Lotus leaf specific promoter pNnCYP80G and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering and molecular biology, and particularly discloses a specific promoter pNnCYP80G of a lotus leaf. A recombinant plant expression vector of the promoter pNnCYP80G is used for constructing an arabidopsis thaliana transgenic plant, specific high expression of the GUS gene in arabidopsis thaliana leaves is achieved, the promoter pNnCYP80G can respond to jasmonic acid methyl ester and mechanical damage treatment, and expression of the GUS gene in the leaves is further enhanced. The promoter pNnCYP80G can specifically express an exogenous gene in a plant leaf tissue, so that the leaf quality character improvement is realized, and the influence of the expression of the exogenous gene on other organs and tissues is effectively reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and molecular biology, and specifically relates to a lotus leaf tissue-specific promoter pNnCYP80G and an application thereof. Background Art

[0002] The promoter plays a key role in transgenic engineering. It is responsible for initiating and finely regulating the transcription initiation process of downstream genes. At the same time, the promoter also controls the expression site, expression period and expression intensity of the gene.

[0003] A promoter is a DNA sequence located upstream of the gene coding region. According to the expression pattern, it can be divided into three categories: constitutive promoters, inducible promoters, and tissue-specific promoters. Among them, tissue-specific promoters only work in specific tissues or organs. In transgenic engineering, the rational use of tissue-specific promoters can more accurately control gene expression in specific tissues, safely improve plant traits, and effectively reduce the impact of exogenous genes on the normal growth and development of plants. Therefore, the exploration of tissue-specific promoter functions has become a research hotspot in the field of plant genetic engineering and has important scientific significance.

[0004] Promoters usually contain some cis-acting elements that control gene expression. Among them, hormone and stress response elements are one of the main components of promoters. Some tissue-specific promoters are regulated by hormones and stress signals. For example, the soybean root-specific promoter Glyma12g02240 responds to salt stress to enhance downstream gene expression and improve plant salt tolerance. The rose RhPIP promoter responds to GA hormone treatment to enhance the expression activity of the GUS gene in plant leaves. The lily DXR promoter regulates the expression of the target gene only in flower tissues, and the expression is induced by injury and methyl jasmonate.

[0005] Lotus is an important aquatic economic crop in my country, with a wide cultivation area and rich germplasm resources. Lotus leaves are important nutritional tissue organs of lotus, rich in a variety of secondary metabolites, and have been developed into a popular weight loss health food. The development of lotus leaf tissue-specific promoters plays a very important role in the production and utilization of leaf secondary metabolites. At the same time, it also has broad application prospects in the research of improving plant leaf organ quality traits. Summary of the invention

[0006] The purpose of the present invention is to provide a leaf-specific promoter pNnCYP80G, which enables the target gene to be specifically and efficiently expressed in leaf tissue.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The lotus leaf-specific promoter pNnCYP80G, the sequence of which is shown in SEQ ID NO. 1. The sequences of primers used to amplify the promoter pNnCYP80G are shown in SEQ ID NOs. 2 and 3.

[0009] A recombinant expression vector comprises a promoter pNnCYP80G. Preferably, the recombinant expression vector is constructed using the plant expression vector pBI101 as a vector.

[0010] Transgenic plants containing the promoter pNnCYP80G.

[0011] The use of pNnCYP80G promoter or a recombinant expression vector containing the promoter pNnCYP80G in driving the specific expression of a target gene in plant leaves. Preferably, the plant is lotus and Arabidopsis thaliana.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] 1. The promoter described in the present invention is a specific promoter pNnCYP80G in lotus leaves obtained by screening through transcriptome sequencing technology. This method has high accuracy and strong credibility.

[0014] 2. The promoter pNnCYP80G of the present invention drives the target gene to be highly expressed specifically in leaves, so the expression of the target gene will not affect the plant's nutritional growth and development. In the future, its tissue-specific expression characteristics can be used to transform plant leaf traits, such as improving plant photosynthetic efficiency, resisting pests and diseases, or changing metabolic pathways in leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the expression analysis results of NnCYP80G gene regulated by promoter pNnCYP80G in different lotus tissues.

[0016] Figure 2 This is the plasmid map of the plant expression vector pBI101.

[0017] Figure 3 Schematic diagram of the restriction sites for constructing a recombinant expression vector for the promoter pNnCYP80G fused to the GUS gene.

[0018] Figure 4 These are the results of GUS staining in different parts of Arabidopsis thaliana and GUS gene fluorescence quantitative analysis.

[0019] Figure 5 The results of GUS staining after Arabidopsis leaf damage and methyl jasmonate treatment. DETAILED DESCRIPTION

[0020] The present invention screens a lotus leaf-specific promoter based on transcriptome analysis of different lotus tissues, clones the promoter using PCR technology, constructs a fusion vector of the promoter and a GUS reporter gene through a homologous recombination method, obtains transgenic Arabidopsis positive plants through Agrobacterium transformation, and analyzes the promoter expression site and promoter activity through GUS histochemical staining technology and quantitative PCR technology of different plant tissues.

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and implementation methods. The specific embodiments are only for illustrating the present invention and do not limit the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, the present invention content is modified and replaced with the embodiments as a guide, which belongs to the scope of the present invention.

[0022] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents and instruments used in the following examples are all commercially available unless otherwise specified. The primers and sequencing used in the following examples were completed by Wuhan Qingke Co., Ltd.; the experimental material lotus variety 'Autumn Star' came from the lotus germplasm resource conservation platform of Wuhan Botanical Garden, Chinese Academy of Sciences; wild-type Arabidopsis Col-0, Escherichia coli competent strain TOP10, Agrobacterium competent strain GV3101 and the modified plant genetic expression vector pBI101 were all preserved in the inventor's laboratory.

[0023] Example 1

[0024] Obtaining the full-length sequence of the lotus pNnCYP80G promoter:

[0025] By analyzing the transcriptome data of various tissues of the lotus variety 'Qiuxing', it was found that the NnCYP80G gene was specifically expressed in lotus leaf tissues ( Figure 1 ). According to the NnCYP80G sequence predicted from the whole genome sequence of lotus, the 1418bp fragment upstream of the gene was selected as the promoter sequence, and the promoter was named pNnCYP80G, and its nucleotide sequence is shown in SEQ ID NO.1. The amplification primers were designed as shown in SEQ ID NO.2 and 3. In the upstream primer (SEQ ID NO.2), the first 10 bases were the homologous recombination arm of the plant genetic expression vector pBI101, followed by the SalI restriction site, and the remaining sequence was the upstream amplification primer sequence; in the downstream primer (SEQID NO.3), the first 8 bases were the homologous recombination arm of the plant genetic expression vector pBI101, followed by the XbaI restriction site, and the remaining sequence was the downstream amplification primer series.

[0026] The Tiangen kit was used to extract the genomic DNA of the lotus variety 'Qiuxing', and the DNA was used as a template to amplify the promoter pNnCYP80G sequence with the above primers. The PCR amplification reaction system is as follows:

[0027]

[0028] The PCR amplification program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, 12°C∞.

[0029] 5 μl of PCR product was taken and subjected to 1% agarose gel electrophoresis to verify the size of the amplified sequence. After electrophoresis, the PCR amplified product showed only one band of about 1500 bp in size. The PCR product recovery kit was used to recover and purify the DNA product.

[0030] Example 2

[0031] Transformation of Arabidopsis thaliana with pNnCYP80G promoter and GUS gene fusion vector:

[0032] (1) Construction of plant expression vector containing pNnCYP80G fused with GUS gene

[0033] The plant expression vector pBI101 was double-digested with SalI and XbaI. The double-digestion system is as follows:

[0034]

[0035] The enzyme digestion reaction was continued in a 37°C water bath for 2 hours. The digestion products were separated by 1% agarose gel electrophoresis, and the plant expression vector pBI101 after digestion was purified using an agarose gel electrophoresis recovery kit. The purified PCR product fragment was connected with the pBI101 linear vector recovered by digestion by homologous recombination to construct the plant expression vector pBI101 fused with the pNnCYP80G promoter and the GUS gene. The connection system is as follows:

[0036]

[0037] Add 5μl of the ligation product to the EP tube containing 50μl of competent cells TOP10, gently pipette and mix, incubate on ice for 30min, then heat shock in a 42℃ water bath for 1min, incubate on ice for 2min, add 200μl of antibiotic-free LB liquid culture medium to the EP tube in the clean bench, incubate the bacterial solution at 37℃ shaker at 220rpm for 1h, and evenly spread it on a solid LB plate containing 50μg / L kanamycin, and then place it in a 37℃ incubator for inversion culture for 12-16h. Add 500μl LB liquid culture medium containing 50μg / L kanamycin to the EP tube in the clean bench, pick up a healthy single colony on the solid LB plate with a clean and sterile 10μl pipette tip, gently mix it in the LB liquid culture medium, incubate the bacterial solution at 37℃ shaker at 220rpm for 24h, take the bacterial solution for PCR verification, and the amplification primers are shown in SEQ ID NO.2 and 3. The reaction system is as follows:

[0038]

[0039]

[0040] The single colonies that tested positive were sent to a sequencing company for sequencing. The plasmids were extracted from the single colonies that were sequenced correctly, and the recombinant vector was named pBI101-pNnCYP80G-GUS based on the promoter it contained.

[0041] (2) Transform the recombinant vector into Agrobacterium

[0042] 100ng of the recombinant vector was added to an EP tube containing 30μl of Agrobacterium competent cells GV3101, and the mixture was gently pipetted and mixed. All the liquid was transferred to a clean and sterile electroporation cup, and the lid was placed in an electroporator. Electric shock was performed once with 2500V / 6ms. After the electric shock, 500μl of non-antibiotic LB liquid culture medium was added to the electroporation cup, and the mixture was gently pipetted and mixed. All the liquid was transferred to a clean and sterile EP tube. After culturing for 3h at 28°C shaker at 220rpm, the mixture was evenly coated on a solid LB plate containing 50μg / L kanamycin and 50μg / L rifampicin, and the mixture was placed in a 28°C incubator and inverted for 36-48h. A single colony was picked for colony PCR verification, and the reaction system was the same as the colony PCR verification system in Example 2.

[0043] (3) Transformation of Arabidopsis thaliana by floral dipping method

[0044] Select Col-0 Arabidopsis plants at the flowering stage, water them sufficiently, cut off the siliques, and infect them with the resuspended Agrobacterium infection solution (OD 600 =0.8) to soak Arabidopsis inflorescence for 1 min 30 s, and culture the infected Arabidopsis in dark conditions for 24 h, then transfer to normal light cycle culture until T0 generation seeds mature.

[0045] The harvested T0 generation seeds were screened with 1 / 2MS seed germination medium containing 50μg / L kanamycin. The resistant seedlings with normal growth were selected for planting, and the T1 generation seeds were harvested and continued to be screened in 1 / 2MS seed germination medium containing 50μg / L kanamycin. The ratio of normal growth seedlings to abnormal growth seedlings was 3:1, that is, this was a single copy insertion plant. The resistant seedlings with normal growth were selected for planting, and the genomic DNA of the wild type and the pBI101-pNnCYP80G-GUS strains were extracted after the plants grew up, and the promoter amplification primers were used for PCR positive identification. Arabidopsis thaliana with positive PCR identification continued to be cultured and harvested T2 generation seeds, and the T2 generation plants with normal growth were screened by the same germination medium method, which was a homozygous single copy insertion transgenic strain.

[0046] Example 3

[0047] GUS identification of transgenic plants:

[0048] Specifically, a GUS staining kit (Zhongke Ruitai, catalog number: RTU4032) was used for staining analysis. Leaves, inflorescences, and fruit pods of wild Col-0 and T2 transgenic Arabidopsis strains were subjected to GUS histochemical staining. The results showed that no blue was observed in all tissues of wild-type Arabidopsis. The results of tissue staining of the pBI101-pNnCYP80G-GUS strain showed that the GUS gene was mainly expressed in the leaf tissue of Arabidopsis ( Figure 4 A). Quantitative detection of GUS gene was performed on different tissues of Arabidopsis thaliana, and the quantitative primers are shown in SEQ ID NO.4 and 5. The specific operation is as follows: RNA was extracted from roots, stems, leaves, inflorescences, and seeds of Arabidopsis thaliana transfected with pBI101-pNnCYP80G-GUS, and then reverse transcribed into cDNA. Fluorescence quantitative PCR analysis of GUS gene was performed using cDNA as a template, and 3 biological replicates were set. The results showed that the expression of GUS gene in leaves of transfected pBI101-pNnCYP80G-GUS strains was significantly higher than that in other tissues ( Figure 4 B).

[0049] Example 4

[0050] Transgenic plant damage and methyl jasmonate treatment:

[0051] The leaves of the pBI101-pNnCYP80G-GUS transgenic plants were damaged using a sterilized needle, and the Col-0 type Arabidopsis was used as a control. GUS staining was performed 24 hours later. Col-0 type and pBI101-pNnCYP80G-GUS transgenic Arabidopsis T2 seeds were germinated using 1 / 2MS medium. When the seeds germinated with two true leaves, 100 μM methyl jasmonate (MeJA) was sprayed, and GUS staining was performed three days later. The GUS staining results showed that after the damage treatment, the blue color of the leaves in the damaged area was significantly darker than that in the undamaged area ( Figure 5 A). Spraying MeJA after seed germination can promote the expression of GUS protein in transgenic Arabidopsis leaves, making the Arabidopsis leaves darker ( Figure 5 B). This indicates that the pNnCYP80G promoter activity in leaves is regulated by damage and the methyl jasmonate signaling pathway.

[0052] In summary, the present invention provides a lotus leaf-specific promoter pNnCYP80G, the promoter nucleotide sequence of which is shown in SEQ ID NO.1; the promoter drives the target gene to be highly expressed in lotus leaves. The promoter pNnCYP80G is connected to the GUS gene and transformed into Arabidopsis thaliana, and the promoter can be used to drive the high expression of GUS in Arabidopsis thaliana leaves. The lotus leaf-specific promoter pNnCYP80G has good application value in the functional research of genes related to plant leaf development and metabolic synthesis and the research on plant metabolic engineering regulation.

[0053] It should be understood that the above specific implementation method is only used to explain the basic principle of the present invention, and is not intended to limit the present invention in any form. Changes and improvements made according to the present invention still fall within the protection scope of the present invention.

Claims

1. Lotus leaf-specific promoter pNnCYP80G, characterized in that: The sequence of the promoter is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: It comprises the promoter pNnCYP80G as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The recombinant expression vector was constructed using the plant expression vector pBI101.

4. A transgenic plant containing the promoter pNnCYP80G according to claim 1.

5. The amplification primers for the promoter pNnCYP80G according to claim 1, characterized in that: The primer sequences are shown in SEQ ID NOs. 2 and 3.

6. Use of the promoter pNnCYP80G according to claim 1 to drive the specific expression of a target gene in plant leaves.

7. Use of the recombinant expression vector according to claim 2 to drive the specific expression of a target gene in plant leaves.

8. The use according to claim 6 or 7, characterized in that: The plants are lotus and Arabidopsis thaliana.

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