CN112760322B - Rice constitutive strong promoter and application thereof - Google Patents

Abstract

The invention relates to a rice constitutive strong promoter and application thereof. Specifically, the invention provides a promoter Nubi5 nucleic acid sequence capable of being expressed in various tissues of plants and a preparation and use method thereof. The invention discloses that the 3' terminal reverse sequence of the plant gene has a strong constitutive promoter functional element for the first time. Also relates to an expression cassette containing the promoter, a recombinant expression vector and a method for obtaining a corresponding transgenic plant. The Nubi5 promoter disclosed by the invention has strong activity in organs such as roots, stems, leaves, flowers and seeds of plants and the like, and has good application prospect in the field of plant transgenosis.

Description

Rice constitutive strong promoter and application thereof

Technical Field

The present invention belongs to the field of biotechnology and genetic engineering technology. In particular to a rice constitutive strong promoter and application thereof.

Background

With the development of transgenic technology, promoters have been used in many different agriculturally important species. The success of transgenic plant development depends largely on several factors, including optimization of cell culture conditions, selection of appropriate transformation methods, and the use of appropriate promoters to become a crucial factor for the success of transgenic plant transformation (McElroy et al, 1990). Heretofore, cauliflower mosaic virusCaMV35SConstitutive promoters have been widely used in plant transformation systems for dicotyledons and monocotyledons (Odell et al, 1985; zhang et al, 1988), although it also has the problem of low viability in monocotyledons (Peterhans et al, 1990). The other two are currently widely usedPan-constitutive promoter isactin(He et al, 2009) andubiquitin(Christensen et al, 1996). Many Polyubiquitin (Polyubiquitin) promoters have been isolated from different crops, including wheat (Christensen et al, 1996), tobacco (Plese et al, 2001), arabidopsis (Callis et al, 1990), sunflower (Binet et al, 1990), potato (Garbarino et al, 1995), tomato (Rollfinke et al, 1998), rice (Wang et al, 2003; sivamani et al, 2006), sugarcane (Wei et al, 2003), soybean (Chiera et al, 2007). The expression level of the exogenous gene driven by most of the promoters is obviously higher than that of the exogenous gene driven by the promotersCaMV35SA promoter. For example, studies have shown that the ubi-1 promoter of wheat, which contains 5' -UTR intron elements, is highly expressed in monocotyledonous plants (Wei et al, 2003; atkinson et al, 2004). Although some promoters have been cloned from different crops, the constitutive promoters for high expression in rice are not cloned much. The invention utilizes molecular biology technology and method to separate a constitutive gene upstream 1676bp promoter region from the genome of wild type Nipponbare variety of rice, fuses reporter gene GUS, transfers the gene into Nipponbare variety of japonica rice by agrobacterium transformation method, and further determines the constitutive expression function by performing GUS staining analysis on different tissues.

Monocot constitutive promoters are an important resource for plant transgenic engineering. The promoter is generally a DNA sequence located upstream of the 5' end of a structural gene and capable of directing RNA polymerase to bind to a template correctly and initiate gene transcription. Its core promoter element generally refers to the minimal DNA sequence necessary for RNA polymerase to initiate transcription, including the TATA box and the CAAT box. Most promoter constructs today include a 5' intron and a non-coding region (UTR), which helps to obtain enhanced and specific expression of transgenic plants (Roger et al, 2009). The gene corresponding to LOC _ Os02g45770 isOsMADS6The gene is mainly expressed strongly in early floral meristem by OsMADS6, transcripts can be detected in peridium and receptacle of palea, serosa, carpel, ovule (Li et al, 2010 dual et al, 2012), and the 3' end of the gene hasConstitutive strong promoter elements have not been reported.

Disclosure of Invention

The invention aims to provide a rice constitutive strong promoter and application thereof. By creating a full-length promoter. The promoter is fused with a reporter gene beta-glucuronidase gene (GUS gene), and is introduced into a Nipponbare variety of rice for GUS histochemical staining analysis.

In order to realize the purpose, the following technical scheme is adopted:

according to bioinformatics means, a 1676bp long reverse sequence fragment of the 3' end region of LOC _ Os02g45770 gene was analyzed by using promoter prediction website (http:// www.dna. Affrc. Go. Jp/htdocs/PLACE /), and functional elements of common promoters including TATA box, CCAAT box, YACT, TTATTT and the like were found (see Table 1). And finally, amplifying the sequence, constructing a vector, and verifying transformation expression. The finally obtained promoter is Nubi5, and the sequence is shown as SEQ ID NO. 1.

The method comprises the following specific steps:

according to the expression region of the previous promoter, a specific primer is designed, the promoter is amplified from the genome of the rice Nipponbare variety, and is connected with a GUS coding sequence of a reporter gene to construct a fusion gene, and then the fusion gene is loaded into a Ti vector pCAMBIA1300, wherein the map, the multiple cloning sites and other information of the vector are shown in figure 2, and the constructed vector is transformed into Nipponbare rice through agrobacterium. GUS histochemical staining detection shows that the promoter is expressed in roots, stems, leaves, glumes, seed coats, embryos and endosperm, so that the promoter is verified to be a constitutive promoter.

The invention has the advantages that:

the invention provides a strong constitutive promoter for rice, which is different from the traditional promoter in that the promoter functional element is derived from a 3' terminal reverse sequence of a gene, shows strong activity in organs such as roots, stems, leaves, flowers and seeds of plants and has good application prospect in the field of plant transgenosis.

Drawings

FIG. 1 is a sequence diagram of the whole length of the promoter, and the whole length of the Nubi5 promoter is 1676bp in the LOC _ Os02g45770 gene region, from 7882bp backward to 6206 bp.

FIG. 2 is a diagram of RT-PCR semiquantitative analysis of LOC _ Os02g45770 gene OsMADS6 and reference gene ACTIN, showing that the expression of the target gene OsMADS6 compared with ACTIN shows that it is a gene specifically expressed in seeds, but not expressed in roots, stems, leaves, glumes.

FIG. 3 is a diagram of a plant expression vector.

FIG. 4 is a diagram of the RT-PCR detection result of GUS gene of a transgenic positive plant of plant expression vector pCAMBIA1300-Nubi 5.

FIG. 5 shows the GUS tissue staining analysis result of transgenic positive plant of plant expression vector pCAMBIA1300-Nubi 5.

Detailed Description

Example 1:1 promoter full-length fragment was obtained.

Extracting the genomic DNA of the Nipponbare rice: extracting the genome DNA of fresh leaves of Nippon variety rice in the tillering stage by a CTAB method, and placing the leaves in a refrigerator at the temperature of 20 ℃ below zero for dissolving and storing for later use.

LOC _ Os02g45770 genome information is extracted from a bioinformatics website (http:// rice. Plant biology. Msu. Edu /), gene sequences of 3' reverse regions (figure 1) of genes are obtained, the length of 1676bp is calculated, 1 pair of specific primers (shown in table 2) is designed, and PCR amplification conditions are as follows: at 94 ℃ for 5min, at 98 ℃ for 30s, at 58.5 ℃ for 30s, at 72 ℃ for 2min, for 35 cycles; 10min at 72 ℃. And (3) PCR reaction system: 10 KOD buffer 2ul, 10mdNTP 1.5ul, 10mM primer PrF 2ul, 10mM primer PrR 2ul, genomic DNA 2ul, KOD polymerase 1ul, and the balance of double distilled water to 50ul.

TABLE 1 analysis of full-Length promoter core elements

TABLE 2 primer sequences designed according to the invention

The PCR product was TA cloned into pMD-18T, and the detailed procedures are described in the Takara pMD-18T kit instructions. The ligation products were left in 16 ℃ overnight water bath for 12 hours or more, transformed into E.coli DH5a by heat shock at 42 ℃ and spread with LB medium containing ampicillin (Amp), and overnight at 37 ℃ to select a single colony which was then expanded and cultured with LB medium containing Amp. Plasmids were extracted, sequenced and vectors of the correct sequence were selected.

Finally, enzyme cutting sites are arranged at two ends of the rice genomeBamHI amplification of the promoter Nubi5 sequence, its blunt end insertionHindIII+NcoI double enzyme digestion plant expression vector pCAMBIA1301, and final expression vector pCAMBIA1301-Nubi5 (shown in figure 3) is formed.

Example 2: RT-PCR analysis and GUS histochemical staining analysis of transgenic rice

The construction vector pCAMBIA1301-Nubi5 transformation japonica rice (Nipponbare) callus is used as an acceptor material for genetic transformation, agrobacterium is utilized to mediate rice immature embryo seed callus to realize genetic transformation, and positive plants are grown through the steps of screening, differentiating, rooting and the like of resistant callus, and the specific steps are as follows:

1. explant: taking young embryos pollinated for about 10-14D, peeling, treating with 75% absolute ethyl alcohol for 3 min, then sterilizing with 3% sodium hypochlorite solution for 10min, cleaning with sterile water for 3-4 times, sucking dry with filter paper, picking embryos, inoculating on N6+2.0 mg/L2, 4-D culture medium, and dark culturing at 27 ℃ for 7-15D.

2. 1-2 mm pale yellow compact callus was picked for Agrobacterium transformation.

3. Infection of agrobacterium: selecting Agrobacterium from the culture medium, picking single colony in YEB liquid medium containing 50mg/ml rifampicin and 50mg/ml kanamycin after 2 days (beef extract 5g/L, yeast extract 1g/L, peptone 5g/L, sucrose 5g/L, mgSO 5) ₄ .7H ₂ O1 g/L, pH 7.2.) and cultured overnight at 250 rpm on a shaker, and the OD600 reading is adjusted to be between 0.6 and 1.0; centrifugal force of 4000 Xg 5mThe bacteria were collected in and resuspended in N6+ DS liquid medium (N6 medium +1 mg/L2, 4-D + 100. Mu. Mol/L acetosyringone) to obtain an Agrobacterium infection with an OD600 reading of about 1.0. Transferring 20g of embryogenic callus into a 50 mL centrifuge tube, adding 50 mL of agrobacterium infection solution, standing in an incubator at 28 ℃ for 20 min for full infection, pouring out liquid culture, drying the embryogenic callus on filter paper, transferring the embryogenic callus onto an N6-AS solid medium (N6 solid medium +100 mu mol/L As), and performing dark culture at 26 ℃ for 3-4 d.

4. And (3) resistant callus screening: transferring the infected callus from co-culture to a 250 mL triangular flask, adding a carbenicillin solution with a final concentration of 250 mg/L, dark-culturing at 28 ℃, standing for 30 min, washing with sterile water for 3-5 times, transferring to a screening culture medium N6-S (N6 medium +50mg/mL hygromycin), and dark-culturing at 28 ℃ to grow the resistant callus.

5. And (3) growing the regenerated plant: transferring the resistant callus to an N6-R (MS basal medium +0.5 mg/L6-BAP +0.2mg/L NAA) plant regeneration medium, carrying out light culture at 28 ℃ for 15-25 days, then transferring to a rooting medium MS medium, carrying out light culture at 28 ℃ to grow plants, and finally obtaining two mutant plants, namely D86-1 and D86-2.

To further confirm whether the Nubi5 promoter is constitutively strong, we performed RT-PCR analysis and GUS histochemical staining analysis on the transgenic seedlings. The expression condition of a GUS reporter gene driven by a Nubi5 promoter in transgenic rice (figure 4) is analyzed by taking a rice ACTIN gene as an internal reference and collecting roots, stems, leaves, glumes and seeds of a pure line of a transgenic Nipponbare rice plant, extracting RNA, carrying out reverse transcription to form cDNA (complementary deoxyribonucleic acid) as a template, and the result in figure 4 shows that the target gene GUS driven by the Nubi5 has expression in the roots, stems, leaves, glumes and seeds of the rice plant and belongs to a constitutive promoter. The roots, stems, leaves, glumes and seeds of the pCAMBIA1301-Nubi5 transgenic rice plants are cut (the seeds are die-cut by a scalpel), immersed in a staining solution, insulated for 3-12 h at 37 ℃, and transferred into 70% ethanol for decolorization for 2-3 times. When the plants were observed under a dissecting mirror and photographed (FIG. 5), the roots, stems, leaves, glumes and seeds were all stained.

RT-PCR detection primers: wherein the ACINT gene primer is actinF 5-type TGTGCTTGATTTCTGGTGATGG-3 ', actinR 5-type ATCTTTCATTGAGGCAGTCAGTCAG-3', the GUS gene primer is GusF 5-type CGGTGATGATATATCGGCTGATGAT-3 ', and GusR 5-type CCTGCGTCGTAATGTTCTG-3'. The size of the ACTIN amplified fragment is 123bp; the size of the GUS gene amplified fragment is 186bp.

Procedure of PCR detection system: 10X Tiangen Mix buffer 12.5, 10mM GusF (ActinF for control) 2ul, 10mM GusR (ActinR for control) 2ul, taq polymerase 0.5ul, cDNA 1ul, and the rest is supplemented with double distilled water to 25 ul. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, 28 cycles; extension at 72 ℃ for 10min.

The results show that the promoter Nubi5 and exogenous gene GUS can make the target gene expressed in the whole plant, so that the Nubi5 promoter has good application prospect in transgenic engineering. The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims (2)

1. A constitutive strong promoter of rice is characterized in that: the promoter is Nubi5, and the sequence is shown in SEQ ID NO. 1.

2. The use of the constitutive strong promoter of rice as claimed in claim 1 in transgenic rice.

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