WO2011130893A1

WO2011130893A1 - Promoter sbubi2, preparation method and uses thereof

Info

Publication number: WO2011130893A1
Application number: PCT/CN2010/001772
Authority: WO
Inventors: 张耕耘; 倪雪梅; 黄刚; 张印新
Original assignee: 深圳华大基因科技有限公司
Priority date: 2010-04-23
Filing date: 2010-11-04
Publication date: 2011-10-27
Also published as: CN102206640A; CN102206640B

Abstract

Promoter of sweet sorghum BT×623 and its variants having promoter functions are provided, wherein the promoter has the nucleotide sequence shown in SEQ ID NO: 1, and the variants are variants 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 SbUbi2, 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 sweet sorghum, and a preparation method and use of the promoter. Background technique

A promoter is a component of a gene, usually located upstream of the structural gene 5, which is a DNA sequence that RNA polymerase recognizes, binds, and initiates transcription. The promoter directs the holoenzyme to properly bind to the template, activating RNA polymerization. The enzyme, which initiates transcription of the gene, thereby controlling the initiation time and degree of 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.

According to the transcriptional pattern of the promoter, it can be divided into three categories: a constitutive promoter, a tissue or organ-specific promoter, and an inducible promoter. The so-called constitutive promoter refers to the fact 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 higher efficiency in both monocotyledonous and dicotyledonous plants, but Ajith Anand et al. transferred the chitinase gene of rice to wheat and found CaMV35S. As a promoter, chitinase showed complete gene silencing in the second-generation plants, 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).

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

Ubiquitin (Ubi) 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 stable genetic traits. And it is favored (Xie Wei, Le Chaoyin. Journal of China Three Gorges University, 2007, 29 (2): 176-179). The Ubi promoter is effective in promoting long-term stable and high-efficiency expression of foreign genes. Many Ubi promoters have been used in monocots and dicots. Moreover, the Ub丄 promoter has a higher level of transcriptional regulation than the nos, ocs, mas derived from T-DNA and the promoters such as CaMV35S and CsVMV derived from viruses. Guo Dianjing et al. (Acta Genetics, 1999, 26 (2): 168-173) found that the Ubi 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 Ubi. U4 into transgenic tobacco to initiate GUS expression. The results showed that the expression level of the GUS gene in the UM. U4 promoter was CaMV35S. 7 times.

With the development of plant genetic engineering, people are no longer satisfied 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 the lack of ideal transgenic plants, and promoters play a key role in determining gene expression (Hou Bing et al., Inheritance, 2001, 23 (5): 492-497). Utilizing the Ubi 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.

Currently, promoter sequences have been isolated from many ubiquitin genes, including: Ubi-1 promoter in the maize genome, 7j rice ubiquitin RUBQ ² promoter, Arabidopsis ubiquitin promoter, sunflower ubiquitin UbBl promoter , tobacco ubiquitin Ubi. U4 promoter, potato ubiquitin Ubi7 promoter, tomato ubiquitin Ubi l-1 promoter, barley ubiquitin Mubl promoter and so on. 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 has also been used in rice and sugar cane. Summary of the invention

In order to provide a new tool and choice for the regulation of target gene expression in plant research, the present inventors have provided a novel ubiquitin promoter by intensive research on the sweet sorghum 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 2330 bases, as shown in SEQ ID NO: 1:

TAAGGACTGTCGTTTACTTGCATTTCAGTTGATCGTATCTAGATAGTCTGAGATG GCCCGCCGGCCCC CCCTTTTATGGGTTGTTGGTCTTTTTTTTTATGATTTTAAAAAAAA

CCCCCCTTTGGCCGTGTTGGCGGTTGTC ATGGGATTGTTTGTTAAAATTAAATATTTAA CCCCCCCCCCCTGGTGGTGTCTTTTTGTGATTGGGGG TTTGAAATAAAAAAAAATAAAA GGGGCCCGGGCGGCCGCGGGATTGCTTTTCAGAAAAGTTTG TAATAAAAAAAAAAAAAA CCCCGCCGTCCCCGTGGGTGCGTTGC AGATTTGTTAAGGAATAATAAGAAAAAAAAAAA C GCGGCCCGGCCGCTTCGATATATTGTTTTATAGTTGTATATTATATATATTAATATAA CGGCGCCCCGGCCCCC TTTGTGGATTATGTTCATTATTTGGTTTATTTGTTTATTATAA CCCCCCTGGTCGGCCTTTTCCTGTTTTTCCTATTGGTTG ATATATTATGAATAATAAAT CCCC GGCGTGCCTTGTCGGGTTCTGGTGGTAATAATAAATTGTATGTTTTATATGAAAA CCCCCGGCGGGCCCGCGGGGCCC TATTTTGGGAATTTTTTAAATGTAGTTGAAAAAAAA CCTGGGGGGGGGCCGTGGCCTTGGCGGCGG AAAAAATATTTATTAATATGATTTGTGTA CCCGCCCC TGTGGTTTTGTTTGGCTCCGGGCATAAATAAAATGTTAATAAAGTGTATAA CCCCGGCCCC AAATTGTAATGTTTTATGGAATTTCGGAAAAAAATAATTTGAATTAATG CCCCC ATCTGCGTGCCCGCCCAAAAAAAAAATGTAATGATTTTAAAATGAATGTCAAAA CCCC TGTCCACTCCGGCCCATTTTTAAAAAATAATTTTGAAAATTTTCATTTTGAAAAA CCGCC ATTTATTGTTTGCGATTCGGTTTTCGCCCGGATTATTAAATTAATAAATAAGAA CGCCCC TTGTTGGGATCCTGCCCCATTAAATAATATAATTTTAATTTTTTTATTTTGTA

GGGCCCCCCCCCCCAGTGGTGGTTAGGTTTTGAGGGTTGGGAAATAAAATAAAAATAAT

CCCGGCCCTTTTGTTGTTGCAAATTTTGCCGTGTGTTATAATTTGTGAATAATATAATT CCGCCCGCCGCGCCTGC ATTTAAAGGTTGTTTTTTGTCTATATAATTTATTTAAAAAAA CGCCCCCCCC TTGTTTTTTATAAAGTTTCAGTGTTCATTAAGTAAATATTAAAAAAAAA

CCCCCCCGGGTGTTGTGTTTTGTCATTGTTTGGAAATAAATAATGATAATAAAATTAAA

CCGCGCCCGCCGGGTTTCTTAGTTTATTGTAAATTTTGTTTATAATTATTATAATAAAA CGCCCCGCCCC TGTGGTATTTGTGAATTTTTCTTGATGTGATAAATATAAAATAATAAA CCCCCCCCCGGCCC TTGCTTTCGGTTGCAGTTTGTTGAATTAAATAAATAATAAAAAAT CCCTCCGTTGCCTCGGAGGTGC TTGATATAATATAGGAAATTTGAAATAAATGATTAAA CC CCCTGTCGCGCTTGCGGTGAGGTAATTTTTTTGATTATTTTTTTTTAAAATAAAAAA CCCCGCCGGGCCGCCCGGGGGCCTGCCG TTGTTATTTATAATGTAAAAATTAAAAATTA CCCCGGGCGGC CGTTTCGTGTGTGCAGGTTTGTTTTTTTGATTTTATAAATAAAAAATA CCCCGCCCCCGTTGGTTGGGATATTTGCGTTTGTGTAAAAATT TATTTAAATATAAAAT CCCCGGCCCCTTTGTCGCTATTGTGCGGGTAAAGAATTATATTAATTATTTTA AAAAAA CGCGGCGGGCTCGCGGGTTGTTTAGTTGTAATTATATAAT TTATATAAAAAAATAAAAT GCTAATGTAACTATGTTCCTGAAGGAAACCTTCTTTTGGATTAACCTGGATTTAGGGAT AAAACCTTCTTGTGGTAGCCTTTACCACAACATGCATCCTCCTGCTGATTAGCTAGATA TGCATGCTTGCTACTGTTCAATGATTCTTTAGTATACCTGATCATGCATGCTCTTGTTA CTTAGCTTGATATACTTGGATGATGACATGCTGCTGTCGTTCACTGTTTCTATACCTGA TGATCATGCATGCTCTTGTTACTTGTTTTGATATACTTGGTTCGATGGTGATGAACACA GAACACACATGACATGATGTTGCTGTTTGCATGAGGCTGTTTCGTTGGTCTACTGCCAG ATACTTCCCCTGTTGTTTGGTTATCTTCTGCAG (SEQ ID NO: 1)

In the present invention, the promoter sequence shown by SEQ ID NO: 1 is referred to as promoter SbUbi2, also referred to as P605 for short.

The promoter is a constitutive promoter, and is also a ubiquitin promoter, a rice callus with the promoter and GUS, and a GUS staining experiment of the transgenic rice seedling, the root of the rice callus and the transgenic rice seedling, Stems, leaves, etc. all 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 plant 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 (below probe Tm5); "higher stringency" occurs below Tm about 5 - "medium stringency" occurs about 10 below probe Tm - 20; "Low tightness" occurs about 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 χ SSC = low to medium tightness; 1 X SSC = medium tightness; 0. 5 χ SSC = higher stringency. 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 the conditions are determined using high stringency conditions. The probe has a nucleic acid sequence of about 80% or more sequence identity.

For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form hybrids, for example, to select relatively low salt and/or high temperature conditions. Sambrook et al. (Sambrook, J. et al. (1989) Molecular Cloning, Laboratory Handbook, Co ld Spring Harbor Pres s, Pla inview, N. Y.) provide hybridization conditions including medium stringency and high stringency.

For ease of explanation, suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the present invention with other polynucleotides include: pre-washing with a solution of 5 x SSC, 0.5% SDS, 1.0 M EDTA (pH 8.0); Hybridization was carried out overnight in 5xSSC at 50-65; then washed twice with 2x, 0.5x and 0.2x SSC containing 0.1% SDS for 20 minutes at 65 TC. 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 or 65-70"€.

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: 1. 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 nucleoside is separately or simultaneously The 5, end and/or 3, end, and/or sequence of the acid 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 of the substitutions, deletions, and additions of the bases represented by successive integers.

In the present invention, the nucleotide sequence of the nucleotide sequence shown by SEQ ID NO: 1 is substituted, deleted or added with one or more bases as described above and has the nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence is identical or similar to the promoter activity.

Described by a polynucleotide having a nucleotide sequence such as at least 95% "identity" with the reference nucleotide sequence of SEQ ID NO: 1 means: Reference in SEQ ID NO: 1. In every 100 nucleotides of the nucleotide sequence, the nucleotide sequence of the polynucleotide is identical to the reference sequence except that it contains up to 5 nucleotides. In other words, in order to obtain a polynucleotide whose nucleotide sequence is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or otherwise a nucleotide substitution; or a nucleotide may be inserted into a reference sequence, wherein the inserted nucleotide may be up to 5% of the total nucleotide of the reference sequence; or in some nucleotides, there is deletion, insertion And a combination of 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 (eg, BLAST analysis using standard parameters), it contains at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, compared to the polynucleotide sequences of the invention. Those sequences of sequence identity of 95%, 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, a sweet sorghum, such as sweet sorghum BT 623 ( deposited on April 1, 2010 in Wuhan, Wuchang, Wuhan, Wushan, Hubei Province, Wuhan, China. The China Type Culture Collection (CCTCC), with the accession number CCTCC P201005).

A further aspect of the invention also relates to a recombinant vector comprising the plant 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 Simple Vecter, pMD19-T Simple Vecter, and the like.

Expression vectors suitable for construction of the invention include, but are not limited to, for example: pBI121, Pl 3W4, pGEM, etc.

In one embodiment of the invention, the recombinant vector is a P8+P605 recombinant vector. In still another aspect of the invention, the invention relates to a recombinant cell comprising the recombinant vector of the plant promoter of the invention. The recombinant cell can be obtained by transforming the recombinant vector containing the plant 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, EHA1 05, GV3101 and the like.

In one embodiment of the invention, the recombinant cell is Agrobacteriu tu efaciens EHA105-P605.

In still another aspect of the invention, the invention relates to a plant callus transformed with a promoter of the invention. In one embodiment of the invention, the plant is rice. The rice includes, but is not limited to, for example: Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yanyou 63, Shanyou 608, Fengyou 22, 黔You 88, Yu You 416, Yan You 1 07, Yan You 128, Yan You 718, Zhun Liang You 527, Chuan Nong No. 1, Miscellaneous 0152, Japonica Rice 88, Japonica Rice 90, Japonica Rice 92, Japonica Rice 94, Rice 96, Japonica 185, Japonica 187, Japonica 189, Japonica 191, Japonica 193, Japonica 195, Japonica 197, Japonica 199, Japonica Rice, Japonica 203, Japonica 205, Japonica 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 曰本晴 ( Oryza sa t iva L. ss p. japoni ca cv. N i pponbare , deposited on December 18, 2009 in Wuchang, Wuhan, Hubei Province Shan Wuhan University Depository Center, China Type Culture Collection (CCTCC), with the accession number CCTCC P200910).

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 sweet sorghum BT 623 genomic DNA as a template, using the design in step 1)? The CR amplification primer pair was subjected to PCR amplification.

It is well known to those skilled in the art that the corresponding PCR amplification primer pair can be designed according to the principle of base complementation 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.

In a further aspect of the invention, a method for regulating expression of a gene of interest in a plant, In one embodiment of the invention, the transformation of the plant callus utilizes recombinant cells comprising a plant promoter of the invention. In one embodiment of the present invention, the aforementioned recombinant Agrobacterium tumefaciens is utilized in the transformation of the plant callus

EHA105-P605. In a specific embodiment of the present invention, the callus of the plant is a rice callus, and specifically, the rice is Nipponbare.

In the present invention, the gene of interest can be inserted into the plant genome by plant genetic transformation techniques, including 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 of 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, the method of transforming monocots which can also be used is protoplast transformation. After gene transformation, a general method is used to screen and regenerate plants integrated with expression units.

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

In a further aspect of the invention, the invention further relates to the use of a plant promoter of the invention for regulating expression of a gene of interest 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 a rice, and in particular the rice is Nipponbare.

For the purpose of achieving the above-described regulation of gene expression of interest, the promoter of the present invention may be used in the form of single copy and/or multiple copies, or may be used in combination with a promoter and an 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, Yuyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Yuyou 416, Π优1 07, Π优128, Πyou 718, Zhunliangyou 527, Chuanong No.1, Miscellaneous 0152, Japonica 88, Japonica 90, Japonica 92, Japonica 94, Japonica 96, Japonica 185, japonica rice 187, japonica rice 189, japonica rice 191, japonica rice 193, japonica rice 195, japonica rice 197, japonica rice 199, japonica rice, japonica rice 203, japonica rice 205, japonica rice 207, and Jinyuan 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 selection, plant flower organ Molecular breeding research such as abortion 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, sweet sorghum, barley, and the like. Advantageous effects of the invention

The inventors obtained a ubiquitin promoter derived from sweet sorghum by bioinformatics research, and verified the function of the promoter P605 by biological experiments, and the promoter has the function of regulating the expression of the target gene across species. The constitutive promoter. Specifically, the promoter is capable of regulating GUS gene expression in rice: high expression of the gus gene can be regulated in rice callus, roots, stems and leaves of rice transgenic seedlings. DRAWINGS

Fig. 1 is the PCR amplification test result of the promoter P605, wherein the lane M: 200 bp DNA Ladder Marker, the number on the left side indicates the size of the stripe of the Ladder Marker pointed, and the unit is bp; Lane 1: PCR amplification products.

Fi g. 2 is a schematic diagram of the pCAMBIA-1301 plasmid used to construct the p8 plasmid.

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

Fig. 4 is a schematic diagram of the p8 plasmid.

Fig. 5 is the result of GUS staining of transformed rice callus. Among them, rice calli (left) transformed with recombinant root cancer Agrobacterium P8+P605 carrying the promoter P605 sequence of the present invention showed blue color after GUS staining; no recombination with the promoter sequence of the present invention Rice callus of Agrobacterium tumefaciens p8 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 seedlings transformed with the recombinant Agrobacterium tumefaciens p8+P605 carrying the promoter P605 sequence of the present invention (left) are stained with GUS, and the roots, stems and leaves thereof are blue; Subsequence of recombinant Agrobacterium tumefaciens p8 transformed rice seedlings (control, right) stained with GUS After color, the color of its roots, stems and leaves did not change. detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, Those who do not specify specific techniques or conditions in the examples, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., "Molecular Cloning Guide", Third Edition , Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially. Example 1: PCR amplification of P605 promoter fragment and construction of PMD18-T+P605 recombinant vector

Take the kit, catalog number: DP320- 02 ) Extract the genomic DNA of the sweet sorghum BT x 623 seed, and design a pair of PCR-specific amplification primers in the first and the last according to the sequence of the promoter in the sweet sorghum BT x 623 gDNA ( The upstream primer F1, the restriction enzyme site Kpn l and the protective base, the downstream primer R1, the restriction enzyme site Sbf l and the protective base). The gDNA of the sweet sorghum BT X 623 extracted above was used as a template, and PCR amplification was carried out using a high-fidelity Ex Taq (TaKaRa, DRR100B) polymerase. As shown in Table 1.

Table 1: PCR system for gene promoter amplification

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 taq 0. 2

ddHjO fills up to the total volume 25 μ 1

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

Among them, the upstream primer F1:

GGggtaccTAAGGACTGTCGTTTACTTGCAT (SEQ ID NO: 2), wherein the lower case letters represent the Kpn I restriction site.

Downstream primer R1:

GCcctgcaggCTGCAGAAGATAACCAAACAACA (SEQ ID NO: 3), wherein lower case letters represent Sbf I restriction sites.

The PCR amplification product was separated by 1.0% agarose gel electrophoresis to obtain a band of 2348 bp ( Fig. 1 ), which was purified using TIANGEN Sepharose DNA Recovery Kit (Catalog No.: DP209-03). Recycling.

Construction of PMD18-T+P605 Recombinant Vector

The PCR amplification product obtained as described above was subjected to T/A cloning (pMD18-T plasmid, TaKaRa, D103A), transformed into Escherichia coli, and the positive clone was picked for sequencing (as shown in SEQ ID NO: 4), which proved to be accurate.

Among them, the connection conditions of the T/A clone are as follows:

T/A connection system: 10 μ 1

PMD18-T 1 μ 1

2 * solut i on 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

Connected to the energy-saving intelligent thermostat (Ningbo Xinzhi, SDC-6) for more than 8 h at 16 , to obtain pMD18-T+P605 recombinant vector. The ligated product was transformed into E. coli according to the following method:

Remove the competent cells prepared by the calcium chloride method shown in the Guide to Molecular Cloning (Third Edition, Science Press) 100 μ 1 DH5 a 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 the water is melted, add 10 μM of the ligated product obtained above, namely PMD18-T+P605 recombinant vector, gently stir, water bath for 30 min, 42. C heat shock for 60 s, water bath for 5 min, add 600 μl 4 4 Ό pre-cooled SOC medium (for details, see “Molecular Cloning Guide”, Third Edition, Science Press), 37 °C 220 rpm recovery Centrifuge at 8000 rpm for 30 s at 45 min, remove the supernatant, and take 150 μl. Resuspend the pellet with the remaining 150 μl supernatant. Mix, gently blow the hook, glass beads coated LB (Carnastatin) plate (for details, see "Molecular Cloning Experimental Guide", third edition, Science Press), 37 inverted culture for 16 h ~ 24 h. Recombinant Escherichia coli containing the pMD18-T+P605 cloning vector was obtained, and named as DH5 α-Ρ605 _β Shenzhen Huada Gene Technology Co., Ltd. to sequence Ρ605 in the pMD18-T+P605 cloning vector, the results are as follows:

GGGGTACC | TAAGGACTGTCGTTTACTTGCAT] TTCAGTTGATCGTATCTAGATAGTCT GAGATGTTACTGCTAGTAAAGAAAAGTTTGTCTTAGTTGATGACATGTAGATAACATGAGA TGCTACTGCGGGTAAAGAAAATCAATAGTATGCTCAACCTATTGTCTTGCATTTTCATCTT GATTTATTTAGGCACTATTGGATGATATTAAATGCCGTGCTCTGTCGTATAAAAATCCTGC CCCTTGGACCATTTTTTCTATGGTGGAATGTGCATAGAGTTAATGAAATTGTCGTGTTCTG TGAATATCCCATTTAACTTTGGAGCACTGCAGTCCACAAGGAGGGCCATCTGCCTGACAGG ATCAAACTTTGTTTTTGTCATTAGCAGTCATTTGTCTTGTTTATGCTAACAATGATTCAGA ATTATAATCATACGGGTTAAGATTCTGCACAATATAAACTGGGAGGTAGTTCCTTGAAACT CTTCAAATAATCTAGCAAGTCTGCAACAGCTTCTGTCATGTCAGGCACTTCTGCTGAGTTA AATAATTGTTGAATTAGCATTTCTCTCCTGATAGAGTGACCTCAAATACCTATCTCTGCTA TGAGCATTTCCTAGTTAAGCTACAGAATGTTTGGTTTATAATTATTACTAAATAGATTGAT CTGAATGTTTCCTAGTTAATCAACATAACCTTTGAGTTGAGATGGTCAAATTGGTCTTCCA TTTTTATAAAGTTAATCACAGACATGCTTAACATTACAAGCTAAGTCTCCAGTCCAAAAGG TTGTCCTTTTGCATGTCTATGATAATCAAACTTTATGCATTCTACTCATTTGTATGTTGAC TGAAAATTTTGCCTGGTGCATGCATTTTATAATTTGCTGGAAAGATAGGCTGGCATGGTAC TAGAGTTTCTTGACAAGCACACCGAGTCTTGACGAGTTACTGTTAGTATCAGGTAAGTCAC CATTAATCCT TTTGACACCTTTTTTTATTTTGCATAACTTTTTAACTTGTTTGTTCGATGC TACAGGATTCTTTTACATGCACCAATAGGAAGATGTACCTTACCCCTTTTACAAACAATAA TTTTGACAAGGACACTTATTCCATTTTAAAGATCAAAACAATGCAAAACTGTCAGTCACTG CGCCAATTTTAAAGATCAGAACCTAAGTCAAATAATGTCAATAGCTCCTATGTGAACCTAA GTGAAATTACTTCGTTGACATTAAGTGTGATGTAGCTTCTTCAAGTAATGCCTATCAGTGT TAAGTCTACAAGTGCTCAGGGTAACATGGGGGAAAAGGGAGCTACTGTGCTTAGTTAACCT CTAGGCTGGATGCAGTGTTGTGTTATCCTCATCCTGGACGGAGGGGAACCCTTGATATTCT GAGGAAGAATGTACCAGTCTGGTAATGACGATCATGACACAATTGTCGGAGTTGATTTCCT GACTGGTTTTCTGATATGGATTATATTGGTCCCGAGCCTGCTTCATTACTAATCTGTATTT CTCCCAATATTGGTTGTTTATGTGGAGTGTATGCTTCTGATTATTTGGACCCTCTTGAGTC TTGACCTTTACTCCTGAATTTCGATATATATGCTTTGGTTATCACGTTGCTGGATCATTAT AACCATATATGCTAGAATTTGCTTGTCAAAAAGGACCATCAACTAGAGCCTGGAGTAGACG AGTTCCAGAACTGAGATAGGATGGTAGAAACAAAGCACATTTTCCAGAGGGAAACAAAGTG GCCTGTGCATTCAATGCTGAGACTATAAAAGGTAAAAACAGTTCACCTTTTTGCTCCGACT TGGCGGGATGTGTGCATTTTCTGATTTGTTAAGAAGCTCTAAGTGACTCTAGGTCGGATCT GCTTCCATCTTTCTTTGTATGAGGAACCTTGTTGAAAGTCCGCTCGTTGCTTATTCCTTAT CAGGCTAATGTAACTATGTTCCTGAAGGAAACCTTCTTTTGGATTAACCTGGATTTAGGGA TAAAACCTTCTTGTGGTAGCCTTTACCACAACATGCATCCTCCTGCTGATTAGCTAGATAT GCATGCTTGCTACTGTTCAATGATTCTTTAGTATACCTGATCATGCATGCTCTTGTTACTT AGCTTGATATACTTGGATGATGACATGCTGCTGTCGTTCACTGTTTCTATACCTGATGATC ATGCATGCTCTTGTTACTTGTTTTGATATACTTGGTTCGATGGTGATGAACACAGAACACA CATGACATGATGTTGCTGTTTGCATGAGGCTGTTTCGTTGGTCTACTGCCAGATACTTCCC

ClTGTTGTTTGGTTATCTTCTGCAGlCCTGCAGGGC (SEQ ID NO: 4) In the above sequence, the undercut is the restriction site (kpn l + Sbf I ), and the frame is the primer sequence.

The sequencing results showed that the P605 promoter sequence was correct in the obtained PMD18-T+P605 cloning vector. Example 2: Construction of vector-P8+P605 recombinant vector Escherichia coli DH5 CX transformed with the promoter P605 constructed from Example 1 according to the manual of the TIANGEN Generic Plasmid Mini Kit (Cat. No. DP103-03) The cloning vector pMD18-T+P605 carrying the P605 promoter sequence of the present invention was extracted from P605; after purification, the corresponding restriction enzymes Kpn l (NEB) and Sbf I (NEB) were used for digestion, and the corresponding promoter was recovered. The fragment was inserted and ligated with the large fragment of the vector which was digested with the same restriction endonuclease as the p8 plasmid.

The resulting ligated product P8+P605 recombinant vector was transformed into competent cell DH5 α, 37 prepared by calcium chloride method according to the Guide for Molecular Cloning (third edition, Science Press), inverted culture for 16-24 h, to be transformed The child grows out of bacteria, picks up the monoclonal for PCR detection and enzyme digestion.

D8 plasmid construction

The p8 plasmid used in the present invention is composed of pCAMBIA-1301 plasmid (Chinese Academy of Sciences) Provided by Dong Yang from Kunming Institute of Zoology; or available from, for example, Shanghai Guorui Gene Technology Co., Ltd., the original source of the company's pCAMBIA-1301 plasmid is The CAMBIA Bios (bio log ica l open source) Licensee, Aus t ra l Ia ) Remodeled and constructed as follows, as follows:

The plasmid pCAMBIA-1301 (see Fig. 2) was digested with Kpn I /Nco I (NEB) to recover large fragments. The following sequences were synthesized based on the restriction enzyme sites used:

5) (Included enzyme cleavage site is Kpn I /Hind m /Spe I /Sbf I /Ps t I /Xba I /BamH I /Sa Π /Nco I ), recovered by double digestion with Kpn I /Nco I Toxic cells were transformed with the large fragments recovered as described above (provided by Dong Yang, Kunming Institute of Zoology, Chinese Academy of Sciences; or available from, for example, Beijing Suo Laibao Technology Co., Ltd.). The transformant was screened with the primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO: 6) / GAGTCGTCGGTTCTGTAAC (SEQ ID NO: 7), and the transformant with the amplified fragment of 350 bp was the multiple cloning site containing the desired construction by PCR detection method. A transformant of the p8 plasmid of the GUS sequence (see Fig. 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 Fig. 3):

GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGA TTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAAC

TA GAAA CCCCAA CCCGTGAAA TCAAAAAA CTCGA CGGCCTGTGGGCA TTCA GTCTGGA T CGCGAAAA CTGTGGAA TTGA TCA GCGTTGGTGGGAAA GCGCGTTA CAA GAAA GCCGGGC AA TTGCTGTGCCA GGCA GTTTTAA CGA TCA GTTCGCCGA TGCA GA TA TTCGTAA TTA TG CGGGCAACGTCTGGTA TCAGCGCGAAGTCTTTA TACCGAAAGGTTGGGCAGGCCAGCGT A TCGTGCTGCGTTTCGA TGCGGTCA CTCA TTA CGGCAAA GTGTGGGTCAA TAA TCA GGA A GTGA TGGA GCA TCA GGGCGGCTA TA CGCCA TTTGAA GCCGA TGTCA CGCCGTA TGTTA TTGCCGGGAAAA GTGTA CGTA TCA CCGTTTGTGTGAA CAA CGAA CTGAA CTGGCA GA CT A TCCCGCCGGGAA TGGTGA TTA CCGA CGAAAA CGGCAA GAAAAA GCA GTCTTA CTTCCA TGA TTTCTTTAA CTA TGCCGGAA TCCA TCGCA GCGTAA TGCTCTA CA CCA CGCCGAA CA CCTGGGTGGACGATATCACCGTGGTGACGCATGTCGCGCAAGACTGTAACCACGCGTCT GTTGA CTGGCA GGTGGTGGCCAA TGGTGA TGTCA GCGTTGAA CTGCGTGA TGCGGA TCA ACAGGTGGTTGCAACTGGACAAGGCACTAGCGGGACTTTGCAAGTGGTGAATCCGCACC TCTGGCAA CCGGGTGAA GGTTA TCTCTA TGAA CTCGAA GTCA CA GCCAAAA GCCA GA CA GA GTCTGA TA TCTA CCCGCTTCGCGTCGGCA TCCGGTCA GTGGCA GTGAA GGGCCAA CA GTTCCTGA TTAA CCA CAAA CCGTTCTA CTTTA CTGGCTTTGGTCGTCA TGAA CA TGCGG A CTTA CGTGGCAAA GGA TTCGA TAA CGTGCTGA TGGTGCA CGA CCA CGCA TTAA TGGA C TGGA TTGGGGCCAA CTCCTA CCGTA CCTCGCA TTA CCCTTA CGCTGA A GA GA TGCTCGA CTGGGCA GA TGAA CA TGGCA TCGTGGTGA TTGA TGAAA CTGCTGCTGTCGGCTTTCA GC TGTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAG GCA GTCAA CGGGGAAA CTCA GCAA GCGCA CTTA CA GGCGA TTAAA GA GCTGA TA GCGCG TGA CAAAAA CCA CCCAA GCGTGGTGA TGTGGA GTA TTGC CAA CGAA CCGGA TA CCCGTC CGCAA GGTGCA CGGGAA TA TTTCGCGCCA CTGGCGGAA GCAA CGCGTAAA CTCGA CCCG A CGCGTCCGA TCA CCTGCGTCAA TGTAA TGTTCTGCGA CGCTCA CA CCGA TA CCA TCA G CGA TCTCTTTGA TGTGCTGTGCCTGAA CCGTTA TTA CGGA TGGTA TGTCCAAA GCGGCG A TTTGGAAA CGGCA GA GAA GGTA CTGGAAAAA GAA CTTCTGGCCTGGCA GGA GAAA CTG CATCAGCCGATTATCATCACCGAATACGGCGTGGATACGTTAGCCGGGCTGCACTCAAT GTA CA CCGA CA TGTGGA GTGAA GA GTA TCA GTGTGCA TGGCTGGA TA TGTA TCA CCGCG TCTTTGA TCGCGTCA GCGCCGTCGTCGGTGAA CA GGTA TGGAA TTTCGCCGA TTTTGCG A CCTCGCAA GGCA TA TTGCGCGTTGGCGGTAA CAA GAAA GGGA TCTTCA CTCGCGA CCG CAAA CCGAA GTCGGCGGCTTTTCTGCTGCAAAAA CGCTGGA CTGGCA TGAA CTTCGGTG AAAAA CCGCA GCA GGGA GGCAAA 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 in the multiple cloning site

I /Sac I /Kpn I /Hind m /Spe I /Sbf I /Ps t I /Xba I /BamH I /Sa il /Nco I Restriction sites are indicated by boxing and underline, respectively, for screening transformants The primers GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTAAC (i.e., SEQ ID NOS: 6 and 7) are indicated by double underlining, the GUS sequences are shown in italics, and the intron sequences are shown in italics and shading, respectively. Construction of Recombinant Vector P8+P605

The cloning vector pMD18-T+P605 obtained in Example 1 and the p8 plasmid constructed as described above were treated according to the following conditions according to the restriction enzymes Kpnl (NEB) and Sbf I (NEB).

Among them, the cloning vectors pMD18-T+P605 and p8 plasmid were digested as follows: Enzyme digestion system: 50 μ 1

Sterile water 34.8 μ ΐ

10* buffer H 5 μ 1

Κρη I 0.1 μ 1 ( 10 U)

Sbf I 0.1 μ 1 ( 10 U) cloning vector MD18-T+P605 or 8 plasmid 10 μ 1 ( <1000 ng ) Separately digested with TIANGEN agarose gel DNA recovery kit (catalog number: DP209-03) The p8 plasmid, and the promoter P605 insert, were ligated according to the following conditions according to the T4 ligase (TaKaRa, D2011A) instructions:

Connection system: 10 μ ΐ

10*T4 buffer 1 μ 1

After cleavage of ρ8 plasmid 1 μ 1 (20 ng) Recovered promoter P605 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 P605 fragment in the present method was added in 10 ng. Connected to the energy-saving intelligent thermostat (Ningbo Xinzhi, SDC-6) for more than 8 hours.

The competent cell DH5a prepared by the 100 μl calcium chloride method was taken out from the ultra-low temperature water tank, and after melting on the water, 10 μ ΐ of the above-mentioned ligation product was added, and the mixture was gently stirred, ice-cooled for 30 min, 42 heat shocked for 60 s, and water bathed for 5 min. Add 600 μl 4 pre-cooled S0C, resuscitate for 45 min at 37 degrees 220 rpm, centrifuge for 30 s at 8000 rpm, remove the supernatant, leave 150 μl, gently spread, glass beads coated LB (kan), 37 V inverted culture for 16~24 h. The recombinant vector p8+P605 was obtained.

The resulting recombinant vector P8+P605 was subjected to PCR detection using F1 (i.e., SEQ ID NO: 2) and R1 (i.e., SEQ ID NO: 3) as primers, respectively, to confirm that the resulting recombinant vector p8+P605 contained the desired promoter P605. Screening for recombinant vector containing Kpn I /Sbf I P8+P605 transformants. Example 3: Preparation of recombinant Agrobacterium tumefaciens EHA105-P605 cells

The p8+P605 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were separately transformed according to the calcium chloride method described in the Molecular Cloning Real Face Guide (Third Edition, Science Press). Agrobacterium tumefaciens EHA105 (Dedicated cells deposited at the Wuhan University Wushan Fushan Wuhan University Depository Center, Wuhan, Hubei Province, December 24, 2009, China National Type Culture Collection (CCTCC), accession number CCTCC M 209315) Methods as below:

The Agrobacterium tumefaciens competent cell EHA105 was taken out in an ultra-low temperature freezer and thawed on ice. After thawing, add 5 μΐ of ρ8+Ρ605 recombinant vector and ρ8 plasmid and ρ8 empty vector as control, gently mix the hook, water bath for 10 min, freeze in liquid nitrogen for 5 min, 37 thaw for 5 min, add 800 μ 1 Normal temperature LB liquid medium, resuscitated at 28"C 160 rpm for 3 h, centrifuged at 8000 rpm for 30 s, aspirate the supernatant, leave 200 μ 1 and mix well, and apply with kan-r if (canazan) - rifampicin) on the YM medium plate of the double antibody (50 mg / 1 Kan, 10 mg / 1 Rif, see Table 4 for the specific formulation). 28 Inverted for 2-3 days.

PCR detection was carried out using F1 (i.e., SEQ ID NO: 2) and R1 (i.e., SEQ ID NO: 3) as primers, and the transformants were digested by Kpn I /Sbf I.

Recombinant Agrobacterium tumefaciens, which is a recombinant vector p8+P605, was amplified by PCR with a 2348 bp band and a 2337 bp band.

In the present invention, the recombinant Agrobacterium tumefaciens having the recombinant vector P8+P605 obtained by the above method is designated as Recombinant Agrobacterium tumefaciens EHA105-P605.

According to the method of the present invention, a control recombinant Agrobacterium tumefaciens strain carrying 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-P605 and recombinant Agrobacterium tumefaciens EHA105-p8, respectively.

1) Shelling Nipponbare Seeds, 70% ethanol surface disinfection for 30 s, then disinfecting with effective chlorine 1.5% sodium hypochlorite for 30 min, shaking vigorously, disinfecting and sterilizing water 5 times; disinfecting seeds Placed in N6D medium (see Table 2 for specific formulations), Seal with a sealing film; 29.5 Ό light culture for 3 to 4 weeks;

2) Select the actively growing callus (yellow-white, dry, diameter l~3 mm), and incubate for 3 days on the new N6D medium at 29.5 °C;

3) Pick a single colony of recombinant Agrobacterium tumefaciens constructed as in Example 3 (recombinant Agrobacterium tumefaciens EHA105-P605 or recombinant Agrobacterium tumefaciens EHA105-p8), and add antibiotics (50 mg/1 Kan, 10 mg) /l Rif) YM medium (see Table 3, Table 4 for specific formulation) was streaked for 3 days at a culture temperature of 28. C; scraping the above recombinant Agrobacterium tumefaciens separately in 30 ml AAM medium supplemented with 30 μΐ 100 mM AS (Acetosyringone, acetosyringone) (see Table 5 for specific formulation), gently resuspending the recombinant roots Agrobacterium tumefaciens cells (recombinant Agrobacterium tumefaciens EHA105-P605 or recombinant Agrobacterium tumefaciens EHA105-p8);

4) the subcultured callus is placed in a sterile culture; the recombinant Agrobacterium tumefaciens suspension prepared as in step 3 is poured into the culture sub, and the callus is immersed therein for 15 min;

5) Pour off the recombinant Agrobacterium tumefaciens suspension, and use the sterilized absorbent paper to remove the excess liquid from the callus; place a sterile filter paper on the N6-AS medium (see Table 6 for the formulation), add 1 ml. The AAM medium containing AS is transferred to the filter paper; the culture medium is sealed, and 28 dark culture is carried out for 48 to 60 hours;

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

(Carb) in sterile water to kill recombinant Agrobacterium tumefaciens; remove excess water from callus with sterile absorbent paper, then transfer to 1 mg/1 hygromycin B (HmB) and 50 mg /1 Carb on 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, according to the method described in Chen SY et al., Journal of Integrative Plant Biology, 2008, 50(6): 742-751, The rice calli transformed with the recombinant Rhizoctonia solani EHA105-P605 or the recombinant Agrobacterium tumefaciens EHA105-p8 were stained.

Formulation of GUS staining solution (l ml ): 610 μ 10.2 Μ Na ₂ HP0 ₄ solution ( ρ Η = 7.0 ); 390 μ 1 0.2 M NaH ₂ P0 ₄ solution and 10 μl 0.1 M X-gluc. The rice calli transformed with recombinant Agrobacterium tumefaciens EHA105-P605 or recombinant Agrobacterium tumefaciens EHA105-P8 were immersed in GUS staining solution, and then incubated at 37 出现 to appear blue, and the results were as shown in Fig. 5. The recombinant callus of Agrobacterium tumefaciens mediated by the P8+P605 recombinant vector containing the promoter (Fig. 5 left) is blue after staining, and is reconstituted by the p8 plasmid without the promoter. Bacillus-mediated transformation of rice calli (as a control, Fig. 5 right) did not change color after GUS staining. The results show that the P605 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 a MS-R differentiation medium containing 50 mg / 1 hygromycin B (HmB) (see Table 7 for specific formulation) to differentiate the seedlings; the culture dish was sealed with a parafilm. 5 Light culture for 3-4 weeks; when the seedlings grow 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 screening.

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 Fig. 6. The roots, stems and leaves of the rice seedlings mediated by recombinant Agrobacterium tumefaciens mediated by the P8+P605 recombinant vector containing the promoter (Fig. 6 left) were stained blue. The roots, stems and leaves of rice seedlings mediated by Agrobacterium tumefaciens-free transformed p8 plasmid without promoter (as a control, Fig. 6 right) did not change color after GUS staining. The results show that the P605 promoter of the present invention has a regulatory effect on GUS gene expression.

The relevant medium formulations used in the examples of the present invention are described as follows:

The following "conventional sterilization" as used in the medium refers to sterilization under the following conditions: 121 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 (Pro l ine ) 2. 88 g 1. 44 g 5. 76 g Hydrolyzed casein (CH ) 0. 30 g 0 15 g 0. 6 g sucrose 30 g 15 g 60 g plant gel ( Phytage l ) 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 Save the spare.

N ₆ micro mother liquor (1000X): Potassium recalcification 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: stored for later use.

Iron salt (Fe ₂ EDTA) stock solution (100X): 3.73 g of disodium edetate (Na ₂ EDTA - 2H ₂ 0 ) and 2.78 g of FeS0 ₄ .7H ₂ 0 were separately dissolved and mixed. Make up to 1000 ml with distilled water, 2 h at 70 °C, and 4 for less than 1 month 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 storage for no more 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

Conventional sterilization, add to room temperature after cooling

Kanal (Kan) (50mg/ml) 1 ml 0.5 ml Rifampicin (50 mg/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 (1000X) 1ml 0.5ml

Inositol (500X) 2ml 1ml

Hydrolyzed casein (CH) 0.5g 0.25g

Potassium chloride (KC1) 3. Og 1.5g

L-Arginine 0.176g 0.088g

L-Glut amine 0.9g 0.45g

Aspartic acid (L-Aspartic acid) 0.3g 0.15g

Sucrose (sucrose) 68g 34g

Glucose 36g 18g

Conventional sterilization, join when used

1ml 0.5ml

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

AAM macro ( 10X) : 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 .2H ₂ 0 ), dilute to 1 L of distilled water, 4 and store for future use.

AAM micro (100X): 0.7 g manganese sulfate monohydrate (MnS0 ₄ · H ₂ 0 ), 0.2 g 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 Mo0 ₄ .2H ₂ 0 ), 2.5 mg copper sulfate pentahydrate (CuS0 ₄ .5H ₂ 0 ), 2.51^ cobalt chloride hexahydrate ((0(:1 ₂ .611 ₂ 0) , dilute to 1 L of distilled water, 41 for storage.

AAM organic (1000X): 0.75 g glycine (Glycine), 0.1 g nicotinic acid (Nicotinic acid), 0. Ig VB ₆ ( Pyridoxine ), lg VB, ( Thiamine ), distilled water to 100 ml, 4 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 _s 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 _s vitamin stock solution ( 1Q0QX) 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 Sucrose (Sucrose) 30 g 15 g 60 g Glucose (Glucose) 10 g 5 g 20 g Phytagel 4 g 2 g 8 g Conventional sterilization, added when used

Acetyl syringone US) ( lOOmM) 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 (100X), N ₆ vitamin stock solution (1000X): see Table 2. Table 7: MS-R differentiation medium

1 L 0.5 L 2 L

MS macro ( 2 OX ) 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 Qingzun (500 mg/ml) 0.5 ml 0.25 ml 1 ml tibogene (50 mg/ml) 1 ml 0.5 ml 2 ml

Tune? 11 to 5.8, sterilized in the usual way.

MS macro ( 20X) : Ammonium nitrate 33.0 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 distilled water at room temperature Make up to 1 L, 4 'C to save.

MS micro ( 1000X) : manganese sulfate 16.90 g, zinc sulfate 8.60 g, boric acid 6.20 g, potassium 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 with distilled water and store at 4 "C.

MS Vitamin Storage Solution (1000X): Vitamin B, 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 and sterilize, 4 'C for no more 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 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 Titroblast (50mg/ml) 1 ml 0. 5 ml 2 ml 11 value to 5.8.

MS macro (20X) is shown in Table 7.

MS micro (1000X) MS vitamin stock solution (1000X) is shown in Table 7.

Iron salt (Fe ₂ EDTA) stock solution (100X): See Table 2.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations may be made to those details in accordance with the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

Rights request

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:

A recombinant vector comprising the promoter according to claim 1, in particular, the recombinant vector is a P8+P605 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-P605.

A plant callus characterized in that the callus is transformed with the promoter according to claim 1, in particular, the callus is a rice callus, more specifically, the rice 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 199, japonica rice 203, japonica rice 205, japonica rice 207, and Jinyuan 101, in particular, The rice is Nipponbare.

6. A method of preparing the promoter of claim 1, comprising the steps 1) designing a PCR amplification primer pair 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 sweet sorghum BT x 623 genomic DNA as a template, the PCR amplification primer set designed in step 1) was used for PCR amplification.

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, specifically, the callus is a rice callus, particularly The rice is Nipponbare.

The method according to claim 7, wherein the recombinant Agrobacterium tumefaciens EHA105-P605 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 specifically, the rice is Nipponbare.

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