CN102146392B - BgIosP516 promoter, preparation method and application of BgIosP516 promoter - Google Patents

BgIosP516 promoter, preparation method and application of BgIosP516 promoter Download PDF

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CN102146392B
CN102146392B CN201010614976XA CN201010614976A CN102146392B CN 102146392 B CN102146392 B CN 102146392B CN 201010614976X A CN201010614976X A CN 201010614976XA CN 201010614976 A CN201010614976 A CN 201010614976A CN 102146392 B CN102146392 B CN 102146392B
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promoter
plant
rice
recombinant
seq id
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CN102146392A (en
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张丰丰
张耕耘
束礼平
蔡雪梅
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深圳华大基因科技有限公司
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Abstract

The invention belongs to the field of molecular biology, and relates to a BgIosP516 promoter, a preparation method and application of the BgIosP516 promoter. Specifically, the promoter contains a nucleotide sequence shown as SEQ ID NO: 1, or has a variant with promoter function selected from the following nucleotide sequences: 1) a nucleotide sequence hybridized with the nucleotide sequence shownas SEQ ID NO: 1 under a high strict condition, 2) a nucleotide sequence for substitution, deletion and adding modification of one or more bases for the nucleotide sequence shown as SEQ ID NO: 1, and 3) a nucleotide sequence with at least 90 percent of sequence identity as the nucleotide sequence shown as SEQ ID NO: 1. The invention also relates to the preparation method of the promoter and application of the promoter in regulation and control of expression of target genes in a monocotyledonous plant or a dicotyledonous plant and in rice or tobacco breeding.

Description

—种启动子BglosP516、其制备方法及用途 - seed promoters BglosP516, their preparation and use

技术领域[0001] 本发明属于分子生物学领域,涉及一种启动子,具体地,涉及一种来源于水稻的启动子。 Technical Field [0001] The present invention belongs to the field of molecular biology, it relates to a promoter, in particular, relates to one kind of a promoter derived from rice. 本发明还涉及含有该启动子的重组载体、含有该重组载体的重组细胞、转化有所述启动子的愈伤组织、一种制备转基因植物的方法、以及所述启动子的制备方法及用途。 The present invention further relates to a recombinant vector containing the promoter, recombinant cells containing the recombinant vectors, calli transformed with the promoter of the promoter, preparation and use method of preparing a transgenic plant, and a promoter.

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(Plantbiotechnology, 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 ( Plantbiotechnology, 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(Plantubiquitinpromoter)、Actin 启动子(Plant Actin promoter)和Adh-I 启动子(Maize alcohol dehydrogenase [0005] monocot genes common promoters are: Ubi promoter (Plantubiquitinpromoter), Actin promoter (Plant Actin promoter) and Adh-I promoter (Maize alcohol dehydrogenase

I promoter)。 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,例如玉米基因组中的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,等等。 Currently, promoter sequences have been isolated from many ubiquitin gene, for example, 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 Mubl ubiquitin promoter, and so on. 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它单子叶植物寻找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 find Actin promoter by other monocots, 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.

[0011] 尽管已经有上述已知的单子叶植物的启动子,本发明人通过对水稻基因组的深入研究,提供了一种新的来源于单子叶植物的启动子,所述启动子能够用于调控单子叶植物甚至是双子叶植物中目的基因表达,为研究单子叶植物和双子叶植物中目的基因表达提供了一种新的工具和选择。 [0011] Despite the above known monocot promoters, the present inventors have intensive studies on the rice genome, there is provided a novel promoter derived from monocots, the promoter can be used regulation of monocots or even the gene expression dicots, provides a new tool for the study and select monocots and target gene expression in dicots.

发明内容 SUMMARY

[0012] 本发明人经过大量的试验和创造性的劳动,从水稻日本晴(Oryzasativa L. ssp.japonica cv. Nipponbare)中得到了一种启动子,并且本发明人惊奇地发现,该启动子不仅能够调控单子叶植物(例如水稻)中目的基因的表达,而且能够调控双子叶植物(例如烟草)中目的基因的表达。 [0012] The present invention after a large number of tests and creative work, from Nipponbare rice (Oryzasativa L. ssp.japonica cv. Nipponbare) obtained in a promoter, and the inventors surprisingly found that not only the promoter regulation of the expression of monocot plants (e.g., tobacco) gene of interest (e.g., rice) the expression of the gene of interest, but also capable of modulating a dicot. 由此提供了下述发明: Thus the invention provides the following:

[0013] 本发明的一个方面提供了一种具有SEQ ID NO :1所不核苷酸序列的启动子。 [0013] One aspect of the invention provides a method having the SEQ ID NO: 1 is not initiated promoter nucleotide sequence. 在本发明中,所述启动子的具体碱基序列长度为2533个碱基,如SEQ ID NO :I所示: In the present invention, the promoter nucleotide sequence of the specific length of the sub is 2533 bases, such as SEQ ID NO: I shown below:

[0014] TCTCTTGGTACTTCAGTTCGCTCATTTATTTCTCATAATTTAAATTTCAAAGTAATAAGCGATAGATCAGGATCCATGATTTGAACATGCAACTTTAAAGTAATGCAGTAAGATATAGATCGGTGTCCATGACTTGAACATGCATGAACAGTAACCCCACTCGTGAGTTAAAAAGATAGATCCATGATTTAAACATACATTCGCAGATATTTGATCATTATATGTTGTAATTATATTCTCATAAAAAAAACTAAAAATGCTTCATAGCATTTGTATATTATGCCAATATTGAGATAATTTCGATATCTCATATGTTTTAGATTGGAACAGATCACGAAAGTTTTTCTTCGAAAAGTTATGGTCATTAGGACATGATGGATTTGAGGTAACAAAATAGATGAGATTTATTAAAACCCTTGAAATAGTCCAACAATCCAGCGCTAACATTTAGCACAGTGTAATTCAAAAGAAGACAACCACAGAAACACACAGGGGATGTTTTAAACTCCAATTTTTATATATTTGGCATTATTCAAATTGCAAACAAATATGATGACTCTCTTTCTCAAAGAAACATATGCCGTTAGGGTGATCCCAGCGCTTCACTTAGAATGGTTTTTCATAGCATTAAATGATATGCCACGTAGGAAAAAACATGATGTGCCATTTTATTAACTTAGGAAAGAGAAGAAAATTGGGTCTTCTCAAAATGATCATCATCTAACAAAATGTCTCTTTGTTTATTTGTGCAAAGAGTAAATGTGCATCGTGCAATGTCATGCATGCTGCCAACCAAGATTAATTTCTATTTAGTTGAACCTGTGCAACATTTAAAGTGTGAAGTAATTTTTGTTGGACTTGAAGCGACAAAGCACCTTGGTCGATTAGGCCATCCCCAACCCATGGTGTCCATGGGTGGTGTCTAGAGCAGTGAGAGATCAATAAATGCATATATGGACACTACCTCCCCATTGCATCGTTTATATACCAGTT [0014] TCTCTTGGTACTTCAGTTCGCTCATTTATTTCTCATAATTTAAATTTCAAAGTAATAAGCGATAGATCAGGATCCATGATTTGAACATGCAACTTTAAAGTAATGCAGTAAGATATAGATCGGTGTCCATGACTTGAACATGCATGAACAGTAACCCCACTCGTGAGTTAAAAAGATAGATCCATGATTTAAACATACATTCGCAGATATTTGATCATTATATGTTGTAATTATATTCTCATAAAAAAAACTAAAAATGCTTCATAGCATTTGTATATTATGCCAATATTGAGATAATTTCGATATCTCATATGTTTTAGATTGGAACAGATCACGAAAGTTTTTCTTCGAAAAGTTATGGTCATTAGGACATGATGGATTTGAGGTAACAAAATAGATGAGATTTATTAAAACCCTTGAAATAGTCCAACAATCCAGCGCTAACATTTAGCACAGTGTAATTCAAAAGAAGACAACCACAGAAACACACAGGGGATGTTTTAAACTCCAATTTTTATATATTTGGCATTATTCAAATTGCAAACAAATATGATGACTCTCTTTCTCAAAGAAACATATGCCGTTAGGGTGATCCCAGCGCTTCACTTAGAATGGTTTTTCATAGCATTAAATGATATGCCACGTAGGAAAAAACATGATGTGCCATTTTATTAACTTAGGAAAGAGAAGAAAATTGGGTCTTCTCAAAATGATCATCATCTAACAAAATGTCTCTTTGTTTATTTGTGCAAAGAGTAAATGTGCATCGTGCAATGTCATGCATGCTGCCAACCAAGATTAATTTCTATTTAGTTGAACCTGTGCAACATTTAAAGTGTGAAGTAATTTTTGTTGGACTTGAAGCGACAAAGCACCTTGGTCGATTAGGCCATCCCCAACCCATGGTGTCCATGGGTGGTGTCTAGAGCAGTGAGAGATCAATAAATGCATATATGGACACTACCTCCCCATTGCATCGTTTATATACCAGTT TTCATAGGCAAAAAAAAATTAGTAGCTCTTTGCATGTAACATTAACAGAGGAGGGTCAGCTGTGGCAAGCAAGCAAGCAGACGCGGGAGGGGAGGCGCGCAGCAGGCATGGTGACGGGGTAGGGCGCAGCGGATCGAGATGCGAGGATTTTTTTTTGGGGGGGTGGGGTGGGGGGAGAGAAACGAGTCATCCAACCCAACGCGGATTTAGCTAGTTTCCAGTGCCTTGGAACAGCCGCCGAAACGGTGTCGCGCATGCGACCTGGTTTCTATGTTTTTGCTTCTTTCTCTTCTTTTATTCCGTTGCCACATCAACTTTTTGTCTAGTTGGCAGCCTAATTAATTCTATGGAAACCAGGTGACATGGAGGGTTGGGGACATGGTGGAAAAAACCGGAACGGGCCGACAGTTCAACCGGAAAAAACCAGAACCCGTTCAGTTCAAAAGAAAGACCGGACATGCATATGACCCGCTTTGAACCGGCAGAACCGGTCGGTTTTTCTATGAACCGGTCATTAAACCGTCCCCGGTTAGACCGAACAAGCCACAATAATCTTGAAATGGGCCTTGATGTGGCCCAATTGGTCTGCCTAGAGCGTTTTGGTTGGCAAAAATCAATCTCCTATTCTCGGCACGTGTGATATACAATGGTAAGTGAGATATACAATTCTCGGCACGGCTACATTACAAGGTGTCGCATTGTGTCAATGTTTGGTTAATTTGCTAGATTCACATAATACATGCCAGGAAGTTCAGAACAATGTGTTGCCTTTCACCGGAAAACTTTGTTGGAGCAAATGCCTTCTTCTTTTTTGCTTCTGCTTCTTGAGTCCATGTGGAGGAAGCAGTAGATAGCTGATGATATCAGGATTCCTTCTGTGTCTGTGTAGGTGTAGCAACACCACTATAATTTTTATTTAGCAACACAATATCAATTTGGTCTATAAAAGTATGAATTAAATCAATCCCCAACCACAATTAGAGTAAGTTGGTGAGTTATT TTCATAGGCAAAAAAAAATTAGTAGCTCTTTGCATGTAACATTAACAGAGGAGGGTCAGCTGTGGCAAGCAAGCAAGCAGACGCGGGAGGGGAGGCGCGCAGCAGGCATGGTGACGGGGTAGGGCGCAGCGGATCGAGATGCGAGGATTTTTTTTTGGGGGGGTGGGGTGGGGGGAGAGAAACGAGTCATCCAACCCAACGCGGATTTAGCTAGTTTCCAGTGCCTTGGAACAGCCGCCGAAACGGTGTCGCGCATGCGACCTGGTTTCTATGTTTTTGCTTCTTTCTCTTCTTTTATTCCGTTGCCACATCAACTTTTTGTCTAGTTGGCAGCCTAATTAATTCTATGGAAACCAGGTGACATGGAGGGTTGGGGACATGGTGGAAAAAACCGGAACGGGCCGACAGTTCAACCGGAAAAAACCAGAACCCGTTCAGTTCAAAAGAAAGACCGGACATGCATATGACCCGCTTTGAACCGGCAGAACCGGTCGGTTTTTCTATGAACCGGTCATTAAACCGTCCCCGGTTAGACCGAACAAGCCACAATAATCTTGAAATGGGCCTTGATGTGGCCCAATTGGTCTGCCTAGAGCGTTTTGGTTGGCAAAAATCAATCTCCTATTCTCGGCACGTGTGATATACAATGGTAAGTGAGATATACAATTCTCGGCACGGCTACATTACAAGGTGTCGCATTGTGTCAATGTTTGGTTAATTTGCTAGATTCACATAATACATGCCAGGAAGTTCAGAACAATGTGTTGCCTTTCACCGGAAAACTTTGTTGGAGCAAATGCCTTCTTCTTTTTTGCTTCTGCTTCTTGAGTCCATGTGGAGGAAGCAGTAGATAGCTGATGATATCAGGATTCCTTCTGTGTCTGTGTAGGTGTAGCAACACCACTATAATTTTTATTTAGCAACACAATATCAATTTGGTCTATAAAAGTATGAATTAAATCAATCCCCAACCACAATTAGAGTAAGTTGGTGAGTTATT GTAAAGCTCTGCAAAGTTAATTTAAAAGTTATTGCATTAACT TATTTCGTATCACAAACAAGTTTTCACAAGAGTATTAATGGAACAATGAAAACCATTGAACATACTATAATTTTTTTTCTTACTGAAATTATATAATTCAAAGAGCATAAACCCACACAGTCGTAAAGTTCCACGTGTAGTGCATTATCAAAATAATAGCTTACAAAACATAACAAACTTAGTTTCAAAAGTTGCAATCCTTATCACATTGACACATAAAGTGAGCGATGAGTCATGTCATTATTTTTTTGCTCACCATCATGTATATATGATGGGCATAAAAGTTACTTTGATGATGATATCAAAGAACATTTTTAGGTGCACCTAACAGAATATCCAAATAATATGACTCACTTAGATCCTAATATAGCATCAAGCAAAACTAACACTCTAAAGCAACCGATAGGGAAACATCTATAAATAGACAAGCATAATGAAAACCCTCCTCATCCTTCACACAATTCAAACATTATAGTTGAAGCATAGTAGTAGAATCCT(SEQ ID NO :1) GTAAAGCTCTGCAAAGTTAATTTAAAAGTTATTGCATTAACT TATTTCGTATCACAAACAAGTTTTCACAAGAGTATTAATGGAACAATGAAAACCATTGAACATACTATAATTTTTTTTCTTACTGAAATTATATAATTCAAAGAGCATAAACCCACACAGTCGTAAAGTTCCACGTGTAGTGCATTATCAAAATAATAGCTTACAAAACATAACAAACTTAGTTTCAAAAGTTGCAATCCTTATCACATTGACACATAAAGTGAGCGATGAGTCATGTCATTATTTTTTTGCTCACCATCATGTATATATGATGGGCATAAAAGTTACTTTGATGATGATATCAAAGAACATTTTTAGGTGCACCTAACAGAATATCCAAATAATATGACTCACTTAGATCCTAATATAGCATCAAGCAAAACTAACACTCTAAAGCAACCGATAGGGAAACATCTATAAATAGACAAGCATAATGAAAACCCTCCTCATCCTTCACACAATTCAAACATTATAGTTGAAGCATAGTAGTAGAATCCT (SEQ ID NO: 1)

[0015] 在本发明中,将SEQ ID NO :1所示的启动子序列称为启动子BgIosP516,或简称为P516启动子。 [0015] In the present invention, SEQ ID NO: 1 shown in the promoter sequence called the promoter BgIosP516, or simply referred to as P516 promoter.

[0016] 带有该启动子和⑶S(0 -葡萄糖苷酸酶,其英文名称为β-glucuronidase,简称为GUS)基因的水稻愈伤组织以及转基因水稻苗经GUS染色实验后,所述水稻愈伤组织和转基因水稻苗的根、叶等变为蓝色。 [0016] with the promoter and ⑶S - after (0-glucuronidase, which is the English name for β-glucuronidase, simply referred to as GUS) gene in rice callus and rice plants by transgenic GUS staining experiments, the more rice injured tissue and transgenic rice seedlings roots and leaves turn blue. 带有该启动子和GUS基因的转基因烟草苗经GUS染色实验后,所述转基因烟草苗的根、叶等也变为蓝色。 Transgenic tobacco seedlings with the promoter and the GUS gene after GUS staining experiments, transgenic tobacco seedlings the roots, leaves and the like also becomes blue.

[0017] 本发明的又一方面涉及具有与SEQ ID NO :1所示核苷酸序列互补的序列的启动子。 [0017] Yet another aspect of the present invention relates to SEQ ID NO: 1 nucleotide sequence complementary to the promoter sequence.

[0018] 本发明的又一方面还涉及SEQ ID NO :1所示启动子的具有启动子功能的选自如下的变体: [0018] Yet another aspect of the present invention also relates to SEQ ID NO: 1 selected from the following variants shown promoter having a promoter function:

[0019] I)在高等严紧条件下与SEQ ID NO :1所示的核苷酸序列杂交的核苷酸序列, [0019] I) at higher stringency conditions with SEQ ID NO: hybridizing to the nucleotide sequence shown in Figure 1,

[0020] 2)对SEQ ID NO :I所示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,和 [0020] 2) of SEQ ID NO: I shown in the nucleotide sequence of one or more base substitutions, deletions, additions modified nucleotide sequence, and

[0021] 3)与SEQ ID NO :1所不的核苷酸序列具有至少90%的序列同一'丨生的核苷酸序列。 [0021] 3) and SEQ ID NO: 1 having a nucleotide sequence is not the same 'nucleotide sequence Shu raw sequence at least 90%.

[0022] 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。 [0022] 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;O. 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;而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;. O 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; and the use of higher stringency conditions is determined 80% or more sequence identity to a nucleic acid sequence about the probe.

[0023] 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/或高温度条件。 [0023] For applications requiring high selectivity, typically desirable to employ relatively stringent conditions to form the hybrids, e.g., 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 HarborPress, 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 HarborPress, Plainview, NY) to provide a moderate stringency conditions include hybridization and including higher stringency.

[0024] 为便于说明,用于检测本发明的多核苷酸与其它多核苷酸杂交的合适的中度严紧条件包括: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。 [0024] For convenience of explanation, a polynucleotide hybridizing with a polynucleotide other suitable moderately stringent conditions for testing of the present invention comprises: a 5XSSC, 0 5% SDS, 1 OmM EDTA (. PH8 O) solution pre-wash; hybridization overnight at 50-65 ° C in 5XSSC; and subsequently by O. 1% SDS containing 2X is, 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,在另一个实施方案中,合适的高度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, in another embodiment, suitable highly stringent hybridization conditions include the conditions described above, except that the hybridization temperature was raised to 60-65 ° C, or for example 65-70 ° C.

[0025] 在本发明中,所述在高等严紧条件下与SEQ ID NO :I所示的核苷酸序列杂交的核苷酸序列,其具有与SEQ ID NO :1所示的核苷酸序列相同或相似的启动子活性。 [0025] In the present invention, the at higher stringency conditions to SEQ ID NO: hybridizing to the nucleotide sequence shown in I, having the SEQ ID NO: 1 nucleotide sequence shown in identical or similar promoter activity.

[0026] 在本发明中,所述对SEQ ID NO :I所示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2-30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。 [0026] In the present invention, the pair of SEQ ID NO: I shown in the nucleotide sequence of one or more base substitutions, deletions, additions modified nucleotide sequence, refers to separately or simultaneously in the 5 'and / or 3' end of said nucleotide sequence, or an internal and / sequences, for example, no more than 2-45, 2-30 or no more than, or no more than 3 to 20, or no more than 4 unsubstituted -15, or no more than 5-10, or no more than 6-8, respectively, in base-by consecutive integers represented by deletions, additions, modifications.

[0027] 在本发明中,所述对SEQ ID NO : I所示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 IDNO :1所示的核苷酸序列相同或相似的启动子活性。 [0027] In the present invention, the pair of SEQ ID NO: nucleotide sequence I is substituted as shown in the one or more bases, deletions, additions, and having a modified nucleotide sequence of SEQ IDNO: 1 identical or similar to the nucleotide sequence shown promoter activity.

[0028] 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SEQID NO :1的参考核苷酸序列至少具95%的“同一性”是指:在SEQ IDNO :1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5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。 [0028] will be described by a polynucleotide having a nucleotide sequence which it is, for example, and SEQID NO: 1 reference nucleotide sequence having at least 95% "identity" refers to: in SEQ IDNO: 1 is per each 100 nucleotides of the reference nucleotide sequence, the nucleotide sequence of the polynucleotide in addition contains up to 5 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95%相同的多核苷酸,参考序列中多达5%的核苷酸可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5% ;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5%。 In other words, to obtain nucleotide sequences with the same reference nucleotide sequence of a polynucleotide at least 95%, up to 5% 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 5% 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 5% 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,或以一个或多个邻近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.

[0029] 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BLAST 2. 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. Biol. 215 :403_410。 [0029] In the present invention, for determining the percent similarity of sequence identity and sequence algorithm such as BLAST and BLAST 2. O algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25 = 3389- . 3402 and Altschul et al. (1990) J. Mol Biol 215:. 403_410. 采用例如文献中所述或者默认参数,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。 Or described in the literature, for example, using default parameters, BLAST and BLAST 2. O may be used to determine percent identity of a nucleotide sequence of the invention. 执行BLAST分析的软件可以通过国立生物技术信息中心(NCBI)为公众所获得。 Performing BLAST analysis software can be obtained to the public by the National Center for Biotechnology Information (NCBI).

[0030] 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。 [0030] 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.

[0031] 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具有与SEQ ID NO :1所示的核苷酸序列相同或相似的启动子活性。 [0031] In the present invention, with the SEQ ID NO: nucleotide sequence identity to the nucleotide sequence having at least 90% of the 1 having SEQ ID NO: 1 nucleotide sequence shown in identical or similar promoter activity.

[0032] 本发明中,所述的启动子来源于单子叶植物;具体地,为禾本科;更具体地,为稻属或水稻,例如所述水稻为日本晴(Oryza sativaL. ssp. japonica cv. Nipponbare)。 [0032] In the present invention, the promoter is derived from a monocot; specifically, Poaceae;. More specifically, as ssp japonica cv Oryza or rice, for example, is the Nipponbare rice (Oryza sativaL.. Nipponbare). 水稻日本晴种子已经于2009年12月18日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCCNO :P200910,保藏单位地址为中国.武汉.武汉大学,邮编430072 ;水稻日本晴的保藏记载在公开号为CN 101864418A的中国专利申请(申请号为200910249575. 6,公开日为2010年10月20日)中。 Rice Nipponbare seeds already on December 18, 2009 preserved in China Type Culture Collection (CCTCC), under the accession number CCTCCNO: P200910, the depository institution address China Wuhan, Wuhan University, zip code 430072, China; rice Nipponbare preserved record in. Publication No. CN 101864418A of Chinese patent application (application No. 200910249575.6, publication date October 20, 2010) in.

[0033] 本发明的又一方面,涉及一种核酸构建体,包含本发明的启动子,以及与启动子可操作地连接的基因,其中启动子与基因的来源相同或者不同。 [0033] In yet another aspect the present invention relates to a nucleic acid construct genome, according to the present invention comprising a promoter and operably linked to a promoter, wherein the promoter is the same or different gene sources.

[0034] 在本发明中,术语“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。 [0034] In the present invention, the term "operably linked" refers to two or more spatial regions or functional nucleotide sequence of a nucleic acid alignment. 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.

[0035] 所述“可操作地连接”可以通过基因重组的手段实现,具体地,所述核酸构建体为重组核酸构建体。 [0035] The "operably connected" may be implemented by means of genetic recombination, in particular, the nucleic acid construct is a recombinant nucleic acid construct thereof. 在本发明一个具体的实施方式中,所述基因为⑶S基因。 In a particular embodiment of the present invention, the gene is a gene ⑶S.

[0036] 本发明的又一方面,还涉及一种含有本发明的启动子或核酸构建体的载体。 [0036] In yet another aspect of the present invention further relates to a promoter comprising a nucleic acid of the present invention or a vector construct. 具体地,为重组载体。 Specifically, a recombinant vector. 所述重组载体可以通过将上述启动子或核酸构建体插入到克隆载体或表达载体而得到。 The recombinant vector can be obtained by the above-mentioned promoter or a nucleic acid construct is inserted into a cloning vector or an expression vector obtained.

[0037] 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 SimpleVecter、pMD19_T Simple Vecter 等。 [0037] The vector construct suitable for cloning the recombinant vector of the present invention include, but are not limited to, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T SimpleVecter, pMD19_T Simple Vecter like.

[0038] 适于构建本发明所述的表达载体包括但不限于,例如:pBI121、pl3W4、pGEM等。 [0038] Expression vectors suitable for constructing the present invention include, but are not limited to, for example: pBI121, pl3W4, pGEM like.

[0039] 在本发明的一个实施方案中,所述重组载体为pMD18-T+P516重组载体或p8+P516 [0039] In one embodiment of the invention, the recombinant vector pMD18-T + P516 is a recombinant vector or p8 + P516

重组载体。 The recombinant vector.

[0040] 本发明的又一方面还涉及含有本发明的启动子或核酸构建体或重组载体的重组细胞。 [0040] Yet another aspect of the present invention further relates to a promoter or a nucleic acid construct or a recombinant cell containing the recombinant vectors of the present invention. 所述重组细胞可以通过将本发明的启动子或核酸构建体或重组载体转化至宿主细胞而得到。 The recombinant cell can be a promoter of the present invention or a nucleic acid construct or recombinant vector was transformed into host cells obtained.

[0041] 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:根癌农杆菌细胞LBA4404、EHA105、GV3101 等。 [0041] The present invention is suitable for constructing the recombinant cell host cells include but are not limited to, for example: cells Agrobacterium tumefaciens LBA4404, EHA105, GV3101 and the like.

[0042] 在本发明的一个实施方案中,所述重组细胞为重组大肠杆菌细胞DH5 α -Ρ516或重组根癌农杆菌(Agrobacterium tumefaciens) EHA105-P516。 [0042] In one embodiment of the invention, the recombinant cell is a recombinant cell E. coli DH5 α -Ρ516 or recombinant Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105-P516. 根癌农杆菌EHA105Agrobacterium tumefaciens EHA105已经于2009年12月24日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO :M 209315,保藏单位地址为中国.武汉.武汉大学,邮编430072 ;农杆菌EHA105的保藏记载在公开号为CN 101864418A的中国专利申请(申请号为200910249575. 6,公开日为2010年10月20日)中。 Agrobacterium tumefaciens EHA105Agrobacterium tumefaciens EHA105 already in December 24, 2009 deposited at the China Center for Type Culture Collection (CCTCC), under the accession number CCTCC NO: M 209315, the depository institution address China Wuhan University, Wuhan, zip code 430072, China;. Agrobacterium EHA105 deposited record in Publication No. CN 101864418A of Chinese patent application (application No. 200910249575.6, publication date October 20, 2010) in. [0043] 本发明的又一方面还涉及一种含有本发明的启动子或本发明的核酸构建体或本发明的载体或本发明的重组细胞的植物愈伤组织或植物外植体或植物。 [0043] Yet another aspect of the present invention further relates to a nucleic acid or the promoter of the invention constructed according to the present invention contains a plant callus or plant explant or a plant cell or recombinant vector of the invention or the present invention. 具体地,所述植物为被子植物,更具体地为单子叶植物或双子叶植物。 In particular, the plant is the angiosperms, more particularly a monocot or a dicot. 在本发明的一个实施方案中,所述单子叶植物为水稻。 In one embodiment of the invention, the monocotyledonous plant is rice. 所述水稻包括但不限于,例如: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 (上述水稻品种均可购自安徽徽商农家福有限公司)等。 The rice include, but are not limited to, for example: 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, 88 Qianyou , II preferably 416, II preferably 107, II preferably 128, II preferably 718, Zhunliangyou 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, Wandao 195, Wandao 197, Wandao 199, Wandao 201, Wandao 203, Wandao 205, Wandao 207, and Jinyuan 101 (above rice varieties are available from Anhui Huishang farmer Fu Co., Ltd.) and so on. 在本发明的又一实施方案中,所述水稻为日本晴。 In yet another embodiment of the present invention is the rice Nipponbare. 所述双子叶植物为烟草,具体为烟草K326、K346、K394、NC82、NC89、G140、G28、G80、中烟90、Cokerl76、或CV87。 Said dicot tobacco, particularly tobacco K326, K346, K394, NC82, NC89, G140, G28, G80, smoke 90, Cokerl76, or CV87. 其中,烟草NC89 种子已经于2010年11月12日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO :P201017,保藏单位地址为中国.武汉.武汉大学,邮编430072。 Among them, tobacco NC89 seed has been on November 12, 2010 preserved in China Center for Type Culture Collection (CCTCC), under the accession number CCTCC NO: P201017, the depository institution address, Wuhan University, Wuhan, China, Post Code 430 072...

[0044] 本发明的又一发明,涉及用于扩增本发明的启动子的引物对,其具有Seq ID NO :2和Seq ID NO :3所示的核苷酸序列;具体地,所述引物对为在Seq ID NO :2和Seq ID NO: 3所示核苷酸序列的5'端分别连接有限制性酶切位点和/或保护碱基;更具体地,所述引物对具有Seq ID No :4和Seq ID No :5所示的核苷酸序列。 [0044] The present invention further relates to the promoter of the present invention for amplification of the primer having Seq ID NO: 2 and Seq ID NO: 3 is the nucleotide sequence shown; in particular, the primer pair as Seq ID NO: 2 and Seq ID NO: 3 nucleotide sequence 5 'ends are connected restriction sites and / or protection bases; more specifically, the primer pair having Seq ID No:. 4 and Seq ID No: 5 nucleotide sequence.

[0045] TCTCTTGGTACTTCAGTTCG(SEQ ID NO :2)。 [0045] TCTCTTGGTACTTCAGTTCG (SEQ ID NO: 2).

[0046] AGGATTCTACTACTATGCTTC(SEQ ID NO :3)。 [0046] AGGATTCTACTACTATGCTTC (SEQ ID NO: 3).

[0047] CCGgaattcTCTCTTGGTACTTCAGTTCG(SEQ ID NO :4),其中小写字母代表EcoRI 酶切位点。 [0047] CCGgaattcTCTCTTGGTACTTCAGTTCG (SEQ ID NO: 4), wherein the lower case letters represent EcoRI restriction sites.

[0048] GCctgcagAGGATTCTACTACTATGCTTC(SEQ ID NO :5),其中小写字母代表PstI 酶切位点。 [0048] GCctgcagAGGATTCTACTACTATGCTTC (SEQ ID NO: 5), wherein the lower case letters represent PstI restriction sites.

[0049] 本发明的又一发明,涉及一种制备本发明的启动子的方法,包括下述步骤: [0049] The present invention further relates to a process for the preparation of a promoter of the present invention, comprising the steps of:

[0050] I)根据SEQ ID NO :1所示的核苷酸序列,设计PCR扩增引物对;具体地,PCR扩增引物对为SEQ ID NO :2 和SEQ ID NO :3,或者为SEQ ID NO :4 和SEQ ID NO :5 ; [0050] I) according to SEQ ID NO: 1 nucleotide sequence, designing PCR amplification primer pair; Specifically, PCR amplification of the primer pair is SEQ ID NO: 2 and SEQ ID NO:. 3, or as SEQ ID NO: 4 and SEQ ID NO: 5;

[0051] 2)以水稻日本晴基因组DNA为模板,使用步骤I)中所设计的PCR扩增引物对进行PCR扩增。 [0051] 2) In transgenic rice genomic DNA Partly Japan as a template, in step I) designed for the PCR amplification primers for PCR amplification.

[0052] 本领域技术人员周知,可以根据待扩增的目的核苷酸序列按照碱基互补原则设计相应的PCR扩增引物对。 [0052] known to those skilled in the art can design the nucleotide sequences of the corresponding PCR amplification primers according to the principle of base complementarity to be amplified according to the purpose. 在本发明的一个实施方案中,所述PCR扩增引物对如SEQ ID NO: In one embodiment of the invention, the PCR amplification primer pair as SEQ ID NO:

2 和SEQ ID NO :3 所示。 2 and SEQ ID NO: 3 shown in FIG.

[0053] 本发明的又一方面,还涉及一种调控植物中基因表达的方法,所述方法包括将本发明的启动子或本发明的核酸构建体或本发明的载体或本发明的重组细胞导入植物的步骤,具体地,导入植物愈伤组织;具体地,包括使用本发明的启动子转化植物愈伤组织的步骤。 [0053] In yet another aspect of the present invention further relates to a method for regulation of gene expression in plants, the recombinant cell comprising a vector nucleic acid promoter of the invention or constructs of the invention or the invention or the invention the step of introducing a plant, in particular, into a plant calli; specifically, the present invention includes the use of a promoter in the transformed plant callus step. 在本发明的一个实施方案中,所述植物愈伤组织的转化利用了含有本发明的启动子的重组细胞。 In one embodiment of the present invention, the transformed plant calli using a recombinant cell containing the promoter according to the present invention. 在本发明的一个实施方案中,所述植物愈伤组织的转化过程中利用了前述的重组农杆菌EHA105-P516。 In one embodiment of the present invention, the conversion process plant callus utilizes the recombinant Agrobacterium EHA105-P516. 具体地,所述植物为被子植物,更具体地,为单子叶植物或双子叶植物。 In particular, the plant is the angiosperms, and more specifically, a monocot or a dicot. 在本发明的一个具体实施方案中,所述单子叶植物为水稻,具体地,所述水稻为日本晴。 In one particular embodiment of the invention, the monocotyledonous plant is rice, in particular, is the Nipponbare rice. 在本发明的一个具体实施方案中,所述双子叶植物为烟草,例如烟草NC89。 In one particular embodiment of the invention, the dicot tobacco, such as tobacco NC89.

[0054] 在本发明中,可采用植物基因转化技术将目的基因插入到植物基因组中,包括农杆菌介导转化、病毒介导的转化、显微注射、粒子轰击、基因枪转化和电穿孔等。 [0054] In the present invention, can be inserted into the plant transformation the gene technology into a plant genome, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation, etc. . 本领域周知,农杆菌介导的基因转化常被用于单子叶植物或双子叶植物的基因转化,但其它转化技术也可用于本发明的启动子的转化。 Known in the art, the gene is often Agrobacterium mediated genetic transformation of monocotyledonous plants or dicotyledonous plants, but other transformation techniques may also be used to transform the promoter of the present invention. 当然,适于本发明的转化单子叶或双子叶植物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, the transformation of monocotyledonous or dicotyledonous plant another method of the present invention is suitable for particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos. 另外,还可以采用的转化单子叶或双子叶植物的方法是原生质体转化。 A method for transforming monocots or dicots addition, may be employed 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.

[0055] 本发明中,可利用本发明的启动子调控目的基因表达的所述单子叶植物包括但不限于,例如:水稻、小麦、玉米、小米、甘蔗、高粱、大麦等。 [0055] In the present invention, the present invention can utilize promoters regulating the expression of the gene of monocots include, but are not limited to, for example: rice, wheat, maize, millet, sugarcane, sorghum, barley, and the like.

[0056] 烟草是典型的基因工程模式植物。 [0056] 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. 本发明中,可利用本发明的启动子调控目的基因表达的所述双子叶植物包括但不限于,例如:烟草、大豆,马铃薯、蚕豆、萝卜、花生等。 In the present invention, the present invention can utilize promoters regulating the expression of the gene dicots include, but are not limited to, for example: tobacco, soybean, potato, beans, carrots, peanuts and the like.

[0057] 本发明的又一方面还涉及本发明的启动子或本发明的核酸构建体或本发明的载体或本发明的重组细胞或本发明的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中的用途。 [0057] Yet another aspect of the present invention also the outer plant calli or plant promoter of the present invention relates to a nucleic acid of the present invention or the construct or vector of the invention or a recombinant cell of the invention of the present invention or plants or explants regulation of expression of genes in plants in use. 在本发明的一个实施方案中,利用本发明所述启动子调控的目的基因是GUS。 In one embodiment of the invention, the use of the present invention is the promoter of the gene is regulated GUS. 在本发明的一个实施方案中,所述单子叶植物为水稻,具体地,所述水稻为日本晴。 In one embodiment of the invention, the monocotyledonous plant is rice, in particular, is the Nipponbare rice. 在本发明的一个实施方案中,所述双子叶植物为烟草,例如烟草NC89。 In one embodiment of the invention, the dicot tobacco, such as tobacco NC89.

[0058] 为实现上述调控目的基因表达的目的,本发明所述启动子可以以单拷贝和/或多拷贝的形式应用,也可以与现有技术中已知的启动子联用。 [0058] To achieve the above object of the purpose of the regulation of gene expression, the promoter of the present invention may be applied as a single copy and / or copying, may be used in combination with known prior art promoters.

[0059] 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本发明的重组细胞或本发明的植物愈伤组织或植物外植体或植物的步骤。 [0059] A method of preparing a transgenic plant, comprising culturing a recombinant cell of the invention or the outer plant callus or plant explants, or plant according to the present invention under conditions effective to produce plants step.

[0060] 本发明的又一方面还涉及本发明的启动子或本发明的核酸构建体或本发明的载体或本发明的重组细胞或本发明的植物愈伤组织或植物外植体或植物在植物育种中的用途;具体地,所述植物为单子叶植物或双子叶植物;更具体地,所述单子叶植物为稻属植物或水稻,所述双子叶植物为烟草属植物;进一步具体地,所述水稻为日本晴、中花9、中花 [0060] Yet another aspect of the present invention also the outer plant calli or plant promoter of the present invention relates to a nucleic acid of the present invention or the construct or vector of the invention or a recombinant cell of the invention of the present invention or plants or explants breeding of plants; in particular, the plant is a monocot or dicotyledonous plants; more specifically, the rice plant is a monocotyledonous plant or rice, said dicot tobacco plants; more specifically the rice Nipponbare is, the flowers 9, Zhonghua

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、或津原101,所述烟草属植物为烟草K326、K346、K394、NC82、NC89、G140、G28、G80、中烟90、Cokerl76、或CV87。 10, 11 Zhonghua, 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 , Zhunliangyou 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, Wandao 195, Wandao 197, Wandao 199, Wandao 201, Wandao 203, Wandao 205, Wandao 207, or Jinyuan 101, the genus Nicotiana tobacco K326, K346, K394, NC82 , NC89, G140, G28, G80, smoke 90, Cokerl76, or CV87. 在本发明的一个实施方案中,所述水稻为日本晴。 In one embodiment of the present invention is the rice Nipponbare. 在本发明的一个实施方案中,所述烟草为烟草NC89。 In one embodiment of the present invention, the tobacco is tobacco NC89.

[0061] 本发明的启动子可成为一种新的启动子作为单子叶植物(尤其是水稻)或双子叶植物(尤其是烟草)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,从而极大的缩短优良品种的选育时间。 [0061] The promoter of the invention may become a new promoter as a tool for monocot plants (particularly in rice) or dicotyledonous plants (especially tobacco) transgenic promoter screening for carrying out genetic transformation seedlings low expression in plants breeding floral organs and other molecules to facilitate abortion, which 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、小麦、玉米、小米、甘蔗、高粱、大麦等单子叶植物以及烟草、大豆、马铃薯、蚕豆、萝卜、花生等双子叶植物。 Promoter of the present invention can be widely used in cultivation of rice, wheat, maize, millet, sugarcane, sorghum, barley and other monocotyledons and tobacco, soybean, potato, beans, carrots, peanuts and other dicots.

[0062] 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,例如日本晴、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。 [0062] In the present invention, the term "monocots", in particular, may be a gramineous plant, more specifically, the case of rice plants such as rice, including but not limited to, e.g. Nipponbare, Zhonghua 9, Zhonghua 10, 11 Zhonghua, 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 , Zhunliangyou 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, Wandao 195, 197 Wandao, Wandao 199, 201 Wandao, Wandao 203, 205 Wandao, Wandao 207, 101 and the original Tianjin.

[0063] 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,可以为烟草属植物例如烟草,包括但不限于烟草K326、K346、K394、NC82、NC89、G140、G28、G80、中烟90、Cokerl76、*CV87。 [0063] In the present invention, the term "dicotyledonous plant", specifically, Solanaceae, more specifically, for example tobacco plants, including but not limited to tobacco K326, K346, K394, NC82, NC89 tobacco , G140, G28, G80, smoke 90, Cokerl76, * CV87.

[0064] 发明的有益效果 [0064] Advantageous Effects of Invention

[0065] 本发明提供了一种新的启动子。 [0065] The present invention provides a new promoter. 所述启动子不仅能够调控单子叶植物(例如水稻)中目的基因的表达,而且能够调控双子叶植物(例如烟草)中目的基因的表达。 The promoter is not only capable of regulating the expression of monocot plants (e.g. rice) in the gene of interest, and capable of regulating the expression of dicotyledonous plants (e.g., tobacco) in the gene of interest. 具体地,所述启动子能够在水稻和基因工程模式植物-烟草中均能够调控GUS基因的表达。 In particular, the promoter capable of rice plants and genetic engineering model - GUS gene expression in both tobacco can be regulated.

附图说明 BRIEF DESCRIPTION

[0066] 图I :用于构建p8质粒的pCAMBIA-1301质粒示意图。 [0066] FIG. I: a schematic diagram of plasmid pCAMBIA-1301 p8 plasmid construct.

[0067] 图2:p8质粒示意图。 [0067] FIG. 2: p8 plasmid representation.

[0068] 图3 :p8质粒示意图中多克隆位点和⑶S序列部分的示意图。 [0068] FIG 3: a schematic view of the multiple cloning site and the sequence portion ⑶S plasmid p8 FIG.

[0069] 图4 :经转化的水稻愈伤组织的GUS染色结果。 [0069] Fig 4: GUS staining was transformed rice callus. 其中,由带有本发明所述启动子P516序列的重组根癌农杆菌P8+P516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 Agrobacterium tumefaciens P516 promoter sequences of the present invention by having P8 + transformed rice callus P516 (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.

[0070]图5 :经过转化的转基因水稻苗的根的GUS染色结果。 [0070] FIG 5: Transgenic rice plants through the root transformed results of GUS staining. 其中,由带有本发明所述启动子P516序列的重组根癌农杆菌P8+P516转化的水稻苗的根(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转化的水稻苗的根(对照,左)经⑶S染色后根的颜色未发生变化。 Wherein the P516 promoter sequence with the present invention by recombinant Agrobacterium P8 + P516 transformed rice seedling root (right) after ⑶S stained a blue color; without recombinant promoter sequence of the invention rice seedling root tumefaciens transformation P8 (control, left) after the root ⑶S dyed color does not change.

[0071]图6 :经过转化的转基因水稻苗的叶的GUS染色结果。 [0071] Figure 6: After GUS staining of transgenic rice plants transformed leaves. 其中,由带有本发明所述启动子P516序列的重组根癌农杆菌P8+P516转化的水稻苗的叶(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转化的水稻苗的叶(对照,左)经GUS染色后叶的颜色未发生变化。 Wherein, starting from the leaves with the present invention, the recombinant Agrobacterium tumefaciens promoter sequence P8 + P516 P516 transformed rice plants (right) after ⑶S stained a blue color; without recombinant promoter sequence of the invention P8 rice plants transformed with Agrobacterium tumefaciens leaf (control, left) after GUS staining leaf color change does not occur.

[0072]图7 :经过转化的转基因烟草苗的叶的GUS染色结果。 [0072] Figure 7: after GUS staining of transgenic tobacco leaves transformed seedlings. 其中,由带有本发明所述启动子P516序列的重组根癌农杆菌p8+P516转化的烟草苗的叶(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转化的烟草苗的叶(对照,左)经GUS染色后叶的颜色未发生变化。 Wherein, starting from the leaves of the seedlings of tobacco with the present invention, the recombinant Agrobacterium tumefaciens promoter sequence p8 + P516 P516 transformed (right) after ⑶S stained a blue color; without recombinant promoter sequence of the invention P8 transformed Agrobacterium tumefaciens leaf tobacco seedlings (control, left) after GUS staining leaf color change does not occur.

[0073]图8 :经过转化的转基因烟草苗的根的GUS染色结果。 [0073] Figure 8: After the root of transgenic tobacco seedlings transformed with the GUS staining. 其中,由带有本发明所述启动子P516序列的重组根癌农杆菌p8+P516转化的烟草苗的根(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转化的烟草苗的根(对照,左)经GUS染色后叶的颜色未发生变化。 Wherein the present invention is initiated by tobacco seedlings with Agrobacterium tumefaciens recombinant promoter sequence p8 + P516 P516 transformed root (right) after ⑶S stained a blue color; without recombinant promoter sequence of the invention P8 root Agrobacterium tumefaciens transformed tobacco seedlings (control, left) after GUS staining leaf color change does not occur.

[0074] 本发明涉及保藏的生物材料: [0075] 烟草NC89种子,2010年11月12日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO :P201017,保藏单位地址为中国.武汉.武汉大学,邮编430072。 [0074] The present invention relates to deposited biological material: [0075] tobacco NC89 seed, November 12, 2010 preserved in China Type Culture Collection (CCTCC), under the accession number CCTCC NO: P201017, preservation Address for the Chinese. Wuhan, Wuhan University, zip code 430072. 具体实施方式 Detailed ways

[0076] 下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,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。 [0076] The following embodiments in conjunction with embodiments of the present invention will be described in detail, 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.

[0077] 实施例中涉及的N6D培养基、YM液体培养基、YM固体培养基、AAM培养基、N6-AS共培养基、MS-R分化培养基、1/2MS生根培养基,其具体配方可以参考《分子克隆实验指南》或者参考公开号为CN101864418A的中国专利申请(申请号为200910249575. 6,公开日为2010 年10 月20 日)。 [0077] Example involved N6D medium, YM broth, YM solid medium, the AAM medium, N6-AS medium were, MS-R differentiation medium, 1 / 2MS rooting medium, the specific formula you can refer to "molecular cloning a Laboratory Manual" or refer to Publication No. CN101864418A of Chinese patent application (application No. 200910249575.6, publication date October 20, 2010).

[0078] 实施例I :P516启动子片段的PCR扩增和dMD18_T+P516重鉬载体的构律 PCR amplification and dMD18_T P516 + P516 promoter fragment reconstructed carrier molybdenum law: [0078] Example I

[0079] I) PCR 扩增 [0079] I) PCR Amplification

[0080] 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EcoRI和保护碱基,下游引物R1,加限制性酶切位点PstI和保护碱基)。 [0080] using plant genomic DNA extraction kit (TIANGEN novel plant genomic DNA extraction kit, catalog number: DP320-02) extracting clear genomic DNA of rice of Japan, according to the promoter sequence of rice Nipponbare gDNA in, respectively, inclusive design one pair of specific PCR amplification primers (upstream primer F1, adding restriction enzyme sites EcoRI and protection bases downstream primer R1, adding restriction enzyme sites PstI and protection bases). 以上述提取的水稻日本晴的gDNA为模板,使用高保真ExTaq (TaKaRa,DRR100B)聚 Mostly of the extracted Japanese rice gDNA as template, high fidelity ExTaq (TaKaRa, DRR100B) poly

[0081] 合酶进行PCR扩增。 [0081] PCR amplification synthase. 扩增启动子的PCR体系如下: PCR amplification of the promoter system as follows:

[0082] 组成 体积(μ I) [0082] Composition Volume (μ I)

[0083]基因组 DNA O. 2 [0083] Genomic DNA O. 2

[0084] dNTPs (2. 5mM) 2 [0084] dNTPs (2. 5mM) 2

[0085] IOXEx Buffer (含续离子) 2.5 [0085] IOXEx Buffer (Continued containing ions) 2.5

[0086]引物 Fl (50 μ M) I [0086] primers Fl (50 μ M) I

[0087]引物 Rl (50 μ Μ) I [0087] Primer Rl (50 μ Μ) I

[0088] Ex taq O. 2 [0088] Ex taq O. 2

[0089] ddH20 补满至总体积25 μ I [0089] ddH20 fill up to a total volume of 25 μ I

[0090] PCR扩增程序为:94°C预变性5min,然后以94°C变性45s,55°C退火50s,72°C延伸90s,进行35个反应循环,最后72°C延伸7min。 [0090] PCR amplification program: 94 ° C denaturation for 5min, then 94 ° C denaturation 45s, 55 ° C annealing 50s, 72 ° C 90s extends, by 35 reaction cycles, 72 ° C final extension 7min.

[0091]其中,上游引物Fl :CCGgaattcTCTCTTGGTACTTCAGTTCG(SEQ ID NO :4),其中小写字母代表EcoRI酶切位点。 [0091] wherein, upstream primer Fl: CCGgaattcTCTCTTGGTACTTCAGTTCG (SEQ ID NO: 4), wherein the lower case letters represent EcoRI restriction sites.

[0092]下游引物 Rl :GCctgcagAGGATTCTACTACTATGCTTC(SEQ ID NO :5),其中小写字母代表PstI酶切位点。 [0092] Lower Primer Rl: GCctgcagAGGATTCTACTACTATGCTTC (SEQ ID NO: 5), wherein the lower case letters represent PstI restriction sites.

[0093] PCR扩增产物经1.0%琼脂糖凝胶电泳分离,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。 [0093] PCR amplification products were separated on 1.0% agarose gel electrophoresis, agarose gel TIANGEN using DNA extraction kit (catalog number: DP209-03) recovering purified.

[0094] 2)pMD18_T+P516重组载体的构建 [0094] 2) Construction of recombinant vector pMD18_T + P516

[0095] 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,测序结果证明得到的序列准确。 [0095] 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, sequencing results demonstrate sequence obtained accurately.

[0096] 其中,T/A克隆的连接条件如下: [0096] where, T / A cloning connection conditions were as follows:

[0097] T/A 连接体系: 10 μ I[0098] pMD18-T I μ I [0097] T / A connection system: 10 μ I [0098] pMD18-T I μ I

[0099] 2*solution I 5 μ I [0099] 2 * solution I 5 μ I

[0100] PCR扩增产物(回收插入片段) 10_20ng,根据其浓度定 [0100] PCR amplification product (recovery insert) 10_20ng, according to a given concentration of

[0101] ddH20 补齐至10 μ I [0101] ddH20 filled to 10 μ I

[0102] 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+P516重组载体。 [0102] In connection to the 16 ° C thermostat bath Intelligent energy (Ningbo Chicago, SDC-6) in more than 8h to give PMD18-T + P516 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:

[0103] 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+P516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。 [0103] 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 + P516 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+P516克隆载体的重组大肠杆菌,命名为DH5 a -P516。 E. coli containing the recombinant pMD18-T + P516 cloning vector, designated as DH5 a -P516. 深圳华大基因科技股份有限公司对pMD18_T+P516克隆载体中的P516进行测序,证明获得的PMD18-T+P516克隆载体中P516启动子序列正确。 BGI Shenzhen Technology Co., Ltd. sequenced pMD18_T + P516 cloning vector P516, PMD18-T + P516 cloning vector proof obtained P516 promoter sequence correctly.

[0104] 实施例2 :p8+P516重纟目载体的构律 [0104] Example 2: configuration law p8 + P516 heavy mesh support Si

[0105] 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P516的大肠杆菌DH5a -P516中提取带有本发明P516启动子序列的克隆载体PMD18-T+P516 ;纯化后用相应的限制性内切酶EcoRI (NEB)和PstI (NEB)进行酶切,回收相应的启动子插入片段,并分别与P8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。 [0105] according to general TIANGEN plasmid mini kit: Manual (Cat. No. DP103_03), the conversion from Example I Construction of a promoter of E. coli DH5a -P516 P516 extracted with clones P516 promoter sequence of the invention vector PMD18-T + P516; purified enzyme EcoRI (NEB) using the corresponding restriction and PstI (NEB) was digested recovered corresponding promoter insert fragment, and cut with the same restriction plasmid P8 respectively after enzyme digestion recovery of large vector fragments were ligated.

[0106] 将所得连接产物p8+P516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。 [0106] The resulting ligation product was p8 + P516 recombinant vector according to "Molecular Cloning A Laboratory Manual" (third edition, Academic Press) prepared as shown in Method calcium chloride competent cells of DH5a, 37 ° C culture inverted 16-24h , after the transformant to grow until colonies were picked for PCR analysis of monoclonal and enzyme digestion.

[0107] 具体操作如下: [0107] Specific operation is as follows:

[0108] 1)ρ8质粒构建 [0108] 1) ρ8 plasmid

[0109] 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 CAMBIABios (biological open source) Licensee, Australia)按照如下方式改造并构建的,具体说明如下: [0109] 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 CAMBIABios (biological open source) Licensee, Australia) renovation and construction in the following ways, as follows:

[0110] 用Kpn I/Nco I (NEB)双酶切质粒pCAMBIA-1301 (参见图I),回收大片段。 [0110] 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:GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGTCGACCATGG(SEQ ID NO :6)(包含的酶切位点是Kpn I/Hind ΙΙΙ/Spe I/Sbf I/Pst I/XbaI/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 sequence: GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGTCGACCATGG (SEQ ID NO: 6) (contains the restriction site Kpn I / Hind ΙΙΙ / Spe I / Sbf I / Pst I / XbaI / 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 cable Leybold Ltd. purchased). 用引物GCTTCCGGCTCGTATGTTGT(SEQ ID NO :7)和GAGTCGTCGGTTCTGTAAC(SEQIDNO :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(参见图2)。 Primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO: 7) and GAGTCGTCGGTTCTGTAAC (SEQIDNO: 8) transformants were selected by PCR detection method is amplified fragment with 350bp of transformants is the multiple cloning site and the need to build GUS sequence p8 plasmid transformants (see FIG. 2). [0111] 所述p8质粒中的多克隆位点和⑶S序列的长度2353碱基,如SEQID NO :9所示(参见图3): [0111] The plasmid p8 2353 bases in length and ⑶S cloning site sequences, such as SEQID NO: 9 shown (see FIG. 3):

[0112] [0112]

Figure CN102146392BD00141

GAGGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGA TCTTTCTTTAAA CTGA TCTA TTTTTTAA TTGA TTGGTTA TGGTGTAAA TATTA CA TA GCTTTAA CTGA TAA TCTGA TTA CTTTA TTTCGTGTGTCTA TGA TGA TGA TGA TAGTTA CA GAA CCGA CGA CTCGTCCGTCCTGTA GAAA CCCCAA CCCGTGAAA TCAAAAAA CTCGACGGCCTGTGGGCA TTCAGTCTGGA TCGCGAAAA CTGTGGAA TTGA TCA GCGTTGGTGGGAAA GCGCGTTA CAA GAAA GCCGGGCAA TTGCTGTGCCA GGCA GTTTTAA CGA TCA GTTCGCCGA TGCA GA TA TTCGTAA TTA TGCGGGCAA CGTCTGGTA TCA GCGCGAA GTCTTTA TA CCGAAA GGTTGGGCA GGCCA GCGTA TCGTGCTGCGTTTCGA TGCGGTCA CTCA TTA CGGCAAA GTGTGGGTCAA TAA TCA GGAA GTGA TGGA GCA TCA GGGCGGCTA TA CGCCA TTTGAA GCCGA TGTCA CGCCGTA TGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GCA GTCTTA CTTCCA TGA TTTCTTTAA CTA TGCCGGAA TCCA TCGCA GCGTAA TGCTCTA CA CCA CGCCGAA CA CCTGGGTGGA CGA TA TCA CCGTGGTGA CGCA TGTCGCGCAA GA CTGTAA CCA CGCGTCTGTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA TGCGGA TCAA CA GGTGGT GAGGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGA TCTTTCTTTAAA CTGA TCTA TTTTTTAA TTGA TTGGTTA TGGTGTAAA TATTA CA TA GCTTTAA CTGA TAA TCTGA TTA CTTTA TTTCGTGTGTCTA TGA TGA TGA TGA TAGTTA CA GAA CCGA CGA CTCGTCCGTCCTGTA GAAA CCCCAA CCCGTGAAA TCAAAAAA CTCGACGGCCTGTGGGCA TTCAGTCTGGA TCGCGAAAA CTGTGGAA TTGA TCA GCGTTGGTGGGAAA GCGCGTTA CAA GAAA GCCGGGCAA TTGCTGTGCCA GGCA GTTTTAA CGA TCA GTTCGCCGA TGCA GA TA TTCGTAA TTA TGCGGGCAA CGTCTGGTA TCA GCGCGAA GTCTTTA TA CCGAAA GGTTGGGCA GGCCA GCGTA TCGTGCTGCGTTTCGA TGCGGTCA CTCA TTA CGGCAAA GTGTGGGTCAA TAA TCA GGAA GTGA TGGA GCA TCA GGGCGGCTA TA CGCCA TTTGAA GCCGA TGTCA CGCCGTA TGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GCA GTCTTA CTTCCA TGA TTTCTTTAA CTA TGCCGGAA TCCA TCGCA GCGTAA TGCTCTA CA CCA CGCCGAA CA CCTGGGTGGA CGA TA TCA CCGTGGTGA CGCA TGTCGCGCAA GA CTGTAA CCA CGCGTCTGTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA TGCGGA TCAA CA GGTGGT TGCAA CTG GA CAA GGCA CTA GCGGGA CTTTGCAA GTGGTGAA TCCGCA CCTCTGGCAA CCGGGTGAA GGTTATCTCTATGAACTCGAAGTCACAGCCAAAAGCCAGACAGAGTCTGATATCTACCCGCTTCGCGTCGGCA TCCGGTCA GTGGCA GTGAA GGGCCAA CA GTTCCTGA TTAA CCA CAAA CCGTTCTA CTTTA CTGGCTTTGGTCGTCA TGAA GA TGCGGA CTTA CGTGGCAAA GGA TTCGA TAA CGTGCTGATGGTGCA CGA CCA CGCA TTAA TGGA CTGGA ttggggccaa CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGAA GA GA TGCTCGA CTGGGCA GA TGAA CA TGGCA TCGTGGTGA TTGA TGAAA CTGCTGCTGTCGGCTTTCAGCTGTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCGATTAAAGAGCTGA TA GCGCGTGA CAAAAA CCA CCCAA GCGTGGTGA TGTGGA GTA TTGCCAA CGAA CCGGA TACCCGTCCGCAA GGTGCA CGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGCGTAAA CTCGA CCCGA CGCGTCCGA TCA CCTGCGTCAA TGTAA TGTTCTGCGA CGCTCA CA CCGA TA CCA TCA GCGA TCTCTTrGA TGTGCTGTGCCTGAA CCGTTA TTA CGGA TGGTA TGTCCAAA GCGGCGA TTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGCATCAGCCGATTA TCA TCA CCGAA TA CGGCGTGGA TA CGTTA GCCGGGCTGCA CTCAA TGTA CA CCGA CA TGCAA CTG GA CAA GGCA CTA GCGGGA CTTTGCAA GTGGTGAA TCCGCA CCTCTGGCAA CCGGGTGAA GGTTATCTCTATGAACTCGAAGTCACAGCCAAAAGCCAGACAGAGTCTGATATCTACCCGCTTCGCGTCGGCA TCCGGTCA GTGGCA GTGAA GGGCCAA CA GTTCCTGA TTAA CCA CAAA CCGTTCTA CTTTA CTGGCTTTGGTCGTCA TGAA GA TGCGGA CTTA CGTGGCAAA GGA TTCGA TAA CGTGCTGATGGTGCA CGA CCA CGCA TTAA TGGA CTGGA ttggggccaa CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGAA GA GA TGCTCGA CTGGGCA GA TGAA CA TGGCA TCGTGGTGA TTGA TGAAA CTGCTGCTGTCGGCTTTCAGCTGTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCGATTAAAGAGCTGA TA GCGCGTGA CAAAAA CCA CCCAA GCGTGGTGA TGTGGA GTA TTGCCAA CGAA CCGGA TACCCGTCCGCAA GGTGCA CGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGCGTAAA CTCGA CCCGA CGCGTCCGA TCA CCTGCGTCAA TGTAA TGTTCTGCGA CGCTCA CA CCGA TA CCA TCA GCGA TCTCTTrGA TGTGCTGTGCCTGAA CCGTTA TTA CGGA TGGTA TGTCCAAA GCGGCGA TTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGCATCAGCCGATTA TCA TCA CCGAA TA CGGCGTGGA TA CGTTA GCCGGGCTGCA CTCAA TGTA CA CCGA CA TGTGGA GTGAA GA GTA TCA GTGTGCA TGGCTGGA TA TGTA TCA CCGCGTCTTTGA TCGCGTCA GCGCCGTCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCGA CCTCGCAA GGCA TA TTGCGCGTTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTGAAAAA CCGCA GCA GGGA GGCAAA CAA GCTAGCCACCACCACCACCACCACGTGTGA (SEQ ID NO :9) TGTGGA GTGAA GA GTA TCA GTGTGCA TGGCTGGA TA TGTA TCA CCGCGTCTTTGA TCGCGTCA GCGCCGTCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCGA CCTCGCAA GGCA TA TTGCGCGTTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTGAAAAA CCGCA GCA GGGA GGCAAA CAA GCTAGCCACCACCACCACCACCACGTGTGA (SEQ ID NO: 9)

[0113] 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EcoRI/Sac I/Kpn I/Hind ΙΙΙ/Spe I/Sbf I/Pst I/Xba I/BamH I/Sall/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SEQID 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。 [0113] As shown in sequence in the present invention is constructed plasmid p8, which is the multiple cloning sites of EcoRI / Sac I / Kpn I / Hind ΙΙΙ / Spe I / Sbf I / Pst I / Xba I / BamH I / Sall / Nco I restriction sites are indicated by boxed and underlined, used for selection of transformants primers GCTTCCGGCTCGTATGTTGT / GAGTCGTCGGTTCTGTAAC (i.e., SEQID NO: 7 and 8) represented by a double underline, italics the GUS sequence, intron sequences are shown in italics to shade.

[0114] 2)重组载体p8+P516的构建[0115] 根据限制性内切酶EcoRI (NEB)和PstI (NEB)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+P516,以及如上所述构建的p8质粒。 [0114] 2) Construction of recombinant vector p8 + P516 [0115] The cut (NEB) and Pstl (NEB) restriction enzyme EcoRI instructions, in accordance with the following process conditions cloning vector obtained in the Example I embodiment PMD18-T + P516, and p8 plasmid constructed as described above.

[0116] 其中克隆载体PMD18-T+P516及p8质粒的酶切条件如下: [0116] wherein the cloning vector PMD18-T + P516 p8 plasmid and restriction digestion conditions are as follows:

[0117]克隆载体 pMD18-T+P516 或p8 质粒10 μ I (< IOOOng) [0117] cloning vector pMD18-T + P516 plasmid p8 or 10 μ I (<IOOOng)

[0118] EcoRI O. I μ I(IOU) [0118] EcoRI O. I μ I (IOU)

[0119] PstI O. I μ I(IOU) [0119] PstI O. I μ I (IOU)

[0120] 10*buffer H 5 μ I [0120] 10 * buffer H 5 μ I

[0121]无菌水 34·8μ1 [0121] Sterile water 34 · 8μ1

[0122] 酶切体系 50 μ I [0122] Digestion of 50 μ I

[0123] 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P516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: [0123] TIANGEN using agarose gel DNA extraction kit (catalog number: DP209_03) P8 were digested plasmid was recovered, and the P516 promoter insert, according T4 ligase (TaKaRa, D2011A) instructions, in accordance with the following conditions connect:

[0124] 酶切后的p8质粒 lyl(20ng) [0124] p8 lyl The digested plasmid (20ng)

[0125] 回收的启动子P516插入片段 10_20ng,根据其浓度确定 [0125] The recovered P516 promoter insert fragment 10_20ng, determined according to the concentration

[0126] T4 ligase (TaKaRa, D2011A) O. 5 μ I [0126] T4 ligase (TaKaRa, D2011A) O. 5 μ I

[0127] 10*T4 buffer I μ I [0127] 10 * T4 buffer I μ I

[0128] 无菌水 补齐至9· 5 μ I [0128] Sterile water filled to 9 · 5 μ I

[0129] 连接体系 10 μ I [0129] Connection system 10 μ I

[0130] Τ4 buffer冰上融化,酶切后的ρ8质粒载体加入量约20ng,本发明中的Ρ516片段加10ng。 [0130] Τ4 buffer thawed on ice, ρ8 digested plasmid vector after addition of about 20ng, Ρ516 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.

[0131] 将100 μ I氯化钙法制得的感受态细胞DH5 α从超低温冰箱取出,冰上融化后,力口入10 μ 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600μ I4°C预冷的SOC, 37度220rpm复苏45min, 8000rpm离心30s,去上清,留取150 μ I,轻轻吹勻,玻璃珠涂布LB (kan),37°C倒置培养16_24h。 [0131] The 100 μ I DH5 α competent cells chloride method were removed from the ultra-low temperature freezer, thawed on ice, the ligation products force mouth 10 I [mu] above, stir gently, the ice bath was 30min, 42 ° heat shock C 60s, the ice bath was 5min, 600μ I4 ° C was added a pre-cooled SOC, 37 recovery of 220rpm for 45 min, centrifuged at 8000 rpm for 30s, the supernatant was discarded, specimens 150 μ I, uniform blowing gently, glass beads coated with an LB ( kan), 37 ° C culture inverted 16_24h. 得到重组载体p8+P516。 Recombinant vector p8 + P516.

[0132] 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+P516进行PCR检测,以确证所得重组载体P8+P516中含有所需启动子P516。 [0132] With Fl (i.e., SEQ ID NO: 4) and Rl (i.e., SEQ ID NO: 5) as the primer pair obtained recombinant vector P8 + P516 by PCR to confirm the resulting recombinant vector P8 + P516 containing the desired promoter sub-P516. 通过酶切筛选含有重组载体P8+P516转化子。 Screening a recombinant vector by digestion P8 + P516 transformants.

[0133] 实施例3 :重组根癌农杆菌EHA105-P516细胞的制备 Preparation of recombinant cells EHA105-P516 tumefaciens: [0133] Example 3

[0134] 将如实施例2所述方法构建的p8+P516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癌农杆菌EHA105的感受态细胞,具体方法如下: [0134] As p8 to the method of Example 2 + P516 embodiment constructed as recombinant vectors and control plasmids were transformed p8 prepared in accordance with "Molecular Cloning A Laboratory Manual" (third edition, Academic Press) in the calcium chloride method Agrobacterium tumefaciens EHA105 competent cells, as follows:

[0135] 将根癌农杆菌感受态细胞EHA105于超低温冰箱中取出,至于冰上解冻。 [0135] Agrobacterium tumefaciens EHA105 competent cells taken at cryogenic refrigerator, as thawed on ice. 融化后,分别加入5 μ I的ρ8+Ρ516重组载体和ρ8质粒以及作为对照的ρ8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 μ I常温的LB液体培养基,28 V160rpm复苏3h, 8000rpm离心30s,吸去上清,留下200 μ I吹勻,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,10mg/l Rif)。 After thawing, were added 5 μ I of ρ8 + Ρ516 recombinant plasmid vector and ρ8 ρ8 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 μ I LB broth room temperature, 28 V160rpm recovery 3h, 8000rpm centrifugal 30s, the supernatant was aspirated, leaving 200 μ I blow uniformly coated to a plus kan-rif (kanamycin - rifampin) bis on YM medium plates antibody (50mg / lKan, 10mg / l Rif). 28°C倒置培养2-3天。 28 ° C for 2-3 days inverted.

[0136] 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EcoRI/Pst I酶切筛选转化子。 [0136] In Fl (i.e., SEQ ID NO: 4) and Rl (i.e., SEQ ID NO: 5) by PCR primers and by EcoRI / Pst I digested selection of transformants.

[0137] 本发明中,按照如上述方法得到的带有重组载体P8+P516的重组农杆菌,命名为重组根癌农杆菌EHA105-P516。 [0137] In the present invention, the recombinant Agrobacterium P8 + P516 in accordance with a recombinant vector obtained by the method as described above, the recombinant designated as Agrobacterium tumefaciens EHA105-P516.

[0138] 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,命名为重组根癌农杆菌EHA105-p8。 [0138] The method according to the present invention, obtained with the control recombinant Agrobacterium p8 plasmid, designated as recombinant Agrobacterium tumefaciens EHA105-p8.

[0139] 实施例4 :水稻愈伤组织的诱导和转化 [0139] Example 4: Induction of transformed callus and rice

[0140] 按照如下步骤诱导水稻愈伤组织,并分别用重组根癌农杆菌EHA105-P516和重组根癌农杆菌EHA105-p8转化所述愈伤组织。 [0140] the following steps rice callus induction, respectively, and the recombinant Agrobacterium tumefaciens EHA105-P516 and recombinant Agrobacterium tumefaciens EHA105-p8 transformed calli.

[0141] I)将水稻日本晴种子去壳,70%乙醇表面消毒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培养基上,用封口膜封口;29. 5°C光照培养3-4周; [0142] 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; [0141] I) The seeds of rice Nipponbare peeled, surface-sterilized with 70% ethanol 30s, and then shaken vigorously 30min, during with 5% available chlorine I. hypochlorite disinfection, washing with sterilized water 5 times after disinfection; after sterilization seeds were placed on N6D medium, sealed with parafilm;. 29 5 ° C for 3-4 weeks lighted culture; callus [0142] 2) selecting actively growing (yellowish white, dried, diameter I~3mm), in new N6D medium on light 29. 5 ° C for 3 days;

[0143] 3)分别挑取如实施例3所构建的重组根癌农杆菌单菌落(重组根癌农杆菌EHA105-P516或重组根癌农杆菌EHA105-p8),于添加抗生素(50mg/l Kan,IOmg/1 Rif)的YM培养基上划线培养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30μ IIOOmM的AS(Acetosyringone,乙酰丁香酮)的30ml AAM培养基中,温和重悬所述重组根癌农杆菌细胞(重组根癌农杆菌EHA105-P516或重组根癌农杆菌EHA105_p8); [0143] 3) were picked constructed as described in Example 3 Recombinant Agrobacterium tumefaciens Single colonies (recombinant Agrobacterium tumefaciens EHA105-P516 or recombinant Agrobacterium tumefaciens EHA105-p8) embodiment, the addition of antibiotics (50mg / l Kan , streaked onto IOmg / 1 Rif) cultured for 3 days in YM medium, culture temperature 28 ° C; were scraped off with the recombinant Agrobacterium tumefaciens disposed added 30μ IIOOmM of AS (acetosyringone, acetosyringone) in 30ml AAM medium, gently resuspended the recombinant Agrobacterium tumefaciens cells (recombinant Agrobacterium tumefaciens EHA105-P516 or recombinant Agrobacterium tumefaciens EHA105_p8);

[0144] 4)将继代培养的愈伤组织置于灭菌培养皿中;将如步骤3制备的重组根癌农杆菌悬液倒入培养皿中,将愈伤组织浸入其中15min ; [0144] 4) The following cultured callus was placed in a sterile Petri dish; recombinant Agrobacterium suspension as prepared in step 3 is poured into a Petri dish, the calli immersed in 15min;

[0145] 5)倒掉重组根癌农杆菌悬液,将愈伤组织用灭菌吸水纸吸掉多余的液体;于N6-AS培养基上放一张灭菌滤纸,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 ; [0145] 5) Discard the recombinant Agrobacterium tumefaciens suspension, the callus siphoning off excess liquid with sterile absorbent paper; put on a sterilized filter paper N6-AS medium containing AS as described above was added Iml an AAM medium, callus was transferred onto filter paper; sealed Petri dish, 28 ° C dark culture 48-60h;

[0146] 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癌农杆菌;用灭菌吸水纸除去愈伤组织上多余的水分,然后将其转移至含lmg/1潮霉素B(HmB)和50mg/l Carb的N6-AS培养基上;用封口膜密封培养皿,29. 5°C光照培养3_4周。 [0146] 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 the medium;. the dishes were sealed with parafilm, 29 5 ° C the lighted culture 3_4 weeks.

[0147] 实施例5 :水稻愈伤组织中的⑶S的表汰 Rice Calli ⑶S jig table: [0147] Example 5

[0148] 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的表达情况,按照Chen SY 等在Journal of Integrative Plant Biology, 2008, 50 (6) :742_751 所述的方法,对分别用重组根癌农杆菌EHA105-P516或重组农杆菌EHA105_p8转化的水稻愈伤组织进行染色。 Is [0148] After detecting the expression of the gene of Example 4 ⑶S rice calli transformed embodiment according to Chen SY et al Journal of Integrative Plant Biology, 2008, 50 (6): The method, according to 742_751 were stained with the recombinant Agrobacterium tumefaciens EHA105-P516 or rice callus transformed with the recombinant Agrobacterium EHA105_p8.

[0149]⑶S 染色液的配方(Iml) :610 μ I O. 2Μ Na2HPO4 溶液(pH = 7. O) ;390 μ I O. 2ΜNaH2PO4 溶液和10 μ I O. IM X-gluc。 [0149] ⑶S formulation staining solution (Iml): 610 μ I O. 2Μ Na2HPO4 solution (pH = 7. O); 390 μ I O. 2ΜNaH2PO4 solution and 10 μ I O. IM X-gluc.

[0150] 将分别用重组根癌农杆菌EHA105-P516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,拍照记录,结果如图4所示,经含有启动子的P8+P516重组载体的重组根癌农杆菌介导转化的水稻愈伤组织(图4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愈伤组织(作为对照,图4左)经⑶S染色后颜色未发生变化。 [0150] respectively with the recombinant Agrobacterium tumefaciens EHA105-P516 EHA105_p8 or recombinant Agrobacterium tumefaciens transformed rice callus was immersed in the GUS staining solution, 37 ° C incubation to appear blue, camera recording, the results shown in Figure 4 as shown by P8 + P516 recombinant vector comprising a promoter of rice callus mediated by Agrobacterium tumefaciens transformed with the recombinant (FIG. 4 right) after staining appear blue, the promoter does not contain the recombinant plasmid p8 tumefaciens Agrobacterium-mediated transformation of rice callus (as a control, left in FIG. 4) after staining ⑶S it does not change. 结果显示,本发明的P516启动子对⑶S基因表达具有调控作用。 The results show, P516 promoter of the present invention has a regulatory effect on gene expression ⑶S.

[0151] 实施例6 :转基因水稻苗中⑶S的表汰[0152] 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分化培养基分化苗;用封口膜密封培养皿,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生根培养基进行生根筛选。 [0151] Example 6: Transgenic rice plants in Table ⑶S elimination [0152] The embodiment of the callus obtained in Example 4 was transferred to a differentiation medium MS-R differentiated seedlings containing 50mg / l hygromycin B (HmB) of ;. the dishes were sealed with parafilm, 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) of rooting filter.

[0153] 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。 [0153] GUS staining of transgenic rice plants during the dyeing process of Example 5 with the callus embodiment. 结果如图5和图6所示。 The results shown in Figures 5 and 6. 经含有启动子的P8+P516重组载体的重组根癌农杆菌介导转化的水稻苗的根(图5右)、叶(图6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5左)、叶(作为对照,图6左)经GUS染色后颜色未发 Via promoter-containing P8 + root (the right in FIG. 5) of rice plants by Agrobacterium mediated transformation of a recombinant vector recombinant P516 tumefaciens, the leaf (the right in FIG. 6) after staining appear blue, the promoter does not contain p8 the recombinant plasmid root surface of rice plants by Agrobacterium mediated transformation (as a control, left in FIG. 5), leaves (as a control, left in FIG. 6) after GUS staining color is not made

生变化。 Health changes.

[0154] 结果表明,本发明的P516启动子对水稻中的基因(例如GUS基因)表达具有调控作用。 [0154] The results show that, P516 promoter of the present invention has a regulatory effect on the rice genes (e.g., GUS gene).

[0155] 实施例7 :转基因烟草苗中GUS的表达 7 [0155] Example: Expression of GUS in transgenic tobacco seedlings of

[0156] I.烟草无菌苗获得: [0156] I. tobacco-free vaccine obtained:

[0157] 烟草NC89 (Nicotiana tobaccum L.)种子浸泡:将烟草种子装入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。 [0157] tobacco NC89 (Nicotiana tobaccum L.) seeds are soaked: tobacco seed was charged in I. 5ml centrifuge tube (<50 / tube), Iml of sterile water was added, pipetted several times, replacing after sterile water, soaked at room temperature for 24 hours.

[0158] 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。 [0158] Tobacco seed sterilization: using a pipette seed soaking water, adding 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 .

[0159] 洗漆:用无菌水清洗五次,每次加入Iml无菌水摇动lmin。 [0159] removers: washed five times with sterile water, sterile water was added to each shake Iml lmin.

[0160] 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(表I)上萌发,每皿约10粒,置于28°C光照培养室(16h光/8h暗)培养一周,光照强度为20001χο [0160] Seeding: dry seeds sterile filter water surface, then the tip or sterile toothpick inoculated germinated on MS medium (Table the I), about 10 per dish and placed in the lighted culture room 28 ° C (16h light / 8h dark) week of culture, light intensity 20001χο

[0161] 转接:待长出幼苗后,转入新鲜MS固体培养基,每瓶(直径6cm,高9cm,30ml培养基/瓶)3株烟草小苗,28°C光照培养室(16h光/8h暗,光照强度为20001x)培养约5周,获得烟草无菌苗。 [0161] Adapter: seedling to be grown, transferred to fresh MS solid medium bottle (diameter 6cm, high 9cm, 30ml medium / flask) 3 tobacco seedlings, 28 ° C the lighted culture room (16h light / 8h dark, light intensity of 20001x) culturing about 5 weeks, no vaccine tabacum.

[0162] 2.烟草无菌苗的继代和扩繁 [0162] 2. tobacco aseptic subculture and propagation

[0163] 剪去烟草无菌苗的叶片和根,将茎杆剪成带有腋芽的茎段(2-3cm),然后将其形态学下端垂直插入到新鲜MS固体培养基(腋芽不能插入培养基里); [0163] Cut aseptic tobacco leaves and roots, the stalk cut stem segments with axillary (2-3cm), which is then inserted into the vertically lower morphology fresh MS solid medium (not insert axillary bud culture Kiri);

[0164] 每瓶接种一个带有腋芽的茎段外植体,置于28°C光照(20001x)培养室培养约5周,作为待转化材料使用。 [0164] a bottle with a stem segments were inoculated explants suckers, placed in the light 28 ° C (20001x) culturing the culture chamber for about 5 weeks to be used as conversion material.

[0165] 3.侵染前菌液的制备: [0165] 3. The front bacteria infested prepared:

[0166] 分别挑取含潮霉素抗性目的质粒的重组根癌农杆菌EHA105-P516或重组根癌农杆菌EHA105-p8 单菌落,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,还未出现沉淀时,将此菌液置4°C保存; [0166] were picked tumefaciens containing the recombinant plasmid of interest hygromycin resistance Agrobacterium EHA105-P516 or recombinant Agrobacterium tumefaciens EHA105-p8 single colony was inoculated into IOml YM (containing Kan 50mg / L, Rif 30mg / L when the) liquid medium, 28 ° C, 250rpm shaking overnight, until broth turbidity, precipitation occurred yet, this bacterium is set at 4 ° C;

[0167] 取保存于4°C的菌液20 μ 1,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; [0167] Take stored at 4 ° C. Bacteria 20 μ 1, inoculated IOml YM (containing Kan 50mg / L, Rif 30mg / L) liquid cultures 50ml centrifuge tube based on 28 ° C, 250rpm shaking overnight, be bacteria turbidity, sediment is yet to come when the train is stopped;

[0168] 取上述菌液3ml加入到50ml YM(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 = O. 5左右时,作为侵染菌液使用。 [0168] Take the above-described bacterial suspension were added to 3ml 50ml YM (without antibiotics) in liquid culture in flasks 28 ° C, 250rpm shaking shake culture for about 2h, at around OD600 = O. 5, as bacteria infection.

[0169] 4.侵染: [0169] 4. Infection:

[0170] 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; [0170] scissor blade from the large tobacco cultured for 5 weeks on aseptic, stored in a petri dish with YM (antibiotic-free) liquid medium;

[0171] 用直径6mm的打孔机将烟草叶片打成叶圆盘,保存在另一个装有YM(不含抗生素)液体培养基的培养皿中; [0171] with 6mm diameter punch blade tobacco leaf discs labeled, stored in Petri dishes with YM another (antibiotic-free) liquid medium;

[0172] 将烟草叶圆盘转移到装有侵染菌液的培养皿中; [0172] The tobacco leaf disc was transferred to a Petri dish containing bacteria infection;

[0173] 轻轻摇动培养皿,确保农杆菌接触到叶盘边缘,浸泡IOmin ; [0173] Petri dish gently shaken, to ensure that the contact of Agrobacterium leaf disc edge, soaking IOmin;

[0174] 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; [0174] The tobacco leaf discs infested been transferred to the Agrobacterium suspension and dried on sterile filter paper, until dry tobacco leaf discs without bacteria dropwise until the bacteria;

[0175] 将烟草叶盘接种到RMOP固体培养基(表2)上,叶面朝上,每皿约10块; [0175] The inoculated to tobacco leaf discs on RMOP solid medium (Table 2), facing leaf per dish, about 10;

[0176] 倒置培养皿,28°C暗培养3天。 [0176] inverted dish, 28 ° C dark for 3 days.

[0177] 5.筛选: [0177] 5. Screening:

[0178] 将步骤4的叶圆盘转移到RMOP-TCH(IOmg/L Hyg)培养基(表3)上,每皿10块,叶面朝上,28°C光照(20001x)培养2周; [0178] Step 4 leaf discs were transferred to RMOP-TCH on (IOmg / L Hyg) medium (Table 3), each plate 10, leaf up, 28 ° C light (20001x) for 2 weeks;

[0179] 每2周继代一次,大约4周出现丛生芽。 [0179] ZHOU Ji passaged once every 2, buds appear about four weeks.

[0180] 6.生根: [0180] 6. Rooting:

[0181] 当再生芽长至约l-2cm时,切下芽接种到MST-TCH(10mg/L Hyg)培养基(表4),每瓶I株烟草苗; [0181] When reproducing the shoot to about l-2cm, shoots were cut to the MST-TCH (10mg / L Hyg) medium (Table 4), I strain bottle tobacco seedlings;

[0182] 28°C光照(20001x)培养室培养2周; [0182] 28 ° C Light (20001x) for 2 weeks culture chamber;

[0183] 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。 [0183] 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.

[0184] GUS 染色液的配方(Iml) :610 μ I O. 2Μ Na2HPO4 溶液(pH = 7. O) ;390 μ I O. 2ΜNaH2PO4 溶液和10 μ I O. IM X-gluc。 [0184] GUS staining solution formulation (Iml): 610 μ I O. 2Μ Na2HPO4 solution (pH = 7. O); 390 μ I O. 2ΜNaH2PO4 solution and 10 μ I O. IM X-gluc.

[0185] 将转基因和对照(转入空的P8质粒)分别浸泡在⑶S染色液中,37°C保温至出现蓝色,拍照记录。 (P8 into empty plasmid) [0185] Transgenic and control were soaked in dyeing liquid ⑶S, 37 ° C incubation to appear blue, is photographed.

[0186] 结果如图8和图9所示。 [0186] The results shown in FIG. 8 and FIG 9. 经含有启动子的p8+P516重组载体的重组根癌农杆菌介导转化的烟草苗的根(图8右)、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8左)、叶(作为对照,图7左)经GUS染色后颜色未发生变化。 p8 + via promoter-containing recombinant Agrobacterium tumefaciens recombinant vector P516 mediated transformation of tobacco seedlings root (the right in FIG. 8), leaves (Figure 7 right) presented by the blue staining, by containing no promoter p8 root recombinant plasmid of Agrobacterium tumefaciens mediated transformation of tobacco seedlings (as a control, left in FIG. 8), leaves (as a control, left in FIG. 7) after GUS staining it does not change.

[0187] 结果表明,本发明的P516启动子对烟草中的基因(例如GUS基因)表达具有调控作用。 [0187] The results show that, P516 promoter of the present invention has a regulatory effect on tobacco gene (e.g., GUS gene).

[0188] 本发明实施例中所使用的相关培养基配方说明如下: [0188] Examples related media formulations used in embodiments of the present invention will be described as follows:

[0189] 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。 [0189] For the medium called "conventional sterilization" refers to a condition of sterilization: 121 ° C for steam sterilization for 20 minutes.

[0190] 表1:MS固体培养基[0191] [0190] Table 1: MS solid medium [0191]

Figure CN102146392BD00201

[0192] MS 有机(IOOOx):维生素BI,O. Olg,维生素B6,O. 05g,烟酸BI,O. 05g,甘氨酸, [0192] MS organic (IOOOx):. Vitamin BI, O ​​Olg, vitamin B6, O 05g, niacin BI, O ​​05g, glycine.

O. 2g,加蒸馏水定容至100ml,过滤除菌,4°C保存不超过I周。 O. 2g, distilled water volume to 100ml, filter sterilized, 4 ° C to save no more than I weeks.

[0193] 表2 : RMOP培养基 [0193] Table 2: RMOP medium

[0194] [0194]

Figure CN102146392BD00202

[0196] pH 5. 8 [0196] pH 5. 8

[0197] 121°C下灭菌20分钟。 Sterilization [0197] 121 ° C 20 min.

[0198] Myo-inositol (500x) :5g 肌醇溶解于H2O 后,定容至100ml, 4°C保存。 [0198] Myo-inositol (500x): After dissolving 5g inositol H2O, volume to 100ml, 4 ° C storage.

[0199]表 3 : RMOP-TCH 培养基 [0199] Table 3: RMOP-TCH medium

Figure CN102146392BD00211

[0201 ]表4 =MST-TCH 培养基 [0201] TABLE 4 = MST-TCH medium

Figure CN102146392BD00212

[0203] 尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解。 [0203] While specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate. 根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。 According to the teachings disclosed herein, various modifications and alternatives to those details are within the scope of the invention these changes. 本发明的全部范围由所附权利要求及其任何等同物给出。 The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (25)

1. 一种启动子,其核苷酸序列为SEQ ID NO: I所示的核苷酸序列或者为与SEQ ID NO:I互补的核苷酸序列。 A promoter nucleotide sequence is SEQ ID NO: or a nucleotide sequence I is shown in the SEQ ID NO: I is complementary to a nucleotide sequence.
2. —种核酸构建体,包含权利要求I所述的启动子,和与启动子可操作地连接的基因序列,其中所述启动子与所述基因序列来源相同或者不同。 2. - nucleic acid construct comprising a promoter as claimed in claim I claim 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.
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, wherein said vector comprises 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PMD18-T或p8质粒经重组得到的重组载体。 4. The vector of claim 3, wherein said promoter or Claim I which is a nucleic acid according to claim claim 2 recombinant vectors and plasmids p8 PMD18-T or obtained by recombinant construct.
5. 一种重组细胞,其特征在于,所述细胞含有权利要求I所述的启动子或者权利要求2所述的核酸构建体或者权利要求3或4所述的载体,并且所述重组细胞为重组大肠杆菌细胞或重组农杆菌细胞。 A recombinant cell, wherein said cell comprising said claim I or claim promoter nucleic acid construct of claim 2 or the vector of claim 3 or 4, and the recombinant cell is a The recombinant E. coli cells or recombinant Agrobacterium cells.
6. 一组引物对,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的核苷酸序列分别为SEQ IDNO :2所示的序列或在其5'端连接有限制性酶切位点和/或保护碱基,和SEQ ID NO :3所示的序列或在其5'端连接有限制性酶切位点和/或保护碱基。 6. a set of primers used for amplification of the promoter obtained Claim I in claim 1, characterized in that for the primer: nucleotide sequence of the two primers are the primer pair is SEQ IDNO: 2 shown in FIG. or a sequence at its 5 'end with a connector restriction sites and / or protection of bases, and SEQ ID NO: 3 of the sequence shown or in its 5' end with a connector restriction sites and / or protection bases.
7.权利要求6的引物对,其特征在于,所述引物对的两条引物分别如SEQ ID N0:4和SEQ ID NO :5 所示。 Primer pair of claim 6, wherein the primer pair of two primers, such as SEQ ID N0: 4 and SEQ ID NO: 5 shown in FIG.
8.制备SEQ ID NO :1所示的启动子的方法,包括下述步骤: 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SEQID NO :1在水稻日本晴基因组DNA中的序列分别针对首尾进行设计。 8. Preparation of SEQ ID NO: 1 shown promoter, comprising the steps of: Nipponbare rice genomic DNA as a template, using a pair of amplification primers for amplification, the amplification primers according to SEQID NO: 1 Nipponbare rice genomic DNA sequences were designed for end to end.
9.根据权利要求8所述的方法,其中,所述引物对为权利要求6或7所示的引物对。 9. The method according to claim 8, wherein the primer pair as claimed in claim 6 or 7 primer pairs.
10. 一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所述的启动子或者权利要求2的核酸构建体或者权利要求3或4的载体或者权利要求5的重组农杆菌细胞导入植物的步骤。 10. A method for the regulation of gene expression in plants, 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 claim 5, a recombinant Agrobacterium to claim the step of introducing a plant cell.
11.根据权利要求10所述的方法,其中,所述导入植物为导入植物愈伤组织。 11. The method according to claim 10, wherein the plant is introduced into a plant callus.
12.根据权利要求10或11所述的方法,其中,所述植物为单子叶植物或双子叶植物。 The method according to claim 11 or claim 10, wherein the plant is a monocot or a dicot.
13.根据权利要求10或11所述的方法,其中,所述植物为稻属或烟草属。 13. The method of claim 10 or claim 11, wherein said plant is a rice or genus Nicotiana.
14.根据权利要求10或11所述的方法,其中,所述植物为水稻或烟草。 14. The method according to claim 10 or claim 11, wherein the plant is tobacco or rice.
15.根据权利要求10或11所述的方法,其中,所述植物为水稻日本晴或烟草NC89。 15. The method as claimed in claim 11 or claim 10, wherein the plant is tobacco or rice Nipponbare NC89.
16. 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植物愈伤组织或植物外植体或植物的步骤,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的重组农杆菌细胞。 16. A method of preparing a transgenic plant, plant calli or explants step plants or plants comprising culturing under conditions effective to produce a plant, wherein the plant callus or plant explants, or plant outer claim comprising 2 nucleic acid construct or vector as claimed in claim 3 or claim 4 or claim 5, wherein a recombinant Agrobacterium cells I promoter or the claims.
17.根据权利要求16所述的方法,其中,所述植物为单子叶植物或双子叶植物。 17. The method of claim 16, wherein the plant is a monocot or a dicot.
18.根据权利要求16所述的方法,其中,所述植物为稻属或烟草属。 18. The method according to claim 16, wherein said plant is a rice or genus Nicotiana.
19.根据权利要求16所述的方法,其中,所述植物为水稻或烟草。 19. The method according to claim 16, wherein the plant is tobacco or rice.
20.根据权利要求16所述的方法,其中,所述植物为水稻日本晴或烟草NC89。 20. The method of claim 16, wherein the plant is tobacco or rice Nipponbare NC89.
21.权利要求I所述的启动子、或者权利要求2的核酸构建体、或者权利要求3或4的载体、或者权利要求5的重组农杆菌细胞、或者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I的启动子或者权利要求2的核酸构建体或者权利要求3或4的载体或者权利要求5的重组农杆菌细胞。 21. The promoter according to claim I, or a nucleic acid construct of claim 2, 3 or 4 or a vector as claimed in claim 5 or a recombinant Agrobacterium cells outside plant callus or plant explant of claim or or plant expression of the gene in plant breeding or in the regulation of the use of a plant, wherein the plant or outside the plant callus or plant explant comprising the promoter of claim I or claim 2, a nucleic acid or construct as claimed in claim 3 or recombinant Agrobacterium cells 5 or a vector of claim 4.
22.根据权利要求21所述的用途,其中,所述植物是单子叶植物或双子叶植物。 22. The use according to claim 21, wherein said plant is a monocot or a dicot.
23.根据权利要求21所述的用途,其中,所述植物为稻属或烟草属。 23. The use according to claim 21, wherein said plant is a rice or genus Nicotiana.
24.根据权利要求21所述的用途,其中,所述植物为水稻或烟草。 24. The use according to claim 21, wherein the plant is tobacco or rice.
25.根据权利要求21所述的用途,其中,所述植物为水稻日本晴或烟草NC89。 25. The use according to claim 21, wherein the plant is tobacco or rice Nipponbare NC89.
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