CN108070595A - The specific expressed promoter POsFT1 of Rice Anther and its application - Google Patents

Abstract

The invention discloses the specific expressed promoter POsFT1 of Rice Anther and its applications.The specific expressed promoter POsFT1 of Rice Anther provided by the invention be it is following a) c) in any DNA molecular：A) DNA fragmentation shown in SEQ ID No.1；B) and a) nucleotide sequence of restriction has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology, and derive from rice and the DNA fragmentation with promoter function；C) under strict conditions with nucleotide sequence hybridization a) or b) limited, and the DNA fragmentation with promoter function.It is experimentally confirmed：The promoter POsFT1 of the present invention can effectively start target gene it is specific expressed in flower pesticide, so as to effectively avoid foreign gene continuous expression adverse effect in plant its hetero-organization.

Description

Rice Anther-specific Promoter POsFT1 and Its Application

technical field

The invention relates to the field of plant molecular biology, in particular to a rice anther-specifically expressed promoter POsFT1 and its application.

Background technique

The growth and development of higher plants is the result of the orderly expression and synergy of different genes in different time and space. Promoter is an important cis-acting element, which determines the expression pattern and expression intensity of genes by combining with transcription factors. According to the different transcription modes of promoters, they can be divided into three types: constitutive, inducible and tissue-specific promoters.

Tissue-specific promoters refer to organ-specific promoters. Driven by such promoters, the expression of genes is often limited to certain specific organs and tissue parts, and exhibits developmental regulation and other characteristics. Tissue-specific promoters can not only make the expression product of the target gene accumulate in a certain organ or tissue site, but also increase the expression level of the region. Using a tissue-specific expression promoter to drive the expression of the target gene can effectively avoid the effects of using a constitutive expression promoter. Some negative effects such as metabolic burden. However, there are still few tissue-specific expression promoters with clear regulatory mechanisms and which can be used for crop genetic improvement.

Contents of the invention

An object of the present invention is to provide a DNA molecule having a promoter function.

The DNA molecule with promoter function provided by the present invention is named POsFT1 promoter, which is derived from rice (Oryza sativa), and is a DNA fragment of any of the following a)-c):

a) DNA fragment shown in SEQ ID No.1;

b) a DNA fragment having more than 90% homology with the nucleotide sequence defined in a) and having a promoter function;

c) A DNA fragment that hybridizes to the nucleotide sequence defined in a) or b) under stringent conditions and has a promoter function.

The above-mentioned stringent conditions can be hybridization at 65° C. in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

Among them, SEQ ID No.1 consists of 1493 nucleotides in total.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the DNA molecule (promoter) also belong to the protection scope of the present invention.

Wherein, the recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

In one embodiment of the present invention, the recombinant vector is specifically obtained by inserting the DNA molecule shown in SEQ ID No.1 between the PstI and EcoRI restriction sites of the pCAMBIA1391Z vector, and keeping other sequences of the pCAMBIA1391Z vector unchanged carrier.

The expression cassette is composed of the DNA molecule having a promoter function, the gene of interest to be expressed by the DNA molecule, and a transcription termination sequence; the DNA molecule is functionally connected to the gene of interest, and the The gene of interest is linked to the transcription termination sequence.

In one embodiment of the present invention, the target gene is specifically the GUS gene (derived from the pCAMBIA1391Z vector); the transcription termination sequence is specifically the NOS transcription terminator (derived from the pCAMBIA1391Z vector).

Another object of the present invention is to provide POsFT1 promoter or the new application of the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

The invention provides the application of the POsFT1 promoter or the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in promoting the expression of the target gene.

In said application, said promoting the expression of the target gene is promoting the expression of the target gene in plants.

Further, the expression is anther-specific expression.

Further, the plant can be a monocot or a dicot.

Furthermore, the monocotyledonous plant can be a grass plant.

More specifically, the gramineous plant can be rice, such as the rice variety Nipponbare.

The application of the above-mentioned POsFT1 promoter or the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacteria in plant genetic improvement also belongs to the protection scope of the present invention.

Compared with the prior art, the present invention has the following advantages:

(1) The rice anther-specific expression promoter of the present invention is POsFT1, which is derived from the rice japonica variety Nipponbare, and the size of the promoter fragment is 1493bp. The POsFT1 promoter has the following characteristics: a) it is located at the 5' end of the OsFT1 gene and its upstream; b) the base length is 1493bp; c) it has a necessary site for initiating transcription and a transcription initiation point; d) it is specifically expressed in rice anthers. The present invention provides a promoter capable of effectively controlling the specific expression of the target gene in anthers, and not expressing it in other organs and tissues, which can avoid the adverse effects caused by the continuous expression of foreign genes in other tissues of plants, such as: transgene drift and pollen escape. posed biosafety issues.

(2) The recombinant expression vector containing the anther-specific expression promoter POsFT1 of the present invention is transferred to the anther-specific expression promoter, and the downstream gene is positioned on the anther expression under the regulation of the anther-specific expression promoter POsFT1 of the recombinant expression vector, It provides a tool for studying the specific expression of genes in anthers in the field of plant genetic engineering, and has broad application prospects.

It is proved by experiments that the promoter POsFT1 provided by the present invention can effectively promote the specific expression of the target gene in rice anthers, and it does not express in other organs and tissues, which can avoid the adverse effects brought by the continuous expression of the target gene in other tissues of the plant. It can also be used for functional analysis and identification of genes related to plant anther growth and development. Not only serve for transgenic rice breeding, but also reserve resources for long-term promoter transformation and design.

Description of drawings

Figure 1 is a graph showing the molecular detection results of POsFT1 promoter. A is the result of PCR; B is the result of double enzyme digestion. M1 and M2: DL-5000, 1: the band size is 1493bp; 2: PstI and EcoRI double restriction band.

Fig. 2 is a map of the T-DNA region of the expression vector pCAMBIA1391Z-POsFT1. Among them, A is the pCAMBIA1391Z vector, LB and RB represent the left and right borders of T-DNA respectively; Hyg represents the hygromycin resistance gene; MCS represents the multiple cloning site; NOS represents the terminator of the gene; B represents the multiple cloning Locus map (MCS).

Fig. 3 is a graph showing the results of histochemical staining of the GUS gene. Among them: A stands for young root; B stands for stem; C stands for young leaf; D stands for glume; E stands for anther.

Detailed ways

The present invention will be further described in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The quantitative tests in the following examples were all set up to repeat the experiments three times, and the results were averaged.

GUS staining solution (pH7.0) in following embodiment: solvent is 200mM PBS damping fluid, solute and its concentration in staining solution are respectively: 100mM potassium ferrocyanide, 100mM potassium ferricyanide, 0.5mM EDTA (pH8.0), 10 mg/ml X-Gluc, 0.1% (volume ratio) Tween 20.

The primers used in the following examples were all synthesized by Shenzhen Huada Bioengineering Co., Ltd. and sequenced by Shanghai Sangon Company; pTEAY-T1, Taq enzyme, Trans5α competence and related kits were purchased from Beijing Quanshijin Company; Dicer PstI, EcoRI, and _T4 ligase were purchased from TaKaRa Company; antibiotics were purchased from Shanghai Sangong and SIGMA; pCAMBIA1391Z vector and Agrobacterium AGL1 were purchased from Beijing Boai Yonghua Biotechnology Co., Ltd.; other reagents were domestically produced Analytical pure.

Embodiment 1, the cloning of rice anther-specific promoter POsFT1

1. Design of primers

According to the full-length sequence of the POsFT1 promoter published on the NCBI website, primers for PCR amplification of this fragment were designed, with the CTGCAG restriction site of PstI added to the upstream primer, and the GAATTC restriction site of EcoRI added to the downstream primer.

The primer sequences are as follows:

Primer 1 (upstream primer): 5'-CTGCAGCCTTCGTTTACCAATC-3';

Primer 2 (downstream primer): 5'-GAATTCCCGGCTGCCGGCCGAC-3'.

2. PCR amplification

Use the rice Nipponbare genomic DNA extracted from Quanshijin Plant Genome Kit as a template, and perform PCR amplification with the upstream and downstream primers designed in step 1 to obtain PCR products.

The PCR reaction conditions are as follows: pre-denaturation: 98°C 10min; denaturation: 98°C 10s; annealing: 62°C 5s; extension: 72°C 2min30s, 36 cycles; total extension: 72°C 10min.

3. PCR product detection

After the PCR reaction, use 2% agarose gel electrophoresis to detect the PCR product; then recover and purify the target fragment, and then connect the recovered fragment with pEASY T1Cloning Vector to obtain the recombinant vector pEASY T1Cloning Vector-POsFT1, and finally the recombinant vector pEASY T1Cloning Vector -POsFT1 was transformed into Escherichia coli competent Trans5α cells; positive clones were screened by PCR and enzyme digestion detection and verified by sequencing (Figure 1).

The results showed that a DNA fragment with a size of 1493bp was amplified by PCR, and its nucleotide sequence was shown in sequence 1 in the sequence listing. The nucleotide sequence shown in sequence 1 was named POsFT1, which consisted of 1493 bases.

Example 2, Functional Verification of POsFT1 Promoter

1. Obtaining of transgenic strains

1. Construction of expression vector

The recombinant vector pEASYT1CloningVector-POsFT1 in Step 3 of Example 1 was cut with restriction endonuclease PstI and EcoRI to obtain the POsFT1 fragment; the pCAMBIA1391Z vector (Fig. 2) was obtained by double digestion with restriction endonuclease PstI and EcoRI to obtain pCAMBIA1391Z Vector backbone fragment; the POsFT1 fragment was connected with the pCAMBIA1391Z vector backbone fragment to obtain the recombinant plasmid POsFT1-pCAMBIA1391Z.

The recombinant plasmid POsFT1-pCAMBIA1391Z is a vector obtained by inserting the POsFT1 promoter shown in SEQ ID No.1 between the PstI and EcoRI restriction sites of the pCAMBIA1391Z vector, and keeping other sequences of the pCAMBIA1391Z vector unchanged. The POsFT1 promoter was used to activate the GUS gene in the pCAMBIA1391Z vector.

2. Introducing the recombinant plasmid POsFT1-pCAMBIA1391Z into Agrobacterium AGL1 to obtain recombinant Agrobacterium.

3. The recombinant Agrobacterium prepared in step 2 was resuspended in a liquid co-cultivation medium (YEP liquid medium + 100 mg/L acetosyringone, pH 5.2) for culture to obtain a bacterial liquid with OD _600nm =1.0.

4. Get the embryogenic callus of the rice material Nipponbare (the rice material Nipponbare was purchased from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences), soak for 30min with the bacterium liquid obtained in step 3, and then through co-cultivation, screening, rooting, and strong seedlings, to obtain T ₀ generation transgenic plants.

5. Extract the DNA of the transgenic plants of the T ₀ generation, and use the primer 1 and primer 2 in Example 1 to carry out PCR amplification, and the PCR procedure is the same as above. The DNA fragment with a size of 1493bp amplified by PCR is the positive transgenic POsFT1 promoter plant. Among the 11 transgenic plants of the T ₀ generation obtained by PCR detection, there were 10 positive POsFT1 promoter transgenic plants.

2. Staining of plants transfected with POsFT1 promoter

GUS histochemical staining was performed on different parts of positive POsFT1 promoter plants. The specific operation steps are as follows: take the following different tissues of positively transfected POsFT1 promoter plants: young roots, stems, young leaves, glumes and anthers, and put each part into a test tube; then add an appropriate amount of GUS buffer to submerge the tissue block, and then Add GUS staining solution, mix well and store at 37°C for 4-12 hours; after staining, rinse and decolorize the stained tissue in 75% ethanol, then soak in 50% and 20% ethanol for more than 20 minutes, until the material turns white; Finally, when observed under a microscope, the blue dots on the tissue are the GUS expression sites.

The staining results are shown in Figure 3. Among them, A stands for young root; B stands for stem; C stands for young leaf; D stands for glume; E stands for anther. It can be seen from the figure that the GUS gene is only expressed specifically in the anthers of the POsFT1 promoter-transferred plants, but not expressed in the roots, stems and leaves. It shows that the promoter POsFT1 of the present invention is only specifically expressed in rice anthers, and can be used to promote the specific expression of target genes in anthers.

sequence listing

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ccttcgttta ccaatcaccc gtcttgtctc gggtcaccca taattccaga tcctaatacc 60

catatcaaag ggctgctccc tccgtttaaa aaaaattcaa aacaaaacaa agacaaactt 120

attackagag atgatatatt ctaatacaat taatctgaac aaagtctatt cagatttatt 180

ttactataat aaatcacatc caatcctagg attatatttt gtggaacgga tggaatagta 240

atccttacta cttccgtctc aaaatataat acgtttaagc ttgaatacga tacaaataga 300

gtaggtaaaa tcaaatgaga aaatattata tatgattagt tgagaaaaga atgtaagttg 360

agttcatttt gttgtgtcta ctattccaaa gtaagtttgc ttctagcgca agtatcatca 420

atttgtaaat cgttaagttt tactccctcc tttctaatat aggtgcaacc taatatagat 480

gtgacatatc taattttttt ttcacgaacg cgtataagaa ttgcacgtta atataatata 540

ttagatatat ccaatgttac atttatattt ttttaaccag atgttacatt tatatttata 600

ttgagacaga tgagtattca tttagtcctc ttatcgacct accatcaatg ctattttctt 660

attaatgatg ggcactttga tattttcttc tttcacgaac acataggact aaaaagtagg 720

agcaagttat tttgtaatga agggatctaa aacataccta aggcgtgagt atatcaataa 780

ctataaattc agcactgaat tgcaagtttt ctgtttaccc tgcttcttat tattttttct 840

gaattgatct aattatggca taagtgcgag ttatgggat tgacttagta ataaaaagta 900

attgacatat aataaataat catctaagac gacatattaa ttaattactc cctttgcttt 960

atattgtaag gtgttctaag ttcatcttaa gtcaaacttt gtttaactac tatcaaaata 1020

tagaaaaact tagcaacatc tctaacacta tattaatttt attaaaaccg ccattaaata 1080

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actagcagtt tcaaaaattg atacgctatc tgtaaccaat gtaatttcta cccatataac 1200

agctactaat ataggagtat atactactcc gtccgtccca taatacaaga gatcatggcc 1260

ttcaaattta tcccgaaacg ttagagatct tggtatcccg agacaatact tatttattcc 1320

tcacccctaa cttaaagcga gcagccaagc tactgtagct aatggtaggg cgcgtgcgac 1380

agtggagtaa agtgaagcag cagcagagaa gcagagacca tttccctcct ccctcctctc 1440

tcgccaaaca cgacgacgtc gcctcggttt agtcgtcgtc ggccggcagc cgg 1493

Claims (10)

1. A DNA molecule, which is a DNA fragment of any of the following a)-c): a) DNA fragment shown in SEQ ID No.1; b) a DNA fragment that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the nucleotide sequence defined in a), and is derived from rice and has a promoter function; c) A DNA fragment that hybridizes to the nucleotide sequence defined in a) or b) under stringent conditions and has a promoter function. 2. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the DNA molecule of claim 1. 3. The expression cassette according to claim 2, characterized in that: the expression cassette is composed of the DNA molecule with promoter function, the target gene for expression initiated by the DNA molecule, and a transcription termination sequence; A DNA molecule is functionally linked to the gene of interest, and the gene of interest is linked to the transcription termination sequence. 4. The application of the DNA molecule according to claim 1 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 2 in promoting the expression of the target gene. 5. The application according to claim 4, characterized in that: said promoting the expression of the target gene is to start the expression of the target gene in plants. 6. The application according to claim 5, characterized in that: the expression is anther-specific expression. 7. The application of the DNA molecule according to claim 1 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium according to claim 2 in plant genetic improvement. 8. The application according to any one of claims 5-7, characterized in that: the plant is a monocot or a dicot. 9. The application according to claim 8, characterized in that: said monocotyledons are gramineous plants. 10. The application according to claim 9, characterized in that: the gramineous plant is rice.