Tomato LACS2 gene promoter and application thereof

Abstract

The invention discloses a tomato LACS2 gene promoter and application thereof, and belongs to the technical field of biology. On one hand, the invention provides the tomato LACS2 gene promoter, and on the other hand, the invention provides application of the tomato LACS2 gene promoter in promoting target gene expression in plants. According to the invention, a tomato LACS2 gene promoter fragment (788bp) is constructed to drive an EGFP-GUS (Enhanced Green Fluorescent Protein-Glucuronidase) gene fusion expression vector. GUS histochemical staining and laser confocal microscopy observation prove that the promoter fragment can guide normal expression of the reporter gene in the tomato epidermis. Therefore, the LACS2 promoter can be used for researching development, quality improvement and postharvest preservation of tomato peels and specific expression of exogenous genes in the tomato peels.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the promoter of tomato LACS2 gene and its application. Background Art

[0002] Tomato (Solanum lycopersicum) is one of the important vegetable crops widely cultivated globally and is also an important cash crop in China's agricultural production. In recent years, the planting area and yield of tomatoes in China have continued to increase, and China has become one of the major tomato-producing countries in the world.

[0003] However, the tomato industry still faces many challenges at present, including problems such as fruit quality, disease resistance, and post-harvest storage. With the improvement of consumers' requirements for food quality, the taste, flavor, and appearance of tomato fruits have become key factors in market competition. Currently, many tomato varieties perform poorly in terms of fruit sugar content, acidity, and aroma substance content, resulting in insufficient market competitiveness. In addition, the development of the epidermal layer of tomato fruits directly affects their appearance quality and storage and transportation resistance. Too thin or incomplete peel structure will cause the fruits to be easily damaged, affecting post-harvest storage and transportation and causing economic losses. During the tomato planting process, it is often threatened by various diseases, such as late blight, gray mold, bacterial wilt, etc. These diseases not only affect the fruit yield but also reduce the fruit quality. The tomato fruit epidermis is the first line of defense against diseases in tomato fruits. Cultivating tomato varieties with natural disease resistance is of great significance.

[0004] The promoter is a section of DNA sequence located upstream of the 5' end of a gene. As the "switch" for regulating gene expression, it controls the starting time, space, and expression intensity of gene expression.

[0005] Currently, in plant genetic engineering, the constitutive strong promoter CaMV35S is mainly used to express related functional genes, which does not have spatio-temporal specificity and easily leads to adverse phenotypes in plants.

[0006] Therefore, a series of different fruit-specific promoters are needed to achieve specific expression in the epidermal layer of tomato fruits, so as to drive the expression of related functional genes by using such promoters, and further improve aspects such as fruit quality, disease resistance enhancement, and post-harvest transportation and storage performance. Summary of the Invention

[0007] Aiming at the existing problems, the purpose of the present invention is to design and provide a technical solution for the promoter of tomato LACS2 gene and its application. By using this promoter to drive the expression of related functional genes, it is expected to obtain further improvement in aspects such as fruit quality improvement, disease resistance enhancement, and post-harvest transportation and storage performance.

[0008] The present invention is specifically implemented by the following technical solutions:

[0009] In the first aspect of the present invention, a tomato LACS2 gene promoter is provided, which contains the nucleotide sequence described in a), b) or c):

[0010] a) The nucleotide sequence shown in SEQ ID NO.1;

[0011] b) A nucleotide sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the nucleotide sequence defined in a), derived from tomato and having promoter function;

[0012] c) A nucleotide sequence that hybridizes with the nucleotide sequence defined in a) or b) under stringent conditions and has promoter function.

[0013] In the second aspect of the present invention, an expression cassette or recombinant vector containing the above-mentioned tomato LACS2 gene promoter is provided.

[0014] In the third aspect of the present invention, a recombinant microorganism containing the above-mentioned tomato LACS2 gene promoter is provided.

[0015] In the fourth aspect of the present invention, a transgenic cell line containing the above-mentioned tomato LACS2 gene promoter is provided.

[0016] In the fifth aspect of the present invention, the application of the above-mentioned tomato LACS2 gene promoter in initiating the expression of a target gene in plants is provided.

[0017] Furthermore, in this application, the initiation of the expression of the target gene is the specific expression of the target gene in tomato fruit epidermal cells.

[0018] Furthermore, in this application, the target gene is the GFP gene.

[0019] In the present invention, the expression of the EGFP-GUS fusion gene is driven by a promoter fragment (788 bp) of the LACS2 gene. GUS histochemical staining and laser confocal observation confirm that this promoter can drive the normal expression of the reporter gene in tomato pericarp epidermal cells. Therefore, the LACS2 promoter can be used to drive the expression of foreign genes in tomato pericarp to promote the research on improving tomato fruit quality, enhancing disease resistance and improving post-harvest transportation and storage performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the tomato LACS2 promoter sequence.

[0021] Figure 2 It is the expression of tomato LACS2 in different fruit tissues.

[0022] Figure 3 It is the GUS staining of transgenic tomato fruits.

[0023] Figure 4 Results of confocal observation of GFP in transgenic tomato fruits Detailed implementation manners

[0024] The present invention will be further described and explained below with reference to the accompanying drawings and specific embodiments

[0025] The experimental methods used in the following examples are all conventional methods, and the experimental reagent materials used can all be obtained through commercial channels

[0026] Example 1: Clone the promoter sequence of tomato LACS2 gene and construct it into the pKGWFS7 vector

[0027] I. Obtain the upstream promoter sequence of LACS2 gene by polymerase chain reaction

[0028] 1. Obtain the LACS2 promoter sequence

[0029] Since the tomato genome is known, the promoter sequence of the LACS2 gene is downloaded through the online website https: / / solgenomics.net, and the sequence is as Figure 1 shown

[0030] 2. Amplify the promoter sequence

[0031] According to the known LACS2 sequence, the forward primer F is designed as

[0032] 5‘-GCGGCCGCCCCCTTCACCAATAATAAAAAATCACAAAATTAAAA TTTTTCATT-3’(SEQ ID NO.2) and the reverse primer R as

[0033] 5‘-GTCGGCGCGCCCACCCTTTTCCCTGAGATATATAAAAATCCACA-3’(SEQ ID NO.3)

[0034] Using the designed primers, with tomato genomic DNA as the template, PCR amplification is carried out using the high-fidelity enzyme KOD. After identifying the bands by gel electrophoresis, they are recovered and purified to obtain a purified promoter fragment. The specific PCR amplification reaction system is: 25 μl of KOD high-fidelity enzyme, 1.5 μl of each of the forward and reverse primers, 2 μl of genomic DNA template, and water is added to 50 μl; the reaction procedure is: pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, extension at 68 °C for 5 s, 34 cycles of denaturation, annealing, and extension, and extension at 68 °C for 5 min. The amplified promoter sequence is as shown in SEQ ID NO.1

[0035] II. Construction of the LACS2 promoter - pENTR recombinant vector (ProLACS2 - pENTR) by homologous recombination

[0036] 1. Linearize the pENTR vector

[0037] According to the vector sequence of pENTR, forward primer: 5’ - AAGGGTGGGCGCGCCGACC - 3’ (SEQ ID NO.4) and reverse primer: 5’ - GGTGAAGGGGGCGGCCGCG - 3’ (SEQ ID NO.5) were designed. Using the designed primers, with the pENTR plasmid as the template, after PCR amplification with the high - fidelity enzyme KOD, the band was identified by gel electrophoresis and then recovered and purified to obtain the purified linearized pENTR vector fragment. The specific PCR amplification reaction system was: 25 μl of KOD high - fidelity enzyme, 1.5 μl of each forward and reverse primer, 0.5 μl of pENTR vector template, and water was added to 50 μl; the reaction program was: pre - denaturation at 98℃ for 2 min, denaturation at 98℃ for 10 s, annealing at 60℃ for 10 s, extension at 68℃ for 15 s, 34 cycles of denaturation, annealing, and extension, and extension at 68℃ for 5 min.

[0038] 2. Connect the promoter fragment with the linearized pENTR fragment by homologous recombination method

[0039] The specific method was as follows: The purified LACS2 promoter fragment and the linearized vector fragment were ligated with homologous recombination enzyme. The ligation reaction system was 2×onestep homologous recombination enzyme premix, 50 ng of pENTR linearized vector fragment, 100 ng of LACS2 promoter fragment, and water was added to 10 μl; the ligation reaction program was: incubation at 50℃ for 15 min.

[0040] 3. Chemically transform the homologous recombination reaction solution into DH5α competent cells

[0041] The specific method was as follows: 10 μl of the reaction solution was added to 100 μl of DH5α competent cells, gently mixed, incubated on ice for 15 min, then heat - shocked at 42℃ for 1 min, finally ice - bathed for 2 min, 500 μl of LB liquid medium was added, activated at 37℃ for 30 min, and finally the transformed cells were cultured overnight on LB solid medium containing kanamycin.

[0042] 4. Colony identification and sequencing

[0043] The grown colonies were identified by PCR. The forward and reverse primers used for the identification were: 5’-GTAAAACGACGGCCAGT-3’ (SEQ ID NO.6) and 5’-CAGGAAACAGCTATGAC-3’ (SEQ ID NO.7). The bacterial solutions with correct identification were sent to a sequencing company for sequencing, and the recombinant plasmids with correct sequence alignment were used for the LR reaction.

[0044] III. Construction of the final vector containing the LACS2 promoter by LR reaction

[0045] 1. The LACS2 promoter fragment on the pENTR vector was introduced into the pKGWFS7 vector by the LR reaction. The specific method was as follows: 150 ng of the ProLACS2-pENTR recombinant vector, 50 ng of the pKGWFS7 vector, 1 μl of the LR premix enzyme, and TE buffer (pH 8.0) was added to make up to 8 μl; after mixing the reaction system evenly, it was incubated at 25 °C for 1 h; 1 μl of proteinase K was added to terminate the reaction.

[0046] 2. Transformation of the LR reaction solution into DH5α competent cells

[0047] The specific method was as follows: 10 μl of the reaction solution was added to 100 μl of DH5α competent cells, gently mixed, incubated on ice for 15 min, then heat shocked at 42 °C for 1 min, and finally ice-bathed for 2 min. 500 μl of LB liquid medium was added, and it was activated at 37 °C for 30 min. Finally, the transformed cells were cultured overnight on the LB solid medium containing Spe.

[0048] IV. Agrobacterium transformation and identification

[0049] 1. Bacterial solution identification and sequencing

[0050] The grown colonies were identified by PCR. The forward and reverse primers used for the identification were: 5’-TGACTCCCTTAATTCTCCGCTC-3’ (SEQ ID NO.8) and

[0051] 5’-CGGACACGCTGAACTTGTG-3’ (SEQ ID NO.9). The bacterial solutions with correct identification were sent to a sequencing company for sequencing, and the recombinant plasmids with correct sequence alignment were used for Agrobacterium GV3101 transformation.

[0052] 2. Agrobacterium transformation and colony PCR identification

[0053] The correctly sequenced pKGWFS7 vector containing the LACS2 promoter (ProLACS2) was transformed into competent Agrobacterium tumefaciens GV3101 cells, and inoculated onto LB solid medium containing spectinomycin (100 mg / L) and rifampicin (25 mg / L) resistance, and cultured inverted at 28 °C for 2 - 3 days. Colonies that grew were selected for PCR identification.

[0054] The forward and reverse primers for identification were: 5’-TGACTCCCTTAATTCTCCGCTC-3’ and 5’-CGGACACGCTGAACTTGTG-3’.

[0055] Example 2. Functional verification of the tomato LACS2 promoter

[0056] I. Expression pattern of the tomato LACS2 gene in fruit tissues

[0057] On the website https: / / tea.solgenomics.net / expression_viewer / input, the expression data of the LACS2 gene in different tissues of tomato fruits were obtained and further analyzed by plotting. The results are as Figure 2 shown. The LACS2 gene is specifically expressed in the epidermal cells of the exocarp, and has the highest expression during the Mature Green fruit development stage, and then gradually decreases. In addition, the LACS2 gene is hardly expressed or weakly expressed in the remaining tissues of the fruit.

[0058] II. Construction of tomato transgenic materials mediated by Agrobacterium

[0059] 1. Activation of Agrobacterium

[0060] The specific process is as follows: The correct Agrobacterium strain was streaked on an LB solid medium plate containing spectinomycin (100 mg / L) and rifampicin (25 mg / L) resistance, and cultured inverted at 28 °C for 2 - 3 days. On the streaked plate, a single colony was picked and added to a liquid LB medium (2 mL) with the corresponding resistance, and cultured at 28 °C and 220 rpm for 14 - 24 h (overnight). According to a ratio of 1:100, the small culture solution was added to the large flask of LB medium (generally 30 μL:30 mL), and cultured at 28 °C and 220 rpm for 10 - 12 h (overnight) until OD600: 0.8 - 1.0.

[0061] 2. Infection of tomato cotyledons with Agrobacterium

[0062] The specific steps are as follows: After harvesting the bacteria by centrifugation at 5000 rpm and 4 °C for 8 min, the resuspension strain was added in a 1:1 ratio to make an infection solution. The tomato cotyledons were infected for 2.5 min, the excess infection solution was poured off, and the cotyledons were laid flat on the pre-culture medium and cultured for 1 day.

[0063] 3. Callus Induction and Differentiation

[0064] The specific method is as follows: The infected cotyledons are grown on the selection medium and the subculture medium respectively. When the buds grow to 2 - 3 cm, the buds are cut off and inoculated onto the rooting medium until roots grow.

[0065] 4. Transplantation and Identification of Transgenic Seedlings

[0066] The specific steps are as follows: When the roots of the transgenic tomato seedlings grow to 4 - 5 cm and the seedling height is close to the height of the bottle, the seedlings are removed and transferred to nutrient soil for continuous cultivation. When the new tissue culture seedlings are removed, a transparent lid is added to prevent excessive water loss due to the large difference in humidity when the tissue culture seedlings are removed, and they are placed in a plant growth chamber for cultivation. Genomic DNA is extracted from the plant leaves, and transgenic positive seedlings are identified using resistance gene primers. The forward and reverse primers for identification are: 5’-GTGGAGAGGCTATTCGGCTATGACTG-3’ (SEQ ID NO.10) and 5’-AGCTCTTCAGCAATATCACGGGTAGC-3’ (SEQ ID NO.11).

[0067] Then use vector-specific primers:

[0068] 5‘-CTAAATTTGATCTATTCACATATATTTATATATAA-3’ (SEQ ID NO.12) and ProLACS2-specific primer 5‘-CCTTGAAGAAGATGGTGCGCT-3’ (SEQ ID NO.13) for PCR identification.

[0069] III. GUS Staining of Tomato Fruits

[0070] 1. Add 2 ml of GUS staining solution to a 5 ml centrifuge tube.

[0071] 2. Put the tomato fruit tissue into the GUS staining solution and place it in an incubator at 37 °C for overnight incubation.

[0072] 3. Observe and photograph under a stereomicroscope, and the results are as Figure 3 shown. The outer epidermis and inner epidermis cells of the Mature Green fruits of transgenic plants show blue, indicating the expression of the GUS gene and indicating that the DNA molecule of the present invention has promoter activity. In addition, upon further observation, it is found that the epidermal cells of the fruit have blue, while the pulp cells do not show blue, indicating that the LACS2 promoter of the present invention enables specific expression of GUS in epidermal cells.

[0073] IV. Laser Confocal Microscopy of Tomato Fruit Peel Epidermis

[0074] 1. Drop a drop of clear water on a glass slide

[0075] 2. Tear off the epidermis of tomato fruits, place it in water, and press it with a coverslip.

[0076] 3. Observe and take pictures under a laser confocal microscope. The results are as Figure 4 shown. The epidermal cells of Mature Green fruits of transgenic plants have strong fluorescence signals, indicating the expression of the GFP gene. It further indicates that the DNA molecule of the present invention has promoter activity, and at the same time, the LACS2 promoter can specifically express the GFP gene in epidermal cells.

Claims

1. Tomato LACS2 gene promoter, characterized in that, The promoter contains the nucleotide sequence described in a), b) or c): a) The nucleotide sequence shown in SEQ ID NO.1; b) A nucleotide sequence having an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the nucleotide sequence defined in a), derived from tomato and having promoter function; c) A nucleotide sequence that hybridizes with the nucleotide sequence defined in a) or b) under stringent conditions and has promoter function.

2. An expression cassette or recombinant vector containing the tomato LACS2 gene promoter according to claim 1.

3. A recombinant microorganism containing the tomato LACS2 gene promoter according to claim 1.

4. A transgenic cell line containing the tomato LACS2 gene promoter according to claim 1.

5. Use of the tomato LACS2 gene promoter according to claim 1 in initiating the expression of a target gene in a plant.

6. The application according to claim 5, characterized in that, The initiating of the expression of the target gene is the specific expression of the target gene in tomato fruit epidermal cells.

7. The application according to claim 5 or 6, characterized in that, The target gene is the GFP gene.

Images