获得转基因抗虫植物。

The invention discloses a method for reducing the toxicity of Vip3 protein to transgenic plants through gene fusion. 1) The C-terminus of a polypeptide (polypeptide) gene is directly connected to the N-terminus of gene vip3; a connecting peptide consisting of 4 amino acids is added in the middle connection; 2), the fusion protein expressed by the fusion gene in the plant cell is not concentrated on the cell membrane of the plant. The Vip3 protein is any one of the following: it is any one of the Bt vegetative phase insecticidal protein Vip3 protein; it is the amino acid sequence shown in SEQ ID NO: 1; it is an amino acid having at least 70% similarity with SEQ ID NO: 1 sequence. The polypeptide (polypeptide) is green fluorescent protein GFP; the connecting peptide is Gly-Asp-Pro-Thr. The fusion gene obtained in the present invention is transferred into plants to obtain transgenic insect-resistant plants.

Description

Translated fromChinese

通过基因融合降低Vip3蛋白对转基因植物毒性的方法Method for reducing toxicity of Vip3 protein to transgenic plants by gene fusion

技术领域technical field

本发明涉及植物基因工程领域，具体涉及一种构建Vip3类蛋白融合基因的方法及其转入植物中构建抗虫转基因植物的应用。The invention relates to the field of plant genetic engineering, in particular to a method for constructing a Vip3 class protein fusion gene and its application in plants to construct insect-resistant transgenic plants.

背景技术Background technique

苏云金芽孢杆菌 Bt 是世界上应用最广泛的杀虫微生物，它在生长过程中能产生一系列的杀虫毒素，其中研究最为深入、应用最多的是其杀虫晶体蛋白 (Insecticidal Crystal Poteins, ICPs)。 然而，在实际应用中有些重要的农作物害虫(如小地老虎、茶尺蠖)对ICPs并不十分敏感，并且由于Bt产品的不合理使用，已有小菜蛾等害虫对Bt制剂产生了不同程度的抗性（Zhao et al., 2003; Griffitts et al., 2001）。因此，寻找具有与ICPs不同杀虫作用方式的蛋白，对于扩大杀虫谱、延缓害虫抗药性的产生具有重要的理论意义和实用价值。Bacillus thuringiensis Bt is the most widely used insecticidal microorganism in the world. It can produce a series of insecticidal toxins during its growth, among which its Insecticidal Crystal Poteins (ICPs) are the most studied and most widely used . However, some important crop pests (such as cutworms and tea geometrids) are not very sensitive to ICPs in practical applications, and due to the irrational use of Bt products, pests such as diamondback moth have produced varying degrees of sensitivity to Bt preparations. Resistance (Zhao et al., 2003; Griffitts et al., 2001). Therefore, finding proteins with different insecticidal action modes from ICPs has important theoretical and practical value for expanding the insecticidal spectrum and delaying the emergence of pest resistance.

营养期杀虫蛋白（Vegetative Insecticidal Protein, VIPs）是一种新型Bt杀虫蛋白，它是Bt从对数生长中期开始分泌、直至稳定前期达到最高峰的胞外毒素。VIPs主要分为Vip1、Vip2和Vip3三种，Vip1和Vip2构成二元毒素，对鞘翅目叶甲科昆虫具有杀虫特异性，而Vip3对鳞翅目害虫有较广谱的杀虫活性（Schnepf et al., 1998）。Vip3与已知的ICPs没有序列同源性和结构相似性（Estruch et al., 1996），杀虫机理也不尽相同。与ICPs蛋白晶体不同，Vip3为可溶性蛋白，不需通过昆虫中肠的消化即可直接起作用。研究表明，其N端是一段信号肽序列，可以使Vip3蛋白识别并结合在敏感昆虫中肠上皮细胞膜上，造成膜穿孔，通过诱发中肠细胞凋亡，最终导致昆虫死亡(Lee et al., 2006)。 Vip3和广泛使用的ICPs类Cry蛋白无交互抗性。因此，Vip3蛋白作为新的杀虫资源在研究和应用方面将具有巨大的潜力，可用于构建和研制高效、广谱的杀虫蛋白基因来进行抗性害虫的有效防治。Vegetative insecticidal protein (Vegetative Insecticidal Protein, VIPs) is a new type of Bt insecticidal protein, which is an extracellular toxin secreted by Bt from the mid-logarithmic growth phase to the peak in the stable phase. VIPs are mainly divided into three types: Vip1, Vip2, and Vip3. Vip1 and Vip2 constitute a binary toxin, which has insecticidal specificity against Coleoptera Chrysophyllidae insects, while Vip3 has a broad-spectrum insecticidal activity against Lepidoptera pests (Schnepf et al. ., 1998). Vip3 has no sequence homology and structural similarity with known ICPs (Estruch et al., 1996), and the insecticidal mechanism is also different. Unlike ICPs protein crystals, Vip3 is a soluble protein that can function directly without digestion in the insect midgut. Studies have shown that its N-terminus is a signal peptide sequence, which can enable Vip3 protein to recognize and bind to the midgut epithelial cell membrane of sensitive insects, causing membrane perforation, inducing midgut cell apoptosis, and finally leading to insect death (Lee et al., 2006). There is no cross-resistance between Vip3 and widely used ICPs Cry-like proteins. Therefore, Vip3 protein, as a new insecticidal resource, will have great potential in research and application, and can be used to construct and develop efficient and broad-spectrum insecticidal protein genes for effective control of resistant pests.

目前，Vip3转基因作物的研发和应用已成为新的研究热点。美国先正达（Syngenta）公司在全球最早开展了Vip3转基因研究，已将Vip3成功地应用于转基因杀虫植物的构建，并在棉花和玉米中首先得到应用。2003年研制出成功表达 Vip3毒蛋白的棉花品种COT102(Artim, 2003)；2007年培育出转vip3a20的转基因玉米品系MIR162。2010年，先正达公司研制的含Vip3蛋白的棉花和玉米获得美国环境保护署的批准(Syngenta，2010)，可将其用于防治对Cry基因产生抗性的鳞翅目害虫，极大地拓宽了Bt转基因作物的抗虫谱。现在，美国已开始种植含有Vip3蛋白的棉花品种和玉米杂交种。在国家转基因专项的资助下，我国也积极开展了对Vip3基因的研究与利用。浙江大学与山西省农业科学院棉花研究所等单位合作，利用国内获得的新型Vip3基因及其改造成果，构建具有自主知识产权的重组vip3与cry1A融合基因从而获得抗虫转基因植物，可望实现对棉花、玉米、水稻等主要作物鳞翅目害虫广谱高抗的育种目标。At present, the development and application of Vip3 transgenic crops has become a new research hotspot. Syngenta Corporation of the United States was the first to carry out Vip3 transgenic research in the world, and has successfully applied Vip3 to the construction of transgenic insecticidal plants, and was first applied in cotton and corn. In 2003, a cotton variety COT102 (Artim, 2003) successfully expressing Vip3 toxic protein was developed; in 2007, a transgenic corn line MIR162 transgenic vip3a20 was bred. In 2010, the cotton and corn containing Vip3 protein developed by Syngenta obtained approval from the US Environmental Protection Agency. The approval of the Conservation Agency (Syngenta, 2010) allows it to be used to control Lepidoptera pests that are resistant to the Cry gene, which greatly broadens the insect resistance spectrum of Bt transgenic crops. Cotton varieties and corn hybrids containing the Vip3 protein are now grown in the United States. With the support of the national transgenic project, my country has also actively carried out the research and utilization of the Vip3 gene. Zhejiang University cooperated with the Cotton Research Institute of Shanxi Academy of Agricultural Sciences and other units to use the new Vip3 gene and its transformation results obtained in China to construct a recombinant vip3 and cry1A fusion gene with independent intellectual property rights to obtain insect-resistant transgenic plants, which is expected to achieve cotton Breeding targets for broad-spectrum and high resistance to lepidopteran pests in major crops such as , corn, and rice.

尽管如此，在转基因作物构建的实际操作中，仍然存在一些急需解决的技术性问题。例如，研究人员发现单独的vip3基因转入植物后，所获得的高表达Vip3蛋白的转基因植株在生长上较迟缓，容易出现白化现象。这是由于在转基因植物中直接表达Vip3蛋白后，该蛋白由于信号肽的分泌作用大量聚集并结合在植物细胞膜上并形成孔道，从而对植物细胞形成一定的损坏，影响其生长发育。这样还导致的另一后果是，转基因植物的抗虫性能也不高。Nevertheless, there are still some technical problems that need to be solved urgently in the actual operation of the construction of transgenic crops. For example, the researchers found that after a single vip3 gene was transferred into plants, the obtained transgenic plants with high expression of Vip3 protein were slower in growth and prone to albinism. This is because after the Vip3 protein is directly expressed in the transgenic plant, due to the secretion of the signal peptide, the protein aggregates in large quantities and binds to the plant cell membrane to form pores, thereby causing certain damage to the plant cell and affecting its growth and development. Another consequence of this is that the insect resistance of transgenic plants is not high.

所用参考文献具体如下：The references used are as follows:

卢雄斌，龚祖埙 （1998）植物转基因方法及进展。生命科学 3：125-131。Lu Xiongbin, Gong Zuxun (1998) Plant transgenic methods and progress. Life Sciences 3:125-131.

刘凡，王国英，曹鸣庆 （2003）农杆菌介导的植物原味转基因方法研究进展。分子植物育种 1:108-116。Liu Fan, Wang Guoying, Cao Mingqing (2003) Research progress on Agrobacterium-mediated transgenic methods of plant flavor. Molecular Plant Breeding 1:108-116.

Artim L (2003) Petition 03–155–01p for the determination of non-regulated status: Lepidopteran insect protected Vip3 cotton transformation event Cot102（抗鳞翅目昆虫的Vip3转基因棉Cot102）。Artim L (2003) Petition 03–155–01p for the determination of non-regulated status: Lepidopteran insect protected Vip3 cotton transformation event Cot102 (Vip3 transgenic cotton Cot102 resistant to Lepidoptera insects).

Estruch J, Warren G, Mullins M, Nye G, Craig JK (1996) Vip3, a novel Bacillus thuringiensis vegetativeinsecticidal protein with a wide spectrum of activities agrainst lepidopteran insects. Proc Natl Acad SciU S A93: 5389–5394. Vip3（一种对鳞翅目昆虫有广谱抗性的新型苏云金芽孢杆菌营养期杀虫蛋白；美国科学院院刊93: 5389–5394. Vip3）。Estruch J, Warren G, Mullins M, Nye G, Craig JK (1996) Vip3, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities agrains lepidopteran insects. Proc Natl Acad 93U ip 3 5 A Novel Bacillus thuringiensis vegetative insecticidal protein with broad-spectrum resistance to Lepidoptera; Proceedings of the National Academy of Sciences 93: 5389–5394. Vip3).

Griffitts SJ, Whitacre LJ, Stevens ED, Aroian VR (2001) Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science 293: 860-864（由于缺失推定的糖修饰酶而产生的Bt毒素抗性；科学293: 860-864）。Griffitts SJ, Whitacre LJ, Stevens ED, Aroian VR (2001) Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science 293: 860-864 (Bt toxin resistance due to loss of a putative carbohydrate-modifying enzyme; Science 293: 860-864).

Lee KM, Miles P, Chen JS (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Bioph Res Co 339: 1043-1047（苏云金芽孢杆菌Vip3毒素对烟蚜夜蛾和谷实夜蛾的刷状缘膜受体结合性质；生化和生理研究通信339: 1043-1047）。Lee KM, Miles P, Chen JS (2006) Brush border membrane binding properties of Bacillus thuringiensis Vip3toxin to Heliothis virescens and Helicoverpa zea midguts. Biochem Bioph Res Co 339: 1043-1047 Brush border membrane receptor-binding properties of Heliotrope moth; Biochemical and Physiological Research Communications 339: 1043-1047).

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Syngenta. EPA approves Syngenta’s VipCot cotton fornatural refuge[EB/OL]. [2010-06-02] http://news.agropages.com/News/NewsDetail---2482.htm，先正达（EPA通过先正达的VipCot棉用于自然庇护）。Syngenta. EPA approves Syngenta's VipCot cotton fornatural refuge[EB/OL]. [2010-06-02] http://news.agropages.com/News/NewsDetail---2482.htm, Syngenta (EPA approved up to VipCot cotton for natural shelter).

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发明内容Contents of the invention

本发明要解决的技术问题是提供一种通过基因融合以降低苏云金芽孢杆菌（Bacillus thuringiensis，Bt）营养期杀虫蛋白（Vegetative insecticidal protein, Vip3）对转基因植物的毒性的方法。The technical problem to be solved by the present invention is to provide a method for reducing the toxicity of Bacillus thuringiensis (Bt) vegetative insecticidal protein (Vip3) to transgenic plants through gene fusion.

为了解决上述技术问题，本发明提供一种通过基因融合降低Vip3蛋白对转基因植物毒性的方法：In order to solve the above-mentioned technical problems, the present invention provides a method for reducing the toxicity of Vip3 protein to transgenic plants by gene fusion:

1）、将一个多肽（polypeptide）基因的C端与基因vip3的N端直接相连；中间加以4个氨基酸组成的连接肽连接；1) The C-terminal of a polypeptide (polypeptide) gene is directly connected to the N-terminal of the gene vip3; a connecting peptide consisting of 4 amino acids is added in the middle;

2）、融合基因在植物细胞表达的融合蛋白质不集中在植物的细胞膜上。2) The fusion protein expressed by the fusion gene in plant cells is not concentrated on the cell membrane of the plant.

作为本发明的通过基因融合降低Vip3蛋白对转基因植物毒性的方法的改进：As the improvement of the method for reducing Vip3 protein toxicity to transgenic plants by gene fusion of the present invention:

Vip3蛋白为以下任意一种：Vip3 protein is any of the following:

为Bt营养期杀虫蛋白Vip3类蛋白的任意一种；It is any one of the Bt vegetative phase insecticidal protein Vip3 protein;

为SEQ ID NO：1所示的氨基酸序列；Be the amino acid sequence shown in SEQ ID NO: 1;

为与SEQ ID NO： 1具有至少70%相似性的氨基酸序列。is an amino acid sequence having at least 70% similarity to SEQ ID NO: 1.

作为本发明的通过基因融合降低Vip3蛋白对转基因植物毒性的方法的进一步改进：As a further improvement of the method for reducing Vip3 protein toxicity to transgenic plants by gene fusion of the present invention:

多肽（polypeptide）为绿色荧光蛋白GFP；Polypeptide is green fluorescent protein GFP;

绿色荧光蛋白GFP为SEQ ID NO.2所示的氨基酸序列。Green fluorescent protein GFP is the amino acid sequence shown in SEQ ID NO.2.

作为本发明的通过基因融合降低Vip3蛋白对转基因植物毒性的方法的进一步改进：As a further improvement of the method for reducing Vip3 protein toxicity to transgenic plants by gene fusion of the present invention:

连接肽为Gly-Asp-Pro-Thr。The connecting peptide is Gly-Asp-Pro-Thr.

本发明还同时提供了如上述方法制备而得的egfp-vip3融合基因， egfp-vip3融合基因为如SEQ ID NO：3（即序列表NO3）所示的序列。The present invention also provides the egfp-vip3 fusion gene prepared by the above method at the same time, the egfp-vip3 fusion gene is the sequence shown in SEQ ID NO: 3 (ie, sequence listing NO3).

本发明还同时提供了如上述方法制备而得的vip3-egfp融合基因， vip3-egfp融合基因序列为如SEQ ID NO：4（即序列表NO4）所示的序列。The present invention also provides the vip3-egfp fusion gene prepared by the above method, the sequence of the vip3-egfp fusion gene is the sequence shown in SEQ ID NO: 4 (ie, sequence listing NO4).

本发明还同时提供了如上述方法制备而得的egfp-vip3融合基因的用途：将该融合基因（融合蛋白基因）转入植物中，从而获得转基因抗虫植物。The present invention also provides the use of the egfp-vip3 fusion gene prepared by the above method: the fusion gene (fusion protein gene) is transferred into plants to obtain transgenic insect-resistant plants.

作为本发明的融合基因的用途的改进植物为单子叶植物或双子叶植物；单子叶植物为水稻、玉米、小麦或高粱，双子叶植物为棉花、大豆或油菜 。The improved plant used as the fusion gene of the present invention is a monocotyledon or a dicotyledon; the monocotyledon is rice, corn, wheat or sorghum, and the dicotyledon is cotton, soybean or rapeseed.

具体而言，本发明提供一种将一个多肽基因序列的C端与vip3基因的N端直接相连，中间加以4个氨基酸组成的连接肽(Gly-Asp-Pro-Thr)连接，获得的融合基因在植物细胞中表达后不集中在植物细胞膜上。其中Vip3可以是苏云金芽孢杆菌（Bt）营养期杀虫蛋白Vip3家族的任一个蛋白，或者其氨基酸序列为SEQ ID NO. 1和/或与SEQ ID NO.1至少70%相似性的序列；一个多肽可以是本发明实施例中提供的绿色荧光蛋白GFP（其序列为SEQ ID NO.2和/或与SEQ ID NO.2至少60%相似性的序列），也可以是任意一个多肽或蛋白质。Specifically, the present invention provides a fusion gene obtained by directly connecting the C-terminus of a polypeptide gene sequence with the N-terminus of the vip3 gene, and connecting it with a connecting peptide (Gly-Asp-Pro-Thr) consisting of 4 amino acids. After expression in plant cells, it does not concentrate on the plant cell membrane. Wherein Vip3 can be any protein of Bacillus thuringiensis (Bt) vegetative phase insecticidal protein Vip3 family, or its amino acid sequence is SEQ ID NO.1 and/or a sequence with at least 70% similarity to SEQ ID NO.1; a The polypeptide can be the green fluorescent protein GFP provided in the embodiment of the present invention (its sequence is SEQ ID NO.2 and/or a sequence with at least 60% similarity to SEQ ID NO.2), or any polypeptide or protein.

用本发明提供的方法获得的融合基因，可以用多种方法转入植物中，从而构建抗虫转基因植物。这些方法可以是目前已知的，也可以是未来新开发的有效的植物转基因的方法，本领域一般技术人员都可以利用这些方法轻易地将使用本发明提供的方法获得的融合基因导入植物中从而构建转基因植物。本发明实施例中使用的转基因方法是目前较常用的农杆菌介导的植物转化方法，即将启动子-编码蛋白质的基因片段-终止子所组成的能够在植物中表达的表达框构建到农杆菌的T-DNA载体中，通过农杆菌遗传转化到植物的基因组中。农杆菌载体及其遗传转化方法目前技术上已经成熟，一般的技术人员都能够完成。表达框可以使用的启动子有很多，包括组成型启动子，例如常用的花椰菜花叶病毒CaMV的35S启动子、玉米ubiquitin启动子等；也可以利用其他组织特异性的启动子控制目的蛋白质的表达，例如在植物绿色组织表达的启动子等。此外，这种基因的表达也可以利用表达增强子加强。如35S增强子，可以加强在其上游或者下游的目的基因的表达。一般的技术人员都可以通过试验比较使用不同启动子的效果。表达框的终止子可以是不同来源的终止子。在植物转化过程中通常需要筛选基因来筛选转化的细胞。常用的筛选基因包括抗卡那霉素基因、抗潮霉素基因、抗除草剂基因（如抗草甘膦、抗草丁膦基因）等。The fusion gene obtained by the method provided by the invention can be transferred into plants by various methods, so as to construct insect-resistant transgenic plants. These methods can be currently known, and can also be effective plant transgenic methods newly developed in the future, and those skilled in the art can use these methods to easily introduce the fusion gene obtained using the method provided by the present invention into plants so that Construction of transgenic plants. The transgenic method used in the examples of the present invention is the currently more commonly used Agrobacterium-mediated plant transformation method, that is, to construct an expression cassette capable of expressing in plants consisting of a promoter-protein-encoding gene segment-terminator into the Agrobacterium In the T-DNA vector, it is genetically transformed into the genome of the plant by Agrobacterium. The Agrobacterium vector and its genetic transformation method are currently technically mature and can be accomplished by ordinary technicians. There are many promoters that can be used in the expression cassette, including constitutive promoters, such as the 35S promoter of the cauliflower mosaic virus CaMV commonly used, the maize ubiquitin promoter, etc.; other tissue-specific promoters can also be used to control the expression of the target protein , such as promoters expressed in plant green tissues, etc. Furthermore, the expression of this gene can also be enhanced using expression enhancers. For example, the 35S enhancer can enhance the expression of the target gene upstream or downstream of it. Those skilled in the art can compare the effects of using different promoters through experiments. The terminator of the expression cassette may be of different origin. Selection genes are often required during plant transformation to select transformed cells. Commonly used screening genes include kanamycin resistance gene, hygromycin resistance gene, herbicide resistance gene (such as glyphosate resistance, glufosinate resistance gene) and so on.

Vip3蛋白是一种新型Bt杀虫蛋白基因。本发明所提供的Vip3融合基因在植物中过表达后所得到的融合蛋白仍然具有Vip3的杀虫活性，但不会集中结合到受体植物细胞膜上，因此不会对受体植物细胞膜造成伤害而影响受体植物正常生长发育。一般技术人员都可以实现任一多肽序列与Vip3基因序列的融合，并利用融合基因构建转基因植物。Vip3 protein is a new Bt insecticidal protein gene. The fusion protein obtained after the Vip3 fusion gene provided by the present invention is overexpressed in plants still has the insecticidal activity of Vip3, but it will not be concentrated on the recipient plant cell membrane, so it will not cause damage to the recipient plant cell membrane. Affect the normal growth and development of recipient plants. Those skilled in the art can realize the fusion of any polypeptide sequence and Vip3 gene sequence, and use the fusion gene to construct transgenic plants.

综上所述，本发明提供了一种vip3融合基因的构建方法，即将一段多肽基因直接与vip3基因的N端相连接形成融合基因。这样的融合基因在转入植物中表达的融合蛋白不集中在植物细胞膜上，因此对植物生长没有影响，且高表达Vip3融合蛋白的转基因植物具有良好的抗虫活性。In summary, the present invention provides a method for constructing a vip3 fusion gene, that is, a polypeptide gene is directly connected to the N-terminus of the vip3 gene to form a fusion gene. The fusion protein expressed by such a fusion gene in the transfected plant is not concentrated on the plant cell membrane, so it has no effect on plant growth, and the transgenic plant highly expressing the Vip3 fusion protein has good insect resistance activity.

附图说明Description of drawings

下面结合附图对本发明的具体实施方式作进一步详细说明。The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

图1为融合基因水稻转化载体pCambia1300-p35S-gfp -vip3 -pZmUbi-G10结构示意图。35S表示花椰菜花叶病毒CaMV 的35S启动子，融合基因egfp-vip3两侧酶切位点分别为HindIII和KpnI，ZmUbi表示玉米ubiquitin启动子，G10为抗草甘膦基因。LB和RB分别表示载体的左右边界。Figure 1 is a schematic diagram of the structure of the fusion gene rice transformation vector pCambia1300-p35S-gfp-vip3-pZmUbi-G10. 35S represents the 35S promoter of cauliflower mosaic virus CaMV, the restriction sites on both sides of the fusion gene egfp-vip3 are HindIII and KpnI, respectively, ZmUbi represents the maize ubiquitin promoter, and G10 represents the glyphosate resistance gene. LB and RB represent the left and right boundaries of the carrier, respectively.

图2为生根培养7天后转基因水稻植株的生长情况对比图。Fig. 2 is a graph comparing the growth of transgenic rice plants after rooting and culturing for 7 days.

CK：对照，空载体（pCambia1300-p35S-pZmUbi-G10）转基因株；CK: control, empty vector (pCambia1300-p35S-pZmUbi-G10) transgenic strain;

V3：单独vip3（pCambia1300-p35S-vip3-pZmUbi-G10）转基因株；V3: vip3 (pCambia1300-p35S-vip3-pZmUbi-G10) transgenic strain alone;

V3G：含vip3-egfp的（pCambia1300-p35S-vip3-gfp-pZmUbi-G10）转基因株；V3G: transgenic strain containing vip3-egfp (pCambia1300-p35S-vip3-gfp-pZmUbi-G10);

GV3：含egfp-vip3融合基因的（pCambia1300-p35S-gfp -vip3 -pZmUbi-G10）转基因株。GV3: (pCambia1300-p35S-gfp-vip3-pZmUbi-G10) transgenic strain containing egfp-vip3 fusion gene.

图3是转基因水稻植株中GFP蛋白的检测对比图，将预分化好的愈伤组织取少量直接在载玻片上压片后于激光共聚焦显微镜下20倍目镜观察；标尺为10μm；Figure 3 is a comparison chart of the detection of GFP protein in transgenic rice plants. A small amount of pre-differentiated callus was directly pressed on a glass slide and observed under a laser confocal microscope with a 20-fold eyepiece; the scale bar is 10 μm;

V3：单独vip3转基因植株（不含GFP）；V3: vip3 transgenic plants alone (without GFP);

V3G：含vip3-egfp的（pCambia1300-p35S-vip3-gfp-pZmUbi-G10）转基因株；V3G: transgenic strain containing vip3-egfp (pCambia1300-p35S-vip3-gfp-pZmUbi-G10);

GV3：含egfp-vip3融合基因的（pCambia1300-p35S-gfp -vip3 -pZmUbi-G10）转基因株。GV3: (pCambia1300-p35S-gfp-vip3-pZmUbi-G10) transgenic strain containing egfp-vip3 fusion gene.

图4：转基因水稻植株对棉铃虫初孵幼虫的生物测定对比图；Figure 4: Comparison of bioassays of transgenic rice plants against newly hatched larvae of cotton bollworm;

CK：对照，空载体（pCambia1300-p35S-pZmUbi-G10）转基因株；CK: control, empty vector (pCambia1300-p35S-pZmUbi-G10) transgenic strain;

V3：单独vip3（pCambia1300-p35S-vip3-pZmUbi-G10）转基因株；V3: vip3 (pCambia1300-p35S-vip3-pZmUbi-G10) transgenic strain alone;

V3G：含vip3-egfp的（pCambia1300-p35S-vip3-gfp-pZmUbi-G10）转基因株；V3G: transgenic strain containing vip3-egfp (pCambia1300-p35S-vip3-gfp-pZmUbi-G10);

GV3：含egfp-vip3融合基因的（pCambia1300-p35S-gfp -vip3 -pZmUbi-G10）转基因株。GV3: (pCambia1300-p35S-gfp-vip3-pZmUbi-G10) transgenic strain containing egfp-vip3 fusion gene.

具体实施方式Detailed ways

以下结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

若未特别指明，本发明以下实施例所使用的分子生物学和生物化学方法均为本领域技术人员所熟知的常规方法。Unless otherwise specified, the molecular biology and biochemical methods used in the following examples of the present invention are conventional methods well known to those skilled in the art.

实施例1、融合基因的获得Embodiment 1, the acquisition of fusion gene

SEQ ID NO：1为Vip3氨基酸序列（789aa）Genebank：ABB72459.1；SEQ ID NO：2 为 GFP氨基酸序列（241aa）Genebank：AFA52654.1。SEQ ID NO: 1 is the amino acid sequence of Vip3 (789aa) Genebank: ABB72459.1; SEQ ID NO: 2 is the amino acid sequence of GFP (241aa) Genebank: AFA52654.1.

根据SEQ ID NO：1 和SEQ ID NO：2的氨基酸序列所对应的核苷酸序列自行合成vip3和egfp（编码GFP蛋白）基因（上海生工）。According to the nucleotide sequences corresponding to the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, vip3 and egfp (encoding GFP protein) genes were synthesized by ourselves (Shanghai Shengong).

利用重叠区扩增基因拼接法（gene splicing by overlap extension，简称SOE）获得egfp-vip3融合基因。具体如下：The egfp-vip3 fusion gene was obtained by gene splicing by overlap extension (SOE) method. details as follows:

首先第一轮PCR，以合成的egfp基因质粒为模板，使用引物egfpPF1：5’-acg aagctt atgtctagagtgagcaag （加HindIII）和egfpPR1：5’-tgttggatctcccttgtacagctcgtcc扩增egfp基因；同时，以合成的vip3基因质粒为模板，使用引物Vip3PF1：5’-ggagatccaacaatgaacaagaataatac和Vip3PR1：5’-acg ggtacc ctacttaatagagac（加KpnI）扩增vip3基因。其中，在引物egfpPF1和Vip3PR1中分别添加HindIII和KpnI酶切位点（如斜体字母所示），在引物egfpPR1和Vip3PF1中分别引入连接肽序列（如粗体带下划线的字母所示）。First, in the first round of PCR, using the synthetic egfp gene plasmid as a template, use primers egfpPF1: 5'-acg aagctt atgtctagagtgagcaag (plus HindIII) and egfpPR1: 5'-tgttggatctcc cttgtacagctcgtcc to amplify the egfp gene; at the same time, use the synthetic vip3 gene plasmid As a template, the vip3 gene was amplified using primers Vip3PF1: 5'-ggagatccaaca atgaacaagaataatac and Vip3PR1: 5'-acg ggtacc ctacttaatagagac (plus KpnI). Among them, HindIII and KpnI restriction sites were added to primers egfpPF1 and Vip3PR1 (as shown in italic letters), respectively, and connecting peptide sequences were introduced into primers egfpPR1 and Vip3PF1 (as shown in bold underlined letters).

扩增体系为：总共为100ul反应体系。（DNA聚合酶为TAKARA公司生产的Primer star高保真聚合酶，模板DNA为含有合成的目的基因的质粒）The amplification system is: a total of 100ul reaction system. (The DNA polymerase is Primer star high-fidelity polymerase produced by TAKARA company, and the template DNA is a plasmid containing a synthetic target gene)

扩增程序为：The amplification procedure is:

重复Step2-Step4，35个循环；Repeat Step2-Step4, 35 cycles;

Step5：72℃7minStep5: 72℃7min

Step6：16℃∞    。Step6: 16°C∞.

然后第二轮再以上述PCR产物为模板，以引物egfpPF和Vip3PR扩增得egfp-vip3融合基因。所得到的egfp-vip3融合基因序列如SEQ ID NO：3所示。SEQ ID NO：3 为egfp-vip3融合基因，“  ”标出的为egfp基因，“  ”标出的为vip3基因。“  ”标出的为连接肽的核苷酸序列。斜体标出的为引入的酶切位点。Then in the second round, the above PCR product was used as a template, and the egfpPF and Vip3PR primers were used to amplify the egfp-vip3 fusion gene. The resulting egfp-vip3 fusion gene sequence is shown in SEQ ID NO:3. SEQ ID NO: 3 is egfp-vip3 fusion gene, "  "marked as egfp gene,"  "The one marked is the vip3 gene."  "marked is the nucleotide sequence of the linking peptide. Italicized is the introduced enzyme cleavage site.

扩增体系等同于上述扩增体系。The amplification system is equivalent to the amplification system described above.

扩增程序：除了Step4的时间变为2.5min外，其余同上述扩增程序。Amplification procedure: except that the time of Step4 is changed to 2.5 minutes, the rest are the same as the above-mentioned amplification procedures.

另外，作为对照，同样用PCR方法构建vip3-egfp融合基因，即将vip3的C端与egfp的N端以同样的方式相连。第一轮PCR引物为egfpPF2：5’-ggagatccaacaatgtctagagtgagc     /egfpPR2:5’- acg ggtacc ttacttgtacagctcg（加KpnI位点，斜体） 和vip3PF2：5’-acg aagcttatgaacaagaataatac （加HindIII位点，斜体）   /vip3PR2：5’- tgttggatctcccttaatagagacatcg     ；在引物egfpPF2和vip3PR2中分别引入连接肽序列（如粗体带下划线的字母所示）。扩增体系除了引物作了相应替代外，其余同egfp-vip3融合基因的第一轮的扩增体系，扩增程序同egfp-vip3融合基因的第一轮的扩增程序。备注说明：斜体字体部分为酶切位点。In addition, as a control, the vip3-egfp fusion gene was also constructed by PCR, that is, the C-terminus of vip3 was connected with the N-terminus of egfp in the same way. The first round of PCR primers are egfpPF2: 5'-ggagatccaaca atgtctagagtgagc /egfpPR2: 5'- acg ggtacc ttacttgtacagctcg (add KpnI site, italics) and vip3PF2: 5'-acg aagcttatgaacaagaataatac (add HindIII site, italics) / vip3PR2: 5 '-tgttggatctcc cttaatagagacatcg; linker peptide sequences were introduced in primers egfpPF2 and vip3PR2 respectively (indicated by bold underlined letters). The amplification system is the same as the first-round amplification system of the egfp-vip3 fusion gene except that the primers are replaced accordingly, and the amplification procedure is the same as that of the first-round amplification procedure of the egfp-vip3 fusion gene. Remarks: The part in italics is the restriction site.

第二轮PCR引物为vip3PF2和egfpPR2，所得到的vip3-egfp融合基因序列如SEQ ID NO.4所示。扩增体系除了引物作了相应替代外，其余同egfp-vip3融合基因的第二轮的扩增体系，扩增程序同egfp-vip3融合基因的第二轮的扩增程序。SEQ ID NO.4 为vip3-egfp融合基因，“  ”标出的为egfp基因，“  ”标出的为vip3基因。“  ”标出的为连接肽的核苷酸序列。斜体标出的为引入的酶切位点。The primers for the second round of PCR are vip3PF2 and egfpPR2, and the obtained vip3-egfp fusion gene sequence is shown in SEQ ID NO.4. The amplification system is the same as the second-round amplification system of the egfp-vip3 fusion gene except that the primers are replaced accordingly, and the amplification procedure is the same as that of the second-round amplification procedure of the egfp-vip3 fusion gene. SEQ ID NO.4 is vip3-egfp fusion gene, "  "marked as egfp gene,"  "The one marked is the vip3 gene."  "marked is the nucleotide sequence of the linking peptide. Italicized is the introduced enzyme cleavage site.

单独vip3基因作为对照同样用PCR方式在其两端引入HindIII和KpnI酶切位点，引物为vip3PF2：5’- acg aagctt atgaacaagaataatac（斜体为HindIII位点）和vipSR：5’- acg ggtacc ctacttaatagagac（斜体为KpnI位点） 。扩增体系除了引物作了相应替代外，其余同egfp-vip3融合基因的第一轮的扩增体系，扩增程序同egfp-vip3融合基因的第一轮的扩增程序。The separate vip3 gene was used as a control to introduce HindIII and KpnI restriction sites at both ends of it by PCR. is the KpnI site). The amplification system is the same as the first-round amplification system of the egfp-vip3 fusion gene except that the primers are replaced accordingly, and the amplification procedure is the same as that of the first-round amplification procedure of the egfp-vip3 fusion gene.

实施例2、融合基因的水稻表达载体的构建Embodiment 2, the construction of the rice expression vector of fusion gene

T-DNA载体pCambia1300-p35S-pZmUbi-G10的全部DNA序列如SEQ IDNO：5所示。该载体包含一个耐草甘膦基因（EPSPS）作为转化的选择基因，包含一组HindIII和KpnI位点作为目的基因的克隆位点。将融合基因egfp-vip3和pCambia1300-p35S-pZmUbi-G10载体分别都用HindIII和KpnI双酶切消化后，各自利用凝胶电泳分离并胶回收目的片段，将载体和片段连接后获得的新的载体称为pCambia 1300-p35S-gfp-vip3-pZmUbi-G10。载体构建的示意图如图1所示。The entire DNA sequence of the T-DNA vector pCambia1300-p35S-pZmUbi-G10 is shown in SEQ ID NO:5. The vector contains a glyphosate-resistant gene (EPSPS) as the selection gene for transformation, and a set of HindIII and KpnI sites as the cloning site of the target gene. After the fusion gene egfp-vip3 and pCambia1300-p35S-pZmUbi-G10 vectors were digested with HindIII and KpnI respectively, the target fragments were separated by gel electrophoresis, and the new vectors were obtained by ligating the vectors and fragments Called pCambia 1300-p35S-gfp-vip3-pZmUbi-G10. The schematic diagram of vector construction is shown in Figure 1.

同时用上述相同的方法构建作为对照的单独vip3基因和vip3-egfp融合基因载体pCambia 1300-p35S -vip3-pZmUbi-G10和pCambia 1300-p35S -vip3-gfp-pZmUbi-G10。Simultaneously use the same method as above to construct separate vip3 gene and vip3-egfp fusion gene vectors pCambia 1300-p35S-vip3-pZmUbi-G10 and pCambia 1300-p35S-vip3-gfp-pZmUbi-G10 as controls.

实施例3、农杆菌介导的水稻转化Embodiment 3, rice transformation mediated by Agrobacterium

转基因水稻的获得方法是采用现有技术（卢雄斌等，1998；刘凡等，2003）。选取成熟饱满的“秀水134”种子去壳，诱导产生愈伤组织作为转化材料。分别取含目的基因载体pCambia1300-p35S-gfp-vip3-pZmUbi-G10和pCambia1300-p35S-vip3-gfp-pZmUbi-G10、pCambia 1300-p35S -vip3 -pZmUbi-G10以及空载体pCambia1300-p35S-pZmUbi-G10的农杆菌划板，挑单菌落接种准备转化用农杆菌。将待转化的愈伤组织放入适当浓度的农杆菌液中（含乙酰丁香酮），让农杆菌结合到愈伤组织表面，然后把愈伤组织转移到共培养基中，共培养2～3天。用无菌水冲洗转化后的愈伤，转移到含2mM草甘膦的筛选培养基上，筛选培养两个月（中间继代一次）。将筛选后生长活力良好的愈伤转移到预分化培养基上培养20天左右，然后将预分化好的愈伤组织转移到分化培养基，14小时光照分化发芽。2－3周后，把抗性再生植株转移到含有0.1mM草甘膦的生根培养基上壮苗生根，最后将再生植株洗去琼脂移植于温室，作为鉴定材料。The method of obtaining transgenic rice is to use the existing technology (Lu Xiongbin et al., 1998; Liu Fan et al., 2003). The mature and plump "Xiushui 134" seeds were dehulled, and callus was induced as transformation materials. Take the target gene vectors pCambia1300-p35S-gfp-vip3-pZmUbi-G10 and pCambia1300-p35S-vip3-gfp-pZmUbi-G10, pCambia1300-p35S-vip3-pZmUbi-G10 and the empty vector pCambia1300-p35S-pZmUbi-G1 The Agrobacterium plate, pick a single colony inoculation ready for transformation with Agrobacterium. Put the callus to be transformed into the appropriate concentration of Agrobacterium solution (containing acetosyringone), let the Agrobacterium bind to the surface of the callus, then transfer the callus to the co-culture medium, and co-culture for 2 to 3 sky. Wash the transformed callus with sterile water, transfer to the selection medium containing 2mM glyphosate, and select and culture for two months (subculture once in the middle). The calli with good growth vigor after screening were transferred to the pre-differentiation medium and cultured for about 20 days, then the pre-differentiated callus was transferred to the differentiation medium, and 14 hours of light were used to differentiate and germinate. After 2-3 weeks, transfer the resistant regenerated plants to the rooting medium containing 0.1mM glyphosate for strong seedlings to take root, and finally the regenerated plants are washed off the agar and transplanted in the greenhouse as identification materials.

实施例4、转基因植物的检测Embodiment 4, the detection of transgenic plant

转基因植物生长情况：Growth of transgenic plants:

在上述实验过程中，观察记录各转化植株的生长情况，得到含egfp-vip3的转化植株的生根率为62.5%，明显大于含vip3-egfp（51.1%）和单独vip3（52.0%）的，但与空载体的转化植株生根率（63.1%）相当。生根培养7天后转化植株幼苗的生长情况为含egfp-vip3与空载体的转化植株对照无明显差别，而含vip3-egfp和单独vip3的转化植株幼苗生长明显受到抑制（如图2所示）。具体为：无转基因的亲本野生株（CK）和含融合基因egfp-vip3的转化株（GV3）生长情况均正常，无明显差异。而含vip3-egfp基因和单独vip3基因的转化株（V3G和V3）的生长均受到抑制，表现为生长缓慢、植株矮弱甚至出现白化现象等。说明在vip3基因的N端融合GFP多肽后，会减少甚至消除vip3基因对转基因植株的毒性。During the above experiments, the growth of each transformed plant was observed and recorded, and the rooting rate of transformed plants containing egfp-vip3 was 62.5%, which was significantly higher than those containing vip3-egfp (51.1%) and vip3 alone (52.0%), but The rooting rate (63.1%) of transformed plants with the empty vector was comparable. After 7 days of rooting culture, the growth of transformed plant seedlings showed no significant difference between the transformed plants containing egfp-vip3 and the empty vector control, while the growth of transformed plant seedlings containing vip3-egfp and vip3 alone was significantly inhibited (as shown in Figure 2). Specifically: the parental wild strain without transgene (CK) and the transformed strain (GV3) containing the fusion gene egfp-vip3 grew normally, with no significant difference. However, the growth of transformants containing vip3-egfp gene and vip3 gene alone (V3G and V3) were all inhibited, showing slow growth, short and weak plants, and even albinism. It shows that the toxicity of vip3 gene to transgenic plants will be reduced or even eliminated after the N-terminal of vip3 gene is fused with GFP polypeptide.

转基因植株中GFP蛋白的检测：Detection of GFP protein in transgenic plants:

取预分化后的愈伤组织，在载玻片上直接压片至激光共聚焦显微镜下观察GFP绿色荧光情况（图3），结果显示vip3-egfp转基因植株中绿色荧光蛋白均分布在细胞膜上，而egfp-vip3转基因植株中绿色荧光蛋白分布在细胞质中，而没有明显定位在细胞膜上。上述现象表明在vip3基因前面融合了GFP多肽基因后（GV3），其在植物中表达的蛋白已不集中在植物细胞膜上，这样使Vip3蛋白对植物原本的伤害就减弱或消除了。而将GFP多肽基因融合在vip3基因后面（V3G）是为了以GFP荧光示踪作为对照，图片显示绿色荧光集中在植物细胞膜上，说明表达的Vip3-GFP融合蛋白集中在植物细胞膜上。而V3是单独vip3基因的转基因植物的样本照片，因为没有融合GFP蛋白，所以没有荧光显示，可以作为对照。The pre-differentiated callus was taken and pressed directly on the glass slide to observe the green fluorescence of GFP under the laser confocal microscope (Figure 3). The results showed that the green fluorescent protein in vip3-egfp transgenic plants was distributed on the cell membrane, while In the egfp-vip3 transgenic plants, the green fluorescent protein was distributed in the cytoplasm, but not located on the cell membrane. The above phenomenon shows that after the GFP polypeptide gene (GV3) is fused in front of the vip3 gene, the protein expressed in the plant is no longer concentrated on the plant cell membrane, so that the original damage of the Vip3 protein to the plant is weakened or eliminated. The purpose of fusing the GFP polypeptide gene behind the vip3 gene (V3G) is to use GFP fluorescence tracer as a control. The picture shows that the green fluorescence is concentrated on the plant cell membrane, indicating that the expressed Vip3-GFP fusion protein is concentrated on the plant cell membrane. And V3 is a sample photo of transgenic plants with vip3 gene alone, because there is no fusion of GFP protein, so there is no fluorescence display, which can be used as a control.

转基因植株的生物测定：Bioassays of transgenic plants:

取温室培养7天的转基因植株新鲜叶片，置于铺有一层湿润的滤纸保湿的直径75mm的培养皿中，然后每皿接入30头棉铃虫初孵幼虫。将接种后的培养皿置于培养箱中25°C 12L：12D光照培养24h后开盖观察。结果显示，含vip3-egfp和单独vip3的转基因植株对棉铃虫初孵幼虫的抗性较差，与不含vip3基因的空载体对照株比较基本无差别。而含egfp-vip3的转基因植株对棉铃虫初孵幼虫的抗性明显较高（图4）。具体为：Get the fresh leaves of the transgenic plants cultivated in the greenhouse for 7 days, place them in a petri dish with a diameter of 75 mm covered with a layer of moist filter paper, and insert 30 newly hatched larvae of cotton bollworm into each dish. Place the inoculated Petri dish in an incubator at 25°C under 12L:12D light for 24 hours and then open the lid for observation. The results showed that the transgenic plants containing vip3-egfp and vip3 alone had poor resistance to the newly hatched larvae of cotton bollworm, and there was basically no difference compared with the empty vector control plants without vip3 gene. However, the transgenic plants containing egfp-vip3 had significantly higher resistance to the newly hatched larvae of cotton bollworm (Fig. 4). Specifically:

无转基因的亲本植株（CK）和单独vip3转基因植株（V3）以及vip3-egfp融合基因的转基因植株（V3G）均被棉铃虫取食得较多，说明它们对棉铃虫几乎没有抗性。而egfp-vip3融合基因的转基因植株（GV3）基本没有受到棉铃虫幼虫的取食（或伤害），说明它对棉铃虫的抗性较高。The non-transgenic parental plants (CK), vip3 transgenic plants alone (V3) and vip3-egfp fusion gene transgenic plants (V3G) were eaten more by the bollworm, indicating that they had almost no resistance to the bollworm. However, the transgenic plant (GV3) with the egfp-vip3 fusion gene was basically not fed (or injured) by the larvae of the cotton bollworm, indicating that it had higher resistance to the cotton bollworm.

最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (9)

1. reduce Vip3 albumen to the method for transgenic plant toxicity by gene fusion, it is characterized in that:

1), the C end with a polypeptide (polypeptide) gene directly links to each other with the N end of gene vip3; The connection peptides that middle in addition 4 amino acid forms connects;

2), fusion gene does not concentrate on the cytolemma of plant at the fused protein of vegetable cell expression.

2. according to claim 1 by the method for gene fusion reduction Vip3 albumen to transgenic plant toxicity, it is characterized in that:

Described Vip3 albumen be following any one:

Be any one of Bt Vegetative Insecticidal Proteins Vip3 proteinoid;

Be the aminoacid sequence shown in the SEQ ID NO:1;

Be the aminoacid sequence that has at least 70% similarity with SEQ ID NO:1.

3. according to claim 2 by the method for gene fusion reduction Vip3 albumen to transgenic plant toxicity, it is characterized in that:

Described polypeptide (polypeptide) is green fluorescent protein GFP;

Described green fluorescent protein GFP is the aminoacid sequence shown in the SEQ ID NO:2.

4. it is characterized in that according to claim 2 or 3 describedly reduce Vip3 albumen to the method for transgenic plant toxicity by gene fusion:

Described connection peptides is Gly-Asp-Pro-Thr.

5. the egfp-vip3 fusion gene that gets such as either method preparation in the claim 1 ~ 4, it is characterized in that: described egfp-vip3 fusion gene is the sequence shown in SEQ ID NO:3.

6. the vip3-egfp fusion gene that gets such as either method preparation in the claim 1 ~ 4, it is characterized in that: described vip3-egfp fusion gene sequence is the sequence shown in SEQ ID NO.4.

7. such as the purposes of any one fusion gene in the claim 1 ~ 6: this fusion gene is changed in the plant, thereby obtain transgenic anti-insect plants.

8. the purposes of fusion gene according to claim 7: described plant is monocotyledons or dicotyledons.

9. the purposes of fusion gene according to claim 8: described monocotyledons is paddy rice, corn, wheat or Chinese sorghum; Described dicotyledons is cotton, soybean or rape.

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