CN104450708B - Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE - Google Patents

Abstract

The invention discloses a retrotransposon promoter P _Cb-RARE induced by high salt and salicylic acid and its application. The promoter is derived from the retrotransposon Cb-RARE-1 of bamboo tree (Carallia brachiata) The LTR region, its sequence is shown in SEQ ID NO:1. The present invention transforms Arabidopsis thaliana with a recombinant expression vector containing the promoter and the GUS gene, and verifies that the promoter is induced by NaCl and salicylic acid. Using this characteristic, the promoter can be used in the screening of saline-alkali-tolerant plants and molecular breeding. The present invention uses an inducible promoter to induce the expression of foreign genes in plants, and the function of the promoter can be activated or inhibited under the stimulation of high salt or salicylic acid, so high-concentration sodium chloride can be used at a specific time or growth period Or salicylic acid activates or inhibits gene expression and then controls the synthesis of gene products, so as to regulate plant growth purposefully.

Description

A Retrotransposon Promoter Induced by High Salt and Salicylic Acid and Its Application

technical field

The invention belongs to the field of biotechnology, in particular to a retrotransposon (also known as retrotransposon, retrotransposon) promoter P _Cb-RARE induced by high salt and salicylic acid and its application. The promoter is derived from the LTR region of the retrotransposon Cb-RARE-1 of the bamboo tree (Caralliabrachiata).

Background technique

Retrotransposon (RTN) is an element that uses RNA as a medium to proliferate and transpose. Under the action of reverse transcriptase RT and RNaseH, the retrotransposon is reverse transcribed into extrachromosomal DNA, and then inserted into the new position of the chromosome under the action of integrase. The insertion of retrotransposons can cause stable mutations in or near genes. Therefore, the characteristic of retrotransposon jumping can be used for mutation initiation and for germplasm breeding and so on.

Retrotransposons with long terminal repeats (long terminal repeats, LTRs) have a direct alignment repeat sequence at both ends, including a transcription-related promoter (promoter) and terminator (terminator). The promoter can initiate the transcription of the gene inside the retrotransposon, and its products are involved in the control of transposition behavior. Most transpositions are deleterious, and the organism has developed many mechanisms to inhibit their transposition activity. Normally, retrotransposons are quiescent. However, when subjected to various biotic and abiotic stresses, some retrotransposons can be activated and undergo transposition. For example, the expression of retrotransposon TaR T-1 in wheat is up-regulated when it is stressed by MeJA, SA or Powdery Mildew Fungus (Tang et al., 2005); , trauma and pathogen stress, the expression level is up-regulated (Kimura et al., 2001).

The development of plant genetic engineering and biotechnology has made great contributions to the research of resistant crops and plant bioreactors. However, how to control the timing and quantitative high-efficiency expression of exogenous genes is an important factor that limits the effective application of genetic engineering technology in crop genetic improvement. One of the solutions is to find an efficient gene of interest, while another is to find a promoter that can effectively control the expression of the gene of interest.

The promoter controls the temporal and spatial expression of the target gene fused with it in the plant host, so the choice of promoter type determines the time and location of gene expression. According to the different characteristics of promoters driving gene expression, promoters are divided into constitutive promoters and specific promoters. Constitutive promoters can initiate transcription in all cells or tissues regardless of time and space; specific promoters can be further divided into tissue-specific promoters and inducible promoters. Tissue-specific promoters mean that the target gene is only expressed in certain specific organs or tissues; inducible promoters are stimulated by certain physical or chemical signals, which can greatly change the expression level of the target gene Promoter.

Kasuga et al. used the inducible rd29A promoter instead of the constitutive promoter CaMV35S to transform Arabidopsis thaliana, which improved the drought resistance of transgenic Arabidopsis and reduced the occurrence of plant growth stagnation or dwarfing caused by constitutive overexpression (Narusaka et al. , 2003, Plant J 34, 137-148). Inducible promoters can change the expression pattern of the target gene only after receiving the induction signal. Therefore, the use of such promoters in plant molecular breeding can effectively control the gene expression and phenotype of plants, not only will not cause various resources. Waste can also reduce the impact on plant growth and development and other metabolic pathways. Therefore, the study and utilization of inducible promoters is of great significance for the study of the physiological mechanism of plants responding to various stresses and the cultivation of new varieties of crops through genetic engineering. It also has broad application prospects in basic research and plant biotechnology.

Contents of the invention

The primary purpose of the present invention is to provide a retrotransposon promoter P _Cb-RARE induced by high salt and salicylic acid, which is derived from the retrotransposon Cb-RARE-1 of bamboo tree (Carallia brachiata) The LTR area.

Another object of the present invention is to provide an expression cassette containing the above promoter sequence.

Another object of the present invention is to provide a recombinant vector containing the above promoter sequence.

The fourth object of the present invention is to provide the application of the above-mentioned promoter in the screening and molecular breeding of saline-alkali-tolerant plants.

The object of the present invention is achieved through the following technical solutions:

A retrotransposon promoter P _Cb-RARE induced by high salt and salicylic acid, its sequence is shown in SEQ ID NO: 1, the sequence length is 974bp, the promoter is derived from the retrotransposon of the bamboo tree (Carallia brachiata) LTR region of transposon Cb-RARE-1. Experimental results show that the promoter sequence can initiate the expression of downstream genes, and its activity and specificity are regulated by salicylic acid and sodium chloride.

An expression cassette, comprising the above-mentioned promoter sequence and target gene sequence.

A recombinant expression vector, comprising the above-mentioned promoter sequence, or comprising the above-mentioned expression cassette.

Based on the characteristics of the above-mentioned promoter being induced by high salt and salicylic acid, the promoter can be applied to the screening and molecular breeding of saline-alkali-tolerant plants.

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

The present invention uses an inducible promoter to induce the expression of foreign genes in plants, and the function of the promoter can be activated or inhibited under the stimulation of high salt or salicylic acid, so high-concentration sodium chloride can be used at a specific time or growth period Or salicylic acid activates or inhibits gene expression and then controls the synthesis of gene products, so as to regulate plant growth purposefully. So far, no report on the nucleotide sequence of the promoter has been found, nor has the application of the promoter to conditionally activate gene expression properties been found.

Description of drawings

Fig. 1 is a graph showing the response experiment results of P _Cb-RARE ::GUS transgenic Arabidopsis to different concentrations of NaCl.

Fig. 2 is a graph showing the response experiment results of P _Cb-RARE ::GUS transgenic Arabidopsis treated with 100mM NaCl for different times.

Fig. 3 is a graph showing the response experiment results of hypocotyls and root tips of P _Cb-RARE ::GUS transgenic Arabidopsis to different concentrations of salicylic acid.

Fig. 4 is a graph showing the response experiment results of P _Cb-RARE ::GUS transgenic Arabidopsis to 10mM salicylic acid treatment for different time.

detailed description

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

Bamboo tree DNA extraction and retrotransposon Cb-RARE-1 putative LTR sequence P _Cb-RARE cloning:

Total DNA was extracted from bamboo leaves using the improved CTAB method (Doyle and Doyle, 1990). The LTR sequence P _Cb-RARE of Cb-RARE-1 was amplified according to the reaction system of the TaKaRa company kit LA PCR ^™ Kit according to its instructions.

The primer pairs used were:

Cb-promoter-F:5'-GAATTCAGAGGTGTCTATTGAGCATATT-3'(SEQ ID NO:2)

Cb-promoter-R2: 5'-GGTACCCACATCTATAAGCATAAT-3' (SEQ ID NO: 3).

The recognition sequence of restriction endonuclease EcoR I (5'-GAATTC-3') or KpnI (5'-GGTACC-3') was added to the 5' end of the primer.

The PCR reaction program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 45 sec, renaturation at 58°C for 45 sec, extension at 72°C for 3 min, 35 cycles; final extension at 72°C for 10 min. The amplified product was recovered (Axygen Biosciences), connected to the pMDTM18-T vector, transformed into Escherichia coli DH5α competent cells, and positive clones were selected for sequencing. The resulting sequence is SEQ ID NO:1.

Example 2

Construction of recombinant expression vector pBI-pCb-RARE containing P _Cb-RARE ::GUS expression cassette:

Expand the positive clone obtained in Example 1 (the clone contains the recombinant plasmid of the P _Cb-RARE sequence). Using a plasmid extraction kit (AXYGEN AXYPrep ^™ Plasmid Minprep Kit) to extract plasmid DNA, using the plasmid as a template and the following sequence as a primer, the PCb _-RARE sequence was amplified by the PCR reaction procedure in Example 1.

PCb _-RARE- F: 5'-AAGCTTAGAGTGTCTATTGAGCATATT-3' (SEQ ID NO: 4).

PCb _-RARE- R:5'-GTCGAC CACATCTATAAGCATAAT-3' (SEQ ID NO:5).

The resulting PCR reaction product was recovered with an agarose gel recovery kit (AXYGEN AXYPrep ^™ PlasmidMinprep Kit, Shanghai Shenergy Gaming). The recovered fragment was connected with a T-vector (Takara, T-easy vector), and transformed into Escherichia coli JM109 strain to obtain a recombinant JM109 strain containing _PCb-RARE sequence (called JM109- _PCb-RARE ).

The JM109-P _Cb-RARE was expanded and the plasmid DNA was extracted using a plasmid extraction kit (AXYGEN AXYPrep ^TM PlasmidMinprep Kit). Carry out double digestion with restriction endonuclease HindIII and SalI, and reclaim the ^PCb _-RARE fragment (SEQ ID NO: 1 plus a total of 12 bp restriction sites at both ends).

The plant binary expression vector plasmid pBI101 was cut with the same restriction enzymes HindIII and SalI, and the large fragment was recovered by gel. The large fragment of the binary vector plasmid pBI101 and the _PCb-RARE fragment were ligated with T4 DNA ligase to obtain the pBI- _PCb-RARE ::GUS recombinant vector plasmid, which was directly transformed into Escherichia coli JM109. Using the specific primers of P _Cb-RARE , positive transformants were identified by PCR and the strains were preserved. The positive transformant contains the recombinant vector (pBI-P _Cb-RARE ::GUS) of P _Cb-RARE fusion GUS reporter gene.

Example 3

Construction of transgenic Arabidopsis containing P _Cb-RARE ::GUS expression cassette:

Expand the culture of Escherichia coli transformants containing the recombinant vector (pBI-P _Cb-RARE ::GUS), and extract the pBI-P _Cb-RARE ::GUS recombinant vector plasmid after collecting the bacteria. Take the purified pBI-P _Cb-RARE ::GUS recombinant vector plasmid to transform Agrobacterium EHA105, select the colony that can grow on the LB culture plate containing 60mg/L rifampicin and 50mg/L kanamycin and carry out PCR identification , Select positive transformants that can be amplified to _PCb-RARE fragments and save the Agrobacterium strains.

After activating and culturing the above-mentioned Agrobacterium strains for one day, they were inoculated at an inoculation ratio of 1% and expanded for more than 12 hours until OD ₆₀₀ =0.6-0.8. Centrifuged at 4000 rpm for 10 min at room temperature, discarded the supernatant, and collected the bacteria. The freshly prepared 5% sucrose transformation liquid was used to suspend the bacterial cells to make OD ₆₀₀ =0.6-0.8. Add 0.03% Silwet (purchased from GE, USA) and mix well.

Select healthy wild-type plants of Arabidopsis thaliana Columbia ecotype with more flower buds, and transform Arabidopsis plants by inflorescence soaking method. The fruits of the transformed plants were collected, which were the seeds of the T1 generation. Sow T1 generation seeds on MS culture plates containing 30 mg/L hygromycin for screening, and select plants that can grow normally, which are T1 generation plants. The fruits of the T1 generation plants are collected, which are the T2 generation seeds. Sow T2 generation seeds on MS culture plates with 30mg/L hygromycin, select healthy growing seedlings and transfer them to nutrient soil for continued cultivation, and harvest single plant seeds, which are T3 generation seeds. The T3 generation seeds obtained from each individual plant were sowed on MS culture plates with 30 mg/L hygromycin for selection of homozygous plants for single-copy insertion. Count the resistance: non-resistant seedling segregation ratio of all samples when cultured on 30mg/L hygromycin MS culture plates, and the strains with a segregation ratio close to or equal to 3:1 are single-copy insertion strains. Among the single-copy insertion lines, select the single plant that can grow 100% healthily on the MS culture plate with 30mg/L hygromycin as the homozygous plant. The homozygous plant (its genotype is recorded as: P _cb-RARE ::GUS/Col) was used for subsequent work.

Example 4

Analysis of GUS gene expression activity of transgenic Arabidopsis P _cb-RARE ::GUS/Col under high salt:

Seeds of transgenic Arabidopsis thaliana P _cb-RARE ::GUS/Col and wild type (Col) after surface disinfection were sown on MS medium plates, treated at 4°C for 3 days, and then transferred to long-day sunlight (16 hours of light, 8 hours dark) for 10 days. Arabidopsis seedlings were taken and transferred to filter paper wetted with 100mM or 150mM NaCl solution for high-salt stress treatment. Each salt concentration was treated for 0.5, 1, 2 hours. Immediately after the stress treatment, the seedlings were taken out and put into a centrifuge tube with 500 μL of GUS histochemical detection solution, so that the seedlings were completely soaked. Place the above centrifuge tube at 37°C for 6 hours or more. Take out the seedlings, rinse and decolorize with 75% ethanol solution and absolute ethanol. During the decolorization process, ethanol was changed from time to time until the decolorization was complete. The seedlings were observed under a stereo microscope or an ordinary light microscope.

The results showed that the expression level and tissue specificity of the GUS gene changed under high-salt stress: as shown in Figure 1, compared with before treatment, the GUS staining of hypocotyls, young true leaves and roots of transgenic plants after 100mM NaCl treatment Lighten, indicating down-regulation of GUS gene expression, and darkening at the root tip, that is, up-regulation of GUS gene expression, indicating that the transcriptional activity of the _Pcb-RARE promoter in the above tissues or organs can be regulated by high salt.

In addition, when 100mM NaCl was treated for different times, the GUS histochemical staining of hypocotyls gradually weakened with time, and the color was the lightest at 2 hours of treatment (Figure 2), indicating that the activity of the P _cb-RARE promoter increased with high salt The prolongation of the processing time was gradually suppressed.

Example 5

Detection of GUS gene expression in transgenic Arabidopsis P _cb-RARE ::GUS/Col under salicylic acid treatment:

The seeds of transgenic Arabidopsis thaliana P _cb-RARE ::GUS/Col and wild type (Col) after surface disinfection were soaked in 200 μl of distilled water, 0.1mM SA, and 1mM SA solution respectively, and treated at 4°C for 3 days , and then sow it in MS medium, cultivate it in 22±2°C and long-day environment for 7-10 days, and then transfer it to nutrient soil to continue culturing. After 3 weeks, choose uniform plants and spray with 10mM salicylic acid for 0.5h, 1h, 2h, and spray with distilled water at the same time as a negative control. Different parts of the plants were cut for GUS histochemical detection.

The results are shown in Fig. 3, the treatment of different concentrations of salicylic acid made the upper part of the hypocotyl stain lighter, and the stain near the root tip deepened to a certain extent.

When treated with 10mM SA, as the treatment time prolongs, the staining of the leaves becomes lighter and then recovers, the staining is the lightest after 1 hour of treatment, and the staining of non-treatment is restored after 2 hours of treatment (Figure 4), indicating that the P _cb-RARE promoter Activity is modulated by salicylic acid.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (4)

1. A retrotransposon promoter P _Cb-RARE induced by high salt and salicylic acid, the sequence of which is shown in SEQ ID NO:1. 2. An expression cassette, characterized in that it comprises the promoter according to claim 1. 3. The expression cassette according to claim 2, characterized in that: it also comprises a target gene sequence. 4. A recombinant expression vector, characterized in that it comprises the promoter sequence according to claim 1, or comprises the expression cassette according to claim 2 or 3.