The U6 promoter of Castanopsis chinensis and its application

Abstract

The present invention relates to the field of biotechnology. The present invention discloses a U6 promoter of Castanopsis fujiensis, wherein the promoter is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 3. The present invention clones four U6 promoters from Castanopsis fujiensis and connects them to a pGreenII0800 vector containing a LUC reporter gene. By transiently transforming tobacco leaves with Agrobacterium, it is found that the four cloned IpU6 promoters are all active, among which IpU6-2 and IpU6-1 have higher transcriptional activities than the other two IpU6 promoters and the control OsU6a. These promoters can be used as the endogenous U6 promoters of Castanopsis fujiensis in the Castanopsis fujiensis CRISPR / Cas9 editing system, thereby improving the gene editing efficiency of Castanopsis fujiensis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. The present invention relates to a Tung-Yang-Jia U6 promoter and application thereof. Background Art

[0002] Known as the "oil grape of the tree," the Castanopsis tungii (Mountain Castanopsis) is a woody oil-bearing tree. Its fruit is highly oily, with mature flesh containing up to 43.6% oil and seeds containing approximately 22.4%-25.9%. Linoleic acid, an unsaturated fatty acid, accounts for 58%-81% of the total oil content. The tree is also rich in vitamin E and squalene, which play a preventive role in hyperlipidemia and cardiovascular disease. The tree is also known for its high yield, with a single plant yielding 50-70 kg at peak fruiting age. Its perennial nature allows it to maintain peak fruiting season for 15-40 years.

[0003] To further increase the yield of Castanopsis truncatum and identify its functional genes, gene editing in Castanopsis truncatum is imperative. In recent years, the CRISPR / Cas9 system, due to its high efficiency, convenience, and ease of use, has become a key tool for gene function research and germplasm genetic improvement, and has been applied to improve various quality traits in crops (Jiang et al., 2017; Bandyopadhyay, 2019). A key component of the CRISPR / Cas9 system is the U6 promoter. Studies have shown that the transcriptional activity of the U6 promoter determines the expression level of sgRNA, thereby affecting gene editing efficiency (Wei et al., 2016). To improve gene editing efficiency in various species, research has been conducted on the endogenous U6 promoter in plants. Gao et al. (2015) successfully established a CRISPR / Cas9 system suitable for tobacco using its own endogenous promoter. In apple callus, Bian Shuxun et al. (2020) used the endogenous U6 promoter to significantly increase sgRNA expression levels. Tang Zhiqiang et al. (2022) cloned three honeysuckle U6 promoters and screened out a promoter with high transcriptional activity.

[0004] Currently, the U6 promoter has been studied in multiple plant species, but there are no reports on the endogenous U6 promoter in Castanopsis sylvestris. Therefore, the isolation and identification of the endogenous U6 promoter in Castanopsis sylvestris is of great significance for conducting and improving CRISPR / Cas9 gene editing research in Castanopsis sylvestris. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention first provides a DNA molecule, wherein the nucleotide sequence of the DNA molecule is SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.3.

[0006] In the above DNA molecules, the DNA molecules may be derived from Castanopsis sylvestris.

[0007] The present invention also protects an expression cassette, wherein the expression cassette contains a promoter, and the promoter is the DNA molecule.

[0008] The present invention also protects a recombinant vector containing the DNA molecule or the expression cassette.

[0009] The recombinant vector can be constructed using existing plant expression vectors.

[0010] The present invention also protects a recombinant microorganism containing the DNA molecule, the expression cassette, or the recombinant vector.

[0011] The above-mentioned recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0012] The present invention also protects the use of the DNA molecule in initiating target gene expression

[0013] In the above application, the initiating expression of the target gene is initiating expression of the target gene in a plant.

[0014] In the above application, the plant is a dicotyledonous plant, specifically a plant of the genus Nicotiana of the Solanaceae family (such as tobacco (Nicotiana tabacum L.)).

[0015] The present invention also provides the use of the DNA molecule in gene editing.

[0016] In the above application, the gene editing is implemented by CRISPR / Cas9 editing.

[0017] The present invention cloned four U6 promoters from Castanopsis sylvestris and connected them to the pGreenⅡ0800 vector containing the LUC reporter gene. By transiently transforming tobacco leaves with Agrobacterium, it was found that the four cloned IpU6 promoters were all active, among which IpU6-2 and IpU6-1 had higher transcriptional activities than the other two IpU6 promoters and the control OsU6a. They can be used as endogenous U6 promoters of Castanopsis sylvestris in the Castanopsis sylvestris CRISPR / Cas9 editing system to improve the gene editing efficiency of Castanopsis sylvestris. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a sequence alignment diagram of the Castanopsis truncatula U6 promoter in Example 1 of the present invention.

[0019] Figure 2 This is a gel image of the amplified Castanopsis chinensis U6 promoter sequence and the control OsU6a in Example 1 of the present invention.

[0020] Figure 3 Schematic diagram of the carrier structure used in Example 1 of the present invention.

[0021] Figure 4 This is a graph showing the fluorescence detection analysis of LUC driven by different U6 promoters in Example 1 of the present invention. Capital letters indicate significance at p = 0.01, and lowercase letters indicate significance at p = 0.05. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0023] The quantitative tests in the following examples were performed three times unless otherwise specified, and the results were averaged.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0025] The pGreenII0800 vector used in the following examples is described in the non-patent document "Hellens, RP, Edwards, EA, Leyland, NR, Bean, S., & Mullineaux, PM (2000). pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant molecular biology, 42 (6)", which is available to the public from the applicant to repeat the present experiment.

[0026] The competent Agrobacterium in the following examples is GV3101 (pSoup-p19), a product of Beijing Zhuangmeng Biogene Technology Co., Ltd.

[0027] The LB solid medium in the following examples was prepared as follows (taking 1 L as an example): 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, distilled water to 1 L, adjusted to pH 7.2 with 5 mol / L NaOH, and sterilized at 121°C for 30 min.

[0028] The LB liquid culture medium in the following examples was prepared as follows (taking 1 L as an example): 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, distilled water to 1 L, adjusted to pH 7.2 with 5 mol / L NaOH, and sterilized at 121°C for 30 min.

[0029] In the following examples, the concentration of antibiotic kana sulfate (Kana) is 50 mg / L, and the concentration of antibiotic rifampicin (Rif) is 50 mg / L).

[0030] The infection solution formula in the following examples is: 10 mM MgCl2, 10 mM MES (pH 5.7), 100 μM AS.

[0031] Example 1

[0032] 1. Obtaining the U6 promoters IpU6-1, IpU6-2, IpU6-3 and IpU6-4 from the fruit of Castanopsis chinensis

[0033] The homology comparison of the genome of Castanopsis truncatula was carried out based on the coding sequence of AtU6 of Arabidopsis thaliana. The results are as follows: Figure 1 Four sequences similar to AtU6 were obtained, and the promoter sequence of IpU6 was further searched, resulting in the suspected A. truncatula U6 promoters IpU6-1 (nucleotide sequence shown in SEQ ID NO.1), IpU6-2 (nucleotide sequence shown in SEQ ID NO.2), IpU6-3 (nucleotide sequence shown in SEQ ID NO.3), and IpU6-4 (nucleotide sequence shown in SEQ ID NO.4).

[0034] 1. Obtaining the Mountain Star Fruit DNA Template

[0035] The castanopsis material used was castanopsis grown at the Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing, and the castanopsis DNA was extracted.

[0036] 2. Using the DNA obtained in step 1 as a template, amplification was performed as follows using a primer pair consisting of upstream primers IpU6-1-F, IpU6-2-F, IpU6-3-F, and IpU6-4-F and downstream primer IpU6_R. Amplification was performed as follows using a rice DNA template using a primer pair consisting of primers OsU6a_F and OsU6a_R.

[0037] Table 1 Primers used to construct pGreenII0800 vector

[0038]

[0039]

[0040] The reaction system and PCR reaction procedure of the PCR amplification are as follows:

[0041] Table 2 Common PCR reaction system

[0042] Reaction components Dosage 2×TaqMasterMix 10 μL IpU6_1 / 2 / 3 / 4_F 1 μL pU6_R 1 μL <![CDATA[ddH2O]]> 7μL DNA 1 μL TotalVolume 20 μL

[0043] Centrifuge briefly to mix thoroughly. PCR reaction uses a touch-down annealing program, with the annealing temperature decreasing by 0.5°C for the first 10 cycles. The program settings are:

[0044] Table 3 Common PCR amplification procedures

[0045] step Temperature (℃) time Remark Pre-denaturation 95 5min transsexual 95 30s annealing 62 30s -0.5℃ extend 72 30s 10 cycles transsexual 95 30s annealing 57 30s extend 72 30s 24 cycles extend 72 5min save 4 ∞

[0046] 4 μL of PCR product was taken and detected by 1% agarose gel electrophoresis. The target bands of the reaction products of the four U6 promoters of Castanopsis chinensis were about 700 bp, and the target band of the control rice U6a (OsU6a) was about 450 bp. Figure 2 .

[0047] A DNA recovery kit from agarose gel produced by Novozymes was used to recover specific amplified fragments. The fragments were sequenced and the following amplified fragments were obtained:

[0048] The nucleotide sequence of the fragment obtained by amplifying the Castanopsis sylvestris DNA with IpU6-1-F and IpU6_R is shown in positions 271-926 of SEQ ID NO.1, and is called fragment IpU6-1.

[0049] The nucleotide sequence of the fragment obtained by amplifying the Castanopsis sylvestris DNA with IpU6-2-F and IpU6_R is shown in positions 291-926 of SEQ ID NO. 2, and is called fragment IpU6-2.

[0050] The nucleotide sequence of the fragment obtained by amplifying the Castanopsis sylvestris DNA with IpU6-3-F and IpU6_R is shown in positions 359-1026 of SEQ ID NO. 3, and is called fragment IpU6-3.

[0051] The nucleotide sequence of the fragment obtained by amplifying the Castanopsis sylvestris DNA with IpU6-4-F and IpU6_R is shown in positions 341-1027 of SEQ ID NO. 4, and is called fragment IpU6-3.

[0052] The nucleotide sequence of the fragment obtained by amplifying rice DNA with OsU6a_F and OsU6a_R is shown in SEQ ID NO. 5, positions 61-507, and is referred to as fragment OsU6a.

[0053] SEQ ID NO.1

[0054]

[0055]

[0056] SEQ ID NO.2

[0057]

[0058]

[0059] SEQ ID NO.3

[0060]

[0061] SEQ ID NO.4

[0062]

[0063]

[0064] SEQ ID NO.5

[0065]

[0066] 2. Construction of recombinant expression vector of Castanopsis chinensis

[0067] The pGreenII0800 vector was double-digested with restriction endonucleases HindⅢ and PstⅠ produced by NEB. The large fragment was recovered after digestion to obtain pGreenII0800 after digestion. Figure 3 .

[0068] 2.1 Using a one-step recombination kit produced by Novozymes, the IpU6-1 fragment was recombined into the digested pGreenII0800. The operation method was as per the kit instructions to obtain a ligation product. 5uL of the ligation product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana, concentration of 50μg / L) for 12h. Single clones were picked and placed in liquid LB containing Kana (concentration of 50μg / L). After the bacterial liquid was identified as a positive clone by PCR, the plasmid was extracted and double enzyme digestion was performed. The successfully verified plasmid was sent to Qingke Bio for sequencing. After correct sequencing, the expression vector pGreenII0800-IpU6-1 was successfully constructed. The structure of the expression vector pGreenII0800-IpU6-1 is described as follows: the small fragment between the recognition sequences of the restriction endonucleases HindⅢ and PstⅠ of pGreenII0800 is replaced with the fragment shown in positions 271-926 of SEQ ID NO.1, while the other sequences of the vector pGreenII0800 remain unchanged, to obtain the expression vector pGreenII0800-IpU6-1, in which the promoter IpU6-1 drives the expression of the LUC gene.

[0069] 2.2 Using a one-step recombination kit produced by Novozymes, the fragment IpU6-2 was recombined into the digested pGreenII0800. The operation method was as described in the kit instructions to obtain a ligation product. 5uL of the ligation product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana, concentration of 50μg / L) for 12h. Single clones were picked and placed in liquid LB containing Kana (concentration of 50μg / L). After the bacterial liquid was identified as a positive clone by PCR, the plasmid was extracted and double enzyme digestion was performed. The successfully verified plasmid was sent to Qingke Bio for sequencing. After correct sequencing, the successfully constructed expression vector pGreenII0800-IpU6-2 was obtained. The structure of the expression vector pGreenII0800-IpU6-2 is described as follows: the small fragment between the recognition sequences of the restriction endonucleases HindⅢ and PstⅠ of pGreenII0800 is replaced with the fragment shown in positions 291-926 of SEQ ID NO.2, while the other sequences of the vector pGreenII0800 remain unchanged, to obtain the expression vector pGreenII0800-IpU6-2, in which the promoter IpU6-2 drives the expression of the LUC gene.

[0070] 2.3 Using a one-step recombination kit produced by Novozymes, the fragment IpU6-3 was recombined into the digested pGreenII0800. The operation method was as described in the kit instructions to obtain a ligation product. 5 μL of the ligation product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana, concentration of 50 μg / L) for 12 h. Single clones were picked and placed in liquid LB containing Kana (concentration of 50 μg / L). After the bacterial liquid was identified as a positive clone by PCR, the plasmid was extracted and double enzyme digestion was performed. The successfully verified plasmid was sent to Qingke Bio for sequencing. After correct sequencing, the expression vector pGreenII0800-IpU6-3 was successfully constructed. The structure of the expression vector pGreenII0800-IpU6-3 is described as follows: the small fragment between the recognition sequences of the restriction endonucleases HindⅢ and PstⅠ of pGreenII0800 is replaced with the fragment shown in positions 359-1026 of SEQ ID NO.3, while keeping the other sequences of the vector pGreenII0800 unchanged, to obtain the expression vector pGreenII0800-IpU6-3, in which the promoter IpU6-3 drives the expression of the LUC gene.

[0071] 2.4 Using a one-step recombination kit produced by Novozymes, the IpU6-4 fragment was recombined into the digested pGreenII0800 according to the kit instructions to obtain a ligation product. 5 μL of the ligation product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana, concentration of 50 μg / L) for 12 h. Single clones were picked and placed in liquid LB containing Kana (concentration of 50 μg / L). After the bacterial liquid was identified as a positive clone by PCR, the plasmid was extracted and double enzyme digestion was performed. The successfully verified plasmid was sent to Qingke Bio for sequencing. After correct sequencing, the expression vector pGreenII0800-IpU6-4 was successfully constructed. The structure of the expression vector pGreenII0800-IpU6-4 is described as follows: the small fragment between the recognition sequences of the restriction endonucleases HindⅢ and PstⅠ of pGreenII0800 is replaced with the fragment shown in positions 341-1027 of SEQ ID NO.4, while keeping the other sequences of the vector pGreenII0800 unchanged, to obtain the expression vector pGreenII0800-IpU6-4, in which the promoter IpU6-4 drives the expression of the LUC gene.

[0072] 2.5 Using a one-step recombination kit produced by Novozymes, the OsU6a fragment was recombined into the digested pGreenII0800 according to the kit instructions to obtain a ligation product. 5 μL of the ligation product was transferred into Escherichia coli DH5α and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 μg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 μg / L). After positive clones were identified by PCR, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Qingke Bio for sequencing. After correct sequencing, the expression vector pGreenII0800-OsU6a was successfully constructed. The structure of the expression vector pGreenII0800-OsU6a is described as follows: the small fragment between the restriction endonuclease HindⅢ and PstⅠ recognition sequences of pGreenII0800 is replaced with the fragment shown in positions 61-507 of SEQ ID NO.5, while keeping the other sequences of the vector pGreenII0800 unchanged, to obtain the expression vector pGreenII0800-OsU6a, in which the promoter OsU6a drives the expression of the LUC gene.

[0073] 3. The expression of the LUC gene in the recombinant plasmid driven by the U6 promoter of Castanopsis chinensis

[0074] 3.1 The constructed plant recombinant expression vector pGreenII0800-IpU6-1 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenII0800-IpU6-1.

[0075] 3.2 The constructed plant recombinant expression vector pGreenII0800-IpU6-2 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenII0800-IpU6-2.

[0076] 3.3 The constructed plant recombinant expression vector pGreenII0800-IpU6-3 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenII0800-IpU6-3.

[0077] 3.4 The constructed plant recombinant expression vector pGreenII0800-IpU6-4 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenII0800-IpU6-4.

[0078] 3.5 The constructed plant recombinant expression vector pGreenII0800-OsU6a containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenII0800-OsU6a.

[0079] The newly activated Agrobacterium monoclonal clones GV3101 / pGreenII0800-IpU6-1, GV3101 / pGreenII0800-IpU6-2, GV3101 / pGreenII0800-IpU6-3, GV3101 / pGreenII0800-IpU6-4 and GV3101 / pGreenII0800-OsU6a were inoculated into LB containing the corresponding antibiotics (50 μg / L Kana + 50 μg / L Rif), respectively, and the pGreenII0800 vector was used as a negative control. Incubate at 28°C, 200 rpm overnight. When the OD value of the bacterial solution is between 0.6 and 1.0, collect the Agrobacterium by centrifugation at 4000 rpm for 5 minutes. Gently resuspend the Agrobacterium in 2 mL of infection solution and let it stand at room temperature for 1-4 hours. Infect 6-8 week-old Nicotiana benthamiana leaves. After 72 hours, grind the infected Nicotiana benthamiana leaves and perform luciferase activity detection. The fluorescence signal data were statistically analyzed. The results are shown in Figure 4 , it was found that the fluorescence signal was the strongest when IpU6-2 was used as the promoter, followed by IpU6-1, while the fluorescence signal of OsU6a was the weakest.

[0080] The results showed that IpU6-1, IpU6-2, IpU6-3 and IpU6-4 could drive LUC gene expression, and IpU6-2 and IpU6-1 had the best effects.

[0081] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a DNA molecule in initiating target gene expression, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.2, positions 291-926, SEQ ID NO.1, positions 271-926, SEQ ID NO.4, positions 341-1027 or SEQ ID NO.3, positions 359-1026.

2. The use according to claim 1, characterized in that The initiating target gene expression is initiating target gene expression in plants.

3. The use according to claim 2, characterized in that The plant is a plant of the genus Nicotiana of the Solanaceae family.

4. The use according to claim 3, characterized in that The plant of the genus Nicotiana in the Solanaceae family is tobacco.

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