CN102146398B - Promoter BgIosP537, and preparation method and use thereof - Google Patents

Promoter BgIosP537, and preparation method and use thereof Download PDF

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CN102146398B
CN102146398B CN2010106163663A CN201010616366A CN102146398B CN 102146398 B CN102146398 B CN 102146398B CN 2010106163663 A CN2010106163663 A CN 2010106163663A CN 201010616366 A CN201010616366 A CN 201010616366A CN 102146398 B CN102146398 B CN 102146398B
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plant
promoter
rice
recombinant
nucleotide sequence
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CN102146398A (en
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倪雪梅
李宁
杨爽
蔡晶
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深圳华大基因科技有限公司
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Abstract

The invention relates to a promoter BgIosP537, and a preparation method and use thereof. The promoter has a nucleotide sequence which is selected from any of the following groups and has a prompter function: a, a nucleotide sequence represented by Seq ID No.1; b, a nucleotide sequence complementary to the Seq ID No.1; c, a nucleotide sequence which can be crossed with the nucleotide sequence a orthe nucleotide sequence b under a highly strict condition; d, a nucleotide sequence obtained by modifying the nucleotide sequence a or the nucleotide sequence b by substituting, losing and increasingone or more bases; and e, a nucleotide sequence having an identity of at least 90 percent with the nucleotide sequence a or the nucleotide sequence b. The promoter can control gene expression in monocotyledons and dicotyledons, so a new tool and selection are provided for the study on the expression of a target gene in plants.

Description

启动子BglosP537、制备方法及应用 Promoter BglosP537, preparation method and application

技术领域 FIELD

[0001] 本发明涉及基因工程技术领域,特别是涉及一种启动子BgIosP537,含有该启动子的核酸构建体、载体、重组细胞、植物愈伤组织,该启动子的制备方法以及利用该启动子来调控植物中基因表达的方法。 [0001] The present invention relates to the field of genetic engineering, particularly to a promoter BgIosP537, nucleic acid containing the promoter constructs, vectors, recombinant cells, plant calli, prepared by using the promoter and the promoter a plant gene expression is regulated.

背景技术 Background technique

[0002]启动子是基因的一个组成部分,通常位于结构基因5'端上游,是RNA聚合酶识别、 结合和开始转录的一段DNA序列。 [0002] The promoter is part of a gene, a structural gene generally is located 5 'upstream region, a RNA polymerase recognition, binding a DNA sequence and a start of transcription. 启动子能够指导全酶(holoenzyme)同模板正确结合,活化RNA聚合酶,启动基因转录,从而控制基因表达(转录)的起始时间和表达的程度。 Promoter capable of directing the whole enzyme (holoenzyme) template with the right combination, the activation of RNA polymerase gene transcription start to control the degree of (transcription) start time and the expression of gene expression. 在转基因植物中,启动子是影响转基因表达效率的重要因素之一,选择高效率的启动子是高效率表达外源基因的关键。 In transgenic plants, the promoter is one of the important factors that affect the efficiency of gene expression turn, choose high efficiency promoters is the key to efficient expression of foreign genes.

[0003] 根据启动子的转录模式可将其分为3类:组成型启动子、组织或器官特异性启动子和诱导型启动子。 [0003] The transcription promoter mode can be divided into three categories: constitutive promoters, tissue or organ-specific promoters and inducible promoters. 所谓组成型启动子是指在组成型启动子调控下,不同组织器官和发育阶段的基因表达没有明显差异,因而称之组成型启动子。 The so-called constitutive promoter refers to a promoter regulates the constitutive, organ gene expression in different tissues and developmental stages did not differ significantly, so call it a constitutive promoter. 双子叶植物中最常使用的组成型启动子是花椰菜花叶病毒(CaMV) 35S启动子。 Constitutive promoter dicots most commonly used cauliflower mosaic virus (CaMV) 35S promoter. 另一种高效的组成型启动子CsVMV是从木薯叶脉花叶病毒(cassava vein mosaic virus)中分离的。 Another effective constitutive CsVMV promoter was isolated from cassava vein mosaic virus (cassava vein mosaic virus) in.

[0004] 人们高度重视从植物本身克隆组成型启动子。 [0004] It attaches great importance to the promoter from clone itself constitutive plant. 例如肌动蛋白(actin)和泛素(ubiquitin)等基因的启动子已被克隆。 For example, promoters of genes actin (actin) and ubiquitin (the ubiquitin), etc., it has been cloned. 用这些启动子代替CaMV 35S启动子,可以更有效地在单子叶植物中驱动外源基因的转录。 Instead of the CaMV 35S promoter with a promoter, can be more effectively drive transcription of exogenous genes in monocots. Naomi等分别从拟南芥的色氨酸合酶β亚基基因和植物光敏色素基因中克隆了相应启动子,用其代替CaMV 35S启动子,在转基因烟草中也取得了很好的表达效果(Plant biotechnology, 2002,19 (I) :19-26)。 Naomi, respectively, cloned from Arabidopsis β subunit genes and phytochrome genes in the appropriate plant tryptophan synthase promoter, with which instead of the CaMV 35S promoter in transgenic tobacco also achieved good expression of the effect ( Plant biotechnology, 2002,19 (I): 19-26).

[0005] 单子叶植物基因中常见的启动子有:Ubi启动子(Plant ubiquitin promoter) > Actin 启动子(Plant Actin promoter)和Adh-I 启动子(Maize alcohol dehydrogenase I promoter)。 [0005] monocot genes common promoters are: Ubi promoter (Plant ubiquitin promoter)> Actin promoter (Plant Actin promoter) and Adh-I promoter (Maize alcohol dehydrogenase I promoter).

[0006] Ubi启动子以其启动效率高、甲基化程度低、遗传性状稳定等因素而倍受青睐。 [0006] Ubi promoter launch its high efficiency, low degree of methylation, stable genetic traits and other factors favorites. 目前,已经从很多泛素基因中分离得到启动子序列,包括玉米基因组中的Ubi-I启动子、水稻泛素RUBQ2启动子、拟南芥泛素启动子、向日葵泛素UbBl启动子、烟草泛素Ubi. U4启动子、 马铃薯泛素Ubi7启动子、番茄泛素Ubil-I启动子,大麦泛素Mubl启动子。 At present, it has been isolated from many of the ubiquitin gene promoter sequence comprising a maize genome Ubi-I promoter, the rice RUBQ2 ubiquitin promoter, Arabidopsis ubiquitin promoter, UbBl sunflower ubiquitin promoter, tobacco Pan Su Ubi. U4 promoter, the potato ubiquitin promoter Ubi7, tomato ubiquitin Ubil-I promoter, barley ubiquitin Mubl promoter. 玉米泛素Ubi-I 启动子已经广泛地应用于玉米、小麦、水稻等单子叶植物中,水稻泛素RUBQ2启动子在水稻和甘蔗中也有较多的应用。 The maize ubiquitin promoter Ubi-I has been widely applied to monocotyledonous plants corn, wheat, rice, the rice ubiquitin promoter RUBQ2 rice and sugar cane is also more applications.

[0007] Actin启动子1990年由康奈尔大学的McElroy等首次在水稻中发现,属于强组成型启动子。 [0007] Actin promoter was first discovered in 1990 by the McElroy et Cornell University in rice, it belongs to a strong constitutive promoter. Actin启动子在单子叶禾本科中作用显著,但是邻近科属的植物中的基因调控功能却十分不理想。 Actin promoter in monocotyledonous significant role, but the regulation of gene function of plant families and genera in the neighboring was very unsatisfactory. 因此,许多相关研究通过其他单子叶植物寻找Actin启动子,并成功在香蕉、甜瓜、玉米和拟南芥中陆续发现。 Therefore, many studies by other monocots looking Actin promoter, and success have been discovered in banana, melon, maize and Arabidopsis. Actin启动子由于对基因表达的强调控作用,在单子叶植物优良性状的转基因中已经得到越来越广泛的应用。 Actin promoter due to a strong regulatory effect on gene expression in transgenic good traits of monocots has been more widely used.

[0008] Adh-I启动子调控乙醇脱氢酶(alcohol dehydrogenase)基因,对植物在缺氧环境下乙醇脱氢酶的表达至关重要。 [0008] Adh-I promoter regulatory alcohol dehydrogenase (alcohol dehydrogenase) gene, essential for the expression in plant hypoxic environment alcohol dehydrogenase. Adh-I启动子对单子叶植物特别是谷类植物如水稻、燕麦和大麦,和少部分双子叶植物如烟草,基因的调控功能比花椰菜花叶病毒CaMV 35S启动子提高10-50倍。 Adh-I promoter for monocots particularly cereals such as rice, oats and barley, and a small number of dicotyledonous plants such as tobacco-control function, gene than the cauliflower mosaic virus CaMV 35S promoter increase 10-50 times. Adh-I启动子主要应用于单子叶植物,对绝大部分双子叶植物基因表达的调控效果都很有限。 Adh-I promoter is mainly used in monocots, for most dicots regulation of gene expression effects are very limited.

[0009] 单子叶植物是被子植物的主要类群,单子叶植物中的禾本科、百合科、棕榈科和天南星等,是非常重要的农业作物。 [0009] monocots are the main groups of angiosperms, monocots of Gramineae, Liliaceae, palms and Araceae, is a very important agricultural crops. 单子叶植物基因的强效启动子,能够调控植物高效率表达具有特殊性状的外源基因,对优良作物的分子育种研究意义重大。 Potent promoters of monocot genes, a plant capable of regulating expression of foreign genes efficiently with special traits of breeding high molecular significant crops.

[0010] 在强效启动子相关研究领域,发现并验证了许多单子叶植物的启动子。 [0010] Promoter related research in the field of powerful, discovered and validated a number of promoters monocots. 此外,双子叶植物中的一些强效启动子如CsVMV启动子、番茄E8启动子、白藜芦醇合酶基因Vstl启动子等高效启动子,在单子叶植物中也有很强的基因调控作用。 In addition, some dicots potent promoters such as the CsVMV promoter, E8 promoter from tomato, resveratrol synthase gene promoter, etc. Vstl efficient promoters, but also has a strong role in the regulation of genes in monocots.

发明内容 SUMMARY

[0011] 本发明的目的是提供一种新的启动子,能够在植物中调控基因的表达。 [0011] The object of the present invention is to provide a novel promoter capable of regulating gene expression in plants.

[0012] 本发明的另一目的在于提供含有上述启动子的核酸构建体、载体、重组细胞、植物愈伤组织、植物外植体及植物。 [0012] Another object of the present invention is to provide a nucleic acid containing the promoter constructs, vectors, recombinant cells, plant calli, plant explants and plant.

[0013] 本发明的再一目的在于提供一种制备上述启动子的引物、方法,以及一种利用该启动子来调控植物中基因表达的方法。 [0013] A further object of the present invention is to provide a primer prepared by one of the above promoter, and a method utilizing the promoter to regulate gene expression in plants.

[0014] 本发明的再一目的在于提供一种制备转基因植物的方法,以及上述启动子在调控植物中目的基因表达或植物育种中的用途。 [0014] A further object of the present invention is to provide a method of preparing a transgenic plant, and said promoter use or expression of the gene in the regulation of plant breeding plants.

[0015] 为实现上述目的,本发明采用了以下技术方案: [0015] To achieve the above object, the present invention employs the following technical solution:

[0016] 本发明公开了一种启动子,所述启动子含有选自以下任意一组并具有启动子功能的核苷酸序列: [0016] The present invention discloses a promoter comprising a nucleotide sequence selected from any of the following sub-group and having a promoter functional in said promoter:

[0017] a、Seq ID No. I所示的核苷酸序列; [0017] a, nucleotide sequence shown in Seq ID No. I;

[0018] b、与Seq ID No. I互补的核苷酸序列; [0018] b, the nucleotide sequence I complementary to Seq ID No.;

[0019] C、在高等严紧条件下能够与上述a或b的核苷酸序列杂交的核苷酸序列; [0019] C, under conditions of higher stringency nucleotide sequence capable of hybridizing with a nucleotide sequence of a or b;

[0020] d、对上述a或b所示核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列; [0020] d, or of a nucleotide sequence shown above b substitutions of one or more nucleotides, deletions, additions, modified nucleotide sequence;

[0021] e、与上述a或b所示核苷酸序列具有至少90%同一性的核苷酸序列。 [0021] e, above a or b having a nucleotide sequence at least 90% nucleotide sequence identity.

[0022] 本发明还公开了一种核酸构建体,包含上述启动子,以及与启动子可操作连接的基因序列,其中所述启动子与所述基因序列来源相同或者不同。 [0022] The present invention further discloses a nucleic acid construct including the promoter, and a gene sequence operably linked to a promoter, wherein the promoter is the same as or different from the promoter and gene sequences source.

[0023] 本发明还公开了一种载体,所述载体含有上述启动子或上述核酸构建体,优选的, 所述载体为上述启动子与PMD18-T或p8质粒经重组得到的重组载体。 [0023] The present invention also discloses a vector comprising a promoter containing the above-described nucleic acid constructs or, preferably, the vector is a recombinant vector with the promoter described above or p8 PMD18-T recombinant plasmid was obtained.

[0024] 本发明进一步公开了一种重组细胞,所述细胞含有上述启动子、上述核酸构建体或上述载体,优选的,所述重组细胞为重组大肠杆菌细胞或重组农杆菌细胞。 [0024] The present invention further discloses a recombinant cell, the cell containing the promoter of the nucleic acid construct or vector described above, preferably, the recombinant cell is a recombinant E. coli cell or a recombinant Agrobacterium cells.

[0025] 本发明进一步公开了含有上述启动子、核酸构建体、载体或重组细胞的植物愈伤组织、植物外植体或植物,优选的,所述植物为单子叶植物或双子叶植物,再优选的,所述植物为稻属或烟草属,更优选的,所述植物为水稻或烟草,进一步优选的,所述植物为水稻日本晴(Oryza sativa L. ssp. japonica cv. Nipponbare)或烟草NC89。 [0025] The present invention further discloses contains the promoter, a nucleic acid construct, vector or recombinant cell callus plant tissue, a plant explant or a plant outside, preferably, the plant is a monocot or a dicot, then preferably, said plant is a rice or genus Nicotiana, more preferably, the tobacco plant is a rice or, more preferably, the plant is Nipponbare rice (Oryza sativa L. ssp. japonica cv. Nipponbare) or tobacco NC89 .

[0026] 本发明进一步公开了一组引物对,用于扩增得到上述启动子,所述引物对的两条引物分别含有Seq ID No :2和Seq ID No :3所示的序列,优选的,所述引物对的两条引物在5'端还分别连接有限制性酶切位点和/或保护碱基,更优选的,所述引物对的两条引物分别具有Seq ID No :4和Seq ID No :5所示的序列。 [0026] The present invention further discloses a set of primer pairs for amplifying the above-described promoter obtained, the two primers were primer pair each comprising Seq ID No: 2 and Seq ID No: 3 of the sequence shown, preferably , the primer pair of the two primers at the 5 'end are also connected with a restriction site and / or protection of bases, and more preferably, the primer pair having two primers Seq ID No: 4 and Seq ID No: 5 in the sequence shown in FIG.

[0027] 本发明还公开了制备SEQ ID No : I所示启动子的方法,包括:以水稻日本晴基因组DNA为模板,使用一对扩增引物进行扩增,所述扩增引物根据SEQ ID NO :1在水稻日本晴(Oryza sativaL. ssp. japonica cv. Nipponbare) gDNA中的序列分别针对首尾进行设计,优选是上述的引物对。 [0027] The present invention also discloses the preparation SEQ ID No: method shown promoter I, comprising: Mostly Japanese rice genomic DNA as a template gene, using a pair of amplification primers for amplification, the amplification primers according to SEQ ID NO : (... ssp japonica cv Nipponbare Oryza sativaL) 1 gDNA sequences is designed for end-rice Nipponbare, preferably the above-described primer pairs.

[0028] 本发明进一步公开了一种调控植物中基因表达的方法,所述方法包括,将上述启动子、核酸构建体、载体或重组细胞导入植物细胞。 [0028] The present invention further discloses a method of regulation of gene expression in plants, the method comprising the promoter described above, a nucleic acid construct, vector or recombinant cell into plant cells. 优选导入植物愈伤组织。 Preferably introduced into a plant callus. 进一步优选的, 启动子或核酸构建体导入植物细胞是利用农杆菌转化植物愈伤组织,所述植物优选为单子叶植物或双子叶植物,所述植物再优选为稻属或烟草属,所述植物更优选为水稻或烟草,所述植物进一步优选为水稻日本晴(Oryza sativa L. ssp. japonica cv. Nipponbare)或烟草NC89。 Further preferably, the promoter or nucleic acid construct into plant cells using Agrobacterium-mediated transformation of plant callus, the plant preferably is monocotyledonous or dicotyledonous plant, further preferably the plant is a rice or genus Nicotiana, said plant more preferably tobacco or rice, further preferably the plant Nipponbare rice (Oryza sativa L. ssp. japonica cv. Nipponbare) or tobacco NC89. 本发明还公开了一种制备转基因植物的方法,包括在有效产生植物的条件下培养上述的重组细胞或植物愈伤组织或植物外植体或植物。 The present invention also discloses a method of preparing a transgenic plant, comprising culturing the recombinant cell or plant callus or the outer plant or plant explant under conditions effective to produce a plant.

[0029] 本发明还公开了上述启动子、核酸构建体、载体、重组细胞或植物愈伤组织或植物外植体或植物在调控植物中目的基因表达或植物育种中的用途,优选植物是单子叶植物或双子叶植物,再优选的植物为稻属或烟草属,更优选植物为水稻或烟草,进一步优选植物为水稻日本晴(Oryza sativa L. ssp. japonica cv. Nipponbare)或烟草NC89。 [0029] The present invention also discloses a promoter, the nucleic acid Use of a construct, a vector, a recombinant cell or plant callus or plant explant or a plant in the regulation of target gene expression in plants or plant breeding, plant is preferably list leaves of plants or dicotyledonous plant, further preferably the plant is rice or genus Nicotiana, more preferably the plant is tobacco or rice, further preferably the plant is Nipponbare rice (Oryza sativa L. ssp. japonica cv. Nipponbare) or tobacco NC89.

[0030] 由于采用了以上技术方案,使本发明具备的有益效果在于: [0030] With the above technical solution of the present invention have an advantageous effect in that:

[0031] 本发明提供的新的启动子能够在植物中调控基因表达,并且,在单子叶植物如水稻和双子叶植物如模式植物-烟草的根和叶中均具有启动子功能,能够调控外源基因的表达,从而为研究植物(包括单子叶植物和双子叶植物)中目的基因的表达提供了一种新的工具和选择。 [0031] The present invention provides novel promoter capable of regulating gene expression in plants, and monocotyledonous plants such as rice and dicots such as model plants - roots and leaves of tobacco both having a promoter function, capable of modulating an outer gene expression, thus providing a new tool for the study of plant expression and selection (including monocotyledonous and dicotyledonous plants) in the gene of interest.

[0032] 保藏信息 [0032] Information Collection

[0033] 培养物名称:烟草NC89种子 [0033] Culture Name: NC89 tobacco seeds

[0034] 保藏日期:2010年11月12日 [0034] Collection date: November 12, 2010

[0035] 保藏单位: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) [0035] depository institution: Wuhan, Hubei Wuchang Luojia Hill Wuhan University Collection, namely China Type Culture Collection (CCTCC)

[0036]保藏编号:CCTCC No :P201017 附图说明 [0036] Accession number: CCTCC No: P201017 BRIEF DESCRIPTION OF DRAWINGS

[0037] 图I为用于构建p8质粒的pCAMBIA-1301质粒示意图。 [0037] FIG. I is used to construct a plasmid p8 schematic plasmid pCAMBIA-1301.

[0038] 图2为p8质粒示意图。 [0038] FIG. 2 is a schematic diagram of plasmid p8.

[0039] 图3为经转化的水稻愈伤组织的GUS染色结果。 [0039] FIG. 3 is a GUS staining Rice Calli transformed. 其中,由带有本发明所述启动子P537序列的重组根癌农杆菌P8+P537转化的水稻愈伤组织(右)经⑶S染色后呈现蓝色; 不带有本发明启动子序列的重组根癌农杆菌P8质粒的水稻愈伤组织(对照,左)经⑶S染色后颜色未发生变化。 Wherein said recombinant promoter sequence of Agrobacterium tumefaciens by sub-P537 with the present invention P8 + transformed rice callus P537 (right) appear blue staining after ⑶S; recombinant root without a promoter sequence of the invention P8 rice callus tumefaciens plasmid (control, left) does not change color after staining ⑶S.

[0040] 图4和图5分别为经转化的水稻苗根和叶的GUS染色结果。 [0040] Figures 4 and 5 respectively, GUS staining and leaves of rice rhizosphere transformed results. 经含有启动子的P8+P537重组载体的重组根癌农杆菌介导转化的水稻苗的根(图4右)和叶(图5右)经染色后呈现蓝色,经不含有启动子的P8质粒重组根癌农杆菌介导转化的水稻苗的根(作为对照,图4左)和叶(图5左)经⑶S染色后颜色未发生变化。 Root (FIG. 4 right) P8 + P537 rice seedling recombinant vector mediated transformation Agrobacterium tumefaciens by recombinant containing the promoter and the leaves (the right in FIG. 5) After staining appear blue, the promoter does not contain P8 the recombinant plasmid root seedlings of rice mediated by Agrobacterium root transformation (as a control, left in FIG. 4) and a leaf (left in FIG. 5) after staining ⑶S it does not change.

[0041] 图6和图7分别为经转化的烟草苗根和叶的GUS染色结果(三倍光学显微镜下拍照)。 [0041] Figures 6 and 7, respectively (the three optical microscope photograph) of GUS staining and rhizosphere of tobacco leaves transformed. 经含有启动子的P8+P537重组载体的重组根癌农杆菌介导转化的烟草苗的根(图6 右)和叶(图7右)经染色后呈现蓝色,经不含有启动子的p8质粒重组根癌农杆菌介导转化的烟草苗的根(作为对照,图6左)和叶(图7左)经GUS染色后颜色未发生变化。 Via promoter-containing P8 + root (the right in FIG. 6) P537 mediated transformation Agrobacterium tumefaciens recombinant vector recombinant tobacco leaves and seedlings (Figure 7 right) after staining appear blue, the promoter does not contain p8 root recombinant plasmid of Agrobacterium tumefaciens mediated transformation of tobacco seedlings (as a control, left in FIG. 6) and leaf (left in FIG. 7) after GUS staining it does not change.

具体实施方式 Detailed ways

[0042] 本发明涉及一种能够在植物中调控基因表达的启动子序列,本发明的启动子含有选自以下任意一组并具有启动子功能的核苷酸序列: [0042] The present invention relates to a promoter sequence capable of modulating gene expression in a plant, the promoter of the present invention contains any one selected from the group of nucleotide sequences having promoter function:

[0043] a、序列表中Seq ID No. I所示的核苷酸序列; [0043] a, nucleotide sequence shown in Seq ID No. I the Sequence Listing;

[0044] b、与Seq ID No. I互补的核苷酸序列; [0044] b, the nucleotide sequence I complementary to Seq ID No.;

[0045] C、在高等严紧条件下能够与上述a或b的核苷酸序列杂交的核苷酸序列; [0045] C, under conditions of higher stringency nucleotide sequence capable of hybridizing with a nucleotide sequence of a or b;

[0046] d、对上述a或b所示核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列; [0046] d, or of a nucleotide sequence shown above b substitutions of one or more nucleotides, deletions, additions, modified nucleotide sequence;

[0047] e、与上述a或b所示核苷酸序列具有至少90%同一性的核苷酸序列。 [0047] e, above a or b having a nucleotide sequence at least 90% nucleotide sequence identity.

[0048] 在本发明一个具体的实施方式中,本发明的启动子优选具有Seq ID No. I所示的核苷酸序列,并在本发明中被称为启动子BgIosP537,或者简称为P537启动子。 [0048] In one particular embodiment of the present invention, the promoter of the present invention preferably has a nucleotide sequence I shown in Seq ID No., and is referred to in the present invention, a promoter BgIosP537, or simply P537 promoter child.

[0049] 在本发明中,典型地,“杂交条件”根据测量杂交时所用条件的“严紧性”程度来分类。 [0049] In the present invention, typically, "hybridization conditions" are classified according to the degree of "stringency" of the hybridization conditions used in the measurement. 严紧性程度可以以例如核酸结合复合物或探针的解链温度(Tm)为依据。 The degree of stringency can be, for example, nucleic acid melting temperature (Tm) binding complex or probe basis. 例如,“最大严紧性”典型地发生在约Tm-5°C (低于探针Tm 5°C )高等严紧性”发生在Tm以下约5_10°C; “中等严紧性”发生在探针Tm以下约10-20°C ;“低严紧性”发生在Tm以下约20_25°C。作为替代,或者进一步地,杂交条件可以以杂交的盐或离子强度条件和/或一或多次的严紧性洗涤为依据。例如,6XSSC =极低严紧性;3XSSC =低至中等严紧性;1XSSC =中等严紧性;0. 5XSSC =高等严紧性。从功能上说,可以采用最大严紧性条件确定与杂交探针严紧同一或近严紧同一的核酸序列;或采用高等严紧性条件确定与该探针有约80%或更多序列同一性的核酸序列。 For example, "maximum stringency" at higher stringency from about Tm-5 ° C (below probe Tm 5 ° C) is typically "occurs at about Tm less 5_10 ° C;" moderate stringency "probe Tm below about 10-20 ° C; "low stringency" at about 20_25 ° C below the Tm Alternatively, or further, hybridization conditions can be in salt or ionic strength conditions of hybridization and / or one or more stringency. based washing example, 6XSSC = very low stringency;. 3XSSC = low to medium stringency; 1XSSC = medium stringency;. 0 5XSSC = Higher stringency functionally, maximum stringency conditions may be used to determine the hybridization probe. stringent needle near the same stringent or nucleic acid sequence identical; or higher stringency conditions is determined using 80% or more sequence identity to a nucleic acid sequence about the probe.

[0050] 对于要求高选择性的应用,典型地期望采用相对严紧的条件来形成杂交体,例如选择相对低的盐和/或高温条件。 [0050] For applications requiring high selectivity, typically desirable to employ relatively stringent conditions to form the hybrids, for example, selecting relatively low salt and / or high temperature conditions. Sambrook等(Sambrook, J.等(1989)分子克隆,实验室手册,Cold Spring Harbor Press,Plainview,NY)提供了包括中等严紧性和高等严紧性在内的杂交条件。 Sambrook et al (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY) to provide a moderate stringency conditions include hybridization and including higher stringency.

[0051] 为了便于说明,用于检测本发明的杂交的合适的中等严紧条件包括:用5XSSC、 0.5% SDS、1. OmM EDTA(pH8. O)溶液预洗;在50_65°C下在5XSSC中杂交过夜;随后用含O. 1% SDS的2X、0. 5X和O. 2XSSC在65°C下各洗涤两次20分钟。 [0051] For ease of illustration, suitable moderately stringent hybridization conditions for the detection of the present invention comprises: a pre-washed with a solution of 5XSSC, 0.5% SDS, 1 OmM EDTA; in at 50_65 ° C in 5XSSC (O pH8.). overnight hybridization;. followed by 2X of O. 1% SDS containing, 0 5X O. 2XSSC and washed twice each at 65 ° C 20 min. 本领域技术人员应当理解,能容易地操作杂交严紧性,如改变杂交溶液的含盐量和/或杂交温度。 It should be understood by those skilled in the art can easily manipulate the hybridization stringency, such as changing the salt content of the hybridization solution and / or hybridization temperature. 例如,合适的高等严紧杂交条件包括上述条件,不同之处在于杂交温度升高到例如60-65°C或65-70°C。 For example, suitable conditions of higher stringency include hybridization conditions described above, except that the hybridization temperature was raised to 60-65 ° C, or for example 65-70 ° C.

[0052] 在本发明中,所述在高等严紧条件下与Seq ID No. I所示或与其互补的核苷酸序列杂交的核苷酸序列,其具有与Seq ID No. I所示核苷酸序列相同或相似的启动子活性。 [0052] In the present invention, and the nucleotide sequence complementary thereto, or ID No. I hybridizes to the nucleotide sequence shown in Seq, having nucleoside shown under Seq ID No. I Higher stringency conditions acid sequence identical or similar promoter activity.

[0053] 在本发明中,所述对Seq ID No. I或与其互补的核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列,是指分别或同时在所述核苷酸序列的5'端和/或3' 端,和/或序列内部进行例如不超过2-45个,或者不超过3-20个,或者不超过3-20个,或者不超过4-15个,或者不超过5-10个,或者不超过6-8个的分别用逐个连续整数表示的碱基的取代、缺失、添加修饰。 [0053] In the present invention, the performing of Seq ID No. I or a nucleotide sequence complementary thereto or a plurality of bases substituted, deleted, added, modified nucleotide sequence, to separately or simultaneously in the nucleotides 5 'end and / or 3' end of the sequence, or an internal and / sequences, for example, no more than 2-45, or no more than 3 to 20, or no more than 3 to 20, or no more than substituted 4-15, 5-10 or no more than, or no more than 6-8, respectively, in base-by consecutive integers represented by deletions, additions, modifications.

[0054] 在本发明中,所述对Seq ID No. I或与其互补的核苷酸序列进行如上述一个或多个碱基的取代、缺失、添加修饰的核苷酸序列,具有与Seq ID No. I所示的核苷酸序列相同或相似的启动子活性。 [0054] In the present invention, the performing of Seq ID No. I or a nucleotide sequence complementary thereto as described above substituted with one or more bases, deletions, additions, modified nucleotide sequence, having Seq ID a nucleotide sequence identical or similar promoter activity No. I shown.

[0055] 通过一种多核苷酸进行说明,其所具有的核苷酸序列例如与Seq ID No. I的参考核苷酸序列至少90% “同一性”是指:在Seq ID No. I的参考核苷酸序列之每100个核苷酸中,该多核苷酸的核苷酸序列除了含有多达10个核苷酸的不同外,该多核苷酸之核苷酸序列与参考序列相同。 [0055] will be described by a polynucleotide having a nucleotide sequence which it is, for example, with reference to the nucleotide sequence of Seq ID No. I at least 90% "identity" refers to: in Seq ID No. I to per each 100 nucleotides of the reference nucleotide sequence, the nucleotide sequence of a polynucleotide comprising in addition up to 10 different nucleotides, the nucleotide sequence of the polynucleotide and the reference sequence identity. 换句话说,为了获得核苷酸序列与参考核苷酸序列至少90%相同的多核苷酸,参考序列中多达10%的核苷酸可以被删除或被另一核苷酸替代;或可将一些核苷酸插入参考序列中,其中插入的核苷酸可多达参考序列之总核苷酸的10% ;或在一些核苷酸中,存在删除、插入和替换的组合,其中所述核苷酸多达参考序列之总核苷酸的10%。 In other words, to obtain nucleotide sequences with the same reference nucleotide sequence of a polynucleotide at least 90%, up to 10% in the reference sequence may be deleted or nucleotide with another nucleotide substitution; or some reference nucleotide insertion sequence, wherein the polynucleotide is inserted may be up to 10% of the total nucleotides in the reference sequence; or a number of nucleotides in the presence of deletion, insertion and replacement composition, wherein the nucleotides up to 10% of the total nucleotides in the reference sequence. 参考序列的这些突变可发生在参考核苷酸序列的5'或3'末端位置,或在这些末端位置之间的任意地方,它们或单独散在于参考序列的核苷酸中,或以一个或多个相邻的组存在于参考序列中。 These mutations of the reference sequence may occur at 'or 3' terminal positions of the reference nucleotide sequence 5, or anywhere between those terminal positions, or they are scattered in a single nucleotide in the reference sequence or in one or a plurality of adjacent groups present in the reference sequence.

[0056] 在本发明中,用于确定序列同一性和序列相似性百分数的算法是例如BLAST和BLAST2. O 算法,它们分别描述在Altschul 等(1977) Nucl. Acid. Res. 25 :3389-3402 和Altschul等(1990) J. Mol. Bio. 215 :403-410o采用例如文献中所述或默认参数,BlAST和BLAST 2. O可以用于确定本发明的核苷酸序列同一性百分数。 [0056] In the present invention, for determining the percent similarity of sequence identity and sequence algorithm such as BLAST and BLAST2 O algorithms, which are described in Altschul et al. (1977) Nucl Acid Res 25:.... 3389-3402 and Altschul et al. (1990) J. Mol Bio 215:.. 403-410o or described in the literature, for example, using default parameters, BLAST and BLAST 2. O may be used to determine the nucleotide sequence of the present invention percent identity. 执行BLAST分析的软件可以通过国立生物技术信息中心为公众所获得。 Performing BLAST analysis software can be obtained to the public by the National Center for Biotechnology Information.

[0057] 在本发明中,所述与SEQ ID NO :1所示的核苷酸序列具有至少90%的序列同一性的核苷酸序列包括与SEQ ID NO :1所公开序列基本同一的多核苷酸序列,例如当采用本文所述方法(例如采用标准参数的BLAST分析)时,与本发明多核苷酸序列相比含有至少90%序列同一性、优选至少91%、92%、93%、94%、95%、96%、97%、98%或99%或更高的序列同一性的那些序列。 [0057] In the present invention, with the SEQ ID NO: nucleotide sequence identity to the nucleotide sequence having at least 90% and comprises SEQ ID NO: 1 disclosed substantially identical sequence polynuclear nucleotide sequences, such as when using the methods described herein (e.g., BLAST analysis using standard parameters), as compared with the present invention comprises a polynucleotide sequence at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to those sequences of. 在本发明中,所述与SEQ ID No. I或其互补的核苷酸序列具有至少90%的序列同一'丨生的核苷酸序列具有与Seq ID No. I所不的核苷酸序列相同或相似的启动子活性。 In the present invention, the SEQ No. I having a nucleotide sequence complementary to at least the same or ID 'nucleotide sequence of raw Shu 90% sequence having a nucleotide sequence of Seq ID No. I are not the identical or similar promoter activity.

[0058] 本发明还涉及一种核酸构建体,包括基因以及与该基因可操作地连接的上述启动子。 [0058] The present invention further relates to a nucleic acid construct, and said gene comprising a promoter operably linked to the gene. 在本发明中,“可操作地连接”是指两个或多个核苷酸区域或核酸序列的功能性的空间排列。 In the present invention, "operably linked" refers to the spatial functional regions of two or more nucleotides or nucleic acid sequences are arranged. 在本发明的核酸构建体中,例如,启动子被置于所述基因的核酸序列的特定位置,例如启动子位于所述基因核酸序列的上游位置,使得核酸序列的转录受到该启动子区域的引导,从而,启动子区域被“可操作地连接”到该基因的核酸序列上。 In the present invention, the nucleic acid construct, e.g., a promoter sequence is placed in a specific position of a nucleic acid of the gene, such as a promoter located at a position upstream of the nucleic acid sequence of the gene, so that transcription of the nucleic acid sequence by the promoter region the guide, whereby the promoter region is "operably linked" to a nucleic acid sequence of the gene. 所述基因一般是需要提高转录水平的任何核酸序列,或者,可设计本发明所述启动子和基因以便下调特定核酸序列。 The gene is generally necessary to increase the level of transcription of any nucleic acid sequence, or can be designed according to the present invention is the promoter and gene specific nucleic acid sequences to down-regulation. 也就是通过将启动子与反义方向的基因相连来实现。 That is accomplished by gene promoter connected with the antisense orientation. 在本发明一个具体的实施方式中,所述基因优选为GUS基因。 In a particular embodiment of the present invention, the gene is preferably GUS gene.

[0059] 本发明还涉及一种含有上述启动子或上述核酸构建体的载体。 [0059] The present invention further relates to a promoter comprising the above vector or said nucleic acid construct. 本发明的载体可以是通过将上述启动子或上述核酸构建体插入到克隆载体或表达载体而得到的重组载体。 Vectors of the present invention may be a recombinant vector obtained by the above-mentioned promoter or said nucleic acid construct is inserted into a cloning vector or expression vector. 适于构建本发明所述重组载体的克隆载体包括但不限于,例如:pUC18、pUC19、pUC118、 pUC119、pMD19-T、pMD20-T、pMD18-T Simple Vecter、pMD19_T Simple Vecter 等。 Cloning vectors suitable for the recombinant vectors of the present invention include, but are not limited to, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T Simple Vecter, pMD19_T Simple Vecter like. 适于构建本发明所述重组载体的表达载体包括但不限于,例如:pMI121、pl3W4、pGEM等。 The present invention is suitable for expression of the recombinant vector constructs include, but are not limited to, for example: pMI121, pl3W4, pGEM like. 在本发明具体的实施方式中,本发明含有所述启动子的载体为上述启动子与PMD18-T或p8质粒经重组得到的重组载体,优选的,本发明的重组载体为PMD18-T+P537重组载体或者p8+P537重组载体。 In a particular embodiment of the present invention, the present invention is a vector containing the promoter of the above-mentioned promoter or p8 PMD18-T recombinant vector recombinant plasmids were obtained, preferably, recombinant vector of the present invention is PMD18-T + P537 a recombinant vector or recombinant vector p8 + P537.

[0060] 本发明的又一方面,还涉及含有本发明所述启动子的重组细胞。 [0060] In yet another aspect of the present invention further relates to a recombinant cell comprising the promoter of the present invention. 本发明的重组细胞可以通过将上述含有本发明启动子的重组载体转化宿主细胞而得到。 The recombinant cells of the invention can be obtained by the above-described recombinant vectors containing a promoter of the present invention is obtained by transforming a host cell. 适于构建本发明所述重组细胞的宿主细胞包括但不限于,例如:大肠杆菌细胞DH5ci,根癌农杆菌细胞LBA4404、EHA105、GV3101等。 Suitable for constructing a recombinant host cell of the present invention include, but are not limited to cells, for example: E. coli cells DH5ci, cell Agrobacterium tumefaciens LBA4404, EHA105, GV3101 and the like. 在本发明一个具体的实施方式中,所述重组细胞为重组根癌农杆菌EHA105-P537。 In a particular embodiment of the present invention, the recombinant cell is a recombinant Agrobacterium tumefaciens EHA105-P537.

[0061] 本发明的再一方面,还涉及一种植物愈伤组织或植物外植体或植物,所述愈伤组织、植物外植体或植物含有有本发明的上述启动子。 [0061] another aspect of the present invention further relates to a plant callus or outside the plant or plant explant, a callus, a plant explant or a plant comprising the present invention have the above-described promoter. 本发明的植物愈伤组织可以是单子叶植物愈伤组织例如水稻愈伤组织,或者双子叶植物愈伤组织例如烟草愈伤组织。 Plant calli present invention may be a monocot plant such as rice callus Callus, or dicots such as tobacco callus Callus.

[0062] 本发明的再一方面,还涉及用于PCR扩增得到本发明所述启动子的一组引物对, 该组引物对含有序列表中Seq ID No. 2及Seq No.3所示的序列。 [0062] another aspect of the present invention further relates to a set of primers for PCR amplification to obtain the promoter of the present invention, the set of primers containing the Sequence Listing Seq ID No. 2 and Seq No.3 shown the sequence of. 为了便于操作,通常优选在引物的5'端含设计连接有限制性酶切位点和/或保护碱基,在本发明具体的实施方式中,所述引物对的两条引物分别具有Seq ID No :4和Seq ID No :5所示的序列。 For ease of operation, the 5 'end of the primer usually the design is connected preferably contains restriction sites and / or protection of bases in a particular embodiment of the present invention, the primer pair having two primers Seq ID No: 4 and Seq ID No: 5 in the sequence shown in FIG.

[0063]米用上述引物,以水稻日本晴(Oryza sativa L. ssp. japonica cv. Nipponbare) 基因组DNA为模板进行PCR扩增,能够制备得到本发明的启动子。 [0063] m, Nipponbare rice (Oryza sativa L. ssp. Japonica cv. Nipponbare) genomic DNA as a template for PCR amplification with the above primer, a promoter can be prepared according to the present invention.

[0064] 本发明进一步公开了一种调控植物中基因表达的方法,所述方法包括,将上述启动子导入植物细胞。 [0064] The present invention further discloses a method of regulation of gene expression in plants, the method comprising the above-described promoter into plant cells. 优选的是通过将同时含有外源基因以及本发明启动子的核酸构建体导入植物细胞来达到调控基因表达的目的。 Preferably by the simultaneous exogenous nucleic acid containing a promoter and a gene construct of the invention into a plant cell to achieve the object of the regulation of gene expression. 所述核酸构建体中,外源基因与本发明的启动子可操作地连接。 The nucleic acid construct, the exogenous gene of the present invention, a promoter is operably linked. 在本发明优选的实施方式中,可以利用含有目的外源基因与本发明启动子的重组载体转化农杆菌,再将得到的重组农杆菌转化植物愈伤组织,从而实现将所述外源基因与本发明启动子一起导入植物细胞的目的。 In a preferred embodiment of the invention may be utilized recombinant vector containing the exogenous gene promoter of Agrobacterium according to the present invention, the recombinant Agrobacterium then transformed plant calli obtained, thereby achieving the exogenous gene and introducing a plant cell with the purpose of the present invention is a promoter. 在本发明一个具体的实施方式中,外源基因优选为GUS基因。 In a particular embodiment of the present invention, the exogenous gene is preferably GUS gene. 本发明的植物可以是单子叶植物,例如水稻、小麦、玉米、小米、甘蔗、高粱、大麦等,也可以是双子叶植物,例如烟草,大豆,马铃薯、蚕豆、萝卜、花生等。 Plants of the present invention may be a monocot, such as rice, wheat, maize, millet, sugarcane, sorghum, barley and the like, may be dicotyledonous plants, such as tobacco, soybean, potato, beans, carrots, peanuts and the like.

[0065] 烟草是典型的基因工程模式植物。 [0065] Tobacco is the typical pattern of genetic engineering of plants. 故本发明选择烟草进行转基因研究,以研究本发明的启动子在双子叶植物中的效果。 Therefore, the present invention is to select tobacco transgenic research to study the effect of the present invention, the promoter in dicot plants. 实验结果表明,该启动子能在转基因烟草中起作用。 Experimental results show that the promoter can function in transgenic tobacco plants.

[0066] 在本发明中,可采用植物基因转化技术将目的基因及所述启动子插入到植物基因组中,包括农杆菌介导转化、病毒介导转化、显微注射、粒子轰击、基因枪转化和电穿孔等。 [0066] In the present invention, it may be used plant transformation technique and the target gene promoter is inserted into the plant genome, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation and electroporation and so on. 本领域周知,农杆菌介导的基因转化常被用于单子叶植物和双子叶植物的基因转化,但其它转化技术也可用于本发明启动子及外源基因的导入。 Known in the art, Agrobacterium mediated gene is often used for genetic transformation of monocotyledonous and dicotyledonous plants, but other transformation techniques may also be used to introduce the promoter of the present invention and the foreign gene. 当然,适于本发明的启动子及外源基因导入的另一种方法是粒子轰击(显微金或钨粒子包覆转化的DNA)胚性愈伤组织或胚胎开发。 Of course, promoters suitable for the invention and another method for introducing a foreign gene is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos. 另外,还可以采用的转化植物细胞的方法是原生质体转化。 In addition, methods of transforming plant cells can be used is protoplast transformation. 基因转化后,采用通用的方法来筛选和再生整合有表达单元的植株。 After the gene transfer, using a common method for screening and regenerated plants have integrated expression unit.

[0067] 为实现上述调控基因表达的目的,本发明所述启动子可以以单拷贝和/或多拷贝的形式应用,可以与现有技术中已知的启动子联用。 [0067] To achieve the above object of the regulation of gene expression, the promoter of the present invention may be applied as a single copy and / or copy, known in the prior art in combination with the promoter. [0068] 本发明的启动子以及调控植物中基因表达的方法,可以应用于植物品种的选育。 [0068] promoter, and a method of modulating gene expression in a plant of the present invention may be applied to breeding of plant species. 比如,可以用于水稻或烟草的育种。 For example, it is used in breeding rice or tobacco. 所述水稻可以为日本晴、中花9、中花10、中花11、台北309、丹江8号、云稻2号、汕优63、汕优608、丰优22、黔优88、II优416、II优107、II优128,11优718、准两优527、川农I号、杂0152、皖稻88、皖稻90、皖稻92、皖稻94、皖稻96、 皖稻185、皖稻187、皖稻189、皖稻191、皖稻193、皖稻195、皖稻197、皖稻199、皖稻201、 皖稻203、皖稻205、皖稻207,以及津原101等。 The rice may be Nipponbare, Zhonghua 9, 10 in the flower, the flower 11, Taipei 309, and Dan No. 8, No. 2 rice cloud SY63, Shanyou 608, preferably 22 Feng, Qianyou 88, II excellent 416, II preferably 107, II excellent excellent 128,11 718, 527 Zhunliangyou, I, Chuannong, heteroaryl 0152, Wandao 88, 90 Wandao, Wandao 92, 94 Wandao, Wandao 96, 185 Wandao , Wandao 187, Wandao 189, Wandao 191, Wandao 193, Wandao 195, Wandao 197, Wandao 199, Wandao 201, Wandao 203, Wandao 205, Wandao 207, and Jin original 101 or the like. 在本发明一个具体的实施方式中,所述水稻为日本晴。 In a particular embodiment of the present invention, the rice is Nipponbare. 所述的烟草可以为K326、K346、K394、NC82、NC89、G140、G28、G80、中烟90、 Cokerl76,以及CV87等。 The tobacco may K326, K346, K394, NC82, NC89, G140, G28, G80, smoke 90, Cokerl76, CV87, and the like. 在本发明另一个具体的实施方式中,所述烟草为烟草NC89。 In another specific embodiment of the invention, the tobacco is tobacco NC89.

[0069] 本发明的启动子可成为一种新的启动子,作为植物(包括单子叶植物和双子叶植物,尤其是水稻和烟草)转基因的工具启动子,为开展低表达基因转化苗筛选、植物花器官败育等分子育种研究提供便利,从而极大地缩短优良品种的选育时间。 [0069] The promoter of the invention may become a new promoter, a plant (including monocotyledonous and dicotyledonous plants, particularly in rice and tobacco) transgene tools promoter, for carrying out genetic transformation seedlings low expression screening, breeding floral organ abortion and other molecules facilitate greatly shorten the breeding of fine varieties of time. 本发明的启动子可广泛用于培育水稻、烟草、小麦、玉米、小米、甘蔗、高粱、大麦、大豆,马铃薯、蚕豆、萝卜、花生等。 Promoter of the present invention can be widely used in cultivation of rice, tobacco, wheat, maize, millet, sugarcane, sorghum, barley, soybeans, potatoes, beans, carrots, peanuts and the like.

[0070] 在本发明中,术语“单子叶植物”,具体地,可以为禾本科植物,更具体地,可以为稻属植物例如水稻,包括但不限于日本晴、中花9、中花10、中花11、台北309、丹江8号、云稻2 号、汕优63、汕优608、丰优22、黔优88、II优416、II优107、II优128、II优718、准两优527、川农I号、杂0152、皖稻88、皖稻90、皖稻92、皖稻94、皖稻96、皖稻185、皖稻187、皖稻189、皖稻191、皖稻193、皖稻195、皖稻197、皖稻199、皖稻201、皖稻203、皖稻205、皖稻207,以及津原101等。 [0070] In the present invention, the term "monocots", in particular, may be a gramineous plant, more particularly, may be a rice plants such as rice, including but not limited to Nipponbare, Zhonghua 9, the flowers 10, Zhonghua 11, Taipei 309, and Dan No. 8, No. 2 rice cloud SY63, Shanyou 608, preferably 22 Feng, Qianyou 88, II preferably 416, II preferably 107, II preferably 128, II 718 preferably quasi two 527, Chuannong I,, heteroaryl 0152, Wandao 88, Wandao 90, Wandao 92, Wandao 94, Wandao 96, Wandao 185, Wandao 187, Wandao 189, Wandao 191, Wandao 193, 195 Wandao, Wandao 197, Wandao 199, 201 Wandao, Wandao 203, 205 Wandao, Wandao 207, 101 and the like Jin original.

[0071] 在本发明中,术语“双子叶植物”,具体地,可以为茄科植物,更具体地,可以为烟草属植物(烟草),包括但不限于烟草K326、K346、K394、NC82、NC89、G140、G28、G80、中烟90、 Cokerl76,以及CV87 等。 [0071] In the present invention, the term "dicotyledonous plant", specifically, Solanaceae, more particularly, a plant belonging to the genus tobacco (tobacco), including but not limited to tobacco K326, K346, K394, NC82, NC89, G140, G28, G80, smoke 90, Cokerl76, CV87, and the like.

[0072] 下面通过具体实施方式结合附图对本发明作进一步详细说明。 [0072] The following figures present invention will be further described in detail by specific embodiments in combination. 但本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。 Those skilled in the art will appreciate that the following examples merely illustrate the invention and should not be construed as limiting the scope of the present invention. 实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。 Example no specific techniques or those conditions, according to the technical literature, or conditions described in the art (see, for example J. Sambrook waiting, "Molecular Cloning A Laboratory Manual", M. Huang Peitang of, Third Edition, Science Press) or in accordance with product instructions. 所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。 The reagents or equipment not specified by the manufacturer, are the conventional products available through the city.

[0073] 实施例I :P537启动子片段的PCR扩增和dMD18_T+P537重组载体的构津PCR扩增 [0073] Example I: PCR amplification and dMD18_T P537 + P537 promoter fragment of the recombinant vector PCR amplification configuration Tsu

[0074] 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)提取水稻日本晴(Oryza sativa L. ssp. japonica cv. Nipponbare)(已在申请号为200910238992.0、发明名称为“一种启动子BgIosP587、其制备方法及用途”的发明申请中保藏,并于2010年9月22日公开,保藏编号为CCTCC NO :P200910)的基因组DNA, 根据该启动子在水稻日本晴gDNA中的序列,分别在首尾设计一对PCR特异性扩增引物(上游引物F1,加限制性酶切位点KpnI和保护碱基,下游引物R1,加限制性酶切位点SbfI和保护碱基)。 [0074] using plant genomic DNA extraction kit (TIANGEN novel plant genomic DNA extraction kit, catalog number: DP320_02) extracting rice Nipponbare (already Application No. 200910238992.0, entitled (Oryza sativa L. ssp japonica cv Nipponbare..) as "a promoter BgIosP587, their preparation and use" application invention is deposited, and on September 22, 2010 discloses under the accession number CCTCC NO: genomic DNA P200910), according to which the promoter of rice Nipponbare gDNA in sequence, we designed a pair end to end in each PCR specific amplification primers (upstream primer F1, adding restriction sites KpnI and protection bases downstream primer R1, plus SbfI restriction site and protection bases ). 以上述提取的水稻日本晴的gDNA为模板,使用高保真Ex Taq (TaKaRa, DRR100B) 聚合酶进行PCR扩增。 Mostly of the extracted Japanese rice gDNA as a template, high fidelity Ex Taq (TaKaRa, DRR100B) polymerase for PCR amplification. 如表I所示。 As shown in Table I.

[0075] 表I基因启动子扩增的PCR体系[0076] [0075] TABLE I gene promoter amplified PCR system [0076]

Figure CN102146398BD00111

[0077] [0077]

90s,进行35个反应循环,最后72°C延伸7min。 90s, by 35 reaction cycles, 72 ° C final extension 7min.

[0078]其中,上游引物Fl (SEQ ID NO :4) :GGGGTACC|GAGTACATGCATGTTTTGTGAC|,其中下划线代表KpnI酶切位点,方框内为SEQ ID No :2。 [0078] wherein, upstream primer Fl (SEQ ID NO: 4): GGGGTACC | GAGTACATGCATGTTTTGTGAC |, wherein the underline represents KpnI restriction site, the block is SEQ ID No: 2.

[0079]下游引物 Rl (SEQ ID NO :5) :TGCCTGCAGG |AATTAAGCTAGCTAGCTGCTG|,其中下划线代表SbfI酶切位点,方框内为SEQ ID No :3。 [0079] The downstream primer Rl (SEQ ID NO: 5): TGCCTGCAGG | AATTAAGCTAGCTAGCTGCTG |, wherein the underline represents SbfI restriction site, the block is SEQ ID No: 3.

[0080] PCR扩增产物经I. 0%琼脂糖凝胶电泳分离,得到大小为2790bp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。 [0080] PCR amplification products I. 0% agarose gel electrophoresis, to obtain a size of approximately 2790bp band TIANGEN using agarose gel DNA extraction kit (catalog number: DP209-03) recovering purified.

[0081] PMD18-T+P537重组载体的构津 [0081] Jin configuration PMD18-T + P537 recombinant vector

[0082] 将如上述得到的PCR扩增产物进行T/A克隆(pMD18-T质粒,TaKaRa,D103A),转化大肠杆菌,挑取阳性克隆测序,证明准确。 [0082] The resulting PCR as the amplification product was subjected to T / A cloning (pMD18-T plasmid, TaKaRa, D103A), transformed into E. coli, positive clones were sequenced to prove correct.

[0083] 其中,T/A克隆的连接条件如下: [0083] where, T / A cloning connection conditions were as follows:

[0084] T/A 连接体系: 10 μ 1[0085] pMD18-T I μ I [0084] T / A connection system: 10 μ 1 [0085] pMD18-T I μ I

[0086] 2*solutionI 5 μ 1[0087] PCR扩增产物(回收插入片段)IOng〜20ng,根据其浓度定 [0086] 2 * solutionI 5 μ 1 [0087] PCR amplification product (recovery insert) IOng~20ng, according to a given concentration of

[0088] ddH20 补齐至10 μ 1[0089] 于16 °C在节能型智能恒温槽(宁波新芝,SDC-6)中连接8h以上,得到PMD18-T+P537重组载体。 [0088] ddH20 filled to 10 μ 1 [0089] connected to the energy in 16 ° C thermostatic chamber Intelligent (Ningbo Chicago, SDC-6) in more than 8h to give PMD18-T + P537 recombinant vector. 将经过上述连接后的产物按照如下方法转化大肠杆菌: The product was connected through the above-described transformation of E. coli as follows:

[0090] 从超低温冰箱中取出按照《分子克隆实验指南》(第三版,科学出版社)所示氯化钙法制备的感受态细胞100μ I DH5a(中国科学院昆明动物研究所董杨提供;或者可从例如:上海生工购得),冰上融化后,加入10 μ I如上所得的连接产物,即pMD18-T+P537重组载体,轻轻搅匀,冰浴30min,42°C热激60s,冰浴5min,加入600 μ I 4°C预冷的SOC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),37°C 220rpm复苏45min, 8000rpm离心30s,去上清,留取150 μ I,用剩下的150 μ I上清重悬沉淀后的混合物,轻轻吹匀,玻璃珠涂布LB (卡那霉素)平板(具体配方详见《分子克隆实验指南》,第三版,科学出版社),37°C倒置培养16h〜24h。 [0090] removed from the cryogenic freezer according to "Molecular Cloning, A Laboratory Manual" (third edition, Academic Press) prepared as shown in Method calcium chloride competent cells 100μ I DH5a (Dong Yang provided Kunming Institute of Zoology; or for example, from: Shanghai Sangon commercially available), thawed on ice after addition of 10 μ I ligation product obtained above, i.e. pMD18-T + P537 recombinant vector, stir gently, the ice bath was 30min, 42 ° C heat shock 60s , the ice bath was 5min, added 600 μ I 4 ° C pre-cooled SOC medium (specific formulations see "molecular cloning, a Laboratory Manual", Third Edition, Academic Press), 37 ° C 220rpm recovery 45min, 8000rpm centrifugal 30s, the supernatant, 150 μ I specimens, with the remaining 150 μ I supernatant and resuspend pellet mixture gently blowing uniform, glass beads coated with an LB (kanamycin) plates (see particular formulation "molecular cloning A Laboratory Manual ", Third Edition, Academic Press), 37 ° C inverted culture 16h~24h. 获得含有pMD18-T+P537克隆载体的重组大肠杆菌,命名为DH5a-P537。 E. coli containing the recombinant pMD18-T + P537 cloning vector, designated DH5a-P537. 深圳华大基因科技有限公司对pMD18_T+P537克隆载体中的P537进行测序。 BGI Shenzhen Technology Co., Ltd. sequenced pMD18_T + P537 cloning vector of P537.

[0091] 测序结果表明,获得的pMD18-T+P537克隆载体中P537启动子序列正确。 [0091] The sequencing results showed that, pMD18-T obtained in the promoter sequence + P537 P537 correct cloning vector. P537启动子的序列如序列表中Seq ID No :1所示。 P537 promoter sequences of the Sequence Listing as Seq ID No: 1 shown in FIG.

[0092] GAGTACATGCATGTTTTGTGACAAAGGGGAGTAAAAAAACCAOGAGATCAGCACTTGAAATACTAGAGACACAGAGA [0092] GAGTACATGCATGTTTTGTGACAAAGGGGAGTAAAAAAACCAOGAGATCAGCACTTGAAATACTAGAGACACAGAGA

[0093] GAATTTTCAACAAAAAAAATCAGCAATTTTAAAOGTGGTTTCTTCTTTAAAAGTGATGAAGATTAATCTTGTOGGAT [0093] GAATTTTCAACAAAAAAAATCAGCAATTTTAAAOGTGGTTTCTTCTTTAAAAGTGATGAAGATTAATCTTGTOGGAT

[0094] AAMGMGGCAACTGGATCACACAOGCGAAGAGAGAGAATACAAAAOGAAGAAAOGTGCATGGTAOGTGTTGGCATA [0094] AAMGMGGCAACTGGATCACACAOGCGAAGAGAGAGAATACAAAAOGAAGAAAOGTGCATGGTAOGTGTTGGCATA

[0095] TACTCOGTATATATCCTGGGAGTAGTGCOGGGCMCMMAGGGTAGGCOGCAGGCMAGCAOGMCTGATOGATGA [0095] TACTCOGTATATATCCTGGGAGTAGTGCOGGGCMCMMAGGGTAGGCOGCAGGCMAGCAOGMCTGATOGATGA

[0096] ATTGCTTACAAGCAAAGTGAAATGATTAAAAGCAACAATTAAAATTAATCAGTTTGGCTAGTACAATAAAAAAATCT [0096] ATTGCTTACAAGCAAAGTGAAATGATTAAAAGCAACAATTAAAATTAATCAGTTTGGCTAGTACAATAAAAAAATCT

[0097] CATATATAQMTTOTTMTTQTTMTTATTaiTTGAGATTGGMTTTGGATAGGGCOGGGTGGGMGCATATG [0097] CATATATAQMTTOTTMTTQTTMTTATTaiTTGAGATTGGMTTTGGATAGGGCOGGGTGGGMGCATATG

[0098] CTTAGTCATTATTAGTATATTGATTAATGAAATAGGAAAATAGAACAGTAGTGGCTACOGGAAGAATAGAAAACATT [0098] CTTAGTCATTATTAGTATATTGATTAATGAAATAGGAAAATAGAACAGTAGTGGCTACOGGAAGAATAGAAAACATT

[0099] AGMCAT(^Q^TC湯CTATAGATAWMACATTOGCGCATCCCTGGTTTGACOGTGTOGGTAOGTTTGGAT [0099] AGMCAT (^ Q ^ TC soup CTATAGATAWMACATTOGCGCATCCCTGGTTTGACOGTGTOGGTAOGTTTGGAT

[0100] GTOGATOGGATOXA(mTCTTCAOGCGAaXTAa^A(«OTraTTAGTTCCTMTTTTTTCTTCAMATTTTA [0101 ] ACTTTTTTATCACATCAAAACTTTTCTACATACATAAACTTTCAACTTTTCAGTCACATCATTACAATTTCAATTAA [0100] GTOGATOGGATOXA (mTCTTCAOGCGAaXTAa ^ A ( «OTraTTAGTTCCTMTTTTTTCTTCAMATTTTA [0101] ACTTTTTTATCACATCAAAACTTTTCTACATACATAAACTTTCAACTTTTCAGTCACATCATTACAATTTCAATTAA

[0102] ACTTATAATTTTAGCGTGAACTAAACACACCCAGCTACAATTTAATTTCACTAATGAAATATAATCCATGTTTGATT [0102] ACTTATAATTTTAGCGTGAACTAAACACACCCAGCTACAATTTAATTTCACTAATGAAATATAATCCATGTTTGATT

[0103] TTTCTTTTTTTTTTCMTTGTGCMTMAOGTACATACACCACCATACATGCMCACCACACTOGTACACCOGAGM [0103] TTTCTTTTTTTTTTCMTTGTGCMTMAOGTACATACACCACCATACATGCMCACCACACTOGTACACCOGAGM

[0104] T(X:AT(X(XTMCATGTACTAAAAAGACAGAAACCAATAGCACATCTOGATCTGATCTCACTCAAATCATGGTACAA [0104] T (X: AT (X (XTMCATGTACTAAAAAGACAGAAACCAATAGCACATCTOGATCTGATCTCACTCAAATCATGGTACAA

[0105] GCTTCACTTGCATATTTGCTAAGTATTCATGGACATATTTGCTAAAGATATGCATCCAOGACTTTATCATCACATTA [0105] GCTTCACTTGCATATTTGCTAAGTATTCATGGACATATTTGCTAAAGATATGCATCCAOGACTTTATCATCACATTA

[0106] CTATAOGTCMTACCAGTGGTGCTTTCCOGATGTTTTCTTTCTTTTOGGTTGTMATCTTTTTTTTTCTCTCACTCC [0106] CTATAOGTCMTACCAGTGGTGCTTTCCOGATGTTTTCTTTCTTTTOGGTTGTMATCTTTTTTTTTCTCTCACTCC

[0107] ATATATCMTCTGTCTATTAATTGTTAGATATGTGTGTTTTTTTCCTCCCTAGGGAATCTGTGTAGCTAGTCATTTT [0107] ATATATCMTCTGTCTATTAATTGTTAGATATGTGTGTTTTTTTCCTCCCTAGGGAATCTGTGTAGCTAGTCATTTT

[0108] CTTMTCMTTGCTATTMTTTGCTMTMTGCAOGGATATGTATTAOGTCCCMCMGCTAGGGCCMGGCATGCA [0108] CTTMTCMTTGCTATTMTTTGCTMTMTGCAOGGATATGTATTAOGTCCCMCMGCTAGGGCCMGGCATGCA

[0109] TGTGAGTCAGCTAGCTGTGGAGAGTTTTTCTTTTGTTAOGATACAATGTACTAGTTACACACACATATACTATCAGT [0109] TGTGAGTCAGCTAGCTGTGGAGAGTTTTTCTTTTGTTAOGATACAATGTACTAGTTACACACACATATACTATCAGT

[0110] ATAAATGATTCAAAATATTTAAATTGGTTTGTTGTTTGACTTAAATAGAATGTAATATAAAATTGAATGAGGTAAAA [0110] ATAAATGATTCAAAATATTTAAATTGGTTTGTTGTTTGACTTAAATAGAATGTAATATAAAATTGAATGAGGTAAAA

[0111] TTGTCTTTTAGTCCTACCACATGMGGGCGATTOGATTGCATGCGGATGATCMTATTCATATOGGGGTATATATAT [0111] TTGTCTTTTAGTCCTACCACATGMGGGCGATTOGATTGCATGCGGATGATCMTATTCATATOGGGGTATATATAT

[0112] GTGAACATGTGATGGATGCTTGACATGCATATATGTGCAGCAGTTGATACTACTCCAGTTATCTOGATCTACTAATT [0112] GTGAACATGTGATGGATGCTTGACATGCATATATGTGCAGCAGTTGATACTACTCCAGTTATCTOGATCTACTAATT

[0113] GATCAATCAATTATTGTTCOGCTTACACATACAOGTATATATGTGTGTGTACAAOGTGTTTGTTCACTGATAGTGTC [0113] GATCAATCAATTATTGTTCOGCTTACACATACAOGTATATATGTGTGTGTACAAOGTGTTTGTTCACTGATAGTGTC

[0114] GTGTGAWATCMMGGACCAOGTMCCMCAOGTGGCOGGCOGGCCAGTAGCMTATATATATATAGAGTAGMCT [0114] GTGTGAWATCMMGGACCAOGTMCCMCAOGTGGCOGGCOGGCCAGTAGCMTATATATATATAGAGTAGMCT

[0115] TAAAGAGTTGAAAATATCATGTACAATCATOGATCATGCGTATATATTCCTGCATGCATATAGAGTTGGATOGATOG [0115] TAAAGAGTTGAAAATATCATGTACAATCATOGATCATGCGTATATATTCCTGCATGCATATAGAGTTGGATOGATOG

[0116] MTTOGATCAGCACATGMCTACTCTCTTGTTATACATCCACTCATATTTTTATACTCMTCACTCCTGTATGTACC [0116] MTTOGATCAGCACATGMCTACTCTCTTGTTATACATCCACTCATATTTTTATACTCMTCACTCCTGTATGTACC

[0117] TGCTGCCTGCTGCAGGTTCCATOGTGTCCTAGMGTAGATGAGATGGATGTTCCTGTCMCMACMGCCCMTMG [0117] TGCTGCCTGCTGCAGGTTCCATOGTGTCCTAGMGTAGATGAGATGGATGTTCCTGTCMCMACMGCCCMTMG

[0118] AGACMGATGGGCTGGTCATOGCATCCAGTGTTCTATGGGCOGTATCTTGGMTOGGGTAOGGCTATGCGGATTGGG [0118] AGACMGATGGGCTGGTCATOGCATCCAGTGTTCTATGGGCOGTATCTTGGMTOGGGTAOGGCTATGCGGATTGGG

[0119] OI«aXATTTACAT(mTATCATATMGCGTGCATGTGATOGTACMAGCAMAGTTTTGGCCTGGaXTCAMA [0119] OI «aXATTTACAT (mTATCATATMGCGTGCATGTGATOGTACMAGCAMAGTTTTGGCCTGGaXTCAMA

[0120] AAGAGAGAAGAAAAACAGAGAGGTTAGGTTAGTTTGGAGAAAGTTCCTAGCCAGGAACAGGAAGGAAGAAGGCACAT [0121 ] (^A^AGTTCAAMTTATTAGAOiCTGCACATOGCACTCTATCTTGTGCAAGACTOGTGACCATCAAATCCCAATCT [0120] AAGAGAGAAGAAAAACAGAGAGGTTAGGTTAGTTTGGAGAAAGTTCCTAGCCAGGAACAGGAAGGAAGAAGGCACAT [0121] (^ A ^ AGTTCAAMTTATTAGAOiCTGCACATOGCACTCTATCTTGTGCAAGACTOGTGACCATCAAATCCCAATCT

[0122] AACAATTGTGATTCAAOGTATGTGTGGACAACATATATACATAGCAAAAGAAATTAAAGAAAATGTGTGTGACCACA [0122] AACAATTGTGATTCAAOGTATGTGTGGACAACATATATACATAGCAAAAGAAATTAAAGAAAATGTGTGTGACCACA

[0123] aTAaiCITGnmmTTTTTCTOGTGAAAAACAAAATCAATGGAOGGCCATGGGAAAAAGAGATACCAGATCT [0123] aTAaiCITGnmmTTTTTCTOGTGAAAAACAAAATCAATGGAOGGCCATGGGAAAAAGAGATACCAGATCT

[0124] GAATCCTGGAGGTACATGTGATTGGCTCACAAATTATTTTTTTTAAAAAAAAAAGCTCAAAAGAAGTTAAGGTGTGA [0124] GAATCCTGGAGGTACATGTGATTGGCTCACAAATTATTTTTTTTAAAAAAAAAAGCTCAAAAGAAGTTAAGGTGTGA

[0125] AGMGAMACAMMAGAMAGCGAGGCGACCACAOGCMGAOGGCOGGACAGCATOGGCGATGTGCATOGTGTGGT [0125] AGMGAMACAMMAGAMAGCGAGGCGACCACAOGCMGAOGGCOGGACAGCATOGGCGATGTGCATOGTGTGGT

[0126] GGTOGTTGCCCCATCCACTCCCCOGCAGATMGCCMCCAGTOGCOGCTOGCCAOGTGGCACCCTCCACCTCCCTTC [0126] GGTOGTTGCCCCATCCACTCCCCOGCAGATMGCCMCCAGTOGCOGCTOGCCAOGTGGCACCCTCCACCTCCCTTC

[0127] TTATTGCCTCAGCCTCCTTCMCCTCAOGCTCACATTCTTGCTAGCTCACTCCTCTCCTCCTOGTCTACCTTACCTA[0128] GCAGCAGCAGCTAGCTAGCTTAATT (上述整段序列为SEQ ID NO :1) [0127] TTATTGCCTCAGCCTCCTTCMCCTCAOGCTCACATTCTTGCTAGCTCACTCCTCTCCTCCTOGTCTACCTTACCTA [0128] GCAGCAGCAGCTAGCTAGCTTAATT (above the whole sequence SEQ ID NO: 1)

[0129] 实施例2 :载体-D8+P537重纟目载体的构律 [0129] Example 2: Vector -D8 + P537 reconstructed mesh support law Si

[0130] 按照TIANGEN普通质粒小提试剂盒(目录号:DP103_03)的操作手册,从实施例I 构建的转化有启动子P537的大肠杆菌DH5 α -Ρ537中提取带有本发明Ρ537启动子序列的克隆载体PMD18-T+P537 ;纯化后用相应的限制性内切酶KpnI和SbfI进行酶切,回收相应的启动子插入片段,并分别与Ρ8质粒用相同的限制性内切酶酶切后回收的载体大片段进行连接。 [0130] according to general TIANGEN plasmid mini kit: Manual (Cat. No. DP103_03) from Example I Construction of E. coli transformed with a promoter of -Ρ537 DH5 α P537 extracted with promoter sequences of the present invention Ρ537 cloning vector PMD18-T + P537; cut after purification by corresponding restriction enzymes KpnI and SbfI digested recovered corresponding promoter insert, respectively Ρ8 plasmid and enzyme digestion with the same restriction recovered large vector fragment are connected.

[0131] 将所得连接产物ρ8+Ρ537重组载体转化按照《分子克隆实验指南》(第三版,科学出版社)所示氯化钙法制备的感受态细胞DH5a,37°C倒置培养16〜24h,待转化子长出菌落后,挑取单克隆进行PCR检测和酶切鉴定。 [0131] The resulting ligation product was ρ8 + Ρ537 recombinant vector prepared according to Method calcium chloride competent cells of DH5a, 37 ° C as shown in an inverted "Molecular Cloning A Laboratory Manual" (third edition, Academic Press) culturing 16~24h , after the transformant to grow until colonies were picked for PCR analysis of monoclonal and enzyme digestion.

[0132] p8质粒构律 [0132] p8 plasmid configuration law

[0133] 本发明中所使用的p8质粒是由pCAMBIA-1301质粒(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,该公司的pCAMBIA-1301质粒的原始来源是The CAMBIA Bios (biological open source) Licensee, Australia)按照如下方式改造并构建的,具体说明如下: [0133] The present invention is used in a plasmid p8 (Dong Yang Kunming Institute of Zoology by plasmid pCAMBIA-1301; available from, for example, or because of the Shanghai Co., Ltd. Ruiji available, the company plasmid pCAMBIA-1301 original source is the CAMBIA Bios (biological open source) Licensee, Australia) renovation and construction in the following ways, as follows:

[0134] 用Kpn I/Nco I (NEB)双酶切质粒pCAMBIA-1301 (参见图I),回收大片段。 [0134] with Kpn I / Nco I (NEB) digested plasmid pCAMBIA-1301 (see FIG. I), recovered large fragment. 根据所采用的限制性内切酶位点合成如下序列:GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGT CGACCATGG(SEQ ID NO :6)(包含的酶切位点是Kpn I/HindIII/Spe I/Sbf I/Pst I/Xba I/ BamH I/Sal I/Nco I),用Kpn I/Nco I双酶切后回收,与上述所回收的大片段连接后转化toplO细胞(中国科学院昆明动物研究所董杨提供;或者可从例如:北京索莱宝科技有限公司购得)。 The restriction enzyme sites used in the synthesis of the following sequences: GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGT CGACCATGG (SEQ ID NO: 6) (contains the restriction site Kpn I / HindIII / Spe I / Sbf I / Pst I / Xba I / BamH I / Sal I / Nco I), with a Kpn I / Nco I digested after recovery, and the recovered large fragment connection toplO conversion cells (Dong Yang Kunming Institute of Zoology provided; or for example, from: Beijing Soledad Symbol Technologies Ltd. purchased). 用引物GCTTCCGGCTCGTATGTTGT (SEQ ID NO :7) /GAGTCGTCGGTTCTGTAAC(SEQ ID NO :8)筛选转化子,通过PCR检测方法,带有扩增片段为350bp的转化子即为含有需要构建的多克隆位点及GUS序列的p8质粒的转化子。 Transformants were screened by PCR for detection, the amplified fragment with 350bp of transformants is the need to construct containing the multiple cloning site and GUS primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO:: 7) / GAGTCGTCGGTTCTGTAAC (8 SEQ ID NO) p8 transformants plasmid sequences. P8质粒图谱见图2 Figure 2 Plasmid map P8

[0135] 所述p8质粒中的多克隆位点和⑶S序列的长度2353碱基,如SEQ ID N0:9所示: [0135] The length of 2353 base p8 and ⑶S cloning site sequence in the plasmid, such as SEQ ID N0: 9 shown:

[0136] GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC [0136] GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC

[0137] ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC [0137] ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC

[0138] [0138]

Figure CN102146398BD00131

CTTTTTA TTTTTTTGAGCTTTGA TCTTTCTTTAAACTGA TCTA TTTTTTAA TTGA TTGGTTA TGGTGTAAA TA CTTTTTA TTTTTTTGAGCTTTGA TCTTTCTTTAAACTGA TCTA TTTTTTAA TTGA TTGGTTA TGGTGTAAA TA

TTACA TAGCTTTAA CTGA TAA TCTGA TTA CTTTA TTTCGTGTGTCTA TGA TGA TGA TGA TAGTTACAGAACCG TTACA TAGCTTTAA CTGA TAA TCTGA TTA CTTTA TTTCGTGTGTCTA TGA TGA TGA TGA TAGTTACAGAACCG

A CGA CTCGTCCGTCCTGTA GAAA CCCCAA CCCGTGAAA TCAAAAAA CTCGA CGGCCTGTGGGCA TTCA GTCTG A CGA CTCGTCCGTCCTGTA GAAA CCCCAA CCCGTGAAA TCAAAAAA CTCGA CGGCCTGTGGGCA TTCA GTCTG

GA TCGCGAAAA CTGTGGAA TTGA TCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAA TTGCTGTGC GA TCGCGAAAA CTGTGGAA TTGA TCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAA TTGCTGTGC

CAGGCAGTTTTAACGA TCAGTTCGCCGA TGCAGA TA TTCGTAA TTA TGCGGGCAA CGTCTGGTA TCAGCGCGA CAGGCAGTTTTAACGA TCAGTTCGCCGA TGCAGA TA TTCGTAA TTA TGCGGGCAA CGTCTGGTA TCAGCGCGA

AGTCTTTA TACCGAAAGGTTGGGCAGGCCAGCGTA TCGTGCTGCGTTTCGA TGCGGTCACTCA TTACGGCAAA AGTCTTTA TACCGAAAGGTTGGGCAGGCCAGCGTA TCGTGCTGCGTTTCGA TGCGGTCACTCA TTACGGCAAA

GTGTGGGTCAA TAA TCAGGAAGTGA TGGAGCA TCAGGGCGGCTA TA CGCCA TTTGAAGCCGA TGTCACGCCGT GTGTGGGTCAA TAA TCAGGAAGTGA TGGAGCA TCAGGGCGGCTA TA CGCCA TTTGAAGCCGA TGTCACGCCGT

A TGTTA TTGCCGGGAAAA GTGTA CGTA TCA CCGTTTGTGTGAA CAA CGAA CTGAA CTGGCA GA CTA TCCCGCC A TGTTA TTGCCGGGAAAA GTGTA CGTA TCA CCGTTTGTGTGAA CAA CGAA CTGAA CTGGCA GA CTA TCCCGCC

GGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GCAGTCT TA CTTCCA TGA TT TCTT TAACTA TGCCGGA GGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GCAGTCT TA CTTCCA TGA TT TCTT TAACTA TGCCGGA

A TCCA TCGCA GCGTAA TGCTCTA CA CCA CGCCGAA CA CCTGGGTGGA CGA TA TCA CCGTGGTGA CGCA TGTCG A TCCA TCGCA GCGTAA TGCTCTA CA CCA CGCCGAA CA CCTGGGTGGA CGA TA TCA CCGTGGTGA CGCA TGTCG

CGCAA GA CTGTAA CCA CGCGTCTGTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA CGCAA GA CTGTAA CCA CGCGTCTGTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA

TGCGGA TCAA CAGGTGGTTGCAA CTGGA CAAGGCA CTA GCGGGA CTTTGCAA GTGGTGAA TCCGCA CCTCTGG TGCGGA TCAA CAGGTGGTTGCAA CTGGA CAAGGCA CTA GCGGGA CTTTGCAA GTGGTGAA TCCGCA CCTCTGG

CAACCGGGTGAAGGTTA TCTCTA TGAA CTCGAA G TCA CA GCCAAAA GCCA GA CA GA G TCTGA TA TCTACCCG C CAACCGGGTGAAGGTTA TCTCTA TGAA CTCGAA G TCA CA GCCAAAA GCCA GA CA GA G TCTGA TA TCTACCCG C

TTCGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCCAACAGTTCCTGATTAACCACAAACCGTTCTACTTTAC TTCGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCCAACAGTTCCTGATTAACCACAAACCGTTCTACTTTAC

TGGCTTTGGTCGTCA TGAAGA TGCGGA CTTA CGTGGCAAA GGA TTCGA TAACGTGCTGA TGGTGCACGACCAC TGGCTTTGGTCGTCA TGAAGA TGCGGA CTTA CGTGGCAAA GGA TTCGA TAACGTGCTGA TGGTGCACGACCAC

GCA TTAA TGGACTGGA TTGGGGCCAA CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGAA GA GA TGCTCGAC T GCA TTAA TGGACTGGA TTGGGGCCAA CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGAA GA GA TGCTCGAC T

GGGCAGA TGAACA TGGCA TCGTGGTGA TTGA TGAAACTGCTGCTGTCGGCTTTCAGCTGTCTTTAGGCA TTGG TTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCAC GGGCAGA TGAACA TGGCA TCGTGGTGA TTGA TGAAACTGCTGCTGTCGGCTTTCAGCTGTCTTTAGGCA TTGG TTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCAC

TTACAGGCGA TTAAAGAGCTGA TA GCGCG TGA CAAAAA CCA CCCAA GCG TGG TGA TGTGGAGTA T TGCCAACG TTACAGGCGA TTAAAGAGCTGA TA GCGCG TGA CAAAAA CCA CCCAA GCG TGG TGA TGTGGAGTA T TGCCAACG

AACCGGA TACCCGTCCGCAAGGTGCACGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGCGTAAA CTCGA CCC AACCGGA TACCCGTCCGCAAGGTGCACGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGCGTAAA CTCGA CCC

GACGCGTCCGA TCACCTGCGTCAA TGTAA TGTTCTGCGA CGCTCA CA CCGA TACCA TCAGCGA TCTCTTTGA T GACGCGTCCGA TCACCTGCGTCAA TGTAA TGTTCTGCGA CGCTCA CA CCGA TACCA TCAGCGA TCTCTTTGA T

GTGCTGTGCCTGAA CCGTTA T TACGGA TGGTA TGTCCAAAGCGGCGA TT TGGAAA CGGCA GA GAA GGTA CTGG GTGCTGTGCCTGAA CCGTTA T TACGGA TGGTA TGTCCAAAGCGGCGA TT TGGAAA CGGCA GA GAA GGTA CTGG

AAAAA GAA CTTCTGGCCTGGCA GGA GAAA CTGCA TCAGCCGA TTA TCA TCACCGAA TACGGCGTGGA TACGTT AAAAA GAA CTTCTGGCCTGGCA GGA GAAA CTGCA TCAGCCGA TTA TCA TCACCGAA TACGGCGTGGA TACGTT

AGCCGGGCTGCACTCAA TGTACACCGACA TGTGGAGTGAAGAGTA TCAGTGTGCA TGGCTGGA TA TGTA TCAC AGCCGGGCTGCACTCAA TGTACACCGACA TGTGGAGTGAAGAGTA TCAGTGTGCA TGGCTGGA TA TGTA TCAC

CGCGTCTTTGA TCGCGTCA GCGCCGTCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCGA CCTCGCAA G CGCGTCTTTGA TCGCGTCA GCGCCGTCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCGA CCTCGCAA G

GCA TA TTGCGCGTTGGCGGTAA CAAGAAAGGGA TCTTCA CTCGCGA CCGCAAA CCGAAGTCGGCGGCTTTTCT GCA TA TTGCGCGTTGGCGGTAA CAAGAAAGGGA TCTTCA CTCGCGA CCGCAAA CCGAAGTCGGCGGCTTTTCT

GCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTGAAAAA CCGCA GCA GGGA GGCAAA CAA GCTA GCCA CCA C CACCACCACCACGTGTGA (SEQ ID NO :9) GCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTGAAAAA CCGCA GCA GGGA GGCAAA CAA GCTA GCCA CCA C CACCACCACCACGTGTGA (SEQ ID NO: 9)

[0139] 如上序列所示本发明中所构建的p8质粒,其多克隆位点中的EcoR I/Sac I/Kpn I/HindIII/Spe I/Sbf I/Pst I/Xba I/BamH I/Sal I/Nco I 限制性酶切位点分别用加框和下划线表示,筛选转化子所用的引物GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTAAC(即SEQ ID NO :7和8)用双下划线表示,GUS序列用斜体表示,其内含子序列分别用斜体加底纹示出。 As shown in the present invention is constructed plasmid p8 which multiple cloning site of EcoR I / Sac I / Kpn I / HindIII / Spe I / Sbf I / Pst I / Xba I / BamH I / Sal [0139] As the sequence I / Nco I restriction sites are indicated by boxed and underlined, used for selection of transformants primers GCTTCCGGCTCGTATGTTGT / GAGTCGTCGGTTCTGTAAC (i.e., SEQ ID NO: 7 and 8) represented by a double underline, italics the GUS sequence, within intron sequences are shown in italics to shade.

[0140] 重组载体D8+P537的构建[0141] 根据限制性内切酶KpnI和SbfI操作说明,按照如下条件处理实施例I中所得到 的克隆载体PMD18-T+P537,以及如上所述构建的p8质粒。 Construction [0141] The enzymes KpnI and SbfI restriction instructions, in accordance with the following process conditions embodiments cloning vector PMD18-T + P537 obtained in Example I and constructed as described above [0140] The recombinant vector of D8 + P537 p8 plasmid. [0142] 其中,克隆载体PMD18-T+P537及p8质粒的酶切条件如下: [0143] 酶切体系: 50 u I[0144] 无菌水 34. 8 u I[0145] 10*buffer H 5 u I[0146] KpnI 0. I u I(IOU)[0147] SbfI 0. I u I(IOU)[0148] 克隆载体PMD18-T+P537 或p8 质粒10 yl ( < IOOOng) [0149] 使用TIANGEN琼脂糖凝胶DNA回收试剂盒(目录号:DP209-03)分别回收经酶切 的P8质粒,以及启动子P537插入片段,根据T4连接酶(TaKaRa,D2011A)操作说明,按照如 下条件进行连接: [0150] 连接体系: IOu I[0151] 10*T4buffer Iul[0152] 酶切后的p8质粒 IUI (20ng)[0153] 回收的启动子P537插入片段 10〜20ng,根据其浓度确定[0154] 无菌水 补齐至9. 5iil[0155] T41igase(TaKaRa, D2011A) 0. 5u I [0142] wherein the cloning vector PMD18-T + P537 p8 plasmid and restriction digestion conditions are as follows: [0143] digestion system: 50 u I [0144] Sterile water 34. 8 u I [0145] 10 * buffer H 5 u I [0146] KpnI 0. I u I (IOU) [0147] SbfI 0. I u I (IOU) [0148] cloning vector PMD18-T + P537 plasmid p8 or 10 yl (<IOOOng) [0149] using TIANGEN agarose gel DNA extraction kit (catalog number: DP209-03) P8 were recovered plasmid was digested, and the fragments inserted P537 promoter the T4 ligase (TaKaRa, D2011A) instructions, to connect under the following conditions: [0150] the ligation: IOu I [0151] 10 * T4buffer Iul [0152] p8 IUI the digested plasmid (20ng) [0153] P537 promoter recovered insert 10~20ng, OK [0154] the concentration of free sterile water filled to 9. 5iil [0155] T41igase (TaKaRa, D2011A) 0. 5u I

[0156] T4buffer冰上融化,酶切后的p8质粒载体加入量约20ng,本发明中的P537片段加10ng。 [0156] T4buffer thawed on ice, p8 digested plasmid vector after addition of about 20ng, P537 fragments of the present invention plus 10ng. 于16°C在节能型智能恒温槽(宁波新芝,SDC-6)中连接8h以上。 8h at 16 ° C or more in the energy-connection intelligent thermostatic chamber (Ningbo Chicago, SDC-6) in.

[0157] 将100 UI氯化钙法制得的感受态细胞DH5 a从超低温冰箱取出,冰上融化后,加入IOu I上面的连接产物,轻轻搅匀,冰浴30min,42°C热激60s,冰浴5min,加入600iil 4°C预冷的S0C, 37度220rpm复苏45min, 8000rpm离心30s,去上清,留取150 y I,轻轻吹勻, 玻璃珠涂布LB (kan),37°C倒置培养16〜24h。 [0157] The obtained calcium chloride 100 UI DH5 competent cells taken from a SYSTEM ultra-low temperature freezer, thawed on ice, added to the ligation product IOu I above, stir gently, the ice bath was 30min, 42 ° C heat shock 60s , the ice bath was 5min, added 600iil 4 ° C precooled S0C, 37 recovery of 220rpm for 45 min, centrifuged at 8000 rpm for 30s, the supernatant was discarded, specimens 150 y I, uniform blowing gently, glass beads coated with LB (kan), 37 Invert ° C culture 16~24h. 得到重组载体p8+P537。 Recombinant vector p8 + P537.

[0158] 分别以Fl (即SEQ ID NO :4)和Rl (即SEQ ID NO :5)为引物对所得重组载体P8+P537进行PCR检测,以确证所得重组载体P8+P537中含有所需启动子P537。 [0158] With Fl (i.e., SEQ ID NO: 4) and Rl (i.e., SEQ ID NO: 5) P8 P537 containing primer resulting recombinant vector P8 + P537 by PCR to confirm the resulting recombinant vector + desired promoter sub-P537. 通过KpnI 和SbfI酶切筛选含有重组载体P8+P537转化子。 KpnI and SbfI digested by screening a recombinant vector containing P8 + P537 transformants.

[0159] 实施例3重组根癌农杆菌EHA105-P537细胞的制备 Preparation of recombinant Agrobacterium tumefaciens EHA105-P537 cells [0159] Example 3

[0160] 将如实施例2所述方法构建的P8+P537重组载体和作为对照的p8质粒分别转化按照《分子克隆实验指南》(第三版,科学出版社)中所述氯化钙方法制备的根瘤农杆菌EHA105(已在申请号为200910238992. O、发明名称为“ー种启动子BgIosP587、其制备方法及用途”的发明申请中保藏,并于2010年9月22日公开,保藏编号为CCTCC NO :M 209315) 的感受态细胞,具体方法如下: [0160] The method as described in Example 2 Construction of recombinant vectors and P8 + P537 as a control plasmid were transformed p8 prepared according to "Molecular Cloning A Laboratory Manual" (third edition, Academic Press) in the calcium chloride method Agrobacterium tumefaciens EHA105 (already application No. 200910238992. O, entitled "ー seed promoters BgIosP587, their preparation and use" application of the invention deposited, and on September 22, 2010 discloses accession number CCTCC NO: M 209315) competent cells, as follows:

[0161] 将根瘤农杆菌感受态细胞EHA105于超低温冰箱中取出,至于冰上解冻。 [0161] The A. tumefaciens EHA105 competent cells taken at cryogenic refrigerator, as thawed on ice. 融化后,分别加入5 ill的p8+P537重组载体和p8质粒以及作为对照的p8空载体,轻轻混匀,冰浴lOmin,放入液氮中冷冻5min,37°C解冻5min,加入800 yl常温的LB液体培养基,28°C 160rpm复苏3h,8000rpm离心30s,吸去上清,留下200 吹匀,涂布于加有kan-rif (卡那霉素-利福平)双抗的YM培养基平板上(50mg/lKan,IOmg/1 Rif,具体配方參见下文说明)。 After thawing, were added 5 ill p8 + P537 of recombinant vectors and plasmids p8 p8 and empty vector as a control, mix gently, the ice bath was lOmin, frozen in liquid nitrogen 5min, 37 ° C were thawed 5min, added 800 yl of LB liquid medium at room temperature, 28 ° C 160rpm recovery 3h, 8000rpm centrifugal 30s, the supernatant was aspirated, leaving 200 blow uniformly coated to a plus kan-rif (kanamycin - rifampin) bis antibody on YM medium plates (50mg / lKan, IOmg / 1 Rif, see specific formulations described below). 28°C倒置培养2-3天。 28 ° C for 2-3 days inverted. [0162] 以Fl (即SEQ ID NO :4)和Rl (即SEQ ID NO :5)为引物进行PCR检测和通过KpnI/ SbfI酶切筛选转化子。 [0162] In Fl (i.e., SEQ ID NO: 4) and Rl (i.e., SEQ ID NO: 5) by PCR and by KpnI / SbfI digestion Transformants were screened for the primer.

[0163] PCR扩增出约2790bp左右条带和酶切出约2790bp左右条带的为重组载体P8+P537的重组根癌农杆菌。 [0163] PCR amplified band of about 2790bp and digested recombinant vector is a recombinant Agrobacterium P8 + P537 is about 2790bp band.

[0164] 本发明中,按照如上述方法得到的带有重组载体P8+P537的重组农杆菌,命名为重组根癌农杆菌EHA105-P537。 [0164] In the present invention, the recombinant Agrobacterium according to P8 + P537 obtained by the method as described above with a recombinant vector designated recombinant Agrobacterium tumefaciens EHA105-P537.

[0165] 按照本发明所述方法,得到的带有p8质粒的对照重组农杆菌,命名为重组根瘤农杆菌EHA105-p8。 [0165] The method according to the present invention, obtained with the control recombinant Agrobacterium p8 plasmid, designated as recombinant Agrobacterium tumefaciens EHA105-p8.

[0166] 实施例4 :水稻愈伤组织的诱导和转化 [0166] Example 4: Induction of transformed callus and rice

[0167] 按照如下步骤诱导水稻愈伤组织,并分别用重组根瘤农杆菌EHA105-P537和重组根瘤农杆菌EHA105-p8转化所述愈伤组织。 [0167] the following steps rice callus induction, respectively, and the recombinant Agrobacterium tumefaciens EHA105-P537, and the recombinant Agrobacterium tumefaciens EHA105-p8 transformed calli.

[0168] I)将水稻日本睛种子去売,70%こ醇表面消毒30s,然后用有效氯I. 5%的次氯酸钠消毒30min,期间剧烈摇动,消毒后用灭菌水清洗5次;将消毒后的种子置于N6D培养基(具体配方參见下文说明)上,用封ロ膜封ロ;29.5°C光照培养3〜4周; [0168] I) The eyes Japanese rice seeds to bai, surface-sterilized 70% alcohol ko 30s, and then washed with 5% available chlorine I. hypochlorite disinfection 30min, during the vigorous shaking, washed with sterile water five times after disinfection; disinfecting seeds were placed on N6D medium (see specific formulations described below), and with the sealing membrane sealed ro ro; 29.5 ° C lighted culture 3 to 4 weeks;

[0169] 2)选取活跃生长的愈伤组织(黄白色,干燥,直径I〜3mm),在新N6D培养基上29. 5 °C光照培养3天; [0169] 2) selecting actively growing callus (yellowish white, dried, diameter I~3mm), 29. 5 ° C over 3 days in the light of new N6D medium;

[0170] 3)分别挑取如实施例3所构建的重组根瘤农杆菌单菌落(重组根瘤农杆菌EHA105-P537或重组根癌农杆菌EHA105_p8),于添加抗生素(50mg/l Kan, 10mg/lRif)的YM培养基(具体配方參见下文说明)上划线培养3天,培养温度28°C ;分别刮取上述重组根癌农杆菌置于添加了30 ii I IOOmM的AS (Acetosyringone,こ酰丁香酮)的30ml AAM培养基(具体配方參见下文说明)中,温和重悬所述重组根瘤农杆菌细胞(重组根瘤农杆菌EHA105-P537或重组根癌农杆菌EHA105_p8); [0170] 3) were picked recombinant Agrobacterium tumefaciens Single colonies embodiment 3 constructed (recombinant Agrobacterium tumefaciens EHA105-P537 or recombinant Agrobacterium tumefaciens EHA105_p8) embodiment, the addition of antibiotics (50mg / l Kan, 10mg / lRif ) was streaked on YM medium 3 days (see specific formulations described below), culture temperature 28 ° C; were scraped off with the recombinant Agrobacterium tumefaciens disposed added 30 ii I IOOmM the AS (Acetosyringone, acyloxy ko acetosyringone) in 30ml AAM medium (see specific formulations described below), the gently resuspended cells the recombinant Agrobacterium tumefaciens (Agrobacterium tumefaciens EHA105-P537 recombinant or recombinant Agrobacterium tumefaciens EHA105_p8);

[0171] 4)将继代培养的愈伤组织置于灭菌培养皿中;将如步骤3制备的重组根瘤农杆菌悬液倒入培养皿中,将愈伤组织浸入其中15min ; [0171] 4) The following cultured callus was placed in a sterile Petri dish; recombinant A. tumefaciens suspensions as prepared in Step 3 was poured into a Petri dish, the calli immersed in 15min;

[0172] 5)倒掉重组根瘤农杆菌悬液,将愈伤组织用灭菌吸水纸吸掉多余的液体;于N6-AS培养基(配方參见下文说明)上放ー张灭菌滤纸,加Iml如上述含AS的AAM培养基, 将愈伤组织转移至滤纸上;密封培养皿,28°C暗培养48〜60h ; [0172] 5) Discard the recombinant Agrobacterium tumefaciens suspension, the callus siphoning off excess liquid with sterile absorbent paper; Zhang sterile filter paper placed on ー N6-AS medium (see formulation described below), as described above plus Iml AAM medium containing the aS, the callus is transferred to the filter paper; sealed Petri dish, 28 ° C dark culture 48~60h;

[0173] 6)将受感染的愈伤组织置于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瘤农杆菌;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lmg/1潮霉素B (HmB)和50mg/l Carb的N6-AS培养基上;用封ロ膜密封培养皿,29. 5°C光照培养3〜4周。 [0173] 6) The infected callus were then placed in a 50ml sterile tube, shaken washed with sterile water until the supernatant became clear; the calli immersed containing 500mg / l carbenicillin (Carb) sterile water to kill the recombinant Agrobacterium tumefaciens; sterile absorbent paper to remove excess water from the callus and transferred to containing lmg / 1 hygromycin B (HmB) and 50mg / l Carb of N6- AS medium on;. the dishes were sealed with the sealing film ro, 29 5 ° C 3 to 4 weeks the lighted culture.

[0174] 实施例5 :水稻愈伤组织中的⑶S的表汰 Rice Calli ⑶S jig table: [0174] Example 5

[0175] 为检测经过实施例4所述转化的水稻愈伤组织中目的基因⑶S的表达情況,按照Chen SY等在Journal of Integrative Plant Biology, 2008, 50 (6) :742-751 所述的方法, 对分别用重组根瘤农杆菌EHA105-P537或重组农杆菌EHA105_p8转化的水稻愈伤组织进行染色。 After the detection of the expression of the gene ⑶S rice calli transformed Example 4 in accordance with Chen SY et al Journal Integrative Plant Biology, 2008, of 50 (6) [0175]: The method of claim 742-751 , respectively, of rice callus recombinant or recombinant Agrobacterium tumefaciens transformed EHA105-P537 EHA105_p8 stained.

[0176] GUS 染色液的配方(Iml) :610u I 0. 2M Na2HPO4 溶液(pH = 7. 0) ;390u I 0. 2M NaH2PO4 溶液和IOu I 0. IM X-gluc。 [0176] GUS staining solution formulation (Iml): 610u I 0. 2M Na2HPO4 solution (pH = 7. 0); 390u I 0. 2M NaH2PO4 solution and IOu I 0. IM X-gluc.

[0177] 将分别用重组根癌农杆菌EHA105-P537或重组根癌农杆菌EHA105_p8转化的水稻愈伤组织浸泡在GUS染色液中,37°C保温至出现蓝色,拍照,结果如图3所示,经含有启动子的p8+P537重组载体的重组根瘤农杆菌介导转化的水稻愈伤组织(图3右)经染色后呈现蓝色,经不含有启动子的p8质粒重组根瘤农杆菌介导转化的水稻愈伤组织(作为对照,图3左)经⑶S染色后颜色未发生变化。 [0177] respectively with the recombinant Agrobacterium tumefaciens EHA105-P537 or rice callus transformed with the recombinant Agrobacterium tumefaciens EHA105_p8 immersed in the GUS staining solution, 37 ° C incubation to appear blue, photographed, the results shown in Figure 3 shown, the promoter-containing p8 + rice callus a recombinant vector recombinant P537 Agrobacterium mediated transformation (the right in FIG. 3) after staining appear blue, the promoter does not contain the recombinant plasmid of Agrobacterium tumefaciens mediated p8 mediated transformation of rice callus (as a control, left in FIG. 3) after staining ⑶S it does not change. 结果显示,本发明的P537启动子对⑶S基因表达具有调控作用。 The results show, P537 promoter of the present invention has a regulatory effect on gene expression ⑶S.

[0178] 实施例6 :转基因水稻苗中GUS的表达 6 [0178] Example: Expression of GUS in transgenic rice plants in

[0179] 将实施例4中得到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2)分化苗;用封ロ膜密封培养皿,29. 5°C光照培养3-4周;待幼苗长至3-4cm时转移到含50mg/l潮霉素B (HmB)的1/2MS生根培养基(具体配方參见表3)进行生根筛选。 [0179] The embodiment of the callus obtained in Example 4 was transferred to MS-R differentiation medium containing 50mg / l hygromycin B (HMB) (the specific formulations shown in Table 2) into seedlings; dishes were sealed with a sealing film ro ., 29 5 ° C for 3-4 weeks lighted culture; to be transferred to 3-4cm seedlings grow to 1 / 2MS rooting medium containing 50mg / l hygromycin B (HMB) (the specific formulations see Table 3) rooting.

[0180] 转基因水稻苗的GUS染色过程同实施例5中愈伤组织的染色过程。 [0180] GUS staining of transgenic rice plants during the dyeing process of Example 5 with the callus embodiment. 结果如图4 及图5所示,经含有启动子的P8+P537重组载体的重组根瘤农杆菌介导转化的水稻苗的根(图4右)和叶(图5右)经染色后呈现蓝色,经不含有启动子的p8质粒重组根瘤农杆菌介导转化的水稻苗的根和叶(作为对照,图4左和图5左)经GUS染色后顔色未发生变化。 The results shown in FIG. 4 and FIG. 5, the rice plants P8 + P537 recombinant Agrobacterium tumefaciens mediated transformation a recombinant vector containing the promoter of the root (the right in FIG. 4) and the leaves (the right in FIG. 5) After staining exhibits a blue p8 plasmid color, does not contain a promoter by a recombinant Agrobacterium tumefaciens mediated transformation of rice seedlings of the roots and leaves (as a control, the left in FIG. 4 and FIG. 5 left) after GUS staining it does not change. 结果显示,本发明的P537启动子对GUS基因表达具有调控作用。 The results show, P537 promoter of the present invention has a regulatory effect on the expression of gene GUS.

[0181] 实施例I :转某闵烟草小苗中GUS表汰 [0181] Example I: a turn table Min tobacco seedlings in GUS Jig

[0182] I.烟草无菌苗获得: [0182] I. tobacco-free vaccine obtained:

[0183] •烟草种子浸泡:将烟草NC89种子(2010年11月12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 No : P201017)装入I. 5ml的离心管中(< 50粒/管),加入Iml无菌水,用移液器吸打几次,更换无菌水后,在常温下浸泡24小时; [0183] • Tobacco seeds are soaked: NC89 tobacco seeds (November 12, 2010 deposited in Wuhan City, Hubei Province, Wuhan University Wuchang Luojia Hill Collection, namely China Type Culture Collection (CCTCC), under the accession number CCTCC No : P201017) centrifuge tube was charged I. 5ml of (<50 / tube), Iml of sterile water was added, pipetted several times, after the replacement of sterile water, soaked at room temperature for 24 hours;

[0184] •烟草种子消毒:用移液器吸出浸泡种子的水,加入Iml 75%的酒精浸泡30秒, 用移液器吸出酒精;加入Iml 10% H2O2浸泡10分钟后,用移液器吸出H2O2 ; [0184] • tobacco seed disinfection: a pipette soaking the seeds in water, was added Iml 75% alcohol soak for 30 seconds using a pipette alcohol; Iml 10% H2O2 was added soak for 10 minutes, and then the pipette H2O2;

[0185] •洗涤:用无菌水清洗五次,每次加入Iml无菌水摇动Imin ; [0185] • washing: washed five times with sterile water, sterile water was added to each shake Iml Imin;

[0186] •接种:无菌滤纸吸干种子表面的水分,再用吸头或无菌牙签接种于MS固体培养基(配方參见表4)上萌发,姆皿约10粒,置于28°C光照培养室(16h光/8h暗)培养一周, 光照强度为20001x(本实验所有的光照培养材料均在此光照強度下培养); [0186] • vaccination: a sterile filter paper dry seed surface water, or sterile toothpick tip then inoculated in MS solid medium (Formulation see Table 4) germinated, about 10 Farm dish, placed in 28 ° C lighted culture room (16h light / 8h dark) week of culture, the light intensity of 20001x (all lighted culture in this experiment were cultured in this material is light intensity);

[0187] 籲转接:待长出幼苗后,转入新鲜MS固体培养基,每瓶(06cm,H 9cm,30ml培养基/瓶)3株烟草小苗,28°C光照培养室(16h光/8h暗)培养约5周,获得烟草无菌苗。 [0187] Calls adapter: seedling to be grown, transferred to fresh MS solid medium bottle (06cm, H 9cm, 30ml medium / flask) 3 tobacco seedlings, 28 ° C the lighted culture room (16h light / 8h dark) culturing about 5 weeks, no vaccine tabacum.

[0188] 2.烟草无菌苗的继代和扩繁 [0188] 2. tobacco aseptic subculture and propagation

[0189] •剪去烟草无菌苗的叶片和根,将茎杆剪成带有腋芽的茎段(2cm-3cm),然后将其形态学下端垂直插入到新鮮MS固体培养基(腋芽不能插入培养基里); [0189] • Cut aseptic tobacco leaves and roots, the stalk cut stem segments with axillary (2cm-3cm), then inserted into the vertically lower morphology fresh MS solid medium (axillary buds can not be inserted medium in);

[0190] •每瓶接种ー个带有腋芽的茎段外植体,置于28°C光照培养室培养约5周,作为待转化材料使用。 [0190] • a bottle ー seeded stem segment explants with axillary buds, and 28 ° C placed in the lighted culture room cultured for about 5 weeks to be used as conversion material.

[0191] 3.侵染前菌液的制备: [0191] 3. The front bacteria infested prepared:

[0192] •挑取含潮霉素抗性目的质粒的农杆菌菌株EHA105单菌落(实施例3所得的重组根癌农杆菌EHA105-P537或重组根癌农杆菌EHA105-p8),接种到IOml YM(含Kan 50mg/ L,Rif 30mg/L)液体培养基,28°C,250rpm振荡过夜,待菌液混浊,还未出现沉淀吋,将此菌液置4°C保存;[0193] •取保存于4°C 的菌液20 iil,接种于IOml YM(含Kan 50mg/L,Rif 30mg/L)液体培养基于50ml离心管中28°C,250rpm振荡过夜,待菌液混浊,还未出现沉淀时,停止培养; [0192] • picking single colonies of Agrobacterium tumefaciens strain EHA105 containing the hygromycin resistance plasmid object (Example 3 obtained recombinant Agrobacterium tumefaciens EHA105-P537 or recombinant Agrobacterium tumefaciens EHA105-p8), inoculated into IOml YM (containing Kan 50mg / L, Rif 30mg / L) liquid medium, 28 ° C, 250rpm overnight shaking, until turbidity bacteria, yet a precipitate inch, this bacterium is set at 4 ° C; [0193] • taking stored at 4 ° C. bacteria 20 iil, inoculated IOml YM (containing Kan 50mg / L, Rif 30mg / L) liquid cultures 50ml centrifuge tube based on 28 ° C, 250rpm overnight shaking, until turbidity bacteria, yet to come precipitation, the culture is stopped;

[0194] •取上述菌液3ml加入到50mlYM(不含抗生素)液体培养于三角瓶中28 °C, 250rpm摇床震荡培养约2h,OD600 = 0 . 5左右时,作为侵染菌液使用。 [0194] • take the above was added to the bacteria 3ml 50mlYM (without antibiotics) in liquid culture in flasks 28 ° C, 250rpm shaking shake culture for about 2h, OD600 = about 0.5, is used as a bacteria infection.

[0195] 4.侵染: [0195] 4. Infection:

[0196] •从培养5周的烟草无菌苗上剪取较大的叶片,保存在一个装有YM (不含抗生素) 液体培养基的培养皿中; [0196] • scissor blade from the larger tobacco cultured for 5 weeks on aseptic, stored in a petri dish with YM (antibiotic-free) liquid medium;

[0197] •用直径6mm的打孔机将烟草叶片打成叶圆盘,保存在另ー个装有YM(不含抗生素)液体培养基的培养皿中; [0197] • 6mm diameter punch blade tobacco leaf discs labeled, stored in a petri dish with YM another ー (antibiotic-free) liquid medium;

[0198] •将烟草叶圆盘转移到装有侵染菌液的培养皿中; [0198] • The tobacco leaf disc was transferred to a Petri dish with a bacteria infection;

[0199] •轻轻摇动培养皿,确保农杆菌接触到叶盘边缘,浸泡IOmin ; [0199] • dish shaken gently to ensure contact with the Agrobacterium leaf disc edge, soaking IOmin;

[0200] •将已侵染的烟草叶盘从农杆菌悬浮液中转移至干燥的无菌滤纸上,吸干菌液直至烟草叶盘不滴菌液为止; [0200] • which has been infected tobacco leaf discs were transferred to the Agrobacterium suspension and dried on sterile filter paper, until dry tobacco leaf discs without bacteria dropwise until the bacteria;

[0201] •将烟草叶盘接种到RMOP固体培养基(配方參见表5)上,叶面朝上,每皿约10 块; [0201] • tobacco leaf disk was inoculated into a solid medium RMOP (Formulation see Table 5), the leaf facing upward, from about 10 per dish;

[0202] •倒置培养皿,28°C暗培养3天。 [0202] • inverted dish, 28 ° C dark for 3 days.

[0203] 5.筛选: [0203] 5. Screening:

[0204] •将步骤4的叶圆盘转移到RMOP-TCH(IOmg/L Hyg)培养基(配方參见表6)上, 每皿10块,叶面朝上,28°C光照培养2周; [0204] • Step 4 leaf discs were transferred to the RMOP-TCH (IOmg / L Hyg) medium (Formulation see Table 6), each plate 10, leaf up, 28 ° C light for 2 weeks ;

[0205] •每2周继代一次,大约4周出现丛生芽。 [0205] • once every two ZHOU Ji-generation, about four weeks buds appear.

[0206] 6.生根: [0206] 6. Rooting:

[0207] •当再生芽长至约l-2cm吋,切下芽接种到MST-TCH(10mg/L Hyg)培养基(配方參见表7),每瓶I株烟草苗; [0207] • When shoot regeneration inch to about l-2cm, shoots were cut to MST-TCH (10mg / L Hyg) medium (see Table 7 Formulation), tobacco seedlings bottle I strain;

[0208] • 28 °C光照培养室培养2周; [0208] • 28 ° C the lighted culture room for 2 weeks;

[0209] 除去幼苗基部的小叶子,转移到新鲜的MST-TCH培养基,每瓶I株烟草苗,培养2 周。 [0209] removing the small leaves of the seedlings of the base, transferred to fresh medium MST-TCH, tobacco seedlings bottle I strain, cultured for 2 weeks. 之后分别取叶片和根进行GUS染色实验,GUS染色液的配方和方法同水稻。 After the leaves and roots were taken for GUS staining experiments, GUS staining solution with the formulations and methods of rice.

[0210] GUS 染色液的配方(Iml) :610u I 0. 2M Na2HP04 溶液(pH = 7. 0) ;390u I 0. 2MNaH2P04 溶液和IOu I 0. IM X-gluc。 [0210] GUS staining solution formulation (Iml): 610u I 0. 2M Na2HP04 solution (pH = 7. 0); 390u I 0. 2MNaH2P04 solution and IOu I 0. IM X-gluc.

[0211] 将转基因和对照(没有转入目的基因的空载体)分别浸泡在⑶S染色液中,37°C 保温至出现蓝色,拍照记录,结果如图6和7所示。 [0211] Transgenic and control (no gene of interest into the empty vector) were immersed in each ⑶S dyeing solution, 37 ° C incubation to appear blue, camera recording, the results shown in Figures 6 and 7.

[0212] 本发明实施例中所使用的相关培养基配方说明如下: [0212] Examples related media formulations used in embodiments of the present invention will be described as follows:

[0213] 以下有关培养基中所称的“常规灭菌”是指如下条件的灭菌:121°C下蒸气灭菌20 分钟。 [0213] For the medium called "conventional sterilization" refers to a condition of sterilization: 121 ° C for steam sterilization for 20 minutes.

[0214] N6D培养基、YM液体培养基、YM固体培养基、AAM培养基及N6-AS共培养基參见中国专利申请《一种启动子BgIosP587、其制备方法及用途》(申请号200910238992. 0,公布号CN101838647A)说明书中表2至表6。 [0214] N6D medium, YM broth, YM solid medium, AAM medium and co-N6-AS medium see Chinese patent application to "a promoter BgIosP587, preparation and use thereof" (Application No. 200910238992. 0, publication No. CN101838647A) table 6 to table 2 in the specification.

[0215] 表2MS-R分化培养基:IL 0. 5 L 2 LMS macro (20X) 50 ml 25 ml 100 mlMS micro (1000X) I ml 0. 5 ml 2 mlFe2EDTA 贮存液(100X) 10 ml 5 ml 20 ml肌醇(500X) 2 ml I ml 4 ml鹿糖(Sucrose) 30 g 15 g 60 g山梨醇(Sorbitol) 30 g 15 g 60 g植物凝月交(phytagel ) 4 g 2 g 8 g常规灭菌后加入 MS维生素(1000X)过滤除菌 I ml 0. 5 ml 2 ml激动素(I mg/ml)过滤除菌 2 ml I ml 4 ml萘こ酸(I mg/ml)过滤除菌 0. 02 ml 0. 01 ml 0. 04 ml羧节青霉素(500 mg/ml) 0. 5 ml 0. 25 ml I ml潮霉素(50 mg/ml) I ml 0. 5 ml 2 ml [0215] TABLE 2MS-R differentiation medium: IL 0. 5 L 2 LMS macro (20X) 50 ml 25 ml 100 mlMS micro (1000X) I ml 0. 5 ml 2 mlFe2EDTA stock solution (100X) 10 ml 5 ml 20 ml inositol (500X) 2 ml I ml 4 ml deer sugar (Sucrose) 30 g 15 g 60 g sorbitol (sorbitol) 30 g 15 g 60 g plant condensate month post (phytagel) 4 g 2 g 8 g conventional sterilization after addition of MS vitamins (IOOOX) filter-sterilized I ml 0. 5 ml 2 ml of kinetin (I mg / ml) filter-sterilized 2 ml I ml 4 ml naphthyl ko acid (I mg / ml) filter-sterilized 0.02 ml 0. 01 ml 0. 04 ml penicillin carboxylic section (500 mg / ml) 0. 5 ml 0. 25 ml I ml hygromycin (50 mg / ml) I ml 0. 5 ml 2 ml

[0217] 调PH值至5. 8,常规方式灭菌。 [0217] PH value is adjusted to 5.8, sterilized in a conventional manner.

[0218]注:MS macro (20X):硝酸铵33. Og,硝酸钾38. Og,磷酸ニ氢钾3. 4g,硫酸镁7. 4g, 氯化钙8. Sg逐一溶解,然后室温下用蒸馏水定容至1L,4°C保存。 [0218] Note: MS macro (20X): ammonium nitrate 33. Og, potassium nitrate 38. Og, potassium hydrogen phosphate ni 3. 4g, magnesium 7. 4g, calcium chloride dissolved one 8. Sg, then at room temperature volume of distilled water to 1L, 4 ° C storage.

[0219] MS micro (1000X):硫酸锰16. 90g,硫酸锌8. 60g,硼酸6. 20g,碘化钾0. 83g,钥酸钠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°C保存。 [0219] MS micro (1000X): manganese sulfate 16. 90g, zinc sulfate 8. 60g, boric 6. 20g, potassium iodide 0. 83g, sodium key 0. 25g, copper sulfate 0. 025g, 0. 025g cobalt chloride the reagent and dissolved at room temperature with distilled water to 1L, 4 ° C storage.

[0220] MS维生素贮存液(1000X):维生素B1O. OlOg,维生素B6O. 050g,烟酸0. 050g,甘氨酸0. 200g,加蒸馏水定容至100ml,过滤除菌,4°C保存不超过I周。 [0220] MS vitamins stock solution (1000X):.. Vitamin B1O OlOg, vitamin B6O 050g, niacin 0. 050g, glycine 0. 200g, distilled water volume to 100ml, filter sterilized, 4 ° C to save no more than I week. 铁盐(Fe2EDTA)贮存液(100X):见中国专利申请CN101838647A说明书中表2。 Iron salts (Fe2EDTA) stock solution (100X): Chinese patent application CN101838647A see Table 2 specification.

[0221] 表31/2MS生根培养基 [0221] TABLE 31 / 2MS rooting medium

Figure CN102146398BD00201

[0223]调 PH 值至5. 8。 [0223] adjust PH to 5.8.

[0224]注:MS macro (20X)见表2。 [0224] Note: MS macro (20X) shown in Table 2.

[0225] MS micro (1000X) MS 维生素C存液(1000X)见表2。 [0225] MS micro (1000X) MS vitamin C liquid reservoir (IOOOX) Table 2.

[0226]铁盐(Fe2EDTA)贮存液(100X):见中国专利申请CN101838647A说明书中表2。 [0226] iron salt (Fe2EDTA) stock solution (100X): Chinese patent application CN101838647A see Table 2 specification.

[0227]表4MS固体培养基: [0227] Table 4MS solid medium:

[0228] [0228]

Figure CN102146398BD00202

[0229] pH 5. 8121°C 下灭菌20 分钟 [0229] Sterilization pH 5. 8121 ° C 20 minutes

[0230]注:MS macro (20X)见表2。 [0230] Note: MS macro (20X) shown in Table 2.

[0231] MS micro (1000X) MS 维生素C存液(1000X)见表2。 [0231] MS micro (1000X) MS vitamin C liquid reservoir (IOOOX) Table 2.

[0232]铁盐(Fe2EDTA)贮存液(100X):见中国专利申请CN101838647A说明书中表2。 [0232] iron salt (Fe2EDTA) stock solution (100X): Chinese patent application CN101838647A see Table 2 specification.

[0233] MS 有机(IOOOx):维生素Bl,0. 01g,维生素B6,0. 05g,烟酸Bl,0. 05g,甘氨酸, 0. 2g,加蒸馏水定容至100ml,过滤除菌,4°C保存不超过1周。 [0233] MS organic (IOOOx):... Vitamin Bl, 0 01g, vitamin B6,0 05g, niacin Bl, 0 05g, glycine, 0. 2g, distilled water volume to 100ml, filter sterilized, 4 ° C preservation is not more than one week.

[0234]表5RM0P固体培养基: [0234] Table 5RM0P solid medium:

Figure CN102146398BD00211

pH 5. 8121°C下灭菌20分钟注:MS macro (20X)见表2。 Note sterilized 20 minutes at pH 5. 8121 ° C: MS macro (20X) shown in Table 2.

MS micro (IOOOX)MS 维生素贮存液(1000X)见表2。 MS micro (IOOOX) MS vitamins stock solution (IOOOX) Table 2.

Figure CN102146398BD00212

[0246] 铁盐(Fe2EDTA)贮存液(100X):见中国专利申请CN101838647A说明书中表2。 [0246] iron salt (Fe2EDTA) stock solution (100X): Chinese patent application CN101838647A see Table 2 specification.

[0247] Myo-inositol (500x):见表5。 [0247] Myo-inositol (500x): Table 5.

[0248]表 7MST-TCH 培养基: [0248] TABLE 7MST-TCH medium:

Figure CN102146398BD00221

[0250] pH 5. 8 [0250] pH 5. 8

[0251]注:MS macro (20X)见表2。 [0251] Note: MS macro (20X) shown in Table 2.

[0252] MS micro (IOOOX)MS 维生素贮存液(1000X)见表2。 [0252] MS micro (IOOOX) MS vitamins stock solution (IOOOX) Table 2.

[0253] 铁盐(Fe2EDTA)贮存液(100X):见中国专利申请CN101838647A说明书中表2。 [0253] iron salt (Fe2EDTA) stock solution (100X): Chinese patent application CN101838647A see Table 2 specification. 以上内容是结合具体的实施方式对本发明所作的进ー步详细说明,不能认定本发明的具体实施只局限于这些说明。 Above with the specific embodiments of the present invention is described in detail taken into ー step, considered that the specific embodiments of the present invention is not only limited to these descriptions. 对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。 Those of ordinary skill in the art for the present invention, without departing from the spirit of the present invention, can make various simple deduction or replacement, should be deemed to belong to the scope of the present invention.

Claims (24)

1. 一种启动子,所述启动子为选自以下任意一组并具有启动子功能的核苷酸序列:a、Seq ID No. I所示的核苷酸序列;b、与Seq ID No. I互补的核苷酸序列。 A promoter, the promoter is a group of any of the following nucleotide sequence and has promoter functions selected from: a, the nucleotide sequence I shown in Seq ID No.; b, with Seq ID No . a nucleotide sequence complementary to the I.
2. —种核酸构建体,包含权利要求I所述的启动子,与启动子可操作连接的基因序列, 其中所述启动子与所述基因序列来源相同或者不同,所述基因为GUS基因。 2. - nucleic acid construct comprising a promoter as claimed in claim I said gene sequence operably linked to a promoter, wherein the promoter is the same as or different from the promoter and gene sequences source, the gene is a GUS gene.
3. 一种载体,其特征在于:所述载体含有权利要求I所述的启动子或权利要求2所述的核酸构建体。 3. A vector, characterized in that: said vector comprising a promoter as claimed in claim in claim I or claim 2, said nucleic acid construct.
4.根据权利要求3所述的载体,其特征在于:所述载体为权利要求I所述的启动子或权利要求2所述的核酸构建体与pMDlS-Τ或p8质粒经重组得到的重组载体。 4. The vector of claim 3, wherein: said carrier is a promoter of claim I or claim 2, claim nucleic acid construct and a recombinant vector comprising plasmid p8 pMDlS-Τ recombinantly or obtained .
5. 一种重组细胞,其特征在于:所述细胞含有权利要求I所述的启动子或权利要求2 所述的核酸构建体或权利要求3或4所述的载体。 A recombinant cell, characterized in that: said cell comprising a promoter as claimed in claim in claim I or claim 2, a nucleic acid construct or the vector of claim 3 or claim 4.
6.根据权利要求5所述的重组细胞,其特征在于:所述重组细胞为重组大肠杆菌细胞或重组农杆菌细胞。 The recombinant cell according to claim 5, wherein: said recombinant cell is a recombinant E. coli cell or a recombinant Agrobacterium cells.
7. —组引物对,所述引物对用于扩增得到权利要求I所述的启动子,其特征在于:所述引物对的两条引物分别含有Seq ID No :2和Seq ID No :3所示的序列。 7. - pairs of primers, the primer pair for amplification of the promoter obtained Claim I in claim 1, characterized in that: said pair of primers comprises two primers, respectively Seq ID No: 2 and Seq ID No:. 3 the sequence shown.
8.根据权利要求7所述的引物对,其特征在于:所述引物对的两条引物在5'端还分别连接有限制性酶切位点和/或保护碱基。 8. A primer according to claim 7, wherein: the primer pair of the two primers at the 5 'end are also connected with a restriction site and / or protective bases.
9.根据权利要求8所述的引物对,其特征在于:所述引物对的两条引物分别为Seq ID No :4和Seq ID No :5所不的序列。 9. The primer according to claim 8, wherein: the primer pair for the two primers Seq ID No: 4 and Seq ID No: 5 in the sequence are not.
10.制备SEQ ID NO :1所示的启动子的方法,包括以水稻日本晴基因组DNA为模板,使用一对扩增引物进行扩增,所述扩增引物根据SEQ ID NO :1在水稻日本晴gDNA中的序列分别针对首尾进行设计。 10. Preparation of SEQ ID NO: Method 1 shown promoter, including clear genomic DNA as a template transgenic rice Japan, using a pair of amplification primers for amplification, the amplification primers according to SEQ ID NO: 1 in rice Nipponbare gDNA the sequences designed for end to end.
11.根据权利要求10所述的方法,其特征在于:所述扩增引物为权利要求7-9任一所述的引物对。 11. The method according to claim 10, wherein: the amplification primer is a primer pair according to claim any one of claims 7-9.
12. —种调控植物中基因表达的方法,所述方法包括,将权利要求I所述的启动子或权利要求2的核酸构建体或权利要求3或4的载体或权利要求5或6的重组细胞导入植物, 所述植物为单子叶植物或双子叶植物。 12. - The method of regulation of expression of genes in plants of the species, the method comprising the promoter according to claim I or claim 2, a nucleic acid construct or vector as claimed in claim 3 or claim 4 or a recombinant of claim 5 or claim 6, will introducing into a plant cell, the plant is a monocot or a dicot.
13.根据权利要求12所述的方法,其特征在于:所述植物为稻属或烟草属。 13. The method according to claim 12, wherein: said plant is rice or genus Nicotiana.
14.根据权利要求13所述的方法,其特征在于:所述植物为水稻或烟草。 14. The method according to claim 13, wherein: said plant is tobacco or rice.
15.根据权利要求14所述的方法,其特征在于:所述植物为水稻日本晴或烟草NC89。 15. The method according to claim 14, wherein: said plant is tobacco or rice Nipponbare NC89.
16.根据权利要求12〜15所述的方法,其特征在于:所述导入植物是导入植物愈伤组织。 16. The method according to claim 12~15, wherein: the introduction into a plant is a plant callus.
17. 一种制备转基因植物的方法,包括在有效产生植物的条件下培养权利要求5或6的重组细胞或植物愈伤组织或植物外植体或植物,所述植物愈伤组织或植物外植体或植物含有权利要求I的启动子或权利要求2的核酸构建体或权利要求3或4的载体或权利要求5 或6的重组细胞,所述植物为单子叶植物或双子叶植物。 17. A method of preparing a transgenic plant, comprising culturing under conditions effective to produce a recombinant plant cell or plant callus or plant explant or a plant as claimed in claim 5 or 6, said plant or plant explant calli or plants comprising the promoter of claim I or claim 2, a nucleic acid construct or vector as claimed in claim 3 or claim 4 or a recombinant cell of claim 5 or 6, wherein the plant is a monocot or a dicot.
18.根据权利要求17所述的方法,其特征在于:所述植物为稻属或烟草属。 18. The method according to claim 17, wherein: said plant is rice or genus Nicotiana.
19.根据权利要求18所述的方法,其特征在于:所述植物为水稻或烟草。 19. The method according to claim 18, wherein: said plant is tobacco or rice.
20.根据权利要求19所述的方法,其特征在于:所述植物为水稻日本晴或烟草NC89。 20. The method of claim 19, wherein: said plant is tobacco or rice Nipponbare NC89.
21.权利要求I所述的启动子或权利要求2的核酸构建体或者权利要求3或4的载体或权利要求5或6的重组细胞或植物愈伤组织或植物外植体或植物在调控植物中目的基因表达或植物育种中的用途,其中,所述植物愈伤组织或植物外植体或植物含有权利要求1 的启动子或权利要求2的核酸构建体或权利要求3或4的载体或权利要求5或6的重组细胞,所述植物单子叶植物或双子叶植物。 Promoter in claim I or claim 2, a nucleic acid construct or vector as claimed in claim or claim 3 or 4 in the regulation of the recombinant plant cell or plant callus or plant explant or a plant 21. 5 or claim 6, the use or expression of the gene in plant breeding, wherein the outer plant callus or plant explant or a plant comprising a promoter of claim 1 or claim 2 requires the nucleic acid construct or the vector of claim 3 or 4 or 5 or 6 recombinant cell, said plant monocot or dicot claims.
22.根据权利要求21 Claim 22. 21
23.根据权利要求22 23. The method of claim 22
24.根据权利要求23所述的用途,其特征在于所述的用途,其特征在于所述的用途,其特征在于:植物为稻属或烟草属。 24. The use according to claim 23, wherein said use, wherein said use, wherein: the plant is a rice or genus Nicotiana. :植物为水稻或烟草。 : Plant rice or tobacco. :植物为水稻日本晴或烟草NC89。 : Japan plant rice sunny or tobacco NC89.
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