CN107058327A - A kind of application of Oil Rape Tissue specificity promoter in regulation and control target gene specifically expressing in plant anther - Google Patents

Abstract

The invention belongs to the field of plant biotechnology and relates to the isolation and application of a plant tissue-specific promoter, in particular to the application of a rape tissue-specific promoter in regulating the expression of a target gene in plant anthers. The inventor fused the promoter and its truncated fragment with a reporter gene and transformed it into Arabidopsis. GUS staining found that they can drive the specific expression of the reporter gene in anthers. Their sequences are as SEQ ID NO.1, SEQ ID NO.2 , shown in SEQ ID NO.3 and SEQ ID NO.4; the promoter fragment shown in SEQ ID NO.5 with pollen-specific expression was obtained after further truncation. The invention further discloses that the tissue-specific promoter or the truncated promoter fragment provides a new effective regulatory element for controlling plant fertility, and has good application prospects for genetic engineering breeding.

Description

A rapeseed tissue-specific promoter regulates target genes in plant anthers Different expression application

technical field

The invention belongs to the field of plant biotechnology. Specifically, the invention relates to the application of a rapeseed tissue-specific promoter in regulating the specific expression of a target gene in plant anthers. The rapeseed tissue-specific promoter provided by the invention can regulate the target gene Specifically transcribed or expressed in plant anthers or pollen.

Background technique

Gene regulation in higher plants is mainly carried out at the transcriptional level, and is controlled by the mutual coordination of various cis-acting elements and trans-acting factors. The exogenous DNA sequence is linked to a specific promoter to promote expression in the plant host, so the choice of promoter type determines the time and location of gene expression. In recent years, the more popular research objects are tissue-specific promoters. These tissue-specific promoters mainly include leaf, phloem, vascular bundle, root and other organ-specific expression promoters, as well as reproductive organ-specific expression promoters such as pollen, floral organs, fruits, and seeds. (Song Yang et al., Plant tissue-specific promoter research. Biotechnology Bulletin, 2007, (4): 21-24). Geng Anqi et al. isolated four promoters from Chinese cabbage rape, Brassica napus, Chinese cabbage, and cabbage and transformed them into tobacco. GUS staining analysis showed that they were promoters specifically expressed in floral organs (Geng AQ, Zhao Z J, Nie X L, et al .Expression analysis of four flower-specific promoters of Brassica spp. in the heterogeneous host tobacco [J]. African Journal of Biotechnology, 2009, 8(20).). Ariizumi et al. isolated the promoters of LTP12, XTH3 and PGA4 and transformed Arabidopsis. GUS staining showed that these three promoters were anther-specific expression promoters, and there were differences in expression periods (Ariizumi T, Amagai M, Shibata D, et al. Comparative study of promoter activity of three anther-specific genes encoding lipid transfer protein, xyloglucan endotransglucosylase/hydrolase and polygalacturonas e intransgenic Arabidopsis thaliana[J]. Plant Cell Reports, 2002,21(1):90-96.). The ZmGLU1 gene promoter cloned from maize was connected to the GUS reporter gene and then transformed into tobacco. The detection and analysis results showed that the ZmGLU1 promoter of maize drove the high expression of GUS gene in tobacco roots (Gu R, Zhao L, Zhang Y, et al. Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgeneic tobacco [J]. Plant cell reports, 2006,25(11):1157-1165.).

The above reports are all examples of promoter function analysis in different plants by using transgenic technology. The function of promoters is widely recognized and accepted. The functional identification of a tissue-specific promoter isolated from rapeseed in the present invention is accomplished through the model plant Arabidopsis thaliana, and finally verified in rapeseed. Based on the successful application of this method, the function of tissue-specific promoters can be quickly verified in vivo, and more and more tissue-specific promoters have been reported, which may become tissue-specific promoters with practical value in the field of plant genetic engineering. son. One of the examples of the successful application of tissue-specific promoters is the regulation of plant pollen fertility through genetic engineering, the creation of plant male sterile lines, restorer lines and maintainer lines, which have been successful in some crops.

The current strategy of using genetic engineering to create male sterility is mainly to use pollen or anther-specific promoters to fuse with foreign genes, construct expression vectors, transform plants, and block the process of pollen development in a timely manner to achieve the purpose of male sterility. Mariani C et al. used the tobacco anther tapetum-specific promoter TA29 fused with RNaseT1 or Barnase to transform into tobacco and rapeseed through Agrobacterium-mediated transformation, and obtained stable male sterile transformants (Marian C, DeBeuckeleer M, Truettner J, et al. Induction of male sterility in plants by achimaeric ribonuclease gene [J]. Nature, 1990, 347:737-741.). Two years later, Mariani C et al. used TA29 to drive the Barstar gene and created the recovery of the above-mentioned male sterile lines (Mariani C, Gossele V, DeBeuckeleer M, et al. A chimaeric ribo nuclease-inhibitor gene restores fertility to male sterile plants[J ]. Nature, 1992, 357(6377): 384-387.). It can be seen that even if the in vivo environment of different plants is different and there are differences in the interaction between genes, even plants that are quite different from tobacco in nature, as long as they have a conserved core promoter region and corresponding conserved regulatory elements, they can be used in gene expression. In other different plants, it plays the biological function of initiating the stable and continuous expression of downstream genes.

One of the keys to regulating pollen fertility and creating plant sterile lines, maintainer lines and restorer lines by means of genetic engineering is the drive activity and specificity of plant pollen or anther promoters. Currently, there are relatively few rapeseed anther or pollen-specific promoters with high driving activity and good specificity. However, due to the large genome and complex structure of rapeseed, there are not many studies on the molecular mechanism of anther development. More The reference is related research in the model plant Arabidopsis thaliana. Therefore, the cloning and identification of rapeseed anther and pollen-specific expression promoters will provide new regulatory elements for the use of genetic engineering to regulate pollen fertility and create plant male sterile lines in rapeseed, so as to fully utilize rapeseed heterosis resources in rapeseed breeding lay the foundation.

Contents of the invention

The purpose of the present invention is to provide an application of a rapeseed tissue-specific promoter in regulating the specific expression of a target gene in plant anthers, and the rapeseed tissue-specific promoter sequence is SEQ ID NO.1, SEQ ID NO.2 , SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

To achieve the above object, the present invention takes the following technical measures:

An application of a rapeseed tissue-specific promoter in regulating the specific expression of a target gene in plant anthers, including the use of conventional means in the art, will include SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, The promoter shown in SEQ ID NO.4 or SEQ ID NO.5 can transfer the expression vector of the target gene into the plant to regulate the specific expression of the target gene in the anther of the plant; the promoter sequence shown in SEQ ID NO.5 It can regulate the specific expression of the target gene in the plant pollen, and use the above method to create a genetically engineered sterile line, maintainer line or restorer line. In the above scheme, the plant is a Brassica plant, preferably rapeseed or Arabidopsis.

The target gene can be an enzyme or modification enzyme, amylase, debranching enzyme and pectinase that promote carbohydrate degradation, or a key gene that causes the PCD process, more specifically the cysteine protease gene, β - 1, 3-glucanase gene, mammalian cytotoxin gene, apoptosis gene or can be selected from prokaryotic regulatory system, and can also be a dominant male sterility gene.

The recombinant plant expression vector may also contain a selectable marker gene. The marker genes generally include genes providing antibiotic resistance or herbicide resistance, such as: hygromycin resistance gene, glufosinate or glufosinate resistance gene and the like.

Any plant transformation method can be used to transform the recombinant plant expression vector constructed in the present invention into the cells and tissues of the recipient plant to obtain a transformant; then the transformant can be regenerated by a plant tissue culture method to obtain a complete plant and its Clones or their progeny; the transformation method includes: Agrobacterium-mediated transformation, protoplast transformation, Ti plasmid, Ri plasmid, plant virus vector, microinjection, electroporation, particle bombardment, etc.; The recipient plants include Brassica plants; preferably, the recipient plants are rapeseed.

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

In the present invention, the sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 are respectively operably connected to the reporter gene to construct a recombinant plant expression vector; Using Arabidopsis thaliana and rapeseed as recipient materials, the recombinant plant expression vector was transformed into Arabidopsis thaliana and rapeseed through the mediation of Agrobacterium GV3101. The GUS staining of different transformants showed that SEQ ID NO.1, SEQ ID NO. 2. The promoter shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 can drive the specific expression of the reporter gene in anthers, but in other tissue parts such as siliques, roots, stems, leaves, etc. There is no expression activity in it; the sequence shown in SEQ ID NO.5 can drive the specific expression of the reporter gene in pollen, but not in other tissue parts. It shows that the promoter fragments shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 all have the function of tissue-specific promoters. The present invention clones and identifies the specific expression promoters of rapeseed anthers and pollen, and provides new regulatory elements for using genetic engineering to regulate pollen fertility and create plant male sterile lines in rapeseed, thereby providing a basis for the use of rapeseed heterosis resources in rapeseed breeding Get the most out of laying the groundwork.

Description of drawings

Fig. 1 is the detection electrophoresis diagram of promoter fragments SEQ ID NO.1 and SEQ ID NO.5;

Lane M: DNA marker; Lane 1: the promoter shown in SEQ ID NO.1; Lane 2: the promoter shown in SEQ ID NO.5.

Fig. 2 is a schematic diagram of the expression vector pG2NHL-H2BYFP-gusplus-Nost.

Fig. 3 is a schematic diagram of promoter elements of promoter SEQ ID NO.1.

Fig. 4 is the GUS staining of different tissues and organs of the promoter SEQ ID NO.1 transgenic Arabidopsis.

Among them, A is a 14d whole seedling; B is a cauline leaf; C is an inflorescence; D is a flower bud; E is a 5mm silique; F is a 10mm silique.

Figure 5 is a schematic diagram of GUS staining of different tissues and organs of promoter SEQ ID NO.5 transgenic Arabidopsis;

Among them, a is a 14d whole seedling; b is a cauline leaf; c is an inflorescence; d is a flower bud; e is a 5mm silique; f is a 10mm silique.

Fig. 6 is a semi-thin section of GUS-stained anthers of the promoter SEQ ID NO.5 transgenic Arabidopsis thaliana.

Among them, S11 is the 11th stage of anther development; S12 is the 12th stage of anther development; S13 is the 13th stage of anther development; S14 is the 14th stage of anther development, bar=20 μm.

detailed description

The methods used in the following examples are conventional methods unless otherwise specified, and the reagents or materials used are all from commercial sources unless otherwise specified.

Example 1:

Cloning of Rapeseed Tissue-Specific Promoters SEQ ID NO.1～SEQ ID NO.5 and Construction of Plant Expression Vectors

Genomic DNA of double 11 leaves in rapeseed was extracted by CTAB method, and the DNA was used as a template for PCR amplification with a reaction volume of 50 μl.

The primers for amplifying the promoter SEQ ID NO.1 are:

1-F: 5'-(Sal I)CGCgtcgacCCATCTTACTCCAATAGTATTTTGG-3', 1-R: 5'-(SmaI)CcccggggCCGTTCTTCTTTCAATATTTAAAA-3;

The primers for amplifying the promoter SEQ ID NO.2 are:

2-F: 5'-(Sal I)CGCgtcgacATGGAGAACTTAAATAGACAAATAC-3', 2-R: 5'-(SmaI)Ccccg ggCCGTTTCTTTCTTTCAATATTTAAAA-3;

The primers for amplifying the promoter SEQ ID NO.3 are:

3-F: 5'-(Sal I)CGCgtcgacATGGAGAACTTAAATAGACAAATAC-3', 3-R: 5'-(SmaI)Ccccggg AATCAGATTATTTAAGCTGTCTAAA-3;

The primers for amplifying the promoter SEQ ID NO.4 are:

4-F: 5'-(Sal I)CGCgtcgacATGAAACGAAAGCCACCTC-3', 4-R: 5'-(SmaI)CcccggggCCGTTTCTTTCTTTCAATATTTAAAA-3;

The primers for amplifying the promoter SEQ ID NO.5 are:

5-F: 5'-(SalI)CGCgtcgacATGAAACGAAAGCCACCTC-3' and 5-R: 5'-(SmaI)CcccggggAATCAGATTATTTAAGCTGTCTAAA-3.

The 50μl system is as follows:

The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min, then denaturation at 98°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, and 30 cycles, and finally extension at 72°C for 10 min. The PCR product was detected by 1.0% mass-volume ratio agarose gel electrophoresis (Figure 1). After the PCR product recovery kit was purified and recovered, it was digested (Sal I and SmaI) at 37°C for 2 hours, recovered again, and respectively mixed with the plant expression vector pG2NHL- H2BYFP-gusplus-Nost was ligated overnight at 4°C, transformed by heat shock method and single clones were picked, and sent to Wuhan Qingke Innovation Biotechnology Co., Ltd. for sequencing after the PCR test was positive. The sequencing results were compared with the reference genome sequences SEQ ID NO.1 and SEQ ID NO .2. SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are completely consistent, that is, the plant expression vectors containing the target promoters are obtained, which are named pG2NHL-H2BYFP-gusplus-Nost-1, pG2NHL- H2BYFP-gusplus-Nost-2, pG2NHL-H2BYFP-gusplus-Nost-3, pG2NHL-H2BYFP-gusplus-Nost-4 and pG2NHL-H2BYFP-gusplus-Nost-5.

Example 2:

The application of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 to regulate the specific expression of target genes in plant anthers:

1) The expression vector of the promoter fusion reporter gene is transformed into Arabidopsis thaliana

Example 1 The successful construction of monoclonal Escherichia coli containing a promoter plant expression vector was activated at 37°C and the plasmid was extracted, and pG2NHL-H2BYFP-gusplus-Nost-1, pG2NHL-H2BYFP-gusplus-Nost-2, pG2NHL-H2BYFP-gusplus-Nost-3, pG2NHL-H2BYFP-gusplus-Nost-4 and pG2NHL-H2BYFP-gusplus-Nost-5 were respectively transformed into Agrobacterium GV3101 competent cells, in the presence of kanamycin (50μg/mL) and After culturing on a rifampin (50 μg/mL) plate, single clones were picked and detected with promoter cloning primers to determine positive clones. Activate the positive Agrobacterium liquid at 28°C, using the inflorescence dip method (Zhang X, Henriques R, Lin S S, et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floraldip method[J].Nature protocols,2006,1(2) : 641-646.) transform wild-type Arabidopsis thaliana, soak the harvested seeds after 2 times, sterilize and spread evenly on the 1/2MS medium containing 50 μg/mL kanamycin to grow, and the survival green Seedlings were initially identified as positive plants. Transplant them into vermiculite, take a leaf when the plant grows to 5-7 true leaves, extract the genomic DNA of the leaf by CTAB method, and carry out PCR identification on it, the primers are promoter-specific amplification primers, reaction system and procedures As described in Example 1. The PCR reaction product was detected by 1% agarose gel electrophoresis, and the size was consistent with the size of the target promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5. The results showed that the plant expression vectors of five promoters had been successfully transformed into Arabidopsis thaliana, and the T1 generation seeds of each positive individual plant were harvested.

2) GUS staining of different tissues and organs of transgenic Arabidopsis

On the 1/2MS medium containing kanamycin at a concentration of 50 μg/ml, the T1 generation seeds of each positive individual plant were screened, and the expression vector of each promoter was determined to conform to the Mendelian inheritance law 3:1 segregated single-copy strain 3-5, different tissues (14d whole plant seedlings, stems, leaves, inflorescences, 5mm siliques, 10mm siliques) after flowering were carried out in the T3 generation for GUS staining, the steps are as follows:

(1) Collect the Arabidopsis thaliana material into a small centrifuge tube or a small glass bottle (adding pre-cooled 90% acetone in advance), and place it on ice until all the samples are collected.

(2) Place the material on ice for 20 minutes after collection.

(3) After the acetone treatment, rinse the tissue with pre-cooled GUS staining buffer for about 10 minutes. The composition of GUS staining buffer is shown in the table below.

Components of GUS Staining Buffer

(4) Replace the staining buffer, add GUS staining solution containing X-gluc, and vacuum for 15-30min. The composition of GUS staining solution is shown in the table below.

Components of GUS staining solution

(5) Stain overnight at 37°C.

(6) Remove the dye solution. Place the stained samples in 20%, 35%, 50%, and 70% ethanol solutions in turn at room temperature for 30 minutes each. In 70% ethanol, the stained tissue can be preserved or observed for a long time.

Use the OLYMPUS DP72 digital imaging system to take pictures of the staining results and save the pictures. The GUS staining results of each line showed that the promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 could drive the specific expression of GUS gene in the anthers of Arabidopsis, and the other It is not expressed in tissues and organs (Fig. 4); the promoter SEQ ID NO.5 drives the specific expression of GUS gene in Arabidopsis pollen (Fig. 5).

3) Semi-thin section of GUS-stained anthers of promoter SEQ ID NO.5 transgenic Arabidopsis thaliana

In order to determine the specific period when the promoter SEQ ID NO.5 drives the expression of the GUS gene, we performed semi-thin sections on the GUS-stained anthers of the promoter SEQ ID NO.5. Proceed as follows:

1) Dehydration: the GUS-stained inflorescences stored in 70% ethanol were dehydrated step by step in 80%, 95% and 100% ethanol respectively, and each step was dehydrated for 2 hours.

2) After the dehydration is completed, gently cut the inflorescence, divide it into individual small flower buds, and then proceed as follows:

A. Pre-infiltration: Place the small flower buds in the pre-infiltration solution (95% ethanol: base liquid Technovit 7100 = 1:1 mixture) for 3 hours.

B. Infiltration: The small flower buds are transferred to an appropriate amount of infiltration solution (1g hardening agent I dissolved in 100ml base liquid Teclmovit 7100) to infiltrate for 8-12 hours.

C. Embedding: Add 100 μL of embedding agent (hardening agent Ⅱ: permeate = 1:15 mix) into each 200 μL small centrifuge tube, gently insert a single small flower bud into the centrifuge tube with pointed tweezers, and keep as much as possible. The small flower buds are placed vertically downwards, and then the centrifuge tubes are placed vertically on the PCR plate. After a few minutes at room temperature, the coagulation and polymerization began. After 2 hours, the embedded samples were placed in a 37-degree incubator to continue the polymerization. After 1 week, they could be sliced.

D. Slicing: Slice with a Leica Ultracut R-type ultra-thin microtome with a thickness of 8 μm. Microscopic inspection (Nikon eclipse 80i microscope) while slicing to ensure the integrity of the slices.

E, Adhesive film: The cut film is picked up with a small copper net wound with fine copper wire, and directly transferred to a clean glass slide dripping with water, and then spread and dried on an electric heating table.

F, Sealing: Canadian resin sealing, permanent storage.

H, Observation and photography: observation under Nikon eclipse 80i microscope, and digital photography with Ds-Ril.

Semi-thin sections of GUS-stained anthers showed that the GUS signal of the promoter SEQ ID NO.5 specifically appeared at the binucleate stage of pollen and lasted until the pollen grain matured ( FIG. 6 ).

These results show that the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 is an anther-specific promoter, and SEQ ID NO.5 is a pollen-specific promoter. Promoter. These specific promoter fragments provide new effective regulatory elements for controlling plant fertility, and have good application prospects for genetic engineering breeding.

4) GUS staining of anthers of SEQ ID NO.5 transgenic rapeseed

The pG2NHL-H2BYFP-gusplus-Nost-5 was transformed into Brassica napus, and the positive individual plants were stained with GUS, and it was found that the GUS gene was only expressed in pollen, which was consistent with the results in Arabidopsis.

Example 3:

Conservation of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in Brassica cabbage, cabbage and rapeseed

Based on the PCR reaction, use the primers in the embodiment case 1 to clone respectively promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in Chinese cabbage, cabbage and rapeseed The homologous fragments in the sequence were sequenced, and their sequence homology was found to be as high as 100%, and the lowest was 99.78%, with only 1 bp difference, and the difference was not in the core promoter region. This shows that the sequences of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are highly conserved in Brassica cabbage, cabbage and rapeseed. It is predicted that the functions of the promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are also conserved. Just as in Example 2, the promoter of SEQ ID NO.5 drives the expression of GUS gene in Arabidopsis and rapeseed in the same mode. That is to say, the plant expression vectors of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are respectively transferred into cabbage, cabbage and rape, They drive the expression of the target gene in the same way, all of which are anther or pollen-specific expression.

SEQUENCE LISTING

<110> Huazhong Agricultural University

<120> Application of a Rapeseed Tissue-Specific Promoter in Regulating the Specific Expression of Target Genes in Plant Anthers

<130> Application of a Rapeseed Tissue-Specific Promoter in Regulating the Specific Expression of Target Genes in Plant Anthers

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Claims (5)

1.SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, the sequence shown in SEQ ID NO.4 or SEQ ID NO.5 In the application of regulation and control target gene specifically expressing in plant anther.

Sequence shown in 2.SEQ ID NO.5 is regulating and controlling the application of target gene specifically expressing in plant pollen.

3. application according to claim 1 or 2, described plant is brassica plant.

4. application according to claim 1 or 2, described plant is rape.

5. application according to claim 1 or 2, described plant is arabidopsis.