CN102465139A - Method for promoting growth of plant roots by using BGIos389 gene - Google Patents
Method for promoting growth of plant roots by using BGIos389 geneDownload PDFInfo
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Abstract
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Translated fromChinese发明领域field of invention
本发明涉及用于促进植物根的生长的方法。特别地，本发明利用BGIos389基因促进植物根的生长。The present invention relates to a method for promoting the growth of plant roots. In particular, the present invention utilizes the BGIos389 gene to promote plant root growth.
发明背景Background of the invention
水稻是我国重要的经济作物，其栽种面积为4.5亿亩左右。世界上约有60％的人口以稻米作为日常主食。通过水稻的分子育种来提高产量，给全球的粮食生产带来了巨大收益。矮秆基因的利用和多分蘖水稻的育成，使我国的水稻产量取得了巨大飞跃。但在相当长的一段时间里，我国的水稻单产出现了徘徊不前的局面。由此，许多育种工作者从育种理论和方法上提出了新的思路和设想，希望使水稻单产取得新的突破。在水稻的高产育种理论上，国际水稻研究所于20世纪90年代初就提出了基于新株型的超级稻理论，然而，到目前为止，在水稻的育种科研与实践中对水稻地下部分的形态和生理性状的改良却未能在水稻的分子育种中得以具体体现。Rice is an important economic crop in my country, and its planting area is about 450 million mu. About 60% of the world's population uses rice as their daily staple food. Improving yields through molecular breeding of rice has brought huge benefits to global food production. The utilization of dwarf genes and the breeding of multi-tiller rice have made a great leap forward in my country's rice yield. However, for quite a long period of time, my country's rice yield per unit area has been stagnant. As a result, many breeding workers have put forward new ideas and assumptions from the breeding theory and methods, hoping to make new breakthroughs in rice yield. In the theory of high-yield breeding of rice, the International Rice Research Institute put forward the theory of super rice based on a new plant type in the early 1990s. The improvement of physiological traits has not been embodied in the molecular breeding of rice.
目前我国水稻的水肥利用率低，这不仅造成大量人力、物力和财力的浪费，而且大量的化学肥料和农药通过地表径流以及土壤渗透排入江河湖泊和地下水中，对地表和地下水造成严重污染。这已经成为环境污染的一个重要方面。另一方面，全世界近一半的水稻种植面积处在缺水的状态下，严重影响了水稻产量的提高。我国很多地区由于相对缺乏灌溉用水，致使水稻生产难以发展，这在一定程度上影响了耕地资源的充分利用。因此，提高水稻本身的水肥利用率，发展节水型水稻，是新时期可持续农业发展的又一基本要求。而对于提高水稻品种本身的肥料利用率来说，根系的相关形态和生理性状的改良至关重要。随着环保和农业的进一步发展，在经济发达的地区，解决水稻易发生根倒伏，提高水肥利用率已成为提高水稻产量和发展的一个关键问题。从根系角度研究水稻抗根倒伏的生物学、力学机制及其遗传基础，对于从栽培和育种角度减轻水稻根倒伏的危害，具有巨大的现实和长远意义。At present, the water and fertilizer utilization rate of rice in our country is low, which not only causes a lot of waste of manpower, material resources and financial resources, but also a large amount of chemical fertilizers and pesticides are discharged into rivers, lakes and groundwater through surface runoff and soil infiltration, causing serious pollution to surface and groundwater. This has become an important aspect of environmental pollution. On the other hand, nearly half of the rice planting area in the world is in a state of water shortage, which seriously affects the increase of rice yield. Due to the relative lack of irrigation water in many areas of my country, it is difficult to develop rice production, which affects the full utilization of cultivated land resources to a certain extent. Therefore, improving the water and fertilizer use efficiency of rice itself and developing water-saving rice is another basic requirement for sustainable agricultural development in the new era. For improving the fertilizer utilization efficiency of rice varieties, the improvement of the related morphology and physiological traits of the root system is very important. With the further development of environmental protection and agriculture, in economically developed areas, solving rice root lodging and improving water and fertilizer use efficiency have become a key issue to increase rice production and development. Studying the biological, mechanical mechanism and genetic basis of rice root lodging resistance from the perspective of root system has great practical and long-term significance for alleviating the damage of rice root lodging from the perspective of cultivation and breeding.
水稻根系既是吸收养分和水分的重要器官，也是一些物质合成和运输的器官，它的形态发育对水稻产量和品质的影响很早就引起人们的注意。育种学家主要关注于环境对根系生长发育的影响(孙传清等，中国农业科学.1995，28(1)：42-48)以及根系与产量等因素之间的相互关系(凌启鸿等，中国农业科学.1984，(4)：3-11)，然而，关于根系的分子育种研究却很少报道。与根系性状表达相关的分子标记的开发表明，控制水稻根系发育的是数量性状(Yadav等人，Theor Appl Genet.1997，94：619-632；徐吉臣等，遗传学报.2002，29(3)：245-249)，但这并未引起足够的重视。石庆华等(中国农业科学.1997，30(4)：61-67)研究了根系发育与地上部分的相关关系，并指出，在栽培上培育有利于塑造理想株型的根型是水稻高产栽培的新要求，深根系更有利于高产。但是，育种工作者至今未能就什么是理想的水稻根型提出具体明确的指标。The rice root system is not only an important organ for absorbing nutrients and water, but also an organ for the synthesis and transportation of some substances. The influence of its morphological development on rice yield and quality has attracted people's attention for a long time. Breeders are mainly concerned with the influence of environment on root growth and development (Sun Chuanqing et al., Chinese Agricultural Sciences. 1995, 28 (1): 42-48) and the interrelationship between factors such as root system and yield (Ling Qihong, etc., Chinese Agricultural Sciences .1984, (4): 3-11), however, studies on molecular breeding of roots are rarely reported. The development of molecular markers related to the expression of root traits shows that it is quantitative traits that control rice root development (Yadav et al., Theor Appl Genet.1997, 94:619-632; Xu Jichen et al., Acta Genetica Sinica. 2002, 29 (3): 245-249), but this has not received enough attention. Shi Qinghua et al. (Chinese Agricultural Science. 1997, 30 (4): 61-67) studied the correlation between root system development and the aboveground part, and pointed out that cultivating a root type that is conducive to shaping the ideal plant type is the key to high-yield cultivation of rice. New requirements, deep root system is more conducive to high yield. However, so far, breeders have not been able to provide specific and clear indicators on what is the ideal rice root type.
目前，如何从思路和技术上提出一种崭新的方法来定位水稻根系发育的基因已成为水稻育种的关键。相关研究表明，检测自然选择信号可能是一条崭新的、高通量的鉴定有用基因的道路。由于人工选择的时间短，遗传重组事件少，受选择的基因与其邻近区域会紧密连锁，因此，如果观察到一个基因组区域的多态性分布很特别，具有统计学显著性，那么该区域含有的基因和突变位点即有可能与选择的性状相关。研究表明，水稻全基因组大约6％的区域为SNP高变区，这些区域可能富集了与亚种分化和重要复杂农艺性状相关的基因(Tang等人，PLoS Genet.2006，2：e199)。Gao L Z等(BMC Evolutionary Biology.2008，8：11)通过对栽培稻和普通野生稻进行对比分析，建立了理论群体遗传学数学模型，检测了60个微卫星位点所受的人工选择，发现了一些留有显著选择痕迹的位点以及其近邻的可能的功能基因。但这些研究离从全基因组水平全面分析人工选择的基因依然遥远，这主要是因为数据量远远不够，模型和方法也有待提高。迄今为止识别出的水稻性状基因如qSH1、Rc、sh4、hd1(Kovach等人，Trends in Genetics.2007，23(11)：578-587)，以及PROG1(Jin等人，Nature Genetics.2008，40(11)：1365-1369)和GIF1(Wang等人，Nature Genetics.2008，40(11)：1370-1374)仍然是通过传统的图位克隆手段获得的。At present, how to propose a brand-new method to locate the genes of rice root development from the perspective of ideas and technologies has become the key to rice breeding. Related studies have shown that detecting natural selection signals may be a new, high-throughput approach to identifying useful genes. Due to the short time of artificial selection and few genetic recombination events, the selected gene will be closely linked with its adjacent regions. Therefore, if the polymorphism distribution of a genomic region is observed to be very special and statistically significant, then the region contains Genes and mutation sites are likely to be associated with selected traits. Studies have shown that about 6% of the rice genome is SNP hypervariable regions, and these regions may be enriched in genes related to subspecies differentiation and important complex agronomic traits (Tang et al., PLoS Genet. 2006, 2: e199). Gao L Z et al. (BMC Evolutionary Biology. 2008, 8: 11) established a mathematical model of theoretical population genetics through comparative analysis of cultivated rice and common wild rice, and detected the artificial selection of 60 microsatellite loci. Some loci with significant traces of selection and their neighbors' possible functional genes were found. However, these studies are still far away from a comprehensive analysis of artificially selected genes at the genome-wide level, mainly because the amount of data is far from enough, and models and methods need to be improved. Rice trait genes identified so far such as qSH1, Rc, sh4, hd1 (Kovach et al., Trends in Genetics. 2007, 23(11): 578-587), and PROG1 (Jin et al., Nature Genetics. 2008, 40 (11): 1365-1369) and GIF1 (Wang et al., Nature Genetics. 2008, 40(11): 1370-1374) were still obtained by traditional map-based cloning.
新一代测序技术(如Solexa、Solid测序仪)的出现，为廉价高效地获得水稻根系发育基因和基因家族提供了前所未有的机会。利用野生稻与栽培稻的基因组重测序数据和技术，能够比较各栽培种及野生种的基因组结构的异同，这使运用进化基因组学的理论和方法来获得根系发育相关的基因或基因组功能元件成为可能。The emergence of next-generation sequencing technologies (such as Solexa, Solid sequencer) has provided unprecedented opportunities for cheap and efficient access to rice root development genes and gene families. Using the genome resequencing data and technology of wild rice and cultivated rice, it is possible to compare the similarities and differences between the genome structures of cultivated species and wild species, which makes it possible to use the theory and methods of evolutionary genomics to obtain genes or genome functional elements related to root development. possible.
长期以来，对水稻根系发育形态、生理性状的遗传改良一直未能在水稻的分子育种中得以体现。虽然杂交水稻的根系比常规品种在形态和生理上占有优势，使育种家看到了根系改良的重要性和实际效果，但是迄今为止未能提出一套具体的改良指标。因此，需要进一步研究和开发水稻根系发育相关基因，以进一步提高根系对营养的吸收和对水肥的利用率，减少农药的施用，增加地上部分的分蘖和收获等。For a long time, the genetic improvement of rice root development morphology and physiological traits has not been reflected in molecular breeding of rice. Although the root system of hybrid rice has advantages in morphology and physiology compared with conventional varieties, allowing breeders to see the importance and practical effect of root system improvement, but so far failed to propose a set of specific improvement indicators. Therefore, further research and development of rice root development-related genes are needed to further improve root absorption of nutrients and water and fertilizer utilization, reduce pesticide application, and increase aerial tillering and harvesting.
随着水稻分子育种理论与技术的不断成熟和完善，以及遗传转化体系的建立和优化，利用新型测序技术结合基因组学方法来鉴定受人工选择的基因资源，使得利用基因组重测序技术发掘根系发育相关基因和遗传转化成为可能。水稻地下部分发育的改良将进一步提高水稻的产量、品质和抗性等。水稻根系发育相关基因的开发和遗传转化对于提高水稻地上部分的产量、降低农业投入、缓解环境污染、实现农业可持续发展具有重要的意义，其有利于增强我国农业在国际上的竞争力。With the continuous maturity and improvement of rice molecular breeding theory and technology, as well as the establishment and optimization of the genetic transformation system, the use of new sequencing technology combined with genomics methods to identify genetic resources subject to artificial selection makes it possible to use genome resequencing technology to discover root system development. Genes and genetic transformation are possible. The improvement of the development of the underground part of rice will further improve the yield, quality and resistance of rice. The development and genetic transformation of genes related to rice root development is of great significance for increasing the yield of rice aboveground, reducing agricultural input, alleviating environmental pollution, and realizing sustainable agricultural development, which is conducive to enhancing the international competitiveness of my country's agriculture.
发明内容Contents of the invention
本发明通过实验证实，基因BGIos389(其序列可以是SEQ ID NO：7)具有促进植物根系生长的功能。The present invention proves through experiments that the gene BGIos389 (its sequence may be SEQ ID NO: 7) has the function of promoting plant root growth.
因此，在一个方面，本发明提供了含有基因BGIos389的载体。在一个实施方案中，所述载体优选是表达载体，更优选是在植物中高效表达基因BGIos389的载体，例如包含基因BGIos389的重组载体p6，如本发明的重组载体p6+BGIos389。Thus, in one aspect, the invention provides a vector comprising the gene BGIos389. In one embodiment, the vector is preferably an expression vector, more preferably a vector that efficiently expresses the gene BGIos389 in plants, such as a recombinant vector p6 comprising the gene BGIos389, such as the recombinant vector p6+BGIos389 of the present invention.
在另一个方面，本发明提供了含有基因BGIos389或根据本发明的载体的宿主细胞。在一个实施方案中，所述宿主细胞优选是根癌农杆菌，例如根癌农杆菌EHA105-p6+BGIos389。In another aspect, the invention provides a host cell containing the gene BGIos389 or a vector according to the invention. In one embodiment, the host cell is preferably Agrobacterium tumefaciens, eg Agrobacterium tumefaciens EHA105-p6+BGIos389.
在另一个方面，本发明提供了基因BGIos389和/或根据本发明的载体和/或宿主细胞用于促进植物根系生长或用于制备转基因植物或用于植物育种的用途。在一个实施方案中，所述转基因植物与未进行转基因的植物相比，展示更优良的植物根系生长。In another aspect, the present invention provides the use of the gene BGIos389 and/or the vector and/or host cell according to the present invention for promoting plant root growth or for preparing transgenic plants or for plant breeding. In one embodiment, the transgenic plant exhibits superior plant root growth compared to a non-transgenic plant.
在另一个方面，本发明提供了促进植物根系生长的方法，其包括将基因BGIos389和/或根据本发明的载体转化入植物，或用根据本发明的宿主细胞感染植物。In another aspect, the present invention provides a method for promoting plant root growth, which comprises transforming the gene BGIos389 and/or the vector according to the present invention into a plant, or infecting a plant with a host cell according to the present invention.
在一个实施方案中，通过本领域公知的方法，例如根癌农杆菌转化法来将基因BGIos389或根据本发明的载体转化入植物。In one embodiment, the gene BGIos389 or the vector according to the invention is transformed into plants by methods known in the art, eg Agrobacterium tumefaciens transformation.
在一个实施方案中，基因BGIos389可操作地连接至在植物中有效的表达调控元件，例如启动子和/或终止子，优选玉米泛素启动子和/或NOS终止子。In one embodiment, the gene BGIos389 is operably linked to expression control elements effective in plants, such as promoters and/or terminators, preferably maize ubiquitin promoters and/or NOS terminators.
在另一个方面，本发明提供了产生转基因植物的方法，所述方法包括以下步骤：In another aspect, the present invention provides a method of producing a transgenic plant, said method comprising the steps of:
1)将基因BGIos389和/或根据本发明的载体转化入植物愈伤组织，或用根据本发明的宿主细胞感染植物愈伤组织；1) transforming the gene BGIos389 and/or the vector according to the present invention into the plant callus, or infecting the plant callus with the host cell according to the present invention;
2)从所述愈伤组织再生转基因植物。2) Regeneration of transgenic plants from said callus.
在一个实施方案中，通过上述方法产生的转基因植物与未进行转基因的植物相比，展示更优良的植物根系生长，例如转基因植物的根系更密集更发达。In one embodiment, a transgenic plant produced by the method described above exhibits superior plant root growth, eg, a denser and more developed root system of the transgenic plant, compared to a non-transgenic plant.
在一个实施方案中，通过本领域公知的方法，例如根癌农杆菌转化法来将基因BGIos389和/或根据本发明的载体转化入植物愈伤组织。In one embodiment, the gene BGIos389 and/or the vector according to the invention are transformed into plant calli by methods well known in the art, eg Agrobacterium tumefaciens transformation.
在一个实施方案中，基因BGIos389可操作地连接至在植物中有效的表达调控元件，例如启动子和/或终止子，优选玉米泛素启动子和/或NOS终止子。In one embodiment, the gene BGIos389 is operably linked to expression control elements effective in plants, such as promoters and/or terminators, preferably maize ubiquitin promoters and/or NOS terminators.
在另一个方面，本发明提供了通过上述方法产生的转基因植物。In another aspect, the present invention provides a transgenic plant produced by the above method.
在另一个方面，本发明提供了含有基因BGIos389或本发明的载体或被本发明的宿主细胞感染的植物愈伤组织。In another aspect, the present invention provides a plant callus comprising the gene BGIos389 or a vector of the present invention or infected with a host cell of the present invention.
在另一个方面，本发明提供了上文所述的植物愈伤组织用于制备转基因植物或用于植物育种的用途。In another aspect, the present invention provides the use of the above-mentioned plant callus for preparing transgenic plants or for plant breeding.
在另一个方面，本发明提供了一种转基因植物，其被导入了基因BGIos389和/或根据本发明的载体和/或根据本发明的宿主细胞。In another aspect, the invention provides a transgenic plant into which the gene BGIos389 and/or the vector according to the invention and/or the host cell according to the invention have been introduced.
在一个实施方案中，所述转基因植物，与未导入基因BGIos389或根据本发明的载体或根据本发明的宿主细胞的对照植物相比，展示更优良的植物根系生长，例如转基因植物的根系更密集更发达。In one embodiment, said transgenic plant, compared to a control plant to which the gene BGIos389 or the vector according to the invention or the host cell according to the invention has not been introduced, exhibits better root growth of the plant, e.g. the root system of the transgenic plant is denser more developed.
在一个实施方案中，基因BGIos389可操作地连接至在植物中有效的表达调控元件，所述表达调控元件例如启动子和/或终止子，优选玉米泛素启动子和/或NOS终止子。In one embodiment, the gene BGIos389 is operably linked to expression control elements effective in plants, such as promoters and/or terminators, preferably maize ubiquitin promoters and/or NOS terminators.
在本发明中，重组载体p6是指，包含玉米泛素启动子和NOS终止子的pCAMBIA-1301载体。In the present invention, the recombinant vector p6 refers to the pCAMBIA-1301 vector containing maize ubiquitin promoter and NOS terminator.
在本发明中，植物优选是单子叶植物，更优选为水稻、谷子(Setariaitalica)、小麦、高粱、玉米，特别优选为水稻，例如日本晴。In the present invention, the plant is preferably a monocot, more preferably rice, millet (Setaria italica), wheat, sorghum, corn, particularly preferably rice, such as Nipponbare.
发明的有益效果Beneficial Effects of the Invention
本发明通过实验证实，基因BGIos389具有促进植物根系生长的功能。从而，与现有技术相比，本发明的技术方案将进一步提高根系对营养的吸收和对水肥的利用率，减少农药的施用，增加地上部分的分蘖和收获，进一步提高水稻的产量、品质和抗性。这对于降低农业投入、缓解环境污染、实现农业可持续发展和增强我国农业在国际上的竞争力具有重要的意义。The invention proves through experiments that the gene BGIos389 has the function of promoting the growth of plant roots. Thereby, compared with the prior art, the technical solution of the present invention will further improve the absorption of nutrients and the utilization rate of water and fertilizer by the root system, reduce the application of pesticides, increase the tillering and harvesting of the aboveground parts, and further improve the yield, quality and quality of rice. resistance. This is of great significance for reducing agricultural input, alleviating environmental pollution, realizing sustainable agricultural development and enhancing the competitiveness of my country's agriculture in the world.
关于生物材料保藏的说明Instructions on the Preservation of Biological Materials
本发明涉及以下生物材料：The present invention relates to the following biological materials:
1.普通野生稻元江种子，其于2010年9月6日保藏于湖北省武汉市武昌珞珈山武汉大学保藏中心，即中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC P201011；1. Common wild rice Yuanjiang seeds, which were preserved in the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province on September 6, 2010, that is, the China Type Culture Collection Center (CCTCC), and the preservation number is CCTCC P201011;
2.根癌农杆菌(Agrobacterium tumefaciens)EHA105，其于2009年12月24日保藏于湖北省武汉市武昌珞珈山武汉大学保藏中心，即中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC M 209315；2. Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105, which was preserved on December 24, 2009 in the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, namely the China Type Culture Collection Center (CCTCC), and the preservation number is CCTCC M 209315;
3.水稻日本晴种子，其于2009年12月18日保藏于湖北省武汉市武昌珞珈山武汉大学保藏中心，即中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC P200910。3. Rice Nipponbare seeds, which were preserved on December 18, 2009 in the Wuhan University Collection Center, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, namely the China Type Culture Collection Center (CCTCC), and the preservation number is CCTCC P200910.
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.
附图概述Figure overview
图1是显示愈伤组织GUS染色的检测结果的照片。左边的愈伤组织为未转化BGIos389基因的对照愈伤组织，右边的愈伤组织为转化了BGIos389基因的转基因愈伤组织。Fig. 1 is a photograph showing the detection results of callus GUS staining. The callus on the left is the control callus without the BGIos389 gene transformed, and the callus on the right is the transgenic callus transformed with the BGIos389 gene.
图2是显示水稻苗的根系性状观察结果的照片。其中，左边试管为转化了BGIos389基因的转基因水稻苗，右边试管为未转化BGIos389基因的对照水稻苗。Fig. 2 is a photograph showing the observation results of root system properties of rice seedlings. Wherein, the test tube on the left is the transgenic rice seedling transformed with the BGIos389 gene, and the test tube on the right is the control rice seedling not transformed with the BGIos389 gene.
具体实施方式Detailed ways
实施例1：BGIos389基因的PCR扩增和pMD18-T+BGIos389重组载体的构建Example 1: PCR amplification of BGIos389 gene and construction of pMD18-T+BGIos389 recombinant vector
1.PCR扩增1. PCR amplification
使用植物基因组DNA提取试剂盒(TIANGEN新型植物基因组DNA提取试剂盒，目录号：DP320-02)提取普通野生稻元江(Oryza.rifupongonYuanjiang)(其由中国科学院昆明动物研究所董杨提供)的基因组DNA(gDNA)。根据BGIos389基因的碱基序列，设计一对PCR特异性扩增引物：上游引物F1(SEQ ID NO：1)包含限制性酶切位点BamHI，下游引物R1(SEQ ID NO：2)包含限制性酶切位点XbaI。以上述提取的元江gDNA为模板，利用上游引物F1、下游引物R1和高保真Ex TaqTM(TaKaRa，DRR100B)聚合酶进行PCR扩增。PCR扩增体系如表1所示。Genomic DNA of common wild rice Yuanjiang (Oryza. (gDNA). According to the base sequence of the BGIos389 gene, a pair of PCR-specific amplification primers were designed: the upstream primer F1 (SEQ ID NO: 1) contained the restriction site BamHI, and the downstream primer R1 (SEQ ID NO: 2) contained the restriction site Restriction site XbaI. Using the Yuanjiang gDNA extracted above as a template, PCR amplification was performed using upstream primer F1, downstream primer R1 and high-fidelity Ex TaqTM (TaKaRa, DRR100B) polymerase. The PCR amplification system is shown in Table 1.
表1：BGIos389基因的PCR扩增体系Table 1: PCR amplification system of BGIos389 gene
PCR反应条件为：94℃预变性5分钟；94℃变性45秒，55℃退火50秒，72℃延伸90秒，进行35个反应循环；72℃延伸7分钟。The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 55°C for 50 seconds, extension at 72°C for 90 seconds, and 35 reaction cycles; extension at 72°C for 7 minutes.
上游引物F1(SEQ ID NO：1)：CGCGGATCCGAGGCATGGACGCGGTCA，其中下划线代表BamHI酶切位点。下游引物R1(SEQ ID NO：2)：TGCTCTAGACGAATGCACACGCATCCTC，其中下划线代表XbaI酶切位点。Upstream primer F1 (SEQ ID NO: 1): CGCGGATCCGAGGCATGGACGCGGTCA, where the underline represents the BamHI restriction site. Downstream primer R1 (SEQ ID NO: 2): TGCTCTAGACGAATGCACACGCATCCTC, where the underline represents the XbaI restriction site.
PCR扩增产物经1.0％琼脂糖凝胶电泳分离，得到大小1360bp左右的条带。使用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号：DP209-03)进行纯化回收。The PCR amplification product was separated by 1.0% agarose gel electrophoresis, and a band with a size of about 1360bp was obtained. Use TIANGEN Agarose Gel DNA Recovery Kit (Cat. No.: DP209-03) for purification and recovery.
2.pMD18-T+BGIos389重组载体的构建2. Construction of pMD18-T+BGIos389 recombinant vector
将上述纯化回收的PCR扩增产物进行T/A克隆(pMD18-T质粒，TaKaRa，D103A)，然后转化大肠杆菌，挑取阳性克隆并测序，以验证目的基因的序列。The above-mentioned purified and recovered PCR amplification products were subjected to T/A cloning (pMD18-T plasmid, TaKaRa, D103A), and then transformed into Escherichia coli, and positive clones were picked and sequenced to verify the sequence of the target gene.
其中，T/A克隆的连接体系如下：Among them, the connection system of T/A clone is as follows:
pMD18-T 1μlpMD18-T 1 μl
2*Solution I 5μl2*Solution I 5μl
纯化回收的产物 10-20ngPurified and recovered product 10-20ng
补充至总体积10Refill to total volume 10
ddH2OddH2O
μlμl
于16℃在节能型智能恒温槽(宁波新芝，SDC-6)中连接至少8小时，得到pMD18-T+BGIos389重组载体。按照本领域技术人员熟知的方法，将经过上述连接后的产物转化入大肠杆菌DH5α，从而获得含有pMD18-T+BGIos389克隆载体的重组大肠杆菌，将其命名为DH5α-BGIos389。由深圳华大基因科技有限公司对pMD18-T+BGIos389克隆载体中的BGIos389进行测序。测序结果与SEQ ID NO：7一致。Ligate at 16°C for at least 8 hours in an energy-saving smart thermostat (Ningbo Xinzhi, SDC-6) to obtain the pMD18-T+BGIos389 recombinant vector. According to methods well known to those skilled in the art, the product after the above ligation was transformed into Escherichia coli DH5α to obtain a recombinant Escherichia coli containing the pMD18-T+BGIos389 cloning vector, which was named DH5α-BGIos389. BGIos389 in the pMD18-T+BGIos389 cloning vector was sequenced by Shenzhen Huada Gene Technology Co., Ltd. The sequencing result is consistent with SEQ ID NO:7.
SEQ ID NO：7的序列如下所示：The sequence of SEQ ID NO: 7 is shown below:
ATGGACGCGGTCATGTCCGCCGCCGACGACGCGGGCGCGGCGTCCGGCCGGGAGGACCCACCGCCAGCCGTGGTGCTCGTCTCCGCCGGCGCCAGCCACTCCGTCGCGCTCCTCGCGGGCAATGTGCTGTGCTCGTGGGGGAGAGGGGAGGACGGGCAGCTCGGGCACGGGGACGCGGAGGACCGGCTGGTGCCGACGGTGCTGAGCGGCTTCGACGCCGCCGCGCCGGGGATCACGTCGGTCATCTGCGGCGCCGACCACACCACCGCCTACTCCGAGGACGAGCAGCAGGTGTACAGCTGGGGCTGGGGAGACTTCGGGAGGCTCGGCCATGGCAACTCCAGCGACGTGTTCACTCCTCAGCCAGTCAAGGCCCTGCAGGGGATAAAGATCAAGCAGATAGCTTGCGGCGATAGCCATTGTCTTGCCGTCACTATGGCAGGTGAAGTGCAAAGTTGGGGGCGCAACCAAAACGGACAGCTTGGTCTTGGAACCACTGAAGACTCATTGCTCCCACAAAAGATTCAATCTTTTGAGGGAGTTTGTGTGAAAATGATTGCTGCTGGTGCTGAACATACCGCTGCAGTAACTGAAGATGGTGACCTCTATGGATGGGGCTGGGGTCGATATGGAAACTTGGGGCTTGGTGATCGCAATGATCGGTTGGTACCTGAGAAAGTATCTTCTGTGGAGGGAGAGAAGATGGTGCTTATTGCATGTGGATGGCGCCATACAATTACTGTTTCTTCCTCTGGTAGTTTGTATACTTATGGTTGGAGCAAATATGGTCAACTAGGGCATGGTGATTTTGAAGATCATTTAGTTCCACATAAACTAGAGGCCTTAAAGGATAGCTCTATATCGCAGATTTCAGGTGGATGGAGGCATACAATGGCGCTTACATCAGATGGAAAGCTTTATGGTTGGGGGTGGAACAAGTTTGGGCAAGTCGGAGTTGGTGATACTGATGATCACTGTTTCCCAGTACAGGTCAAGTTTCCAGAGGATCAGAAAGTTGCCCAAGTTGCTTGCGGGTGGAGGCATACTCTTGCATTTACAGAAAAGAAAAATGTTTTCTCATGGGGGAGGGGTACTAGCGGACAACTTGGTCATGGTGAAATAGTTGACAGGAACAAACCTGTGATGATTGATGCCTTAAGCCCGGATGGTCCTGGCTGCAAGAAATTAGAGCCATCAACAGCTGTACCATTTGCAGCCAAAGTCTGGGTCTCGCCATCAGAAAGATATGCCATTGTTCCTGATGAAAAAGTTCCAAATTCAGGTGAAGGCACGGCACGAGGCAACGGGGCGGACGCAAATGTACCAGAGAACGATGTAAAGAGGATGCGTGTGCATTCGTAGATGGACGCGGTCATGTCCGCCGCCGACGACGCGGGCGCGGCGTCCGGCCGGGAGGACCCACCGCCAGCCGTGGTGCTCGTCTCCGCCGGCGCCAGCCACTCCGTCGCGCTCCTCGCGGGCAATGTGCTGTGCTCGTGGGGGAGAGGGGAGGACGGGCAGCTCGGGCACGGGGACGCGGAGGACCGGCTGGTGCCGACGGTGCTGAGCGGCTTCGACGCCGCCGCGCCGGGGATCACGTCGGTCATCTGCGGCGCCGACCACACCACCGCCTACTCCGAGGACGAGCAGCAGGTGTACAGCTGGGGCTGGGGAGACTTCGGGAGGCTCGGCCATGGCAACTCCAGCGACGTGTTCACTCCTCAGCCAGTCAAGGCCCTGCAGGGGATAAAGATCAAGCAGATAGCTTGCGGCGATAGCCATTGTCTTGCCGTCACTATGGCAGGTGAAGTGCAAAGTTGGGGGCGCAACCAAAACGGACAGCTTGGTCTTGGAACCACTGAAGACTCATTGCTCCCACAAAAGATTCAATCTTTTGAGGGAGTTTGTGTGAAAATGATTGCTGCTGGTGCTGAACATACCGCTGCAGTAACTGAAGATGGTGACCTCTATGGATGGGGCTGGGGTCGATATGGAAACTTGGGGCTTGGTGATCGCAATGATCGGTTGGTACCTGAGAAAGTATCTTCTGTGGAGGGAGAGAAGATGGTGCTTATTGCATGTGGATGGCGCCATACAATTACTGTTTCTTCCTCTGGTAGTTTGTATACTTATGGTTGGAGCAAATATGGTCAACTAGGGCATGGTGATTTTGAAGATCATTTAGTTCCACATAAACTAGAGGCCTTAAAGGATAGCTCTATATCGCAGATTTCAGGTGGATGGAGGCATACAATGGCGCTTACATCAGATGGAAAGCTTTATGGTTGGGGGTGGAACAAGTTTGGGCAAGTCGGAGTTGGTGATACTGATGATCACTGTTTCCCAGTACAGGTCAAGTTTC CAGAGGATCAGAAAGTTGCCCAAGTTGCTTGCGGGTGGAGGCATACTCTTGCATTTACAGAAAAGAAAAATGTTTTCTCATGGGGGAGGGGTACTAGCGGACAACTTGGTCATGGTGAAATAGTTGACAGGAACAAACCTGTGATGATTGATGCCTTAAGCCCGGATGGTCCTGGCTGCAAGAAATTAGAGCCATCAACAGCTGTACCATTTGCAGCCAAAGTCTGGGTCTCGCCATCAGAAAGATATGCCATTGTTCCTGATGAAAAAGTTCCAAATTCAGGTGAAGGCACGGCACGAGGCAACGGGGCGGACGCAAATGTACCAGAGAACGATGTAAAGAGGATGCGTGTGCATTCGTAG
实施例2：p6重组载体的构建Embodiment 2: Construction of p6 recombinant vector
1.玉米泛素(ubi)启动子片段的PCR扩增和pMD18-T+Ubi重组载体的构建1. PCR amplification of maize ubiquitin (ubi) promoter fragment and construction of pMD18-T+Ubi recombinant vector
Ubi启动子的PCR扩增PCR amplification of Ubi promoter
使用植物基因组DNA提取试剂盒(TIANGEN新型植物基因组DNA提取试剂盒，目录号：DP320-02)提取玉米品种B73(Zea mays mays cv.B73)的基因组DNA(gDNA)。根据实施例1中描述的方法，使用下列引物，以上述提取的玉米B73的gDNA为模板，用高保真Ex TaqTM(TaKaRa，DRR100B)聚合酶进行Ubi启动子的PCR扩增：Genomic DNA (gDNA) of maize variety B73 (Zea mays mays cv. B73) was extracted using a plant genomic DNA extraction kit (TIANGEN New Plant Genomic DNA Extraction Kit, catalog number: DP320-02). According to the method described in Example 1, the following primers were used to perform PCR amplification of the Ubi promoter with the high-fidelity Ex Taq™ (TaKaRa, DRR100B) polymerase using the above-mentioned extracted gDNA of maize B73 as a template:
上游引物F2(SEQ ID NO：3)：GGCTGCAGTGCAGCGTGACCCGGTCGT，含有限制性酶切位点PstI；Upstream primer F2 (SEQ ID NO: 3): GGCTGCAGTGCAGCGTGACCCGGTCGT, containing restriction enzyme site PstI;
下游引物R2(SEQ ID NO：4)：GGCTGCAGAAGTAACACCAAAC，含有限制性酶切位点PstI。Downstream primer R2 (SEQ ID NO: 4): GGCTGCAGAAGTAACACCAAAC, containing restriction enzyme site PstI.
PCR扩增产物经1.0％琼脂糖凝胶电泳分离后，使用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号：DP209-03)纯化回收。The PCR amplification products were separated by 1.0% agarose gel electrophoresis, and then purified and recovered by using TIANGEN Agarose Gel DNA Recovery Kit (Catalogue Number: DP209-03).
pMD18-T+Ubi重组载体的构建Construction of pMD18-T+Ubi recombinant vector
根据实施例1中描述的方法，将上述纯化回收的PCR扩增产物连接入pMD18-T质粒(TaKaRa，D103A)，然后转化入大肠杆菌DH5，从而获得含有pMD18-T+Ubi克隆载体的重组大肠杆菌，将其命名为DH5α-Ubi。由深圳华大基因科技有限公司进行测序验证，从而确定目的片段的正确插入。According to the method described in Example 1, the PCR amplification product purified and recovered above was ligated into pMD18-T plasmid (TaKaRa, D103A), and then transformed into Escherichia coli DH5, thereby obtaining a recombinant large intestine containing pMD18-T+Ubi cloning vector bacillus and named it DH5α-Ubi. Shenzhen Huada Gene Technology Co., Ltd. conducts sequencing verification to determine the correct insertion of the target fragment.
2.pCAMBIA-1301+Ubi重组载体的构建2. Construction of pCAMBIA-1301+Ubi recombinant vector
根据厂商的说明书，使用TIANGEN普通质粒小提试剂盒(目录号：DP103-03)从DH5α-Ubi重组大肠杆菌提取含有玉米Ubi启动子序列的重组载体pMD18-T+Ubi。用限制性内切酶Pst I(购自NEB)对重组载体pMD18-T+Ubi进行酶切，然后用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号：DP209-03)回收玉米Ubi启动子片段。同样地，用限制性内切酶Pst I酶切pCAMBIA-1301质粒(由中国科学院昆明动物研究所董杨提供；或者可从例如上海国瑞基因科技有限公司购得)，并进行纯化回收。50μl酶切体系如下：According to the manufacturer's instructions, the recombinant vector pMD18-T+Ubi containing the maize Ubi promoter sequence was extracted from DH5α-Ubi recombinant Escherichia coli using TIANGEN common plasmid mini-prep kit (catalogue number: DP103-03). The recombinant vector pMD18-T+Ubi was digested with restriction endonuclease Pst I (purchased from NEB), and then the maize Ubi promoter fragment was recovered with TIANGEN Agarose Gel DNA Recovery Kit (catalog number: DP209-03) . Similarly, the pCAMBIA-1301 plasmid (provided by Dong Yang, Kunming Institute of Zoology, Chinese Academy of Sciences; or can be purchased from, for example, Shanghai Guorui Gene Technology Co., Ltd.) was digested with restriction endonuclease Pst I, and purified and recovered. The 50μl enzyme digestion system is as follows:
ddH2O 34.9μlddH2O 34.9μl
10*Buffer 3 5μl10*Buffer 3 5μl
Pst I 0.1μl(10U)Pst I 0.1μl(10U)
pMD18-T+Ubi或pCAMBIA-1301 10μl(＜1000pMD18-T+Ubi or pCAMBIA-1301 10μl (<1000
ng)ng)
根据厂商的说明书，使用T4连接酶(TaKaRa，D2011A)，连接回收的Ubi启动子片段与回收的pCAMBIA-1301质粒片段。10μl连接体系如下：According to the manufacturer's instructions, the recovered Ubi promoter fragment was ligated with the recovered pCAMBIA-1301 plasmid fragment using T4 ligase (TaKaRa, D2011A). 10μl connection system is as follows:
10*T4Buffer 1μl10*T4Buffer 1μl
回收的pCAMBIA-1301质粒 1μl(20ng)Recovered pCAMBIA-1301 plasmid 1 μl (20ng)
回收的Ubi启动子片段 10-20ngRecovered Ubi promoter fragment 10-20ng
ddH2O 补齐至9.5μlddH2O Fill up to 9.5μl
T4连接酶 0.5μlT4 ligase 0.5μl
将连接体系于16℃在节能型智能恒温槽(宁波新芝，SDC-6)中连接至少8小时。Connect the connection system at 16°C for at least 8 hours in an energy-saving intelligent constant temperature bath (Ningbo Xinzhi, SDC-6).
将经过上述连接后的产物转化入大肠杆菌DH5，从而获得含有重组载体pCAMBIA-1301+Ubi的重组大肠杆菌。根据厂商的说明书，使用TIANGEN普通质粒小提试剂盒(目录号：DP103-03)提取重组载体pCAMBIA-1301+Ubi。The above ligated product was transformed into Escherichia coli DH5 to obtain recombinant Escherichia coli containing the recombinant vector pCAMBIA-1301+Ubi. According to the manufacturer's instructions, the recombinant vector pCAMBIA-1301+Ubi was extracted using the TIANGEN Ordinary Plasmid Mini-Isolation Kit (catalogue number: DP103-03).
用引物F2和R2对所得的重组载体pCAMBIA-1301+Ubi进行PCR检测，从而确证所得的重组载体pCAMBIA-1301+Ubi中含有所需的Ubi启动子。PCR detection was performed on the obtained recombinant vector pCAMBIA-1301+Ubi with primers F2 and R2, thereby confirming that the obtained recombinant vector pCAMBIA-1301+Ubi contained the required Ubi promoter.
3.NOS终止子的PCR扩增和pMD18-T+NOS重组载体的构建3. PCR amplification of NOS terminator and construction of pMD18-T+NOS recombinant vector
根据上文描述的方法，使用下列引物，以pCAMBIA-1301质粒为模板，用高保真Ex TaqTM(TaKaRa，DRR100B)聚合酶进行NOS终止子的PCR扩增：According to the method described above, the following primers were used to perform PCR amplification of the NOS terminator with the high-fidelity Ex Taq™ (TaKaRa, DRR100B) polymerase using the pCAMBIA-1301 plasmid as a template:
上游引物F3(SEQ ID NO：5)：GGGAGCTCGAATTTCCCCGATCGTTCAA，含有限制性酶切位点Sac I；Upstream primer F3 (SEQ ID NO: 5): GGGAGCTCGAATTTCCCCGATCGTTCAA, containing restriction enzyme site Sac I;
下游引物R3(SEQ ID NO：6)：GGGAATTCCCGATCTAGTAACATAGAT，含有限制性酶切位点EcoR I。Downstream primer R3 (SEQ ID NO: 6): GGGAATTCCCGATCTAGTAACATAGAT, containing restriction enzyme cutting site EcoR I.
PCR扩增产物经1.0％琼脂糖凝胶电泳分离后，使用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号：DP209-03)纯化回收。The PCR amplification products were separated by 1.0% agarose gel electrophoresis, and then purified and recovered by using TIANGEN Agarose Gel DNA Recovery Kit (Catalogue Number: DP209-03).
根据上文描述的方法，将上述纯化回收的PCR扩增产物连接入pMD18-T质粒(TaKaRa，D103A)，然后转化入大肠杆菌DH5，从而获得含有pMD18-T+NOS克隆载体的重组大肠杆菌，将其命名为DH5α-NOS。由深圳华大基因科技有限公司进行测序验证，从而确定目的片段的正确插入。According to the method described above, the above-mentioned purified and recovered PCR amplification product was ligated into pMD18-T plasmid (TaKaRa, D103A), and then transformed into Escherichia coli DH5, thereby obtaining recombinant Escherichia coli containing pMD18-T+NOS cloning vector, Name it DH5α-NOS. Shenzhen Huada Gene Technology Co., Ltd. conducts sequencing verification to determine the correct insertion of the target fragment.
4.pCAMBIA-1301+Ubi+NOS即p6重组载体的构建4. Construction of pCAMBIA-1301+Ubi+NOS ie p6 recombinant vector
根据厂商的说明书，使用TIANGEN普通质粒小提试剂盒(目录号：DP103-03)从DH5α-NOS重组大肠杆菌提取pMD18-T+NOS克隆载体。使用上文描述的方法，用限制性内切酶Sac I和EcoR I(购自NEB)对pMD18-T+NOS克隆载体进行酶切，然后用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号：DP209-03)回收NOS终止子片段。同样地，用限制性内切酶Sac I和EcoR I酶切上文制备的pCAMBIA-1301+Ubi重组载体，并进行纯化回收。According to the manufacturer's instruction, the pMD18-T+NOS cloning vector was extracted from DH5α-NOS recombinant Escherichia coli using TIANGEN common plasmid mini-prep kit (catalogue number: DP103-03). Using the method described above, the pMD18-T+NOS cloning vector was digested with restriction enzymes Sac I and EcoR I (purchased from NEB), and then the TIANGEN Agarose Gel DNA Recovery Kit (catalog number: DP209-03) recovered the NOS terminator fragment. Similarly, the pCAMBIA-1301+Ubi recombinant vector prepared above was digested with restriction enzymes Sac I and EcoR I, and purified and recovered.
使用上文描述的方法，用T4连接酶(TaKaRa，D2011A)连接回收的NOS终止子片段与回收的pCAMBIA-1301+Ubi重组载体片段，并将连接产物转化入DH5α，并最终获得重组载体pCAMBIA-1301+Ubi+NOS，即p6。Using the method described above, use T4 ligase (TaKaRa, D2011A) to ligate the recovered NOS terminator fragment and the recovered pCAMBIA-1301+Ubi recombinant vector fragment, and transform the ligated product into DH5α, and finally obtain the recombinant vector pCAMBIA- 1301+Ubi+NOS, namely p6.
实施例3：p6+BGIos389重组载体的构建Embodiment 3: Construction of p6+BGIos389 recombinant vector
使用上文描述的方法，提取pMD18-T+BGIos389重组载体，用限制性内切酶KpnI/XbaI进行酶切并回收BGIos389基因片段。类似地，提取p6重组载体，用相应的限制性内切酶KpnI/XbaI进行酶切并回收大片段。使用上文描述的方法，连接回收的BGIos389基因片段和p6重组载体片段，并将连接产物转化入DH5α，从而最终获得重组载体p6+BGIos389。进行测序验证，从而确定目的片段的正确插入。Using the method described above, the pMD18-T+BGIos389 recombinant vector was extracted, digested with restriction endonuclease KpnI/XbaI, and the BGIos389 gene fragment was recovered. Similarly, the p6 recombinant vector was extracted, digested with the corresponding restriction endonuclease KpnI/XbaI and the large fragment was recovered. Using the method described above, the recovered BGIos389 gene fragment and the p6 recombinant vector fragment were ligated, and the ligated product was transformed into DH5α, thereby finally obtaining the recombinant vector p6+BGIos389. Perform sequencing verification to confirm the correct insertion of the target fragment.
实施例4：重组根癌农杆菌EHA105-p6+BGIos389细胞的制备Embodiment 4: Preparation of recombinant Agrobacterium tumefaciens EHA105-p6+BGIos389 cells
按照本领域技术人员熟知的方法，制备根癌农杆菌EHA105的感受态细胞(参见《分子克隆实验指南》，第三版，科学出版社)。Competent cells of Agrobacterium tumefaciens EHA105 were prepared according to methods well known to those skilled in the art (see "Molecular Cloning Experiment Guide", third edition, Science Press).
将实施例3制备的p6+BGIos389重组载体转化入根癌农杆菌EHA105的感受态细胞，具体方法如下。The p6+BGIos389 recombinant vector prepared in Example 3 was transformed into competent cells of Agrobacterium tumefaciens EHA105, and the specific method was as follows.
将根癌农杆菌感受态细胞EHA105于超低温冰箱中取出，置于冰上解冻。解冻后，加入5μl的p6+BGIos389重组载体，轻轻混匀，冰浴10分钟，放入液氮中冷冻5分钟，37℃温育5分钟，然后加入800μl常温的LB液体培养基(具体配方详见《分子克隆实验指南》，第三版，科学出版社)，并于28℃，160rpm下复苏3小时。复苏后，以8000rpm离心30秒，吸去上清而留下200μl并吹匀、涂布于含有卡那霉素-利福平(kan-rif)的YM培养基平板上(50mg/l Kan，10mg/l Rif，具体配方参见表4)。28℃倒置培养2-3天，获得重组根癌农杆菌单菌落。The Agrobacterium tumefaciens competent cells EHA105 were taken out from the ultra-low temperature refrigerator and thawed on ice. After thawing, add 5 μl of p6+BGIos389 recombinant vector, mix gently, ice bath for 10 minutes, freeze in liquid nitrogen for 5 minutes, incubate at 37°C for 5 minutes, then add 800 μl of normal temperature LB liquid medium (specific formula For details, see "Molecular Cloning Experiment Guide", third edition, Science Press), and recover at 28°C, 160rpm for 3 hours. After recovery, centrifuge at 8000rpm for 30 seconds, suck off the supernatant and leave 200 μl and blow evenly, spread on the YM medium plate containing kanamycin-rifampicin (kan-rif) (50mg/l Kan, 10mg/l Rif, see Table 4 for specific formulation). Inverted culture at 28°C for 2-3 days to obtain a single colony of recombinant Agrobacterium tumefaciens.
通过用引物F1(SEQ ID NO：1)和R1(SEQ ID NO：2)进行PCR检测和通过BamH I/XbaI酶切筛选重组根癌农杆菌转化子。Recombinant Agrobacterium tumefaciens transformants were screened by PCR detection with primers F1 (SEQ ID NO: 1) and R1 (SEQ ID NO: 2) and by BamH I/XbaI digestion.
PCR扩增出约1360bp左右的条带和酶切出约1360bp左右的条带的转化子为包含重组载体p6+BGIos389的重组根癌农杆菌，将其命名为重组根癌农杆菌EHA105-p6+BGIos389。The transformant with a band of about 1360 bp amplified by PCR and a band of about 1360 bp cut out by enzyme is the recombinant Agrobacterium tumefaciens containing the recombinant vector p6+BGIos389, which is named recombinant Agrobacterium tumefaciens EHA105-p6+ BGIos389.
实施例5：水稻愈伤组织的诱导和转化Example 5: Induction and transformation of rice callus
按照如下步骤诱导水稻愈伤组织，并用重组根癌农杆菌EHA105-p6+BGIos389转化所述愈伤组织。Rice callus was induced according to the following steps, and the callus was transformed with recombinant Agrobacterium tumefaciens EHA105-p6+BGIos389.
1)将水稻日本晴(Oryza sativa L.japonica.cv.Nipponbare)种子去壳，用70％乙醇表面消毒30秒，然后用有效氯1.5％的次氯酸钠消毒30分钟，并伴随剧烈摇动；消毒后用灭菌水清洗5次；将清洗后的种子置于N6D培养基(具体配方参见表2)上，用封口膜封口；29.5℃光照培养3-4周；1) Shell the rice Nipponbare (Oryza sativa L.japonica.cv.Nipponbare) seeds, disinfect the surface with 70% ethanol for 30 seconds, and then disinfect with sodium hypochlorite with 1.5% available chlorine for 30 minutes, accompanied by vigorous shaking; Bacteria water was washed 5 times; the washed seeds were placed on N6D medium (see Table 2 for the specific formula), and sealed with parafilm; 29.5 ° C light culture for 3-4 weeks;
2)选取活跃生长的愈伤组织(黄白色，干燥，直径1-3mm)，在新N6D培养基上29.5℃光照培养3天；2) Select actively growing callus tissue (yellow-white, dry, 1-3 mm in diameter), and culture it on a new N6D medium at 29.5° C. under light for 3 days;
3)挑取实施例4获得的重组根癌农杆菌EHA105-p6+BGIos389单菌落，于添加抗生素(50mg/l Kan，10mg/l Rif)的YM培养基(具体配方参见表3、表4)上划线培养3天，培养温度为28℃；刮取上述重组根癌农杆菌，将其置于添加了30μl 100mM的乙酰丁香酮(Acetosyringone，AS)的30ml AAM培养基(具体配方参见表5)中，温和重悬所述重组根癌农杆菌EHA105-p6+BGIos389细胞；3) pick the recombinant Agrobacterium tumefaciens EHA105-p6+BGIos389 single colony obtained in Example 4, and add antibiotics (50mg/l Kan, 10mg/l Rif) to the YM medium (see Table 3, Table 4 for specific formulations) The upper streak was cultured for 3 days, and the culture temperature was 28° C.; the above-mentioned recombinant Agrobacterium tumefaciens was scraped off, and it was placed in 30 ml of AAM medium added with 30 μl of 100 mM Acetosyringone (Acetosyringone, AS) (see Table 5 for the specific formula) ), gently resuspend the recombinant Agrobacterium tumefaciens EHA105-p6+BGIos389 cells;
4)将继代培养的愈伤组织置于灭菌培养皿中；然后将步骤3)制备的重组根癌农杆菌悬液倒入所述培养皿中，将所述愈伤组织浸泡15分钟；4) placing the subcultured callus in a sterilized petri dish; then pouring the recombinant Agrobacterium tumefaciens suspension prepared in step 3) into the petri dish, soaking the callus for 15 minutes;
5)弃去培养皿中的重组根癌农杆菌悬液，用灭菌吸水纸将愈伤组织上多余的液体吸干；在N6-AS培养基(具体配方参见表6)上放置一张灭菌滤纸，并添加1ml含AS的AAM培养基，然后将愈伤组织转移至滤纸上；密封培养皿，28℃暗培养48-60小时；5) Discard the recombinant Agrobacterium tumefaciens suspension in the culture dish, and blot the excess liquid on the callus with sterilized absorbent paper; Bacteria filter paper, and add 1ml of AS-containing AAM medium, then transfer the callus to the filter paper; seal the Petri dish, and culture in the dark at 28°C for 48-60 hours;
6)将步骤5)获得的愈伤组织置于50ml灭菌管中，用灭菌水摇动清洗，直至上清液变澄清；将愈伤组织浸泡于含500mg/l羧苄青霉素(Carb)的无菌水中以杀死重组根癌农杆菌；用灭菌吸水纸除去愈伤组织上多余的水分，然后将其转移至含1mg/l潮霉素B(HmB)和50mg/l Carb的N6-AS培养基上；用封口膜密封培养皿，29.5℃光照培养3-4周。6) Place the callus obtained in step 5) in a 50ml sterile tube, shake and wash with sterilized water until the supernatant becomes clear; soak the callus in a solution containing 500mg/l carbenicillin (Carb) Tumefaciens in sterile water to kill recombinant Agrobacterium tumefaciens; remove excess water from the callus with sterilized absorbent paper, and then transfer it to N6- on AS medium; seal the petri dish with parafilm, and culture in light at 29.5°C for 3-4 weeks.
实施例6：水稻愈伤组织中的GUS表达的检测Example 6: Detection of GUS expression in rice callus
为检测实施例5制备的经转化的水稻愈伤组织中GUS的表达情况，按照Chen S Y等描述的方法(Journal of Integrative Plant Biology，2008，50(6)：742-751)，对用重组根癌农杆菌EHA105-p6+BGIos389转化的水稻愈伤组织进行染色。In order to detect the expression of GUS in the transformed rice callus prepared in Example 5, according to the method described by Chen S Y (Journal of Integrative Plant Biology, 2008, 50 (6): 742-751), the recombinant Rice calli transformed with Agrobacterium tumefaciens EHA105-p6+BGIos389 were stained.
1ml GUS染色液中包含：610μl 0.2M Na2HPO4溶液(pH＝7.0)；390μl 0.2M NaH2PO4溶液和10μl 0.1M X-gluc。1ml GUS staining solution contains: 610μl 0.2M Na2HPO4 solution (pH=7.0); 390μl 0.2M NaH2PO4 solution and 10μl 0.1M X-gluc.
将用重组根癌农杆菌EHA105-p6+BGIos389转化的水稻愈伤组织浸泡在GUS染色液中，在37℃下温育直至出现蓝色。拍照记录染色结果，并示于图1中。结果显示，实施例5制备的经转化的水稻愈伤组织经染色后呈现蓝色(图1右侧)，而未经转化的对照愈伤组织经GUS染色后颜色未发生变化(图1左侧)。这表明，本发明的p6+BGIos389重组载体已转化入水稻愈伤组织。Rice calli transformed with recombinant Agrobacterium tumefaciens EHA105-p6+BGIos389 were soaked in GUS staining solution and incubated at 37°C until blue appeared. Take photos and record the staining results, which are shown in Figure 1. The results showed that the transformed rice callus prepared in Example 5 was stained blue (the right side of Figure 1), while the color of the untransformed control callus remained unchanged after GUS staining (the left side of Figure 1 ). This shows that the p6+BGIos389 recombinant vector of the present invention has been transformed into rice callus.
实施例7：转基因水稻幼苗中的GUS表达的检测Example 7: Detection of GUS Expression in Transgenic Rice Seedlings
将实施例5制备的经转化的愈伤组织转移至含50mg/l潮霉素B(HmB)的MS-R分化培养基(具体配方见表7)，以分化生长幼苗；用封口膜密封培养皿，29.5℃光照培养3-4周；待幼苗长至3-4cm时，将幼苗转移到含50mg/l潮霉素B(HmB)的1/2MS生根培养基(具体配方参见表8)，以进行生根筛选。The transformed callus prepared in Example 5 is transferred to the MS-R differentiation medium containing 50mg/l hygromycin B (HmB) (see Table 7 for specific formulations) to differentiate and grow seedlings; seal the culture with parafilm Dish, 29.5 ℃ of light cultivation 3-4 weeks; When the seedling grows to 3-4cm, the seedling is transferred to the 1/2MS rooting medium containing 50mg/l hygromycin B (HmB) (see Table 8 for specific formula), for rooting screening.
转基因水稻幼苗的GUS染色方法与实施例6中愈伤组织的GUS染色方法相同。拍照记录染色结果，结果显示，经含有p6+BGIos389重组载体的根癌农杆菌转化的水稻幼苗的根和叶经GUS染色后呈现蓝色，未经转化的对照幼苗的根和叶经GUS染色后颜色未发生变化。这表明，本发明的p6+BGIos389重组载体已转化入水稻幼苗中。The GUS staining method of the transgenic rice seedlings is the same as the GUS staining method of the callus in Example 6. Take photos and record the staining results. The results show that the roots and leaves of rice seedlings transformed with Agrobacterium tumefaciens containing the p6+BGIos389 recombinant vector turned blue after GUS staining, and the roots and leaves of untransformed control seedlings were stained with GUS. Color has not changed. This shows that the p6+BGIos389 recombinant vector of the present invention has been transformed into rice seedlings.
实施例8：转基因水稻苗的根系的性状观察Embodiment 8: Observation of the traits of the root system of transgenic rice seedlings
转基因水稻幼苗在1/2MS生根培养基中生长20天后，进行拍照记录，结果示于图2中。结果显示，经含有p6+BGIos389重组载体的根癌农杆菌转化的水稻苗的根系(图2，左边试管)与未经转化的对照水稻苗的根系(图2，右边试管)相比，根系更密集发达。在本实施例中，一共栽培了22株转基因水稻苗，其中的19株显示密集发达生长的根系(与对照水稻苗相比)。这些数据表明，本发明的BGIos389基因有效地促进植物的根系生长，并且用BGIos389基因转化的植物显示更优良的植物根系生长(与对照相比，其根系更密集发达)。After the transgenic rice seedlings were grown in 1/2MS rooting medium for 20 days, they were photographed and recorded, and the results are shown in FIG. 2 . The results showed that the roots of rice seedlings transformed with Agrobacterium tumefaciens containing the p6+BGIos389 recombinant vector (Fig. densely developed. In this example, a total of 22 transgenic rice seedlings were cultivated, and 19 of them showed densely developed root systems (compared with control rice seedlings). These data show that the BGIos389 gene of the present invention effectively promotes the root growth of plants, and the plants transformed with the BGIos389 gene show better plant root growth (the roots are more densely developed compared with the control).
本发明实施例中所使用的各种培养基的配方示于下文中。特别地，在本发明中，培养基的“常规灭菌”是指如下条件的灭菌：在121℃下蒸汽灭菌20分钟。The formulations of various media used in Examples of the present invention are shown below. In particular, in the present invention, "conventional sterilization" of the culture medium refers to sterilization under the following conditions: steam sterilization at 121° C. for 20 minutes.
表2：N6D培养基Table 2: N6D medium
用1N氢氧化钾将pH值调节到5.8，封口后常规灭菌。Adjust the pH value to 5.8 with 1N potassium hydroxide, and routinely sterilize after sealing.
N6 macro母液(20X)：硝酸钾56.60g，磷酸二氢钾8.00g，硫酸铵9.26g，硫酸镁3.70g，氯化钙3.32g，用蒸馏水定容至1L，4℃保存备用。N6 macro mother liquor (20X): Potassium nitrate 56.60g, potassium dihydrogen phosphate 8.00g, ammonium sulfate 9.26g, magnesium sulfate 3.70g, calcium chloride 3.32g, dilute to 1L with distilled water, store at 4°C for later use.
N6 micro母液(1000X)：碘化钾0.80g，硼酸1.60g，硫酸锰3.33g，硫酸锌1.50g，用蒸馏水定容至1L，4℃保存备用。N6 micro mother liquor (1000X): Potassium iodide 0.80g, boric acid 1.60g, manganese sulfate 3.33g, zinc sulfate 1.50g, dilute to 1L with distilled water, store at 4°C for later use.
铁盐(Fe2EDTA)贮存液(100X)：将3.73g乙二铵四乙酸二钠(Na2EDTA·2H2O)和2.78g FeSO4·7H2O分别溶解，并混合。用蒸馏水定容至1000ml，70℃温浴2小时，冷却后4℃保存不超过1个月。Iron salt (Fe2EDTA) stock solution (100X): Dissolve 3.73g disodium edetate (Na2EDTA·2H2O) and 2.78g FeSO4·7H2O respectively and mix them. Dilute to 1000ml with distilled water, incubate at 70°C for 2 hours, and store at 4°C for no more than 1 month after cooling.
N6维生素贮存液(1000X)：维生素B1 0.10g，维生素B6 0.05g，烟酸0.05g，甘氨酸0.20g，用蒸馏水定容至100ml，过滤除菌，4℃保存不超过1周。N6 vitamin storage solution (1000X): vitamin B1 0.10g, vitamin B6 0.05g, niacin 0.05g, glycine 0.20g, dilute to 100ml with distilled water, filter and sterilize, store at 4°C for no more than 1 week.
表3：YM液体培养基(含有50mg/L Kan，10mg/L Rif)Table 3: YM liquid medium (containing 50mg/L Kan, 10mg/L Rif)
表4：YM固体培养基(含有50mg/L Kan，10mg/L Rif)Table 4: YM solid medium (containing 50mg/L Kan, 10mg/L Rif)
表5：AAM培养基Table 5: AAM Medium
用1N氢氧化钾将pH值调节至5.2，常规灭菌。Adjust the pH to 5.2 with 1N potassium hydroxide and routinely sterilize.
AAM macro(10X)：2.5g七水硫酸镁(MgSO4·7H2O)，1.5g二水氯化钙(CaCl2·2H2O)，1.33g二水磷酸二氢钠(NaH2PO4·2H2O)，用蒸馏水定容至1L，4℃保存备用。AAM macro (10X): 2.5g magnesium sulfate heptahydrate (MgSO4 7H2 O), 1.5g calcium chloride dihydrate (CaCl2 2H2 O), 1.33g sodium dihydrogen phosphate dihydrate (NaH2 PO4 ·2H2 O), dilute to 1L with distilled water, store at 4°C for later use.
AAM micro(100X)：0.7g单水硫酸锰(MnSO4·H2O)，0.2g七水硫酸锌(ZnSO4·7H2O)，0.075g碘化钾(KI)，0.3g硼酸(H3BO3)，25mg二水钼酸钠(Na2MoO4·2H2O)，2.5mg五水硫酸铜(CuSO4·5H2O)，2.5mg六水氯化钴(CoCl2·6H2O)，用蒸馏水定容至1L，4℃保存备用。AAM micro (100X): 0.7g manganese sulfate monohydrate (MnSO4 ·H2 O), 0.2g zinc sulfate heptahydrate (ZnSO4 ·7H2 O), 0.075g potassium iodide (KI), 0.3g boric acid (H3 BO3 ), 25mg sodium molybdate dihydrate (Na2 MoO4 2H2 O), 2.5mg copper sulfate pentahydrate (CuSO4 5H2 O), 2.5mg cobalt chloride hexahydrate (CoCl2 6H2 O) , dilute to 1 L with distilled water, and store at 4°C for later use.
AAM有机(1000X)：0.75g甘氨酸(Glycine)，0.1g烟酸(Nicotinicacid)，0.1g VB6(Pyridoxine)，1g VB1(Thiamine)，用蒸馏水定容至100ml，4℃保存备用。AAM Organic (1000X): 0.75g Glycine, 0.1g Nicotinic acid, 0.1g VB6 (Pyridoxine), 1g VB1 (Thiamine), dilute to 100ml with distilled water, store at 4°C for later use.
铁盐(Fe2EDTA)贮存液(100X)：见表2。Iron salt (Fe2 EDTA) stock solution (100X): see Table 2.
表6：N6-AS共培养基Table 6: N6-AS co-culture medium
调节pH至5.2。Adjust the pH to 5.2.
N6macro母液(20X)，N6micro母液(1000X)，铁盐(Fe2EDTA)贮存液(100X)，N6维生素贮存液(1000X)：均见表2。N6 macro mother solution (20X), N6 micro mother solution (1000X), iron salt (Fe2 EDTA) stock solution (100X), N6 vitamin stock solution (1000X): see Table 2.
表7：MS-R分化培养基Table 7: MS-R Differentiation Medium
调节PH值至5.8，常规灭菌。Adjust the pH value to 5.8, and routinely sterilize.
MS macro(20X)：硝酸铵33.0g，硝酸钾38.0g，磷酸二氢钾3.4g，硫酸镁7.4g，氯化钙8.8g，逐一溶解，然后室温下用蒸馏水定容至1L，4℃保存。MS macro (20X): 33.0g ammonium nitrate, 38.0g potassium nitrate, 3.4g potassium dihydrogen phosphate, 7.4g magnesium sulfate, 8.8g calcium chloride, dissolve one by one, then dilute to 1L with distilled water at room temperature, store at 4°C .
MS micro(1000X)：硫酸锰16.90g，硫酸锌8.60g，硼酸6.20g，碘化钾0.83g，钼酸钠0.25g，硫酸铜0.025g，氯化钴0.025g，上述试剂在室温下溶解并用蒸馏水定容至1L，4℃保存。MS micro (1000X): manganese sulfate 16.90g, zinc sulfate 8.60g, boric acid 6.20g, potassium iodide 0.83g, sodium molybdate 0.25g, copper sulfate 0.025g, cobalt chloride 0.025g, the above reagents were dissolved at room temperature and fixed with distilled water Make up to 1L and store at 4°C.
MS维生素贮存液(1000X)：维生素B1 0.010g，维生素B6 0.050g，烟酸0.050g，甘氨酸0.200g，用蒸馏水定容至100ml，过滤除菌，4℃保存不超过1周。MS vitamin stock solution (1000X): vitamin B1 0.010g, vitamin B6 0.050g, niacin 0.050g, glycine 0.200g, dilute to 100ml with distilled water, sterilize by filtration, store at 4°C for no more than 1 week.
铁盐(Fe2EDTA)贮存液(100X)：见表2。Iron salt (Fe2 EDTA) stock solution (100X): see Table 2.
表8：1/2MS生根培养基Table 8: 1/2MS rooting medium
调节PH值至5.8。Adjust the pH to 5.8.
MS macro(20X)：见表7。MS macro(20X): See Table 7.
MS micro(1000X)，MS维生素贮存液(1000X)：见表7。MS micro (1000X), MS vitamin stock solution (1000X): see Table 7.
铁盐(Fe2EDTA)贮存液(100X)：见表2。Iron salt (Fe2 EDTA) stock solution (100X): see Table 2.
参考文献references
本文中用于举例说明本发明或提供关于本发明的实施的另外的详细内容的专利、出版物和其他材料通过引用合并入本文，并且为方便起见按下列文献目录提供。The patents, publications and other materials used herein to illustrate the invention or to provide additional details regarding the practice of the invention are incorporated herein by reference and are provided for convenience in the following bibliography.
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| CN119752934A (en)* | 2024-12-26 | 2025-04-04 | 浙江大学 | A method for improving crop resistance to high temperature stress |
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| US20040123343A1 (en)* | 2000-04-19 | 2004-06-24 | La Rosa Thomas J. | Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement |
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| KIKUCHI.S: "Genbank：AK100857", 《GENBANK》, 4 December 2008 (2008-12-04)* |
| SHOSHI KIKUCHI ET AL: "Collection, Mapping, and Annotation of Over 28,000 cDNA Clones from japonica Rice", 《SCIENCE》, vol. 301, 18 July 2003 (2003-07-18)* |
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| CN116217686A (en)* | 2023-01-05 | 2023-06-06 | 海南大学 | A kind of OsUVR8 gene from rice and its application |
| CN116217686B (en)* | 2023-01-05 | 2024-03-08 | 海南大学 | An OsUVR8 gene from rice and its application |
| CN119752934A (en)* | 2024-12-26 | 2025-04-04 | 浙江大学 | A method for improving crop resistance to high temperature stress |
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