CN118006609A

CN118006609A - Idesia polycarpa U6 promoter and application thereof

Info

Publication number: CN118006609A
Application number: CN202410176834.1A
Authority: CN
Inventors: 王雷; 左毅; 向浩鑫; 李彬; 张冬冬
Original assignee: Salt Alkali Land Comprehensive Utilization Technology Innovation Center; Institute of Botany of CAS
Current assignee: Salt Alkali Land Comprehensive Utilization Technology Innovation Center; Institute of Botany of CAS
Priority date: 2024-02-08
Filing date: 2024-02-08
Publication date: 2024-05-10

Abstract

The invention relates to the field of biotechnology, and discloses a U6 promoter of idesia polycarpa, which is a DNA molecule with a nucleotide sequence shown as SEQ ID NO.2, SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO. 3. According to the invention, 4U 6 promoters cloned from idesia polycarpa are connected to pGreenII0800 vectors containing LUC reporter genes, and tobacco leaves are transiently transformed by agrobacterium, so that the cloned four IpU promoters have activity, wherein the transcriptional activities of IpU-2 and IpU-1 are higher than the activities of other two IpU6 promoters and the contrast OsU a, and the cloned four IpU promoters can be used as idesia polycarpa endogenous U6 promoters for a idesia polycarpa CRISPR/Cas9 editing system, and the gene editing efficiency of idesia polycarpa is improved.

Description

Idesia polycarpa U6 promoter and application thereof

Technical Field

The invention relates to a idesia polycarpa U6 promoter and application thereof in the technical field of biology.

Background

The idesia has the reputation of 'grape with oil on tree', is woody oil tree species, has high fruit oil content, can reach 43.6 percent of ripe pulp oil content to the maximum, has about 22.4 to 25.9 percent of seed oil content, has the unsaturated fatty acid linoleic acid content as high as 58 to 81 percent of the total oil content, is rich in vitamin E, squalene and the like, and has the prevention effect on hyperlipidemia and cardiovascular diseases. The idesia fruit yield is high, the single plant yield in the full bearing period can reach 50-70kg, and the full bearing period can last 15-40 years in addition to the perennial characteristic of the arbor.

In order to further improve idesia output, functional genes of idesia are mined and identified, and idesia gene editing work is imperative. In particular, in recent years, CRISPR/Cas9 systems have become the main tools for gene function research and germplasm genetic improvement due to their advantages of high efficiency, convenience, ease of operation, etc., and are applied to improvement of various quality traits of crops (Jiang et al, 2017; bandyopadyhyay, 2019). One of the important elements in 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 the editing efficiency of the gene (Wei et al, 2016). In order to improve gene editing efficiency in different species, research has been conducted on U6 promoters endogenous to plants. Gao et al (2015) successfully established a CRISPR/Cas9 system suitable for tobacco using its own endogenous promoter. In apple callus, shuxun et al (2020) use of the endogenous U6 promoter can greatly increase the expression level of sgRNA. Tang Zhijiang et al (2022) cloned 3 honeysuckle U6 promoters and screened a promoter with high transcription activity.

At present, the U6 promoter has been studied in a plurality of plants, but no report has been made on the endogenous U6 promoter of idesia. Therefore, the separation and identification of the idesia polycarpa endogenous U6 promoter are of great significance for developing and perfecting idesia polycarpa CRISPR/Cas9 gene editing research.

Disclosure of Invention

In order to solve the technical problems, the invention firstly provides a DNA molecule, and 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.

Among the above DNA molecules, the DNA molecule may be derived from idesia polycarpa.

The invention also protects an expression cassette, which contains a promoter, wherein the promoter is the DNA molecule.

The invention also protects recombinant vectors containing said DNA molecules or containing said expression cassettes.

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

The invention also protects recombinant microorganisms comprising said DNA molecule, or comprising said expression cassette, or comprising said recombinant vector.

The recombinant microorganism can be yeast, bacteria, algae and fungi.

The invention also protects the application of the DNA molecule in the promotion of the expression of the target gene

In the above application, the initiation of the expression of the gene of interest is to initiate expression of the gene of interest in a plant.

In such applications, the plant is a dicotyledonous plant, and may specifically be a plant of the genus Nicotiana of the family Solanaceae (e.g., nicotiana tabacum (Nicotiana tabacum L.)).

The invention also provides application of the DNA molecule in gene editing.

In the above application, the gene editing is performed by CRISPR/Cas9 editing.

According to the invention, 4U 6 promoters cloned from idesia polycarpa are connected to pGreen II 0800 vector containing LUC reporter gene, tobacco leaves are transiently transformed by agrobacterium, and the cloned four IpU promoters are found to have activity, wherein the transcriptional activity of IpU6-2 and IpU6-1 is higher than that of other two IpU6 promoters and contrast OsU6a, and the cloned four IpU promoters can be used as idesia polycarpa endogenous U6 promoters to be applied to an idesia polycarpa CRISPR/Cas9 editing system, so that the gene editing efficiency of idesia polycarpa is improved.

Drawings

FIG. 1 is a sequence alignment of the U6 promoter of idesia polycarpa in example 1 of the present invention.

FIG. 2 is a gel diagram of the amplified idesia polycarpa U6 promoter sequence and a control OsU a in example 1 of the present invention.

FIG. 3 is a schematic diagram of the structure of the carrier used in example 1 of the present invention.

Fig. 4 is a graph of fluorescence detection analysis of different U6 promoter-driven LUCs in example 1 of the present invention, with capital letters indicating p=0.01 significance and lowercase letters indicating p=0.05 significance.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The quantitative tests in the following examples were repeated three times, and the results were averaged unless otherwise specified.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The pGreenII0800 vector in the examples described below is described in non-patent document "Hellens,R.P.,Edwards,E.A.,Leyland,N.R.,Bean,S.,&Mullineaux,P.M.(2000).pGreen:a versatile and flexible binary Ti vector forAgrobacterium-mediatedplant transformation.Plantmolecularbiology,42(6)",, publicly available from the applicant, to repeat the experiment.

Competent Agrobacterium in the examples described below was GV3101 (pSoup-p 19) and was a product of Beijing bang biological gene technologies Co., ltd.

The preparation method of the LB solid medium in the following examples is as follows (1L for example): 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar powder, distilled water is fixed to 1L, pH is adjusted to 7.2 by 5mol/L NaOH, and sterilization is carried out for 30min at 121 ℃.

The preparation method of the LB liquid medium in the following examples is as follows (1L is taken as an example): 10g of peptone, 5g of yeast extract and 10g of sodium chloride, distilled water is fixed to a volume of 1L, pH is adjusted to 7.2 by 5mol/L NaOH, and sterilization is carried out for 30min at 121 ℃.

The antibiotic, kana sulfate (Kana) concentration, and the antibiotic, rifampicin (Rif) concentration in the examples described below were 50 mg/L.

The formulation of the infection liquid in the following examples is: 10mM MgCl ₂, 10mM MES (pH 5.7), 100 μm AS.

Example 1

1. Acquisition of idesia polycarpa U6 promoter IpU-1, ipU6-2, ipU6-3 and IpU6-4

Homologous alignment is carried out on idesia genome according to an Arabidopsis AtU coding sequence, and the alignment result is shown in figure 1. Four sequences similar to AtU6 are obtained, and the promoter sequence of IpU6 is further searched to obtain a suspected idesia polycarpa U6 promoter IpU-1 (the nucleotide sequence is shown as SEQ ID NO. 1), ipU-6-2 (the nucleotide sequence is shown as SEQ ID NO. 2), ipU-6-3 (the nucleotide sequence is shown as SEQ ID NO. 3) and IpU-4 (the nucleotide sequence is shown as SEQ ID NO. 4).

1. Obtaining of idesia DNA template

The idesia materials used are idesia which grow on plant institute of the national academy of sciences of the Beijing incense mountain, and idesia DNA is extracted.

2. Using the DNA obtained in step 1 as a template, the primer pairs consisting of the upstream primer IpU6-1-F, ipU6-2-F, ipU6-3-F and IpU6-4-F and the downstream primer IpU. Sup. 6-R, respectively, were amplified as follows. Primer pairs consisting of primer OsU a_F and primer OsU a_R were amplified using rice DNA templates as follows.

TABLE 1 construction of primers for pGreenII0800 vector

The PCR amplification reaction system and the PCR reaction program are as follows:

TABLE 2 ordinary PCR reaction System

Reaction components	Dosage of
		2×TaqMasterMix	10μL
IpU6_1/2/3/4_F	1μL
		pU6_R	1μL
ddH₂O	7μL
		DNA	1μL
TotalVolume	20μL

The mixture was homogenized by instantaneous centrifugation and the PCR reaction was performed using a step down (Touch down) annealing procedure, with an annealing temperature of 0.5℃for the first 10 cycles. The setting procedure is as follows:

TABLE 3 ordinary PCR amplification procedure

Step (a)	Temperature (. Degree. C.)	Time of	Remarks
				Pre-denaturation	95	5min
Denaturation (denaturation)	95	30s
				Annealing	62	30s	-0.5℃
Extension of	72	30s	10cycles
				Denaturation (denaturation)	95	30s
Annealing	57	30s
				Extension of	72	30s	24cycles
Extension of	72	5min
				Preservation of	4	∞

The PCR product was taken at 4. Mu.L, and detected by 1% agarose gel electrophoresis, the target band of the reaction product of the four U6 promoters of idesia was about 700bp, and the target band (OsU a) of the comparative rice U6a was about 450bp, as shown in FIG. 2.

Cutting gel and recovering by using a common agarose gel DNA recovery kit produced by Novain company, obtaining a specific amplified fragment by referring to a kit instruction, and sequencing to obtain the following amplified fragment:

The nucleotide sequence of the obtained fragment obtained by amplifying idesia polycarpa DNA with IpU6-1-F and IpU6_R is shown as 271-926 of SEQ ID NO.1, and is called fragment IpU-1.

The nucleotide sequence of the obtained fragment obtained by amplifying idesia polycarpa DNA with IpU6-2-F and IpU6_R is shown as 291-926 of SEQ ID NO.2, and is called fragment IpU-2.

The nucleotide sequence of the obtained fragment obtained by amplifying idesia polycarpa DNA with IpU6-3-F and IpU6_R is shown as 359-1026 th site of SEQ ID NO.3, and is called fragment IpU6-3.

The nucleotide sequence of the obtained fragment obtained by amplifying idesia polycarpa DNA with IpU6-4-F and IpU6_R is shown in the 341 th to 1027 th positions of SEQ ID NO.4, and is called fragment IpU6-3.

The nucleotide sequence of the obtained fragment obtained by amplifying rice DNA with OsU a_F and OsU a_R is shown in the 61 st-507 th positions of SEQ ID NO.5, and is called fragment OsU a.

SEQ ID NO.1

SEQ ID NO.2

SEQ ID NO.3

SEQ ID NO.4

SEQ ID NO.5

2. Construction of idesia recombinant expression vector

The pGreenII0800 vector was simultaneously double digested with the restriction enzymes HindIII and PstI produced by NEB, and the large fragment was recovered after the digestion to give pGreenII0800 after the digestion, see FIG. 3.

2.1A one-step recombination kit produced by Novain was used to recombine fragment IpU-1 onto cleaved pGreenII0800, and the ligation product was obtained according to the kit instructions by the procedure. Transferring 5uL of the ligation product into escherichia coli DH5 alpha, culturing for 12 hours on a solid LB medium containing kanamycin (Kana, the concentration is 50 mug/L), picking up a monoclonal colony, placing the monoclonal colony into a liquid LB containing Kana (the concentration is 50 mug/L) for culturing, extracting plasmids after bacterial liquid PCR identification is positive cloning, carrying out double enzyme digestion verification on the plasmids, carrying out sequencing on the plasmids with successful verification, and obtaining the successfully constructed expression vector pGreenII 0800-IpU-1 after sequencing is correct. The structure of expression vector pGreenII0800-IpU6-1 is described as follows: the small fragment between the recognition sequences of the restriction enzymes HindIII and PstI of pGreenII0800 was replaced with the fragment shown at 271-926 of SEQ ID NO.1, leaving the other sequences of vector pGreenII0800 unchanged, resulting in expression vector pGreenII0800-IpU6-1 in which promoter IpU-1 drives LUC gene expression.

2.2A one-step recombination kit from Norwegian corporation was used to recombine fragment IpU-2 onto cleaved pGreenII0800, and the ligation product was obtained according to the kit instructions. Transferring 5uL of the ligation product into escherichia coli DH5 alpha, culturing for 12 hours on a solid LB medium containing kanamycin (Kana, the concentration is 50 mug/L), picking up a monoclonal colony, placing the monoclonal colony into a liquid LB containing Kana (the concentration is 50 mug/L) for culturing, extracting plasmids after bacterial liquid PCR identification is positive cloning, carrying out double enzyme digestion verification on the plasmids, carrying out sequencing on the plasmids with successful verification, and obtaining the successfully constructed expression vector pGreenII 0800-IpU-2 after sequencing is correct. The structure of expression vector pGreenII0800-IpU6-2 is described as follows: the small fragment between the recognition sequences of the restriction enzymes HindIII and PstI of pGreenII0800 was replaced with the fragment shown at positions 291-926 of SEQ ID NO.2, and the other sequences of vector pGreenII0800 were kept unchanged, resulting in expression vector pGreenII0800-IpU6-2 in which promoter IpU-2 drives LUC gene expression.

2.3A one-step recombination kit from Norwegian corporation was used to recombine the fragment IpU-3 onto the digested pGreenII0800, and the ligation product was obtained according to the kit instructions by the procedure. Transferring 5 mu L of the ligation product into escherichia coli DH5 alpha, culturing for 12 hours on a solid LB medium containing kanamycin (Kana, the concentration is 50 mu g/L), picking up a monoclonal colony, placing the monoclonal colony into a liquid LB containing Kana (the concentration is 50 mu g/L) for culturing, extracting plasmids after bacterial liquid PCR identification is positive cloning, carrying out double enzyme digestion verification, verifying successful plasmids, carrying out sequencing on the plasmids by a qinghao organism, and obtaining the successfully constructed expression vector pGreenII 0800-IpU-3 after sequencing is correct. The structure of expression vector pGreenII0800-IpU6-3 is described as follows: the small fragment between the recognition sequences of the restriction enzymes HindIII and PstI of pGreenII0800 was replaced with the fragment shown in SEQ ID NO.3 at 359-1026, and the other sequences of vector pGreenII0800 were kept unchanged, resulting in expression vector pGreenII0800-IpU6-3 in which promoter IpU-3 drives LUC gene expression.

2.4A one-step recombination kit from Norwegian Co., ltd was used to recombine the fragment IpU-4 onto the digested pGreenII0800, and the ligation product was obtained according to the kit instructions by the procedure. Transferring 5uL of the ligation product into escherichia coli DH5 alpha, culturing for 12 hours on a solid LB medium containing kanamycin (Kana, the concentration is 50 mug/L), picking up a monoclonal colony, placing the monoclonal colony into a liquid LB containing Kana (the concentration is 50 mug/L) for culturing, extracting plasmids after bacterial liquid PCR identification is positive cloning, carrying out double enzyme digestion verification on the plasmids, carrying out sequencing on the plasmids with successful verification, and obtaining successfully constructed expression vectors pGreenII0800-IpU6-4 after sequencing is correct. The structure of expression vector pGreenII0800-IpU6-4 is described as follows: the small fragment between the recognition sequences of the restriction enzymes HindIII and PstI of pGreenII0800 was replaced with the fragment shown at positions 341-1027 of SEQ ID NO.4, and the other sequences of vector pGreenII0800 were kept unchanged, resulting in expression vector pGreenII0800-IpU6-4 in which promoter IpU6-4 drives LUC gene expression.

2.5A one-step recombination kit from Norwegian corporation was used to recombine fragment OsU a onto cleaved pGreenII0800, and the ligation product was obtained according to the kit instructions. Transferring 5uL of the ligation product into escherichia coli DH5 alpha, culturing for 12 hours on a solid LB medium containing kanamycin (Kana, the concentration is 50 mug/L), picking up a monoclonal colony, placing the monoclonal colony into a liquid LB containing Kana (the concentration is 50 mug/L) for culturing, extracting plasmids after bacterial liquid PCR identification is positive cloning, carrying out double enzyme digestion verification, verifying successful plasmids, carrying out sequencing on the plasmids by a qinghao organism, and obtaining a successfully constructed expression vector pGreenII0800-OsU a after sequencing is correct. The structure of expression vector pGreenII0800-OsU a is described as follows: the small fragment between the recognition sequences of the restriction enzymes HindIII and PstI of pGreenII0800 was replaced with the fragment shown at positions 61-507 of SEQ ID NO.5, leaving the other sequences of vector pGreenII0800 unchanged, resulting in expression vector pGreenII0800-OsU a in which promoter OsU a drives LUC gene expression.

3. The idesia U6 promoter drives the expression of the LUC gene in recombinant plasmids

3.1A plant recombinant expression vector pGreenII-IpU 6-1 constructed to contain the LUC gene is transformed into Agrobacterium GV3101 (pSoup-p 19), the transformed Agrobacterium is subjected to screening culture (50 mug/L Kana+50 mug/L Rif), and colony PCR identification is performed to obtain positive monoclonal GV3101/pGreenII0800-IpU6-1.

3.2A plant recombinant expression vector pGreenII-IpU 6-2 containing the LUC gene was constructed to transform Agrobacterium GV3101 (pSoup-p 19), the transformed Agrobacterium was subjected to screening culture (50. Mu.g/L Kana+50. Mu.g/L Rif), and colony PCR was performed to identify positive monoclonal GV3101/pGreenII0800-IpU6-2.

3.3A plant recombinant expression vector pGreenII-IpU 6-3 containing the LUC gene constructed was transformed into Agrobacterium GV3101 (pSoup-p 19), the transformed Agrobacterium was subjected to screening culture (50. Mu.g/L Kana+50. Mu.g/L Rif), and colony PCR was performed to identify positive monoclonal GV3101/pGreenII0800-IpU6-3.

3.4A plant recombinant expression vector pGreenII-IpU 6-4 constructed to contain the LUC gene is transformed into Agrobacterium GV3101 (pSoup-p 19), the transformed Agrobacterium is subjected to screening culture (50 mug/L Kana+50 mug/L Rif), and colony PCR identification is performed to obtain positive monoclonal GV3101/pGreenII0800-IpU6-4.

3.5A plant recombinant expression vector pGreenII-OsU a containing the LUC gene is constructed to transform Agrobacterium GV3101 (pSoup-p 19), the transformed Agrobacterium is subjected to screening culture (50 mug/L Kana+50 mug/L Rif), and colony PCR identification is performed to obtain positive monoclonal GV3101/pGreenII0800-OsU6a.

The newly activated Agrobacterium monoclonal GV3101/pGreenII0800-IpU6-1、GV3101/pGreenII0800-IpU6-2、GV3101/pGreenII0800-IpU6-3、GV3101/pGreenII0800-IpU6-4 and GV3101/pGreenII0800-OsU a were inoculated into LB containing the corresponding antibiotics (50. Mu.g/L Kana+50. Mu.g/L Rif), respectively, and the pGreenII0800 vector was used as a negative control. 28 ℃,200rpm overnight; when the OD value of the bacterial liquid is between 0.6 and 1.0, centrifugally collecting agrobacterium at 4000rpm for 5 min; gently suspending the agrobacterium with 2mL of infection liquid, and standing for 1-4h at room temperature; leaf of Nicotiana benthamiana 6-8 weeks old; after 72h, the infected Nicotiana benthamiana leaves are ground, luciferase activity detection is carried out, and the fluorescence signal data are subjected to statistical analysis, and the result is shown in FIG. 4, wherein IpU < 6 > -2 is found to be the strongest in fluorescence signal when being used as a promoter, ipU < 6 > -1 is found to be the weakest in fluorescence signal when being used as a promoter, and OsU a is found to be the weakest in fluorescence signal.

The results show that IpU6-1, ipU6-2, ipU6-3 and IpU6-4 can drive LUC gene expression, and the best effect is IpU6-2 and IpU6-1.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

A dna molecule characterized in that: 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.
2. An expression cassette, characterized in that: the expression cassette contains a promoter, which is the DNA molecule of claim 1.
3. A recombinant vector, characterized in that: the recombinant vector comprises the DNA molecule of claim 1 or the expression cassette of claim 2.
4. A recombinant microorganism characterized in that: the recombinant microorganism comprises the DNA molecule of claim 1, or comprises the expression cassette of claim 2, or comprises the recombinant vector of claim 3.
5. Use of a DNA molecule according to claim 1 for the initiation of the expression of a gene of interest.
6. The use according to claim 5, wherein said promoting expression of the gene of interest is promoting expression of the gene of interest in a plant.
7. The use according to claim 6, wherein the dicotyledonous plant is a plant of the genus nicotiana of the family solanaceae.
8. The use according to claim 7, wherein the plant of the genus nicotiana of the family solanaceae is tobacco.
9. Use of the DNA molecule of claim 1 in gene editing.
10. The use of claim 9, wherein the gene editing is performed by CRISPR/Cas9 editing.