WO2011130895A1 - Promoter siubi1, preparation method and uses thereof - Google Patents

Abstract

Promoter of modified Jizhanggu No.1 millet and its variants, preparation method of the promoter and uses thereof are provided, wherein the promoter has the nucleotide sequence shown in SEQ ID NO: 1. The variants having promoter functions are selected from: 1) nucleotide sequences that hybridize with the sequence shown in SEQ ID NO: 1 under high stringent conditions, 2) nucleotide sequences that comprise a substitution, deletion, and/or insertion modification of one or more bases of the nucleotide sequence shown in SEQ ID NO: 1, and 3) nucleotide sequences that have at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 1. Preparation methods of the promoter and uses thereof in regulating expressions of target genes in plants and in rice breeding are also provided.

Description

 Promoter SiUbi1, preparation method and use thereof

 The present invention belongs to the field of plant molecular biology and relates to a promoter, particularly a promoter of a plant such as millet, and a preparation method and use of the promoter. Background technique

 A promoter is a component of a gene, usually located on the structural gene 5, which is a DNA sequence that RNA polymerase recognizes, binds to and initiates transcription. The promoter can direct the holoenzyme to properly bind to the template, activate the RNA polymerase, and initiate gene transcription to control the start and expression of the gene expression (transcription). In transgenic plants, promoters are one of the important factors affecting the efficiency of transgene expression. Selecting a highly efficient promoter is the key to efficient expression of foreign genes.

 They can be divided into three classes depending on the transcriptional mode of the promoter: a constitutive promoter, a tissue or organ-specific promoter, and an inducible promoter. The so-called constitutive promoter means that under the control of the constitutive promoter, there is no significant difference in gene expression between different tissues and developmental stages, so it is called a constitutive promoter.

A constitutive promoter widely used at present is CaMV35S, which produces high efficiency expression in both monocotyledonous and dicotyledonous plants, but Ajith Anand et al. transferred the chitinase gene of rice to wheat and found that CaMV35S was used. As a promoter, the transferred plants showed gene silencing in the second generation, and the maize ubiquitin Ubiquitin promoter was used. In the fourth generation, the gene expression was still large (Ajith Anand et al., Plant). Biotechnology Journal, 2003 (1): 241-251) _β

It is highly valued to clone constitutive promoters from plants themselves. Promoters such as Ubiquitin and Actin have been cloned. By replacing these CaMV 35S promoters with these promoters, transcription of foreign genes can be driven more efficiently in plants.

 The ubiquitin (Ub iqui t in, Ub i ) promoter is widely distributed in eukaryotes and has significant effects in enhancing the long-term and stability of gene expression, and has high activation efficiency, low methylation level, and genetic It is favored by factors such as stable traits (Xie Wei, Le Chaoyin. Journal of China Three Gorges University, 2007, 29 ( 2 ) : 176-179 ). Currently, promoter sequences have been isolated from many ubiquitin genes, including: Ubi-1 promoter in the maize genome, rice ubiquitin RUBQ2 promoter, Arabidopsis ubiquitin promoter, sunflower ubiquitin UbBl promoter, tobacco Ubiquitin Ub i. U4 promoter, potato ubiquitin Ubi 7 promoter, tomato ubiquitin Ub i l-1 promoter, barley ubiquitin Mubl promoter and the like. Among them, the maize ubiquitin Ubi-1 promoter has been widely used in monocotyledonous plants such as corn, wheat and rice. The rice ubiquitin RUBQ2 promoter is also widely used in rice and sugar cane.

 The Ub i promoter is effective in promoting long-term stable and efficient expression of foreign genes, and many Ub i promoters have been used in monocots and dicots. Moreover, the Ub i promoter has a higher level of transcriptional regulation than the promoters of nos, ocs, mas derived from T-DNA and CaMV35S and CsVMV derived from viruses. Guo Dianjing et al. (Acta Genetics, 1999, 26 (2): 168-173) found that the Ub i promoter was the most efficient in transgenic wheat callus, 4-5 times more than the CaMV35S promoter. Genschik et al. (Gene, 1994, 148: 195-202) introduced the promoter sequence of the isolated tobacco ubiquitin gene UM. U4 into transgenic tobacco to initiate GUS expression, and the results indicated that the Ub i. U4 promoter initiates the expression of the GUS gene. 7 times that of CaMV35S.

With the development of plant genetic engineering, people no longer need to transfer specific foreign genes into recipient plants, but to specifically and efficiently express foreign genes. Insufficient expression of foreign genes is often an important reason for not getting the ideal transgenic plants, and the promoter is It plays a key role in determining gene expression (Hou Bingkai et al. Genetics, 2001, 23 (5): 492-497). Utilizing the UM promoter to maintain a high level of transcriptional regulation in the cell body and integrating it into an episomal vector, a high level of expression system can be obtained, and thus the ubiquitin promoter has great potential for application in transgenic plants. Summary of the invention

 In order to provide a new tool and choice for the regulation of target gene expression in plant research, the inventors have provided a new ubiquitin promoter through in-depth study of the millet genome, which can be used to regulate plants. Target gene expression.

 In one aspect of the invention, a plant promoter having the nucleotide sequence set forth in SEQ ID NO: 1 is provided. In the present invention, the specific base sequence length of the promoter is 2020 bases, as shown in SEQ ID NO: 1:

ACTTAGCTTCTGGCCATCTCCAGAGACAGTGAGTTGATGCTTGATGCTAGACG AGGGGAAAAAAAGCAGAAAATCAGCCATACTAAATCAACTAATGATTTCAAAGAGAG GTACCTAATGCTCAAAAAGGAAGAGATTGGGCGATTTGCGACTAAAGAAGAGAATAA AATAGATTTTTTATAGCGTTAAGAAGTGTGTCACAGCTCTTACAGGAATGCTTGATC TACAAATGGAATAGATGATAATGGCAGCGGATATGGACGGATCGGGGTTGTTTAGTT CCTAATTTTTTAAAAAGTTTTTCGTCACATCGAATCTTATAACACATGTGTGAGTAT TAAATATAGATAAAAAATAACTAATTACATAATTTATTTGTAGTTTGCTAGATAAAA TTTTTGAGCCTAGGGTTAGTTTATGATTGAATAATAATTATCACGAAAGTACTACAG TAGTTAAATTTAAAATTGTTCGCAAACTAAACAAGGCCATGGTGTGTTTTTATTTTA CTCTCTAAAAATCTGCACAAAGGTTTTCTGACTCATGGGCCACACGTCTCAGTGTCG GTAAACACGGACGGAATCACGGGAGAAGGCATTAACAGCGTCGGGTCTAACGGCCAC AAACCAGCGACGAACGAAACAGACGTTCTGACGTCTCCGTGTCCACTCCGTCACTGG TTCCTTCTGGAGAGCTCTGACCTCCTCCGTCTCTATCTACGGCCGGCTCGCCTTCCG TTCCGCGTTCGCGTTGGACTCTTTGCGCTGGCGTGTTCCTGGAATTGCGTGGCGGAG ACGAGGCGGATTTCTCTCGCACGGAACGGAACCGCCACGGGCCCAAAGGCACGGTGA TTCCTTCTCCACCAACATAAATAGCCAGGACCCCTCCTCGCCTTTCCCCAATCTCAT CTCGCATTGTGTTGTTCGGAGCAAGGAGAACCCAGCCCCCCATCGCTCTCAATCCCA ATCGATCTTCTTCTCGTGAGCCTCGTCAATCCATCACCCGCTTCTAAGGTACGGCTC CCCCTCTAATCTTCTCTTCCCATCTCAGATTGGCGAGTTTATGTGATTAGATTAGAT GCTTCTCATCTAGATTGCGAGTTTCTGTTCGTAGATGGCTGGCTTGTAAGCGGTTCC TAGGTGGGTTTCTGTTCGTAGATGACTGGCTTGTAAGCGGTTCCTAGGTGGGATCGT TCTGATGATTTCTTTGGCTGCTGCGTAGAGATAGATCTGGTCCTGCTTTTCTTAATT CTTGGTGCAGATTTTGTGACCTGGTTCTATGTTCTTGTTCCTGCTTTGTAGCTCAAA TAGTTGTCTTAACTAGCTGGGCTTATTATTTGATTTGTACCTGCATGTATTATCACC AAATACAATTACTGTGAAGGAGTCAATATACCCTGCTCTGTACCTTTTACCTGACGA GCCATACTATCATTTTGATTCGTGTCATATGCATGCCAGATACGGAAATTATATGCT GCTACTTGCGTTATTATCATGCTGATTTGTTTCATATGCACGCCTAGATAGATGGAA ATTATATGCTACTGCTGAGCGTTATTATCATGCTGATTCGTTTCATATGCATGCCTA GATAGTTGGAAGTTTTGTTGTTTGCTGAGTGTTACTATCATGTTGATTTGTAATCAT ATGCATGCCTAGATAGATGAAGATACATGAATGTTATTCGTTTCAGATAGATGGAAT ATGCTGCTACTGAGCGTTACTATCATGTTGATTTGTTTCATATACACGCCTAGATAG ATGAAGAGATGGAT GTTGATTTGTTTCATATGCATGCCTAATAGATGAAGATATATG CTGCTACTGATGATTACTTACTACTTCGTGCCCATGCATGCTCTTTGGTTTACTTGG ATGGTGACATGCTGATGCAGTTTTGCTGGTTCTATAGTACCTATGTGCTTAGCATGT ATATCTGTTTCTTGTTGCTGACTGTTTCTTTCCCTCCTTAGTCTACCGCCGTATACT TATCATGTTGCTTGTTTTTTCTTCTACAG (SEQ ID NO: 1)

 In the present invention, the promoter sequence shown by SEQ ID NO: 1 is referred to as promoter S iUbi l , also referred to as promoter P603 for short.

The promoter is a constitutive promoter, rice callus with the promoter and GUS, and transgenic rice seedlings after GUS staining experiments, the rice callus and The roots, stems, leaves, etc. of the transgenic rice seedlings turn blue.

 A further aspect of the invention relates to a promoter having a sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1.

 A further aspect of the invention also relates to a variant of the promoter of SEQ ID NO: 1 having a promoter function selected from the group consisting of:

 1) a nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence shown in SEQ ID NO: 1,

 2) a nucleotide sequence in which one or more bases are substituted, deleted, or added to the nucleotide sequence shown in SEQ ID NO: 1, and

 3) A nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

 Typically, "hybridization conditions" are classified according to the degree of "stringency" of the conditions used in the measurement of hybridization. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 Π (below probe Tm 5 °C); "higher stringency" occurs about 5-10*C below Tm; "medium tightness" occurs Approximately 10 - "low stringency" below the needle Tm occurs approximately 20 - 25 below Tm. Alternatively, or in addition, hybridization conditions can be based on hybridized salt or ionic strength conditions and/or one or more stringency washes. For example, 6 x SSC = very low tightness; 3 x SSC = low to medium tightness; l x SSC = medium tightness; 0.5 x SSC = high tightness. Functionally, the nucleic acid sequence that is identical or nearly identical to the hybridization probe can be determined using the conditions of maximum stringency; and nucleic acids having about 80% or more sequence identity to the probe are determined using conditions of high stringency. sequence.

For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form hybrids, for example, to select relatively low salts and/or high temperature conditions. Sambrook et al. (Sambrook, J. et al. (1989) Molecular Cloning, Laboratory Manual, Cold Spring Harbor Press, Plainview, NY) provides hybridization conditions including medium stringency and high stringency.

 For ease of illustration, suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the invention to other polynucleotides include: pre-washing with 5xSSC, 0.5% SDS, 1. OmM EDTA (pH 8.0) solution; Hybridization was carried out in 5x SSC at -65 °C overnight; then washed twice with 2x, 0.5x and 0.2x SSC containing 0.1% SDS at 65 °C for 20 minutes. Those skilled in the art will appreciate that hybridization stringency can be readily manipulated, such as varying the salt content and/or hybridization temperature of the hybridization solution. For example, in another embodiment, suitable high stringency hybridization conditions include the above conditions, except that the hybridization temperature is raised to, for example, 60-65'C or 65-70 °C.

 In the present invention, the nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence shown by SEQ ID NO: 1 has the same or similar nucleotide sequence as SEQ ID NO: Promoter activity.

In the present invention, the nucleotide sequence which has one or more base substitutions, deletions, and additions to the nucleotide sequence shown by SEQ ID NO: 1 means that the nucleotides are respectively or simultaneously The 5, the end and/or the 3, the end, and/or the sequence of the sequence are, for example, no more than 2 - 45, or no more than 2 - 30, or no more than 3 - 20, or no more than 4 - 15, or no more than 5 - 10, or no more than 6-8 consecutive integers are represented respectively by one base substitutions, deletions, additions, modifications ₀

 In the present invention, the nucleotide sequence shown in SEQ ID NO: 1 is substituted with a nucleotide sequence represented by SEQ ID NO: 1 by a substitution, deletion or addition modification of one or more of the above bases. The nucleotide sequence has the same or similar promoter activity.

Described by a polynucleotide having a nucleotide sequence such as at least 95% "same" to the reference nucleotide sequence of SEQ ID NO: 1 means: a reference core of SEQ ID NO: 1. The polynuclear per 100 nucleotides of the nucleotide sequence The nucleotide sequence of the nucleotide has the same nucleotide sequence as the reference sequence except that it contains up to 5 nucleotides. In other words, in order to obtain a nucleotide sequence and a reference nucleotide sequence of at least 95 ° /. With the same polynucleotide, up to 5% of the nucleotides in the reference sequence can be deleted or replaced by another nucleotide; or some nucleotides can be inserted into the reference sequence, wherein the inserted nucleotides can be as many as 5% of the total nucleotides of the reference sequence; or in some nucleotides, there is a combination of deletions, insertions and substitutions, wherein the nucleotides are up to 5% of the total nucleotides of the reference sequence. These mutations in the reference sequence may occur at the 5, or 3, terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either alone in the nucleotides of the reference sequence, or as an OR Multiple adjacent groups are present in the reference sequence.

 In the present invention, algorithms for determining sequence identity and percent sequence similarity are, for example, BLAST and BLAST 2.0 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. BLAST and BLAST 2.0 can be used to determine the percent nucleotide sequence identity of the invention, as described, for example, in the literature or by default parameters. Software for performing BLAST analyses is available to the public through the National Center for Biotechnology Information (NCBI).

 In the present invention, the nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown by SEQ ID NO: 1 comprises a polynucleotide sequence substantially identical to the sequence disclosed in SEQ ID NO: 1, For example, when employing the methods described herein (e.g., BLAST analysis using standard parameters), it contains at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, 95, as compared to the polynucleotide sequences of the present invention. Those sequences of sequence identity of %, 96%, 97%, 98% or 99% or higher.

In the present invention, the nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown by SEQ ID NO: 1 has the same or similar nucleotide sequence as SEQ ID NO: 1. Promoter activity. In the present invention, the promoter is derived from a monocotyledonous plant, specifically, the monocotyledonous plant is a millet, for example, the millet is a modified 冀 Zhanggu No. 1 (Improved 冀 Zhanggu No. 1 seed in April 2010 On the 1st, it was deposited at the Wuhan University of Wushan City, Hubei Province, Wushan City, Wuhan, China, the China Center for the Collection of Cultures (CCTCC), the preservation number is CCTCC P201 006).

 A further aspect of the invention also relates to a recombinant vector comprising a promoter of the invention. The recombinant vector can be obtained by inserting the above promoter into a cloning vector or an expression vector.

 Cloning vectors suitable for construction of the recombinant vectors of the invention include, but are not limited to, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T S imp le Vec ter, pMD19-T S imp le Vec ter and so on.

 Expression vectors suitable for construction of the invention include, but are not limited to, for example: pBI 121, pl 3W4, pGEM, and the like.

 In one embodiment of the invention, the recombinant vector is a P8+P603 recombinant vector.

 A further aspect of the invention also relates to a recombinant cell comprising said recombinant vector of a promoter of the invention. The recombinant cell can be obtained by transforming the recombinant vector containing the promoter of the present invention into a host cell.

 Host cells suitable for constructing the recombinant cells of the present invention include, but are not limited to, for example, Agrobacterium tumefaciens cells LBA4404, EHA105, GV3101 and the like.

 In one embodiment of the invention, the recombinant cell is Agrobacteriu tumefaciens EHA105-P603.

In still another aspect of the invention, the invention relates to a monocot callus transformed with a promoter of the invention. In one embodiment of the invention, the monocot is rice. The rice includes, but is not limited to, for example: Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yuyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Yuyou 416, Yanyou 107, Yuyou 128, Yanyou 718, Zhunliangyou 527, Chuanong No. 1, Miscellaneous 0152, Japonica 88, Japonica 90 , japonica rice 92, japonica rice japonica 94, japonica rice japonica 96, japonica rice 185, japonica rice 187, japonica rice 189, japonica rice 191, japonica rice 193, japonica rice 195, japonica rice 197, japonica rice 199, japonica rice 201, alfalfa Rice 203, japonica rice 205, japonica rice 207, and Jinyuan 101 (the above rice varieties can be purchased from Anhui Huishang Farming Co., Ltd.) and so on. In still another embodiment of the present invention, the rice is Nipponbare.

 A further aspect of the invention also relates to a method of preparing a promoter of the invention, comprising the steps of:

 1) designing a PCR amplification primer pair according to the nucleotide sequence shown in SEQ ID NO: 1,

 2) Using the modified genomic DNA of Zhanggu No. 1 as a template, PCR amplification was performed using the PCR' amplification primer pair designed in step 1). '

 It is well known to those skilled in the art that a corresponding PCR amplification primer pair can be designed according to the nucleotide complementation principle according to the nucleotide sequence of interest to be amplified. In one embodiment of the invention, the PCR amplification primer pair is as set forth in SEQ ID NO: 2 and SEQ ID NO: 3.

 A further aspect of the invention also relates to a method of regulating expression of a gene of interest in a plant, the method comprising the step of transforming a callus of a plant with the promoter of the invention. In one embodiment of the invention, the transformation of the monocot callus utilizes recombinant cells comprising a promoter of the invention. In one embodiment of the present invention, the aforementioned recombinant Agrobacterium tumefaciens EHA105-P603 is utilized in the transformation of the monocot callus. In a specific embodiment of the present invention, the callus of the monocotyledonous plant is a rice callus, and specifically, the rice is Nipponbare.

In the present invention, plant gene transformation technology can be used to insert the gene of interest into the plant. The genome includes Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, gene gun transformation, and electroporation. It is well known in the art that Agrobacterium-mediated gene transformation is often used for gene transformation of monocots and dicots, but other transformation techniques can also be used for gene transformation of the monocots of the present invention. Of course, another method for transforming monocotyledonous plants suitable for the present invention is particle bombardment (microgold or tungsten particle coated transformed DNA) embryogenic callus or embryo development. In addition, a method for transforming monocots that can also be used is protoplast transformation. After gene transformation, a general method is used to select and regenerate plants integrated with expression units.

 In the present invention, plants which can utilize the monocot promoter to regulate expression of a gene of interest include, but are not limited to, rice, wheat, corn, millet, sugar cane, sorghum, barley, and the like.

 A further aspect of the invention also relates to the use of a promoter according to the invention for regulating the expression of a gene in a plant. In one embodiment of the invention, the plant is a monocot. In one embodiment of the invention, the gene of interest regulated by the promoter of the invention is GUS. In one embodiment of the invention, the monocot is rice, and in particular the rice is Nipponbare.

 For the purpose of achieving the above-described regulation of the expression of the gene of interest, the promoter of the present invention may be used in the form of a single copy and/or multiple copies, or may be used in combination with a promoter and/or enhancer known in the art.

A further aspect of the invention relates to the use of a promoter according to the invention in rice breeding. The rice is Nipponbare, Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yanyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Qiyou 416 , Youyou 107, Youyou 128, Yanyou 718, Zhunliangyou 527, Chuanong 1 , Miscellaneous 0152, Japonica 88, Japonica 90, Japonica 92, Japonica 94, Japonica 96, Japonica 185, Indica 187, japonica 189, japonica rice 191, japonica rice 193, japonica rice 195, japonica rice 197, japonica rice 199, japonica rice 199, japonica rice 203, japonica rice 205, japonica rice 207, and Tsuwara 101. In one embodiment of the invention, the rice is Nipponbare.

 The promoter of the present invention can be a novel promoter, as a plant: for example, rice, a transgenic tool promoter. Since the promoter of the present invention is a constitutive promoter and can regulate the expression of a gene of interest across species, the promoter of the present invention can facilitate a variety of plant breeding, such as low-expression gene transformation seedling screening, plant flower organ failure. Research on molecular breeding such as breeding. It will greatly shorten the breeding time of excellent varieties.

 The promoter of the present invention can be widely used for cultivating plants such as rice, wheat, corn, millet, sugar cane, sorghum, barley, and the like. Advantageous effects of the invention

 The inventors obtained a ubiquitin promoter derived from millet by bioinformatics research, and verified the function of the promoter P603 by biological experiments, which has the function of regulating the expression of the target gene across species. The constitutive promoter. Specifically, the promoter can regulate the expression of GUS gene in rice: the efficient expression of the gus gene can be regulated in the root, stem and leaf of rice callus and rice transgenic seedlings. DRAWINGS

 F ig. 1 is the result of PCR amplification detection of promoter P603, wherein lane M: 200 bp DNA Ladder Marker, the number on the left side indicates the size of the strip of the ladderer pointed to, in bp; lane 1 : PCR amplification products.

 F ig. 2 is a schematic representation of the pCAMBIA-1301 plasmid used to construct the p8 plasmid.

 F ig. 3 is a schematic representation of the multiple cloning site and the GUS sequence portion of the p8 plasmid map.

 F ig. 4 is a schematic representation of the p8 plasmid.

F ig. 5 is the result of GUS staining of transformed rice callus. Among them, by belt Rice callus transformed with recombinant Agrobacterium tumefaciens p8+P603 having the promoter sequence P603 of the present invention (left) exhibits blue color after GUS staining; recombinant Agrobacterium tumefaciens p8 without the promoter sequence of the present invention The rice callus of the plasmid (control, right) did not change color after GUS staining.

 F ig. 6 is the result of GUS staining of transformed transgenic rice seedlings. Wherein, the rice seedling transformed with the recombinant Agrobacterium tumefaciens P8+P603 having the sequence of the promoter P603 of the present invention (right) is stained with GUS, and the roots, stems and leaves thereof are blue; The sub-sequence of recombinant Agrobacterium tumefaciens P8 transformed rice seedlings (control, left) did not change in color of roots, stems and leaves after GUS staining. detailed description

 The embodiments of the present invention are described in detail below with reference to the accompanying drawings. Those who do not specify the conditions in the examples are subject to the usual conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that are commercially available. Example 1 Promoter PCR amplification of P603 fragment and construction of pMD18-T+P603 recombinant vector

 PCR amplification

The genomic DNA (gDNA) of the improved sorghum Zhanggu No. 1 seed was extracted using the plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02), and the improved 冀 谷 谷 1 was modified according to the promoter. The sequence in gDNA is designed with a pair of PCR-specific amplification primers at the beginning and the end (upstream primer Fl, restriction enzyme site EcoR I and protective thiol, downstream primer R1, restriction enzyme site Sbf I and Protect bases). Improved gDNA of Zhanggu No. 1 extracted as described above For template, PCR amplification was performed using high fidelity Ex Taq (TaKaRa, DRR100B) polymerase. As shown in Table 1.

 Gene promoter amplification PCR system

 Composition volume ( μ 1 )

 Genomic DNA 0. 2

 dNTPs ( 2. 5 mM ) 2

 1 0 χ Ex Buf fer (with magnesium ion) 2. 5

 Primer Fl ( 50 μ Μ ) 1

 Primer Rl ( 50 μ Μ ) 1

 Ex t aq 0. 2 .

ddH ₂ 0 fills up to a total volume of 25 μ 1

The PCR amplification procedure was: 94 pre-denaturation for 5 min, then denaturation at 94 for 45 s, 55 Ό annealing for 50 s, 72 extension for 90 s, 35 reaction cycles, and finally 72 X: extension for 7 min.

 Among them, the upstream primer F1: GGGAATTCACTTAGCTTCTGGCCATCTCCAGA (SEQ ID NO: 2), wherein the underline represents the EcoR I restriction site.

 The downstream primer R1: GCCCTGCAGGCTGTAGAAGAAAAAACAAGCAA (SEQ ID NO: 3), wherein the underline represents the Sbf I restriction site.

 The PCR amplification product was separated by 1.0% agarose gel electrophoresis to obtain a band of 2038 bp (Fig. 1), which was purified by TIANGEN agarose gel DNA recovery kit (catalog number: DP209-03). .

 Construction of PMD18-T+P6Q3 Recombinant Vector

 The PCR amplification product obtained above was subjected to T/A cloning (PMD18-T plasmid, TaKaRa, D103A), transformed into Escherichia coli, and the positive clone was picked and sequenced (as shown in SEQ ID NO: 4), which proved to be correct.

Among them, the connection conditions of the T/A clone are as follows: T/A connection system: 10 μ ΐ

 PMD18-T 1 μ 1

 2* so lut ion I 5 μ 1

PCR amplification product (recovered insert) 10 ng ~ 20 ng, according to its concentration, ddH ₂ 0 is added to 10 μ 1

 In the energy-saving intelligent thermostat (Ningbo Xinzhi, SDC-6), it was connected for more than 8 h to obtain PMD18-T+P603 recombinant vector. The product after the above ligation was transformed into Escherichia coli as follows:

 Remove the competent cells 100 μ 1 DH5 o prepared by the calcium chloride method shown in the Guide to Molecular Cloning (Third Edition, Science Press) from the ultra-low temperature freezer (provided by Dong Yang, Kunming Institute of Zoology, Chinese Academy of Sciences; or From, for example, Shanghai Shengong), after melting on ice, add 10 μ ΐ of the ligation product obtained above, ie pMD18-T+P603 recombinant plant, gently stir the hook, ice bath for 30 min, 42 heat shock 60 s , ice bath for 5 min, add 600 μ 1 4 X: pre-cooled SOC medium (for details, see the Guide to Molecular Cloning, Third Edition, Science Press), 37 "C 220 rpm recovery for 45 min, 8000 Centrifuge at rpm for 30 s, remove the supernatant, and take 150 μl. Resuspend the precipitated mixture with the remaining 150 μl of the supernatant, gently spread, and apply LB (kanamycin) plate to the glass beads. For details, see the Guide to Molecular Cloning, Third Edition, Science Press, 37 Inverted culture for 16 h ~ 24 h. Recombinant E. coli containing pMD18-T+P603 cloning vector was obtained and named DH5 ot-P603. Big Gene Technology Co., Ltd. For PMD18-T + P603 P603 cloning vector for sequencing, the results are as follows:

GGGAATTC|ACTTAGCTTCTGGCCATCTCCAG^GACAGTGAGTTGATGCTTGAT GCTAGACGAGGGGAAAAAAAGCAGAAAATCAGCCATACTAAATCAACTAATGATTTC AAAGAGAGGTACCTAATGCTCAAAAAGGAAGAGATTGGGGGATTTGCGACTAAAGAA GAGAATAAAATAGATTTTTTATAGCGTTAAGAAGTGTGTCACAGCTCTTACAGGAAT GCTTGATCTACAAATGGAATAGATGATAATGGCAGCGGATATGGACGGATCGGGGTT GTTTAGTTCCTAATTTTTTAAAAAGTTTTTCGTCACATCGAATCTTATAACACATGT GTGAGTATTAAATATAGATAAAAAATAACTAATTACATAATTTATTTGTAGTTTGCT AGATAAAATTTTTGAGCCTAGGGTTAGTTTATGATTGAATAATAATTATCACGAAAG TACTACAGTAGTTAAATTTAAAATTGTTCGCAAACTAAACAAGGCCATGGTGTGTTT TTATTTTACTCTCTAAAAATCTGCACAAAGGTTTTCTGACTCATGGGCCACACGTCT CAGTGTCGGTAAACACGGACGGAATCACGGGAGAAGGCATTAACAGCGTCGGGTCTA ACGGCCACAAACCAGCGACGAACGAAACAGACGTTCTGACGTCTCCGTGTCCACTCC GTCACTGGTTCCTTCTGGAGAGCTCTGACCTCCTCCGTCTCTATCTACGGCCGGCTC GCCTTCCGTTCCGCGTTCGCGTTGGACTCTTTGCGCTGGCGTGTTCCTGGAATTGCG TGGCGGAGACGAGGCGGATTTCTCTCGCACGGAACGGAACCGCCACGGGCCCAAAGG CACGGTGATTCCTTCTCCACCAACATAAATAGCCAGGACCCCTCCTCGCCTTTCCCC AATCTCATCTCGCATTGTGTTGTTCGGAGCAAGGAGAACCCAGCCCCCCATCGCTCT CAATCCCAATCGATCTTCTTCTCGTGAGCCTCGTCAATCCATCACCCGCTTCTAAGG TACGGCTCCCCCTCTAATCTTCTCTTCCCATCTCAGATTGGCGAGTTTATGTGATTA GATTAGATGCTTCTCATCTAGATTGCGAGTTTCTGTTCGTAGATGGCTGGCTTGTAA GCGGTTCCTAGGTGGGTTTCTGTTCGTAGATGACTGGCTTGTAAGCGGTTCCTAGGT GGGATCGTTCTGATGATTTCTTTGGCTGCTGCGTAGAGATAGATCTGGTCCTGCTTT TCTTAATTCTTGGT GCAGATTTTGTGACCTGGTTCTATGTTCTTGTTCCTGCTTTGT AGCTCAAATAGTTGTCTTAACTAGCTGGGCTTATTATTTGATTTGTACCTGCATGTA TTATCACCAAATACAATTACTGTGAAGGAGTCAATATAGGCTGCTCTGTACCTTTTA CCTGACGAGCCATACTATCATTTTGATTCGTGTCATATGCATGCCAGATACGGAAAT TATATGCTGCTACTTGCGTTATTATCATGCTGATTTGTTTCATATGCACGCCTAGAT AGATGGAAATTATATGCTACTGCTGAGCGTTATTATCATGCTGATTCGTTTCATATG CATGCCTAGATAGTTGGAAGTTTTGTTGTTTGCTGAGTGTTACTATCATGTTGATTT GTAATCATATGCATGCCTAGATAGATGAAGATACATGAATGTTATTCGTTTCAGATA GATGGAATATGCTGCTACTGAGCGTTACTATCATGTTGATTTGTTTCATATACACGC CTAGATAGATGAAGAGATGGATGTTGATTTGTTTCATATGCATGCCTAATAGATGAA GATATATGCTGCTACTGATGATTACTTACTACTTCGTGCCCATGCATGCTCTTTGGT TTACTTGGATGGTGACATGCTGATGCAGTTTTGCTGGTTCTATAGTACCTATGTGCT TAGCATGTATATCTGTTTCTTGTTGCTGACTGTTTCTTTCCCTCCTTAGTCTACCGC

CGTATACTTATCATG|TTGCTTGTTTTTTCTTCTACAG|CCTGCAGGGC (SEQ ID NO: 4)

 In the above sequence, underlined is the restriction site (EcoRI+Sbf l ), and the box is the primer sequence.

 The sequencing results showed that the P603 promoter sequence was correct in the obtained pMD18-T+P603 cloning vector. Example 2 Construction of vector-P8+P603 recombinant vector

The promoter of the Ρ603 promoter of the present invention was extracted from the Escherichia coli DH ⁵ α -Ρ603 transformed with the promoter P603 constructed in Example 1 according to the manual of the TIANGEN Ordinary Plasmid Mini Kit (Cat. No. DP103-03). The cloning vector PMD18-T+P603; after purification, the corresponding restriction enzymes EcoR I ( NEB ) and Sbf I ( NEB ) were digested, and the corresponding promoter inserts were recovered and used with the same restriction as the p8 plasmid. A large fragment of the vector recovered by restriction endonuclease digestion was ligated.

 The resulting ligated product P8+P603 recombinant vector was transformed into competent cells DH5 a , 37 X: inverted culture for 16-24 h according to the method of molecular cloning experiments (third edition, Science Press). After the transformant grows out of bacteria, the monoclonal is picked for PCR detection and enzyme digestion.

 P8 plasmid construction

The p8 plasmid used in the present invention is provided by pCAMBIA-1301 plasmid (Dong Yang, Kunming Institute of Zoology, Chinese Academy of Sciences; or may be derived from, for example, Shanghai Guorui Gene Technology) The company purchased, or can be obtained from The CAMBIA Bios (biological open source) Licensee, Austral ia), modified and constructed as follows, as follows:

 The plasmid pCAMBIA-1301 (see Figure 2) was double-digested with Kpn I /Nco I (NEB) to recover large fragments. The following sequence was synthesized according to the restriction enzyme site used: GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAG GATCCGTCGACCATGG (SEQ ID NO: 5) (The cleavage site included is Kpn I /Hindm/Spe I /Sbf I /Pst I /Xba I /BamH I /Sal I /Nco I ), recovered by double digestion with Kpn I /Nco I, and ligated with the large fragment recovered above to transform topk cells (provided by Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences; or from Beijing, for example: Beijing Solaibao Technology Co., Ltd. purchased). Using the primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO: 6) / GAGTCGTCGGTTCTGTAAC (SEQ ID NO: 7), the transformant was selected by PCR, and the transformant with the amplified fragment of 350 bp was the multiple cloning site containing the desired construction. And the transformant of the p8 plasmid of the GUS sequence (see Figure 4).

 The multiple cloning site in the p8 plasmid and the length of the GUS sequence are 2353 bases, as shown in SEQ ID NO: 8 (see Figure 3):

 GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG CCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTG AGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACA

 ^TTTTTCTCCTTCATTTTCTTGGTTAGGACCCT TCTGTTTTTA TTT TTGAG^ ^f f(iXf TTJWTTAAACTGAi: :ntrTTAATTlG TG^TATGG JAl^

\Α ΤΤΑ CA ΤΑ GCTTTAA CTGA ΤΑΑ TCTGATTA CTTTA TTTCGTGTGTCTA TGA TGA Τ\

\GA TGA TAGT AGAGAA CCGA CGA CTCGTCCGTCCTGTA GAAA CCCCAA CCCGTGA AA TCAAAAAA CTCGA CGGCCTGTGGGCA TTCA GTCTGGA TCGCGAAAA CTGTGGA A TTGA TCA GCGTTGGTGGGAAA GCGCGTTA CAA GAAA GCCGGGCAA TTGCTGTGC CA GGCA GTTTTAA CGA TCA GTTCGCCGA TGCA GA TA TTCGTAA TTA TGCGGGCAA CGTCTGGTA TCA GCGCGAA GTCTTTA TA CCGAAA GGTTGGGCA GGCCA GCGTA TC GTGCTGCGTTTCGA TGCGGTCA CTCA TTA CGGCAAA GTGTGGGTCAA TAA TCA GG AA GTGA TGGA GCA TCA GGGCGGCTA TA CGCCA TTTGAA GCCGA TGTCA CGCCGTA TGTTA TTGCCGGGAAAA GTGTA CGTA TCA CCGTTTGTGTGAA CAA CGAA CTGAA C TGGCA GA CTA TCCCGCCGGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GC A GTCTTA CTTCCA TGA TTTCTTTAA CTA TGCCGGAA TCCA TCGCA GCGTAA TGCT CTA CA CCA CGCCGAA CA CCTGGGTGGA CGA TA TCA CCGTGGTGA CGCA TGTCGCG CAA GA CTGTAA CCA CGCGTCTGTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA TGCGGA TCAA CA GGTGGTTGCAA CTGGA CAA GGCA CTA G CGGGA CTTTGCAA GTGGTGAA TCCGCA CCTCTGGCAA CCGGGTGAA GGTTA TCTC TA TGAA CTCGAA GTCA CA GCCAAAAGCCA GA CA GA GTCTGA TA TCTA CCCGCTTC GCGTCGGCA TCCGGTCA GTGGCA GTGAA GGGCCAA CA GTTCCTGA TTAA CCA CAA A CCGTTCTA CTTTA CTG GCTTTGGTCGTCA TGA AG A TGCGGA CTTA CGTGGCAAA GGA TTCGA TAA CGTGCTGA TGGTGCA CGA CCA CGCA TTAA TGGA CTGGA TTGGGG CCA A CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGA A GA GA TGCTCGA CTGGGC A GA TGAA CA TGGCA TCGTGGTGA TTGA TGAAA CTGCTGCTGTCGGCTTTCA GCTG TCTTTA GGCA TTGGTTTCGAAGCGGGCAA CAA GCCGAAA GAA CTGTA CA GCGAA GA GGCA GTCAA CGGGGAAA CTCA GCAA GCGCA CTTA CA GGCGA TTAAA GA GCTGA TA GCGCGTGA CAAAAA CCA CCCAA GCGTGGTGA TGTGGA GTA TTGCCAA CGAA CCG GA TA CCCGTCCGCAA GGTGCA CGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGC GTAAA CTCGA CCCGA CGCGTCCGA TCA CCTGCGTCAA TGTAA TGTTCTGCGA CGC TCA CA CCGA TA CCA TCA GCGA TCTCTTTGA TGTGCTGTGCCTGAA CCGTTA TTA C GGA TGGTA TGTCCAAA GCGGCGA TTTGGAAA CGGCA GA GAA GGTA CTGGAAAAA G AA CTTCTGGCCTGGCA GGA GAAA CTGCA TCA GCCGA TTA TCA TCA CCGAA TA CGG CGTGGA TA CGTTA GCCGGGCTGCA CTCAA TGTA CA CCGA CA TGTGGA GTGAA GA G TA TCA GTGTGCA TGGCTGGA TA TGTA TCA CCGCGTCTTTGA TCGCGTCA GCGCCG TCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCGA CCTCGCAA GGCA TA TT GCGCGTTGGCGGTAA CAA GAAA GGGA TCTTCA CTCGCGA CCGCAAA CCGAA GTCG GCGGCTTTTCTGCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTGAAAAA CCGC A GCA GGGA GGCA AA CAA GCTA GCCA CCA CCA CCA CCA CCA CGTGTGA ( SEQ ID NO: 8 )

 The p8 plasmid constructed in the present invention as shown in the above sequence, EcoR I /Sac I /Kpn I /H ind m /Spe I /Sbf I /Ps t I /Xba I /BamH I /Sa in the multiple cloning site l I / Nco I restriction sites are indicated by box and underline, respectively, and the primers used for screening transformants are GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTA AC (ie SEQ ID NO: 6 and 7), which are indicated by double underline, and GUS sequences are indicated in italics. The intron sequences are shown in italics and shading, respectively.

 Construction of recombinant vector P8+P603

 The cloning vector pMD18-T+P603 obtained in Example 1 and the p8 plasmid constructed as described above were treated according to the following conditions under restriction enzymes EcoR I (NEB) and Sbf I (NEB).

 Among them, the cloning vectors pMD18-T+P603 and p8 plasmid were digested as follows: Enzyme digestion system: 50 μ 1

 Sterile water 34. 8 μ 1

 10* buffer H 5 μ 1

EcoR I 0. 1 μ 1 ( 10 U ) Sbf I 0. 1 μ 1 ( 10 U ) cloning vector PMD18-T+P603 or p8 plasmid 10 μ 1 ( <1000 ng ) using TIANGEN agarose gel DNA recovery kit (Catalog No.: DP209-03) The digested p8 plasmid and the promoter P603 insert were separately recovered and ligated according to the T4 ligase (TaKaRa, D2011A) operating instructions according to the following conditions: Ligation system: 10 μ 1

 10*T4 buffer 1 μ 1

 Restricted ρ8 plasmid 1 μ ΐ ( 20 ng)

 Recovered promoter P603 insert 10-20 ng, according to its concentration, sterile water is added to 9.5 μ 1

 Τ4 ligase (TaKaRa, D2011A) 0.5 μ 1

 The Τ4 buffer was thawed on ice, and the p8 plasmid vector after digestion was added in an amount of about 20 ng, and the P603 fragment in the present invention was added in an amount of 10-20 ng. Connected in energy-saving intelligent thermostat (Ningbo Xinzhi, SDC-6) for more than 8 h at 16 'C.

 The competent cell DH5ot prepared by the 100 μl calcium chloride method was taken out from the ultra-low temperature freezer, and after melting on ice, 10 μ ΐ of the above-mentioned ligation product was added, and the mixture was gently stirred, water bathed for 30 min, and heat shocked at 42 ° C for 60 s. Water bath for 5 min, add 600 μl 4 pre-cooled S0C, 37. C resuscitate at 220 rpm for 45 min, centrifugation at 8000 rpm for 30 s, remove the supernatant, and take 150 μΐ, gently spread, glass beads coated with LB (kan), 37 X: inverted culture for 16-24 h. The recombinant vector p8+P603 was obtained.

 The resulting recombinant vector P8+P603 was subjected to PCR detection using Fl (SEQ ID NO: 2) and Rl (SEQ ID NO: 3) as primer pairs, respectively, to confirm that the resulting recombinant vector P8+P603 contained the desired promoter P603. The recombinant vector containing the recombinant vector P8+P6Q3 was screened by EcoR I /Sbf I digestion. Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P603 cells

The p8+P603 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were separately transformed into root cancer prepared according to the calcium chloride method described in the Guide to Molecular Cloning (Third Edition, Science Press). Agrobacterium EHA105 (2009 12 On the 24th of the month, it was deposited in the Wuhan University of Wuhan Wushan, Wuhan, Wushan, Hubei Province, the China National Type Culture Collection (CCTCC), the accession number is CCTCC M 209315), the specific methods are as follows:

 The Agrobacterium tumefaciens competent cell EHA105 was taken out in an ultra-low temperature water tank and thawed on ice. After thawing, 5 μΐ of ρ8+Ρ603 recombinant vector and ρ8 plasmid and ρ8 empty vector as control were added, gently mixed, ice-bathed for 10 min, frozen in liquid nitrogen for 5 min, and thawed at 37 °C for 5 min. Add 800 μl of normal temperature LB liquid medium, resuscitate at 28 160 rpm for 3 h, centrifuge at 8000 rpm for 30 s, aspirate the supernatant, leave a 200 μ ΐ blown hook, and apply with kan- r if (Kanamycin) On the YM medium plate (50 mg / 1 Kan, 10 mg / 1 Ri f , see Table 4 for specific formulation). 28 X: Inverted culture for 2-3 days.

 The PCR was carried out using Fl (SEQ ID NO: 2) and Rl (SEQ ID NO: 3) as primers and the transformants were cleaved by Kpn l /Sbf I.

 A recombinant Agrobacterium tumefaciens which is a recombinant vector p8+P603 was amplified by PCR and amplified with a band of about 2038 bp and a band of about 2027 bp.

 In the present invention, the recombinant Agrobacterium tumefaciens having the recombinant vector P8+P603 obtained according to the above method is named Recombinant Agrobacterium tumefaciens EHA105-P603.

 According to the method of the present invention, the obtained recombinant Agrobacterium tumefaciens harboring the p8 plasmid was designated as Recombinant Agrobacterium tumefaciens EHA105-p8. Example 4 Induction and Transformation of Rice Callus

 The rice callus was induced as follows, and the callus was transformed with recombinant Agrobacterium tumefaciens EHA105-P603 and recombinant Agrobacterium tumefaciens EHA105-p8, respectively.

1) Rice Nippon Seed (deposited on December 18, 2009 in Wuhan, Wuchang, Wuhan, Wushan, Wuhan, China, the China Center for the Collection of Cultures (CCTCC), under the accession number CCTCC P200910), 70% Ethanol surface disinfection 30 s, then use effective chlorine 1.5 ° /. The sodium hypochlorite was disinfected for 30 min, during which it was shaken vigorously, and then washed with sterile water for 5 times; the disinfected seeds were placed on N6D medium (see Table 2 for specific formulation) and sealed with a sealing film; 29.5. C light culture for 3-4 weeks;

 2) Select actively growing callus (yellow-white, dry, 1-3 mm in diameter), on new N6D medium 29.5: light culture for 3 days;

 3) Picking a single colony of recombinant Agrobacterium tumefaciens (recombinant Agrobacterium tumefaciens EHA105-P603 or recombinant Agrobacterium tumefaciens EHA105-p8) constructed as in Example 3, and adding antibiotics (50 mg/1 Kan, 10) The YM medium of mg/1 Rif) (see Table 3 and Table 4 for specific formulation) was streaked for 3 days at a culture temperature of 28 °; the above recombinant Agrobacterium tumefaciens was scraped separately and placed in an AS supplemented with 30 μΐ 100 mM. (Acetosyringone, acetosyringone) in 30 ml of A AM medium (see Table 5 for specific formulation), gently resuspend the recombinant Agrobacterium tumefaciens cells (recombinant Agrobacterium tumefaciens EHA105-P603 or recombinant Agrobacterium tumefaciens EHA105) -p8 ) ;

 4) placing the subcultured callus in a sterilized culture dish; pouring the recombinant Agrobacterium tumefaciens suspension prepared in step 3 into the culture dish, and immersing the callus therein for 15 min;

 5) Pour out the recombinant Agrobacterium tumefaciens suspension, and use the sterile absorbent paper to absorb excess liquid from the callus; place a sterile filter paper on N6-AS medium (see Table 6 for formulation), add 1 ml. The AAM medium containing AS is used to transfer the callus to the filter paper; the sealed culture is sub-28 X: dark culture for 48-60 h;

6) Place the infected callus in a 50 ml sterile tube and rinse with sterile water until the supernatant becomes clear; soak the callus in 500 mg/1 carbenicillin (Cb) Killing recombinant Agrobacterium tumefaciens in sterile water; removing excess water from the callus with sterile absorbent paper and transferring it to 1 mg/1 hygromycin B (HmB) and 50 mg/1 Carb N6-AS medium; seal the culture dish with a parafilm and incubate for 2 to 4 weeks at 29.5"C light. Example 5 Expression of GUS in Rice Callus

 To examine the expression of the gene GUS in the rice callus transformed by the method described in Example 4, the method described in Chen SY et al., Journal of Integrative Plant Biology, 2008, 50 (6): 742-751, The rice calli transformed with recombinant Agrobacterium tumefaciens EHA105-P603 or recombinant Agrobacterium tumefaciens EHA105-p8 was stained.

Formulation of GUS staining solution (1 ml): 610 μ 1 0.2Μ Na ₂ HP0 ₄ solution (pH-7.0); 390 μ 10.2MNaH ₂ P0 ₄ solution and 10 μ 10.1 MX-gluc will use recombinant Agrobacterium tumefaciens EHA105 -P603 or recombinant callus of Agrobacterium tumefaciens EHA105-P8 was immersed in GUS staining solution, 37 was incubated until blue appeared, and the results were recorded. The results are shown in Figure 5, and recombined with p8+P603 containing the promoter. The vector recombinant Agrobacterium tumefaciens-mediated transformed rice calli (Fig. 5 left) was stained blue, and the rice callus mediated by Agrobacterium tumefaciens mediated by the p8 plasmid without the promoter (as Control, Figure 5, right) The color did not change after staining with GUS. The results show that the P603 promoter of the present invention has a regulatory effect on GUS gene expression. Example 6 Expression of GUS in Transgenic Rice Seedlings

The callus obtained in Example 4 was transferred to MS-R differentiation medium containing 50 mg/1 hygromycin B (HmB) (see Table 7 for specific formulation). The seedlings were sealed with a parafilm seal, 29.5°. C light culture for 3-4 weeks; when the seedling grows to ^3-4 cm, transfer to 1/2 MS rooting medium containing 50 mg/1 hygromycin B (HmB) (see Table 8 for specific formulation) for rooting.

The GUS staining process of the transgenic rice seedlings was the same as that of the callus in Example 5. The results are shown in Figure 6. The weight of the recombinant plasmid containing p8+P603 containing the promoter The roots, stems and leaves of rice seedlings mediated by Agrobacterium tumefaciens (Fig. 6 right) were stained blue, and the roots of rice transformed by Agrobacterium tumefaciens mediated by recombinant p8 plasmid without promoter , stems, leaves (as a control, Figure 6 left) The color did not change after GUS staining. The results show that the P603 promoter of the present invention has a regulatory effect on GUS gene expression. The formulation of the relevant medium used in the examples of the present invention is as follows: The following "conventional sterilization" as used in the medium refers to sterilization under the following conditions:

121 X: Steam sterilization for 20 minutes.

 Table 2 N6D medium

 1 L 0.5 L 2 L

N ₆ macro mother liquor ( 20X) 50 ml 25 ml 100 ml

Fe ₂ EDTA stock solution ( 100X) 10 ml 5 ml 20 ml

N ₆ micro mother liquor ( 1000X) 1 ml 0.5 ml 2 ml

N ₆ vitamin stock solution ( 1000X) 1 ml 0.5 ml 2 ml Inositol ( 500X) 2 ml 1 ml. 4 ml

2,4-D stock solution ( 1 mg/ml ) 2 ml 1 ml 4 ml Proline 2.88 g 1.44 g 5.76 g Hydrolyzed casein (CH) 0.30 g 0.15 g 0.6 g

 Sugar ( sucrose ) 30 g 15 g 60 g plant gel ( Phytagel ) 3 g 1.5 g 6 g

 The pH was adjusted to 5.8 with IN potassium hydroxide, and then sterilized by a conventional method after sealing.

N ₆ macro mother liquor (20X): potassium nitrate 56.60 g, potassium dihydrogen phosphate 8.00 g, ammonium sulfate 9.26 g, magnesium sulfate 3.70 g, calcium chloride 3.32 g, distilled water to 1 L, 4 X storage.

N ₆ micro mother liquor (1000X): potassium iodide 0.80 g, boric acid 1.60 g, manganese sulfate 3.33 g, zinc sulfate 1.50 g, distilled water to 1 L, 4 X storage.

Iron salt (Fe ₂ EDTA) stock solution (100X) : 3.73 g EDTA Disodium (Na ₂ EDTA · 2H ₂ 0 ) and 2.78 g of FeS0 ₄ .7H ₂ 0 were separately dissolved and mixed. Dilute to 100Q ml with distilled water, warm bath for 2 hours, and store for 4 months after cooling.

N ₆ vitamin stock solution (1000X): Vitamin 0.10 g, vitamin B ₆ 0.05 g, niacin 0.05 g, glycine 0.20 g, add distilled water to 100 ml, filter sterilization, 4 X: save for less than 1 week.

 Table 3 YM liquid medium (containing 50 mg / L Kan, 10 mg / L Rif):

 1 L 0.5 L

 YM Broth Dry Powder 21 g 10, 5 g

 Conventional sterilization, add to the room after cooling to room temperature

 Kanamycin (Kan) (50mg/ml) 1 ml 0.5 ml

 Rifampicin (Rif) (50mg/ml) 0.2 ml 0.1 ml

Table 4 YM solid medium (containing 50 mg / L Kan, 10 mg / L Rif):

 1 L 0.5 L

YM Broth dry powder 21 g 10. 5 g agar powder (Agar) 15 g 7.5 g conventionally sterilized, cooled to room temperature and added

 Kanal (50mg/ml) 1 ml 0.5 ml

Rifampicin (Rif) (50mg/ml) 0.2 ml 0.1 ml

Table 5 AAM medium

 1 L 0.5 L

 AAM macro (10X) 100ml 50ml

 AAM micro (100X) 10ml 5ml

Fe ₂ EDTA stock solution (100X) 10 ml 5ml

 AAM Organic (1Q00X) 1ml 0.5ml

 Inositol (500X) 2ml 1ml

 Hydrolyzed casein (CH) 0.5g 0.25g

 Potassium chloride (C1) 3. Og 1.5g

 L-Arginine 0.176g 0.088g

 L-Glutamine 0.9g 0.45g

 L-Aspartic acid 0.3g 0.15g

 Sugar (sucrose) 68g 34g

 Glucose 36g 18g

 Conventional sterilization, join when used

 AS (lOOmM) 1 ml 0.5ml

 Add pH to 5.2 by adding IN potassium hydroxide and routinely sterilize.

AAM macro ( 1 OX) : 2.5 g magnesium sulfate heptahydrate ( MgS0 ₄ · 7H ₂ 0 ), 1.5 g calcium chloride dihydrate (CaCl ₂ ■ 2H ₂ 0 ), 1.33 g sodium dihydrogen phosphate dihydrate (NaH ₂ P0 ₄ .2Η ₂ 0) , dilute to 1 L of distilled water, and store at 4 °C for later use.

AAM micro ( 100X) : 0.7 g manganese sulfate monohydrate ( MnS0 ₄ · H ₂ 0 ), 0.2 g zinc sulfate heptahydrate ( ZnS0 ₄ · 7H ₂ 0 ), 0.075 g potassium iodide (KI), 0.3 g boric acid (H ₃ B0 ₃ ), 25 mg sodium molybdate dihydrate (Na ₂ MoG ₄ .2H ₂ 0 ), 2.5 mg copper sulfate pentahydrate (CuS0 ₄ .5H ₂ 0), 2.50^ cobalt chloride hexahydrate ((00:1 ₂ , 611 ₂ 0), dilute to 1 L of distilled water, 4 Ό save for use.

AAM organic (1000X): 0.75 g glycine (Glycine), 0.1 g nicotinic acid (Nicotinic acid), 0. Ig VB ₆ (Pyridoxine), lg VB) (Thime), dilute to 100 ml of distilled water, 4 X for storage.

Iron salt (Fe ₂ EDTA) stock solution (100X): See Table 2. Table 6 N6-AS co-culture medium

 1 L 0.5 L 2 L

N ₆ macro mother liquor ( 20X) 50 ml 25 ml 100 ml

Fe ₂ EDTA stock solution ( 100X) 10 ml 5 ml 20 ml

Niinicro mother liquor (1000X) 1 ml 0.5 ml 2 ml

N ₆ vitamin stock solution ( 1000X) 1 ml 0.5 ml 2 ml inositol ( 500X) 2 ml 1 ml 4 ml

2,4-D stock solution (1mg/ml) 2 ml 1 ml 4 ml Hydrolyzed casein (CH) 0.30 g 0.15 g 0.6 g Sugar ( Sucrose ) 30 g 15 g 60 g Glucose ( Glucose ) 10 g 5 g 20 g Plant Gel ( Phytagel ) 4 g 2 g 8 g Conventional sterilization, added when used

 Acetyl syringone (AS) (100 mM) 1 ml 0.5 ml 2 ml Adjust the pH to 5.2.

N ₆ macro mother liquor (20X), N ₆ micro mother liquor (1000X), iron salt (Fe ₂ EDTA) stock solution 0X), N ₆ vitamin stock solution (1000X): See Table ^{2 for} details.

Table 7 MS-R differentiation medium:

 1 L 0.5 L 2 L

MS macro ( 20X) 50 ml 25 ml 100 ml

MS micro ( 1000X) 1 ml 0.5 ml 2 ml

Fe ₂ EDTA stock solution ( 100X) 10 ml 5 ml 20 ml inositol ( 500X) 2 ml 1 ml 4 ml sucrose ( Sucrose ) 30 g 15 g 60 g Sorbitol 30 g 15 g 60 g plant gel ( Phytagel ) 4 g 2 g 8 g added after routine sterilization

MS vitamin (1000X) Filtration and sterilization 1 ml 0.5 ml 2 ml kinetin (1 mg/ml) Filter sterilization 2 ml 1 ml 4 ml Naphthaleneacetic acid (1 mg/ml) Filter sterilization 0.02 ml 0.01 ml 0.04 ml Carboxybenzyl Qing Zun Su ( 500 mg / ml ) 0.5 ml 0.25 ml 1 ml Chao Zun ( 50 mg / ml ) 1 ml 0.5 ml 2 ml Adjust the pH to 5.8 and sterilize in the usual way.

 MS macro ( 20X) : Ammonium nitrate 33. Q g, potassium nitrate 38.0 g, potassium dihydrogen phosphate 3.4 g, magnesium sulfate 7.4 g, calcium chloride 8.8 g dissolved one by one, then dilute to 1 L with distilled water at room temperature, 4 X: Save.

 MS micro ( 1000X) : manganese sulfate 16.90 g, zinc sulfate 8 · 60 g, boric acid 6.20 g, potassium iodide 0.83 g, sodium molybdate 0.25 g, copper sulfate 0.025 g, cobalt chloride 0.025 g, the above reagents are dissolved at room temperature Dilute to 1 L of distilled water and store at 4 °C.

MS vitamin stock solution (1000X): vitamin 0.010 g, vitamin B ₆ 0.050 g, niacin 0.050 g, glycine 0.200 g, dilute to 100 ml with distilled water, filter sterilization, 4 'C for less than 1 week.

Iron salt (Fe ₂ EDTA) stock solution (100X): See Table 2. Table 8 1/2 MS Rooting Medium

 1 L 0.5 L 2 L

MS macro mother liquor (20X) 25 ml 12.5 ml 50 ml

Fe ₂ EDTA stock solution (100X) 5 ml 2.5 ml 10 ml

MS micro mother liquor (1000X) 0.5 ml 0.25 ml 1 ml Inositol (500X) 1 ml 0.5 ml 2 ml Sugar (sucrose) 15 g 7.5 g 30 g Plant gel (Phytagel) 3 g 1.5 g 6 g Add after routine sterilization

 MS Vitamin (1000X) Filtration and sterilizing 0.5 ml 0.25 ml 1 ml Tidalin (50mg/ml) 1 ml 0. 5 ml 2 ml Adjust the pH to 5.8.

MS macro (20X) is shown in Table 7. MS micro (1000X) MS Vitamin Storage Solution (10QQX) See Table 7. Iron salt (Fe ₂ EDTA ) stock solution (100X): See Table 2. Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

A promoter having a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto, or a variant thereof having the function of a promoter selected from the group consisting of:

 1) a nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence shown in SEQ ID NO: 1,

 2) a nucleotide sequence in which one or more bases are substituted, deleted, or added to the nucleotide sequence shown in SEQ ID NO: 1, and

 3) A nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

A recombinant vector, characterized in that the recombinant vector contains the promoter of claim 1, and preferably, the recombinant vector is a P8+P603 recombinant vector.

3. A recombinant cell comprising the recombinant vector of claim 2.

4. The recombinant cell of claim 3 which is recombinant Agrobacterium tumefaciens EHA105-P603.

A plant callus characterized in that the callus is transformed with the promoter of claim 1, preferably, the callus is a rice callus, and more preferably, the rice is For Nipponbare, Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yanyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Yuyou 416, Yuyou 107, Yanyou 128, Yanyou 718, Zhunliangyou 527, Chuanong No.1, Miscellaneous 0152, Japonica 88, Japonica 90, Japonica 92, Japonica 94, Japonica 96, Japonica 185, Japonica 187 , japonica rice 189, japonica rice 191, japonica rice 193, japonica rice 195. Japonica rice 197, japonica rice 199, japonica rice 201, japonica rice 203, japonica rice 205, japonica rice 207, and Jinyuan 101, and particularly preferably, the rice is Nipponbare.

6. A method of preparing the promoter of claim 1 comprising the steps of:

 1) A PCR amplification primer pair is designed according to the nucleotide sequence shown in SEQ ID NO: 1, specifically, the PCR amplification primer pair is SEQ ID NO: 2 and SEQ ID NO: 3.

 2) Using the modified genomic DNA of Zhanggu No. 1 as a template, PCR amplification of the primer pair designed in step 1) was carried out.

A method for regulating gene expression in a plant, the method comprising the step of transforming a callus of a plant with the promoter of claim 1, preferably, the callus is a rice callus, and particularly preferably The rice is Nipponbare.

The method according to claim 7, wherein the recombinant Agrobacterium tumefaciens EHA105-P603 according to claim 4 is utilized in the transformation of the plant callus.

The use of the promoter according to claim 1 for regulating expression of a gene of interest in a plant, wherein the gene of interest is GUS, the plant is rice, and preferably, the rice is Nipponbare.

10. The use of the promoter according to claim 1 in rice breeding, preferably, the rice is Nipponbare, Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2 , Youyou 63, Yuyou 608, Fengyou 22, Yanyou 88, Yuyou 416, Π优107, Π优128, Πyou 718, Zhunliangyou 527, Chuanong No.1, Miscellaneous 0152, Japonica 88, Japonica 90, Japonica 92, Japonica 94, Japonica 96, Japonica 185, 皖Rice 187, japonica rice 189, japonica rice 191, japonica rice 193, japonica rice 195, japonica rice 197, japonica rice 199, japonica rice 201, japonica rice 203, japonica rice 205, japonica rice 207, and Jinyuan 101, particularly preferably The rice is Nipponbare.