CN106191068A - The promoter of Soybean Root specifically expressing and application thereof - Google Patents

Abstract

The invention provides the promoter of a kind of Soybean Root specifically expressing, named GmPHT1.01, its nucleotide sequence, as shown in SEQ ID No.1, present invention also offers the application in regulation and control downstream gene expression of the described promoter.By Agrobacterium rhizogenes instantaneous conversion system, with GmPHT1.01 promoter expression gus gene, result shows that GmPHT1.01 promoter can be obviously promoted the expression of gus gene in plant roots, thus GmPHT1.01 promoter has specificity in plant roots, it is suitable in the root of the plants such as various crops, forest, vegetable, flowers, herbage specifically expressing target gene and improves the expression of genes of interest.

Description

Soybean root-specific promoter and its application

technical field

The invention relates to the field of plant genetic engineering, in particular to a GmPHT1.01 promoter specifically expressed in soybean roots and nodules and its application.

Background technique

The promoter is an important cis-acting element in a gene, generally located tens of bases upstream of the transcription initiation site. Promoters can be divided into three types according to different transcription modes: constitutive promoters, tissue or organ-specific promoters, and inducible promoters. The activity of tissue- or organ-specific promoters is induced and regulated by specific tissue cell structures and chemical and physical signals, that is, the expression of such promoters has specific spatiotemporal characteristics. Driven by such promoters, the expression of genes is often limited to certain specific organs or tissue parts or specific developmental periods. Tissue or organ-specific promoters can not only make the expression product of the target gene accumulate in a certain organ or tissue site, increase the regional expression level, but also avoid the adverse effects caused by the expression of the target gene in other tissues and organs.

So far, a large number of tissue or organ-specific promoters have been isolated, including leaf-specific promoters, flower-specific promoters, seed-specific promoters, and root-specific promoters. The root is an important organ for plants to absorb water and nutrients, and the root-specific expression system can be used to study plant hyperosmotic stress tolerance, phytoremediation, and rhizosphere secretion, etc. Borisjuk et al. used root-specific promoter mas2, GFP and tobacco calreticulin (calreticulin) gene to construct a fusion expression vector. The research results of transgenic tobacco hydroponics show that root cells can not only produce GFP efficiently, but also secrete the target protein into the liquid medium (Borisjuk et al., 1999). In addition, Ying Qicai et al. used the pine root-specific promoter PmPgRP10 to drive the CMO/BADH bivalent gene and transformed it into rice. The activities of CMO enzyme, BADH enzyme and other physiological and biochemical indicators in the roots and leaves of transgenic plants were measured, and the results showed that the CMO/BADH bivalent gene could be specifically expressed in roots (Qicai Ying, 2006).

The GmPHT1.01 gene belongs to the family of plant phosphorus transporters and plays an important role in the regulation of phosphorus transport. Therefore, it is necessary to study and obtain the promoter sequence regulating soybean phosphorus transport gene GmPHT1.01, so as to provide effective means and ways for in-depth study of plant phosphorus transport process.

Contents of the invention

The purpose of the present invention is to provide a promoter sequence for regulating soybean phosphorus transport gene GmPHT1.01, so as to provide an effective way for specific expression of target genes in the roots of various crops, forest trees, vegetables, flowers, pastures and other plants.

To achieve the above object, the present invention provides a GmPHT1.01 promoter specifically expressed in soybean roots, which has:

1) the nucleotide sequence shown in SEQ ID NO.1; or

2) A nucleotide sequence derived from 1) obtained by substituting, deleting and/or adding one or several nucleotides to the nucleotide sequence shown in SEQ ID No. 1 and having the same function.

It should be understood that those skilled in the art can replace, delete and/or add one or several promoters of the soybean phosphorus transport gene GmPHT1.01 promoter (SEQ ID No.1) disclosed in the present invention without affecting its activity. nucleotides to obtain the mutant sequence of the promoter. For example, in the inactive segment, without changing the function of the promoter, the nucleotide sequence obtained by performing at least one of the following substitutions: replace the T at the 110th position with A, replace the A at the 2601st position Substitute T, replace A at position 3004 with T.

The present invention also provides vectors, host cells, transformed plant cells or expression cassettes containing the GmPHT1.01 promoter.

Various vectors known in the art can be used in the present invention, as long as they are suitable for the expression of the GmPHT1.01 promoter, for example, commercially available vectors and plasmids can be used.

The biological material of the present invention is a vector, a host cell, a transformed plant cell or an expression cassette. The present invention also provides the application of the GmPHT1.01 promoter or the above-mentioned biological material containing it in regulating the specific expression of downstream genes in plant roots.

The present invention also provides the application of the GmPHT1.01 promoter or the above-mentioned biological material containing it in increasing the expression of downstream genes in plant roots.

The present invention also provides the application of the GmPHT1.01 promoter or the above biological material containing it in the preparation of transgenic plants.

The present invention also provides the application of the GmPHT1.01 promoter or the above-mentioned biological material containing it in the regulation of plant root development.

The present invention also provides the application of the GmPHT1.01 promoter or the above-mentioned biological material containing it in the improvement of plant germplasm resources.

The present invention also provides the application of the GmPHT1.01 promoter or the above biological material containing it in plant breeding.

Preferably, the above-mentioned plants are crops, trees, vegetables, flowers, pastures.

More preferably, the aforementioned plants are crops. Most preferred is soybean.

The present invention also provides the specific primers used to clone the 5240bp GmPHT1.01 promoter sequence from the soybean genome, including forward primer: 5'-attgGAGCTCGTGTCCCTTTTGTATGATGGAATC-3'; and reverse primer: 5'-gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3 '.

Kits containing the above-mentioned specific primers also belong to the protection scope of the present invention.

The above-mentioned specific primers can also be used to detect the promoter of the soybean phosphorus transport gene GmPHT1.01 described in the present invention.

The present invention isolates the GmPHT1.01 promoter specifically expressed in soybean roots for the first time, and uses the GmPHT1.01 promoter to express the GUS gene. The results show that the GmPHT1.01 promoter can significantly promote the expression of the GUS gene in plant roots. The promoter has specificity in plant roots, and is suitable for specifically expressing target genes in the roots of various crops, forest trees, vegetables, flowers, pastures, etc., and can increase the expression level of the target genes.

Description of drawings

Figure 1 shows the expression results of GmPHT1.01-GUS in soybean roots.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Cloning of Example 1 Soybean GmPHT1.01 Promoter

Using the forward primer forward primer: 5'-attgGAGCTCGTGTCCCTTTGTATGATGGAATC-3' and reverse primer: 5'-gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3' from the soybean genome PCR amplification and sequencing to obtain a length of 5240bp GmPHT1.01 promoter, its nucleoside The acid sequence is shown in SEQ ID NO.1. Sac I and Pst I restriction sites and protective bases are respectively attached to both ends of the PCR product.

The above PCR amplification system is: (20μl system)

PCR program: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 3min, a total of 25 cycles; 72°C for 10min.

Example 2 The soybean GmPHT1.01 promoter regulates the expression of the downstream gene GUS

The GmPHT1.01 promoter was obtained by PCR cloning in Example 1, and the recovered product was double-digested with Sac I and Pst I. The vector pCAMBIA3301 (pCAMBIA3301 was purchased from Cambia, Australia) was cut off the 35S promoter with HindIII and NcoI double enzymes, and then filled with Klenow to form blunt ends. After self-ligating with Solution I, it was transformed into DH5α for amplification. After the plasmid was extracted, it was digested with Sac I and Pst I, and after recovery, it was ligated with the recovered fragment of the GmPHT1.11 promoter with Solution I. The binary expression vector pGmPHT1.01::GUS was obtained, and the plant expression vector pGmPHT1.01::GUS was transformed into Escherichia coli DH5α for propagation.

The transformation of Escherichia coli competent cells includes: (1) preparing LB solid medium containing kanamycin; (2) taking out the competent cells stored at -80°C, melting them in an ice bath, adding 5 μl of the ligation product to lightly Mix lightly and place in ice bath for 30 minutes; (3) Heat shock at 42°C for 30 seconds, then immediately place in ice bath for 3 minutes; add 300 μl LB liquid medium without antibiotics, shake and incubate at 37°C for 1 hour (160rpm); (4) Take an appropriate amount of culture solution and spread it evenly on the selective medium, culture it upside down at 37°C for 16 hours, pick 5-10 (or more) colonies with a sterilized toothpick, and place them in 200 μl selective liquid Shaking bacteria culture in LB medium; (5) Transform the positive clones detected and screened by PCR into Agrobacterium EHA105 after multiplying and extracting plasmids. Through the Agrobacterium rhizogenes-mediated transformation method, pGmPHT1.01::GUS was transformed into soybean Tianlong No. 1, and 50 hairy roots transformed with pGmPHT1.01::GUS were obtained.

GUS activity analysis shows that the GmPHT1.01 promoter is specifically expressed in the roots of plants (Figure 1), and can be used to specifically express target genes in the roots of various plants, including various crops, forest trees, vegetables, flowers, pastures, etc. .

Methods of Agrobacterium rhizogenes-mediated transformation of soybean:

(1) After the soybeans were sterilized by chlorine gas fumigation for 12 hours, they were taken out and placed in an ultra-clean bench to blow off the remaining chlorine, and soaked in sterilized ultrapure water for 16 hours for later use;

(2) After activating the single clone of Agrobacterium K599 with pGmPHT1.01::GUS, shake the test tube slightly and shake it with YEP until the OD of the bacterial solution is between 0.8-1.0, centrifuge at 22°C for 10 minutes at 4000 rpm, Resuspend in LCCM (1/10X Gamborg B ₅ salt, 30g/L sucrose, 3.9g/L MES, pH 5.4. Add 40mg/L acetosyringone after sterilization);

(3) Cut off the radicle of the soaked soybean, use the following hypocotyls as explants, immerse the explants in the resuspension liquid for 30 minutes to complete the infection, blot the liquid on the filter paper, and put the infected The soybean explants were placed in CCM (1/10X Gamborg B ₅ salt, 30g/L sucrose, 3.9g/LMES, 4.25g/L agar, pH 5.4. After sterilization, add Cysteine 400mg/L, and 40mg/L acetyl clove Ketone) for 3 days in the dark;

(4) Insert the hypocotyls of co-cultured soybean explants into hairy root induction medium (1X Gamborg B ₅ salt, 30g/L sucrose, and 0.59g/L MES, 7g/L agar, pH5.7. After sterilization, add Cefotaxime 100mg/L), culture under long-term light for 10-14 days to induce hair roots, and take materials when the hair roots grow to 2cm;

GUS staining of transformants:

(1) Add the tissue sample into 90% acetone and place it on ice for 20-30 minutes;

(2) After being treated with acetone, rinse the tissue with 50mM phosphate buffer (pH7.2), 2mM K ₄ Fe[CN] ₆ and 2mM K ₃ Fe[CN] ₆ solutions;

(3) Put the sample in X-Gluc staining solution (50mM phosphate buffer (pH7.2), 1mM X-gluc, 2mM K ₄ Fe[CN] ₆ , 2mM K ₃ Fe[CN] ₆ ), pump for 10 minutes, and then stained overnight at 37°C;

(4) Decolorize with 70% ethanol the next day;

(5) Add a few drops of HCG solution on the glass slide, and place the decolorized sample in it;

(6) Press the tablet and make it transparent for 15 minutes or overnight (according to the development stage of the tissue).

GUS staining solution formula:

20mM X-gluc (MW=521.8)

Dissolve with DMSO or N,N-dimethylformamide, 100mg X-gluc needs to be dissolved with 9.58ml DMSO, the final concentration is 20mM.

50mM phosphate buffer (pH7.0)

Solution A: Dissolve 3.12g of NaH ₂ PO ₄ 2H ₂ O in sterile distilled water, and dilute to 100ml;

Solution B: Dissolve 7.17g of Na ₂ HPO ₄ 12H ₂ O in sterile distilled water, and make up to 100ml;

Take 39ml of liquid A and mix with 61ml of liquid B.

Preparation of staining solution: 5mM potassium ferricyanide; 5mM potassium ferrocyanide; 10mM EDTA; 50mM phosphate buffer; 1mM X-gluc.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. the promoter of Soybean Root specifically expressing, for GmPHT1.01 promoter, it is characterised in that its sequence is:

1) nucleotide sequence shown in SEQ ID NO.1；Or

2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or adds the tool that one or several nucleotide is obtained Have equal function by 1) derivative nucleotide sequence.

Promoter the most according to claim 1, it is characterised in that described promoter is by following replacement mode extremely Few a kind of obtained nucleotide sequence: the T of the 110th is substituted by A, the A of the 2601st is substituted by T, by the 3004th A be substituted by T.

3. containing the biomaterial of promoter described in claim 1 or 2, described biomaterial is that carrier, host cell, conversion are planted Thing cell or expression cassette.

4. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are regulating and controlling downstream gene at root or root Application in tumor specifically expressing.

5. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are improving downstream gene in plant roots Or the application in root nodule expression.

6. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 answering in preparing transgenic plant With.

7. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 regulate in plant roots and Radical extension In application.

8. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are in plant germplasm resource is improved Application.

9. for cloning the specific primer of the promoter described in claim 1 or 2, it is characterised in that including:

Forward primer: 5 '-attgGAGCTCGTGTCCCTTTGTATGATGGAATC-3 '；Reverse primer: 5 '- gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3’。

10. contain the test kit of specific primer described in claim 9.