CN114703187B - Fraxinus mandshurica U6 gene promoter proFMU6.7, cloning and application thereof - Google Patents

Abstract

The invention relates to a fraxinus mandshurica U6 gene promoter proFMU6.7, and cloning and application thereof. The invention successfully clones the RNA polymerase III type promoter of the fraxinus mandshurica-the fraxinus mandshurica Liu Nayuan U6 gene promoter proFMU6.7 for the first time, successfully constructs the fraxinus mandshurica U6 gene promoter activity detection vector, and proves that the promoter has high-efficiency transcription activity through transient transformation fraxinus mandshurica Liu Youmiao and GUS dyeing verification. Experiments prove that the fraxinus mandshurica U6 gene truncated promoter proFMU6.7.4 has a starting activity and is successfully applied to a CRISPR/Cas9 system. Cloning of the fraxinus mandshurica U6 gene promoter proFMU6.7 and expression analysis of promoter activity provide efficient promoter sequences for research on fraxinus mandshurica and transformation research of related plants, and can also realize efficient and accurate germplasm innovation and variety genetic improvement of fraxinus mandshurica.

Description

A Mandshurica U6 gene promoter proFmU6.7 and its cloning and application

technical field

The invention belongs to the field of biological technology, especially the field of plant transgenic technology, specifically relates to a type III promoter of Mandshurica mandshurica RNA polymerase, more specifically relates to a kind of Mandshurica mandshurica U6 gene promoter proFmU6.7, and further discloses its cloning methods and applications.

Background technique

Fraxinus mandshurica is a large deciduous tree of Oleaceae Oleaceae, and it is listed as a national second-class protected endangered species. The overall strength of the wood is high, the earthquake resistance is good, and the wood is fine. It is often used in construction and furniture, and has important economic value. Since tobacco transgenic plants were obtained for the first time in 1983, the genetic transformation of woody plants has also received attention. important role. In the field of life sciences, mutants play a vital role in the study of gene function. However, due to the long generation cycle of forest trees, high genetic heterozygosity, and complex genome ploidy, traditional random mutagenesis methods often require the construction of a large population of mutant libraries and large-scale screening to obtain mutants with loss of target gene functions. The process requires a lot of manpower and material resources. In contrast, genome-directed editing technology has the great advantage of being able to directly introduce mutations at specific locations in the genome.

The CRISPR/Cas9 system is currently recognized as the gene editing technology with the most development potential. Under the guidance of sgRNA, the Cas9 enzyme cleaves the target site in a targeted manner to precisely edit the target gene. Compared with zinc finger nucleases (zinc finger nucleases, ZFNs) and transcription activator-like effector nucleases (transcription activator-like effector nucleases, TALENs) technologies, the CRISPR/Cas9 system has a simple design process, convenient operation, and high gene editing efficiency. The development is rapidly and widely used in the research of animals, plants and microorganisms. Since CRISPR/Cas9 was first used in model plants Arabidopsis and tobacco in 2013, the gene editing system has been applied to rice (Nekrasov et al., NatBiotechnol, 2013, 31(8):691–693.), wheat (Wang et al. , Nature biotechnology, 2014,32(9):947-951.), corn (Zhen et al., Journal of Genetics and Genomics, 2014,41(2):63-68.), sorghum (Jiang et al., Nucleic acids research, 2013,41(20):e188.), Brachypodium distachyon, tomato (Brooks et al., Plant Physiology, 2014,166(3):1292-1297) and other plants in 24 families and 45 genera (Shan et al. , Applications in plant sciences, 2020, 8(1):e11314.), has good application prospects in improving crop yield, quality, resistance, etc. The emergence of the CRISPR/Cas9 system has also greatly promoted the research on gene function and genetic improvement of forest trees. However, compared with herbaceous plants and food crops, the research and application of the CRISPR/Cas9 system in woody plants lags behind, and most of them are still in the stage of establishing gene editing systems. Citrus plants are the first woody plants to try to apply CRISPR/Cas9 for gene editing. In addition, this technology has also been successfully applied to Actinidia, Vitis vinifera, Apple, Pomegranate, Coffee, Cocoa, and Cassava. At the same time, its successful application in economic woody plants such as poplar has demonstrated the great potential of the CRISPR/Cas9 system in regulating plant stress tolerance and shortening the breeding cycle of forest trees, realizing new varieties of forest trees such as drought resistance and disease resistance Cultivation provides new ways for basic forest tree research and molecular breeding, and provides new ideas for precise improvement of plant traits and breeding of new varieties.

In order to improve the gene editing efficiency of the CRISPR/Cas9 system, people continue to optimize the vector. The sgRNA that determines target specificity is a small RNA with a specific secondary structure, usually driven by a U3/U6 series promoter. The U3/U6 promoter is one of the important elements of the CRISPR/Cas9 gene editing system. Its transcription start sites are A and G, respectively, and its transcriptional activity is high. Among them, U3 is mostly used in monocotyledonous plants, and U6 is mostly used in dicotyledonous plants. Choosing a U3 or U6 promoter with a clear start site can precisely guide the transcription of sgRNA, thereby reducing off-target effects caused by irrelevant DNA transcription. Although U3/U6 has been successfully used in gene editing in multiple species, the same promoter is not necessarily applicable in species with distant homologous relationships, and there are often multiple U3 or U6 promoters in the genes of the same species. There are some differences in transcription efficiency. Therefore, cloning more endogenous U3/U6 promoters of target plants is conducive to the improvement of the CRISPR/Cas9 gene editing system. Huijin Fan et al. cloned two PeU3 promoters in Moso bamboo and truncate them to different lengths, and found that different promoters and different truncation lengths of the same promoter have different transcriptional activities (Fan Huijin et al., Acta Botanica, 2020, 55(03):299-307.). When Pu Yan et al. verified the transcriptional activity of the promoter in tomato, they found that the cloned U3 promoter still had transcriptional activity within 250 bp (Pu Yan, North China Agricultural Science Journal, 2019(1): 33-39.). Based on the need to construct a CRISPR/Cas9 gene editing vector, the U3/U6 promoter used should be as short as possible while ensuring its high transcriptional activity, so as to ensure that it does not contain enzyme cleavage sites as much as possible. Studies have shown that , the U6 promoter used in CRISPR/Cas9 gene editing technology is usually only 200-400bp long (Fauser et al., The Plant Journal, 2014, 79(2):348-359.), even shorter than 100bp (Vladimir et al., Nature biotechnology , 2013, 31(8):691-693.). Long et al. used the cotton endogenous U6 promoter to increase the expression level of sgRNA by 6-7 times, and the gene editing efficiency was also increased by 4-5 times (Longs et al., Plant Methods. 2018, 14(1):80.). Liu Chunxia et al. used the tomato U6 promoter to drive the expression of sgRNA. Compared with the sgRNA driven by the Arabidopsis U6 promoter, the gene editing efficiency of tomato increased from 63% to 73% (Liu Chunxia et al., Molecular Plant Breeding, 2020, 18( 20):6716-6724.).

The successful establishment of the CRISPR/Cas9 gene editing system applied to Mandshurica mandshurica will provide efficient and reliable technical support for the acquisition of the directional mutant library of Mandshurica mandshurica, and provide valuable resources for in-depth research on the gene function of Mandshurica mandshurica and the development and utilization of gene resources. Material basis. However, so far, there is still a lack of research on the U6 promoter in Mandshurica mandshurica, and the lack of an applicable, as short as possible, endogenous U6 promoter with high transcriptional activity has become the basis for the construction of the Mandshurica mandshurica CRISPR/Cas9 gene editing system. Restriction factors also limit the application of the CRISPR/Cas9 system in the genetic breeding and germplasm innovation of Mandshurica mandshurica. Therefore, the endogenous FmU6 promoter of Mandshurica mandshurica with high transcriptional activity was preferentially cloned, and on this basis, its length was truncated, and the sgRNA transcription of the Mandshurica mandshurica target gene was constructed using the endogenous U6 gene promoter of Mandshurica mandshurica. The CRISPR/Cas9 vector is applied to functional gene research and genetic breeding of Fraxinus mandshurica, which has important research significance and application value.

Contents of the invention

In view of the current research status, the purpose of the present invention is to provide a kind of promoter FmU6.7 of Fraxinus mandshurica U6 gene and the cloning method and application of the promoter.

One of the technical schemes adopted by the present invention to solve the technical problems is to provide proFmU6.7, an endogenous U6 gene promoter of Mandshurica mandshurica, the nucleotide sequence of which is shown in SEQ ID NO:1.

Preferably, the promoter proFmU6.7 of the Fraxinus Fraxinus U6 gene belongs to the RNA polymerase type III promoter of the Fraxinus Fraxinus U6 snRNA gene.

Preferably, the nucleotide sequence of the promoter proFmU6.7 of Fraxinus mandshurica U6 gene contains 102bp U6SnRNA.

The second of the technical scheme that the present invention solves its technical problem adopts is: provide a kind of method of cloning described Fraxinus Fraxinus FmU6 gene promoter proFmU6.7, comprise the steps:

(1) Using the DNA of Fraxinus mandshurica aseptic seedlings as a template, design the following specific primers:

proFmU6.7-F:TATACCAACCATTCCTTCTCACTG

proFmU6.7-R:TTCTGGACAAAACCCTGGC

(2) Use LA Taq enzyme to carry out PCR cloning in a 50 μL system. The reaction program of PCR amplification is: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 2 minutes, 35 cycles, 70°C The final extension is 10min.

(3) The amplified product was cloned into the pCloneEZ-TOPO vector, transformed into Escherichia coli DH5α, and the recombinant single clone was picked and sequenced, that is, proFmU6.7 of the ash U6 gene promoter with a length of 1506 bp was obtained respectively.

The third technical solution adopted by the present invention to solve the technical problems is: a method for cloning the truncated promoter of the Fraxinus mandshurica U6 gene is provided, comprising the steps of:

(1) Using the positive recombinant monoclonal plasmid of Mandshurica U6 gene promoter proFmU6.7 as a template, design the following specific primers with different sequence lengths:

proFmU6.7.4-F:TAACATCGTTGGGTAAATG

proFmU6.7.4-R:AATTTTATCGGATGTCCCCG

(2) Use Phanta Max Master Mix high-fidelity enzyme to carry out PCR cloning in a 50 μL system. The reaction program of PCR amplification is: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 15 seconds, 57°C annealing for 15 seconds, and 72°C extension for 30-60 sec/ kb, 30 cycles, final extension at 72°C for 7min.

(3) The amplified product was cloned into the pCloneEZ-TOPO vector, transformed into Escherichia coli DH5α, and the recombinant single clone was picked and sequenced, that is, the truncated promoter proFmU6.7.4 of Mandshurica U6 gene with a length of 334bp was obtained respectively.

Preferably, the Fraxinus Fraxinus U6 gene truncated promoter proFmU6.7.4 contains 27bp SnRNA, and its nucleotide sequence is selected from the nucleotide sequence shown in 937-1270 positions in SEQ ID NO:1;

The fourth technical solution adopted by the present invention to solve the technical problem is to provide the application of the ash U6 gene promoter proFmU6.7 in the field of ash molecular breeding technology.

The present invention has the following beneficial effects: for the first time, the present invention clones and obtains the RNA polymerase type III promoter of the Fraxinus ash U6 snRNA gene in the Fraxinus ash, the endogenous U6 promoter proFmU6.7 of the Fraxinus ash, and combines it with GUS Gene fusion, transient transformation of aseptic seedlings of Mandshurica mandshurica, verified that the promoter can be highly expressed in Mandshurica mandshurica through GUS transient expression, and provided a high-efficiency promoter sequence for the transformation research of Mandshurica mandshurica and related plants. In addition, for the first time, the present invention truncated the promoter proFmU6.7 of the Fraxinus mandshurica U6 gene with high-efficiency activation activity to obtain the truncated promoter proFmU6.7.4 of the Fraxinus mandshurica U6 gene, which was verified by constructing an expression vector fused with the GUS gene The truncated promoter of Mandshurica U6 gene still has high-efficiency promoter activity, and the truncated promoter proFmU6.7.4 of Mandshurica U6 gene was constructed into the gene editing vector to obtain the promoter guide RNA of the endogenous U6 gene promoter of Mandshurica mandshurica Transcribed gene editing vector pSC1-FmU6.7.4-Cas9-HYP. Therefore, the truncated promoter FmU6.7.4 of the cloned Fraxinus ash U6 gene of the present invention can be applied to the CRISPR/Cas9 gene editing system of Fraxinus ash, so as to realize efficient and accurate germplasm innovation and variety genetic improvement of Fraxinus ash.

Description of drawings

Figure 1 is the sequence alignment of the U6 gene promoter proFmU6.7 of Mandshurica mandshurica and the U6 promoters of Arabidopsis thaliana and soybean. Transcription start site, the position marked by the horizontal line is U6 snRNA;

Fig. 2 is the cloning electrophoresis figure of Manchurian Fraxinus U6 gene promoter proFmU6.7, it can be seen that a 1506bp fragment of Mandshurica Mandshurica U6 gene promoter proFmU6.7 has been obtained respectively through PCR amplification;

Figure 3 is a schematic diagram of the truncated promoter proFmU6.7.4 of Fraxinus mandshurica U6 gene, in which the position of the short line is CAAT-box;

Fig. 4 is the cloning electrophoresis diagram of the truncated promoter proFmU6.7.4 of the Mandshurica U6 gene, it can be seen that the 334bp fragment of the truncated promoter proFmU6.7.4 of the Mandshurica U6 gene has been obtained respectively through PCR amplification;

Figure 5 shows the GUS staining of the aseptic seedlings of Mandshurica mandshurica. Control is the control group transiently infected with Agrobacterium GV3101.

Figure 6 is a schematic diagram of the structure of the recombinant gene editing vector pSC1-FmU6.7.4-Cas9-HYP of Mandshurica mandshurica, in which the LguI restriction site is the insertion position of the sgRNA sequence of the target gene.

Figure 7 shows the transcription level of sgRNA under the truncated promoter proFmU6.7.4 of Ash U6 gene

Detailed ways

In order to better understand the present invention, the present invention will be described in further detail below in conjunction with the examples and accompanying drawings, but those skilled in the art should understand that the following examples do not limit the protection scope of the present invention, and any work done on the basis of the present invention All changes and changes mentioned above are within the protection scope of the present invention.

In the following examples, the experimental methods used are conventional methods unless otherwise specified.

In the following examples, the promoter activity detection vector pNC-121-pro is the pNC series vector of NC Biotech Company, and this biological material is only used for repeating related experiments of the present invention, and is not used for other purposes.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1:

To obtain the promoter proFmU6.7 of Fraxinus mandshurica U6 gene, the specific operation is as follows:

(1) According to the conservation of the U6 snRNA sequence among different species, the snRNA sequence of the Arabidopsis AtU6 promoter (GTCCCTTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCATAAATCGAGAAATGGTCCAAAATTTT) and the soybean GmU6 promoter snRNA sequence (GTCCCTTCGGGGACATCCGATAAAAT TGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCACAAATCGAGAAATGGTCCAAAATTTT) was compared with the genome sequence of Mandshurica mandshurica (BLAST ). Check the comparison results, select the position whose sequence homology is higher than or equal to 99%, and use the Plant CARE online analysis software to analyze the promoter cis-acting element of its upstream 1800bp sequence. The analysis results showed that USE (Upstream Sequence Element) and TATA-like Box related to basic transcription were located at 60bp and 30bp upstream of the transcription initiation site of these U6 genes, respectively. One of the promoter sequences was selected and named proFmU6.7.

(2) Using Genomic DNA of Mandshurica bacterium as template, design specific primers on the upstream and downstream of Mandshurica U6 gene promoter proFmU6.7:

proFmU6.7-F:TATACCAACCATTCCTTCTCACTG

proFmU6.7-R:TTCTGGACAAAACCCTGGC

The downstream primers are located at 300bp downstream of the Fraxinus mandshurica snRNA in the genome sequence to ensure that the cloned sequence contains a complete 102bp snRNA.

(3) Use LA Taq enzyme to perform PCR cloning in a 50 μL system. The reaction program of PCR amplification is: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 2 minutes, 35 cycles, 70°C The final extension is 10min.

(4) The amplified product was cloned into the pCloneEZ-TOPO vector, transformed into Escherichia coli DH5α, and the recombinant single clone was picked and sequenced, and finally the 1506bp promoter proFmU6.7 of Mandshurica mandshurica U6 gene was obtained as shown in Figure 2 .

(5) Alignment and analysis of the promoter sequence with Arabidopsis AtU6-1, AtU6-26, AtU6-29 and soybean GmU6-16g-1, GmU6-16g-2, GmU6-19g-2 base sequences using DNAMAN It was found that the proFmU6.7 sequence of the Fraxinus mandshurica U6 gene promoter contains the USE element and the U6 snRNA transcription key site TATA-like Box, as shown in Figure 1, and the two elements in the Fraxinus mandshurica FmU6 gene promoter sequence are relatively The location of the transcription start site is consistent with the promoter sequence of Arabidopsis AtU6-1, AtU6-26, AtU6-29 and soybean GmU6-16g-1, GmU6-16g-2, GmU6-19g-2, which The exercise of function is of great significance.

(6) Using the positive recombinant monoclonal plasmid of Mandshurica U6 gene promoter proFmU6.7 as a template, design the following specific primers:

proFmU6.7.4-F:TAACATCGTTGGGTAAATG

proFmU6.7.4-R:AATTTTATCGGATGTCCCCG

Among them, the proFmU6.7.4 fragment of the truncated promoter of Fraxinus mandshurica U6 gene is located on the U6 snRNA to ensure that the sequence contains 27bp snRNA, and the truncated position is shown in Figure 3 .

(7) Use Phanta Max Master Mix high-fidelity enzyme to carry out PCR cloning in a 50 μL system. The reaction program of PCR amplification is: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 30-60 sec/ kb, 30 cycles, final extension at 72°C for 7min. (8) The amplified product was cloned into the pCloneEZ-TOPO vector, transformed into Escherichia coli DH5α, and the recombinant single clone was picked and sequenced, that is, the truncated promoter proFmU6 of the Fraxinus mandshurica U6 gene with a length of 334bp as shown in Figure 4 was respectively obtained. 7.4.

Example 2:

The promoter activity detection of Mandshurica U6 gene promoter is as follows:

(1) Construction of the detection vector for the promoter activity of Fraxinus mandshurica U6 gene:

Specific primers containing homology arms for homologous recombination cloning with pNC-121-pro (pBI121 frame, GUS reporter gene, NC cloning frame replaced pBI121 35S promoter) were designed as follows:

pNC-proFmU6.7-F: CAGTGGTCTCTGTCCAGTCCT TATACCAACCATTCCTTTCACTG

pNC-proFmU6.7-R: CGGTCTCAGCAGACCACAAGTTTCTGGACAAAACCCTGGC

pNC-proFmU6.7.4-F: CAGTGGTCTCTGTCCAGTCCTTAACATCGTTGGGTAAATG

pNC-proFmU6.7.4-R: CGGTCTCAGCAGACCACAAGT AATTTTATCGGATGTCCCCG (underlined is the homologous sequence with pNC-121-pro vector)

Mandshurica U6 gene promoter FmU6.7 and Mandshurica U6 gene truncated promoter proFmU6.7.4 positive recombinant monoclonal plasmids were used as templates to obtain the Mandshurica U6 gene promoter with homologous arms at both ends For DNA fragments, use Nimble Mix (A) to mix the above fragments with pNC-121-pro plasmid respectively, pipette and mix 10-20 times, and then treat with a PCR instrument at 50°C for 45 minutes, and keep the temperature at 4°C to finally obtain pNC- 121-pro:proFmU6.7, pNC-121-pro:proFmU6.7.4 two promoter activity detection vectors.

(2) Verification of Agrobacterium transformation of Mandshurica U6 gene promoter:

The five constructed promoter activity detection vectors were respectively transformed into the Agrobacterium agrobacterium GV3101 strain, and the aseptic seedlings of Mandshurica mandshurica inoculated for 15 days were used as explant materials for transient transformation, and the Agrobacterium was transiently infected, and the ash was taken out after 3 days of co-cultivation The willow seedlings were observed by GUS staining to evaluate the starting ability and expression activity of the U6 gene promoter of Mandshurica mandshurica.

Add GUS staining solution to the tissue-cultured Mandshurica mandshurica seedlings used for detection, vacuum filter for half an hour, and then stain overnight in a shaker at 37°C in the dark, pour off the staining solution, decolorize with 95% alcohol for 3 days, and observe the GUS staining situation. Fraxinus mandshurica seedlings infected with empty GV3101 Agrobacterium were used as controls,

The dyeing situation is shown in Figure 5.

Example 3:

The construction of the recombinant vector for gene editing of Mandshurica U6 gene truncated promoter proFmU6.7.4, the specific operation is as follows:

(1) Construction of a truncated promoter proFmU6.7.4 gene editing vector of Fraxinus mandshurica U6 gene

Use Asc I and Lgu I to double digest the pSC1-GmU6-GmUbi3 vector, remove the soybean GmU6 promoter on the vector, and recover a large fragment of the 15472bp vector backbone.

Primers with homology arms designed for homologous recombination cloning:

pSC1-proFmU6.7.4-F: ATCTTTCACTGGCGCGCC TAACATCGTTGGGTAAATG

pSC1-proFmU6.7.4-R: TCTAGCTCTAAAACAGAAGAGC AATTTTATCGGATGTCCCCG (underlined is the sequence homologous to pSC1-GmU6-GmUbi3 vector)

Using the positive recombinant monoclonal plasmid of Fraxinus mandshurica U6 gene truncated promoter FmU6.7.4 as template, the DNA fragment of Mandshurica mandshurica U6 gene truncated promoter FmU6.7.4 containing homology arms at both ends was obtained by PCR amplification. ×Master AssemblyMix homologously recombined the above fragments into the linearized pSC1-GmU6-GmUbi3 vector to obtain the Fraxinus mandshurica gene editing vector pSC1-FmU6.7.4-Cas9-HYP. The schematic diagram of the vector structure is shown in Figure 6, where the arrow is the target gene sgRNA insertion site.

(2) Construction of recombinant vector for gene editing of Fraxinus mandshurica U6 gene truncated promoter proFmU6.7.4

In order to verify the functional activity of the truncated promoter proFmU6.7.4 of Mandshurica Mandshurica U6 gene in Mandshurica mandshurica gene editing, the endogenous glycosyltransferase GT8 family gene FmGAUT9 of Mandshurica mandshurica was selected as the target gene for CRISPR/Cas9 editing. target site:

GTTAATCCGCGGGCTTGTGCA.

For the target site, linker sequences are added to both ends of the target sequence, and the designed target site cloning primers are as follows:

pSC1-GAUT9-F: GTTGTTAATCCGCGGGCTTGTGCA

pSC1-GAUT9-R: AAC TGCACAAGCCCGCGGATTAAC (the linker sequence used for vector ligation is underlined)

Mix the above-mentioned pSC1-GAUT9-F and pSC1-GAUT9-R target primers separately, treat them with a PCR instrument at 95°C for 3 minutes, then cool down to 16°C for 1°C/20s and anneal to form a doublet with cohesive ends complementary to the vector. Strand annealed product, at the same time digest the pSC1-FmU6.7.4-Cas9-HYP vector with Lgu I, and then use T4 DNA ligase to connect the target fragment to the restriction vector, finally get pSC1-FmU6.7.4:GAUT9-Cas9- HYP Fraxinus Fraxinus gene editing recombinant vector.

(3) The truncated promoter proFmU6.7.4 of Fraxinus mandshurica U6 gene promotes the transcription of sgRNA

The editing vector pSC1-FmU6-7-4:GAUT2-Cas9-HYP was transformed into the Agrobacterium strain GV3101, and the seedlings of Mandshurica mandshurica were used as the explant material for transient transformation, and Agrobacterium was transiently infected, and placed at 28°C and 35°C respectively. ℃ constant temperature incubator, after 3 days of co-cultivation, take out the Fraxinus mandshurica seedlings and wash them repeatedly with distilled water. After quick freezing in liquid nitrogen, extract RNA, reverse transcribe into cDNA, and perform fluorescence quantitative PCR detection and verification.

Design the following specific quantitative primers according to the sgRNA targeting the FmGAUT9 gene and the sgRNA backbone:

sgRNA9-F:CGGGCTTGTGCAGTTTTA

sgRNA9-R:CGACTCGGTGCCACTTTT

ACTIN-F:AGGACGCTGCCAACAACTTT

ACTIN-R:TTGAGGGGAAGGGTAAATAGTG

Among them, ACTIN is the housekeeping gene of Mandshurica mandshurica, and the relative expression level of sgRNA is shown in Figure 7.

It can be seen that the present invention has obtained the RNA polymerase type Ⅲ promoter of the Fraxinus Fraxinus U6 snRNA gene in Fraxinus Fraxinus - the endogenous U6 promoter FmU6.7 of Fraxinus Fraxinus Fraxinus, and it has been verified to have activation activity. The truncated promoter proFmU6.7.4 of the willow U6 gene has promoter activity and can initiate sgRNA transcription, and it is the first time to apply the endogenous U6 promoter of Mandshurica mandshurica to the CRISPR/Cas9 system. Therefore, the promoter described in the present invention can be applied to the CRISPR/Cas9 gene editing system of Fraxinus ash, so as to realize efficient and precise creation of new germplasm and variety improvement of Fraxinus ash.

Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we have described are only illustrative, rather than used to limit the scope of the present invention. For those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the claims of the present invention.

sequence listing

<110> Northeast Forestry University

<120> A Mandshurica U6 gene promoter proFmU6.7 and its cloning and application

<130> proFmU6.7

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1506

<212>DNA

<213> Fraxinus mandshurica

<400> 1

tataccaacc attccttctc actgacatct tgtttgagct ttgtcgtgac atttttatt 60

acatgtggaa aacatgaata ggttaaggga gaaaatcaat actttttagt caaacacaat 120

cttttctttc gagaatcacg ttatatttat aagatctgca atttaacgac gtggcacaac 180

tgttagtcga tgataaggtt taattgggcc acgttgaatc tggagtgagt ttctaacaga 240

gtagtcaatt atgtaacgaa attaacttta gtgactaaaa tatatttaaa aaataattta 300

aagacaaatt taaaatttta tcgattattt gtaaaattaa ctcgagaatg gtacatgttg 360

gtgtgtacaa tgggtattaa ggcatataat ctaagtaaat ggcaaaagtt atatgaacat 420

gattttcttt tttcaaattt aaatgaatta ttgtcgagcg ttatctgtaa aacagttttg 480

tgaaaaagtt ctgtaagtgt agcacaactc aaggtagatt gtacatccgc ccatgactaa 540

tacaatataa cgttcgtaat gacatgtcac aatcacccaa cttagaaagt taagttgatt 600

ggaagctaat atgtggttga aattaaacgt ctggcgccat aattttgtag aaacttgttg 660

atattgagag aggatctcgc gagaggcccc cgggggggtg aattcccgtg aaagataacg 720

aaaaaaaaaa tattttttat tgattaaaat taaacgtaag tgagtaattg attttgatag 780

taagttttat ataaattttt tacttaaaat taattaatta aaaataagtc ataaacatgt 840

tgtagacaaa agtaaattta ctcataagta atttagtaag ttttaaaata actatttttc 900

agttagtcag agaaaaatat atgtaaattc ttattataac atcgttgggt aaatgaaatt 960

tttaaaattc gtttttagac aaaaataaat ggcaaaagat tgatttcaat tttattccaa 1020

atatttatac ttttttatta ctggaggatt ggaaaactat atggaccaat ccaacaaaat 1080

agctccataa acccatcagc aataaggccc agccaataat ttgaactaaa attaacaaag 1140

cccaagaact tattagttgt atttctcaac tatcccagtc ccacatcgac gaaatggaat 1200

agtttttacc cgtttatata gcaatgtgat agctagatga cttgtccctt cggggacatc 1260

cgataaaatt ggaacgatac agagaagatt agcatggccc ctgcgcaagg atgacacgca 1320

caaatcgaga aatggtccaa atttttttcc tttcattttc tccagttttc tgcagtttca 1380

tccaaactgt tggattgttg tttgagaaat tatcgaccaa tttttatgtt tcttgatttt 1440

caattattga agttttctgt agaatgcttg acggtttggt ttaaattgcc agggttttgt 1500

ccagaa 1506

Claims (4)

1. The promoter proFMU6.7 of the U6 gene of the fraxinus mandshurica is characterized in that the nucleotide sequence of the promoter proFMU6.7DNA is shown as SEQ ID NO. 1.

2. A cloning method of a gene promoter, which is characterized in that the cloning method of the fraxinus mandshurica U6 gene promoter profmu6.7 according to claim 1 comprises the following steps:

(1) Designing a specific primer proFMU6.7-F: TATACCAACCATTCCTTCTCACTG, proFmU6.7-R: TTCTGGACAAAACCCTGGC by taking the fraxinus mandshurica DNA as a template;

(2) The PCR amplification was performed in a 50. Mu.L system using LATaq enzyme, and the reaction procedure for the PCR amplification was: pre-denaturation at 95 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min,35 cycles, and final extension at 70 ℃ for 10min;

(3) Cloning the amplified product onto pCloneEZ-TOPO vector, converting colibacillus DH5 alpha, picking up recombinant monoclonal sequencing to obtain the promoter proFMU6.7 of the fraxinus mandshurica U6 gene with the length of 1506 bp.

3. A method for detecting the promoter activity of fraxinus mandshurica is characterized in that the method is a method for detecting the promoter activity of the U6 gene promoter proFMU6.7 in claim 1, and the promoter activity detection vectors pNC-121-pro: FMU6.7 are respectively and transiently introduced into fraxinus mandshurica seedlings.

4. The application of the mangrove Liu Qi promoter is characterized in that the application of the mangrove U6 gene promoter proFMU6.7 in the technical field of molecular breeding of the mangrove is disclosed in claim 1.

Images