CN114703189B - Fraxinus mandshurica U6 gene promoter proFMU6.3, cloning and application thereof - Google Patents

Abstract

The invention relates to a fraxinus mandshurica U6 gene promoter proFMU6.3, and cloning and application thereof. The invention successfully clones the RNA polymerase III type promoter of the fraxinus mandshurica, namely the fraxinus mandshurica Liu Nayuan U6 gene promoter proFMU6.3 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 instant transformation fraxinus mandshurica Liu Youmiao and GUS dyeing verification, thereby providing high-efficiency promoter sequences for the transformation research of fraxinus mandshurica and related plants.

Description

A Mandshurica U6 gene promoter proFmU6.3 and its cloning and application

technical field

The invention belongs to the field of biological technology, especially the field of plant transgenic technology, and specifically relates to a type III promoter of Mandshurica mandshurica RNA polymerase, more specifically to a proFmU6.3 promoter of Mandshurica mandshurica U6 gene, 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. and transcription efficiencies. 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, cloning the endogenous FmU6 promoter of Fraxinus ash with high transcriptional activity has important research significance and application value for the construction of CRISPR/Cas9 vector of Fraxinus ash, functional gene research and genetic breeding of Fraxinus ash.

Contents of the invention

In view of the current research status, the purpose of the present invention is to provide a kind of promoter FmU6.3 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 proFmU6.3, an endogenous U6 gene promoter of Mandshurica mandshurica, the nucleotide sequence of which is shown in SEQ ID NO:1.

Preferably, the promoter proFmU6.3 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 proFmU6.3 of Fraxinus mandshurica U6 gene contains 102bp U6SnRNA.

Two of the technical scheme adopted by the present invention to solve the technical problems are: a method for cloning the described Fraxinus Fraxinus FmU6 gene promoter proFmU6.3 is provided, comprising the steps of:

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

proFmU6.3-F:ACCACCGTCGTCTCTCTCCA

proFmU6.3-R:CATCCAGAAGTTCACCTAAAGCA

(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, proFmU6.3 of the promoter of Manchurian Fraxinus U6 gene with a length of 1442bp was obtained respectively.

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 proFmU6.3 in the field of ash molecular breeding technology.

The present invention has the following beneficial effects: for the first time, the RNA polymerase type III promoter of the U6 snRNA gene of Mandshurica mandshurica is cloned and obtained - the endogenous U6 gene promoter proFmU6.3 of Mandshurica mandshurica, and the The fusion expression of U6 gene promoter proFmU6.3 and GUS gene transformed Fraxinus mandshurica seedlings, and the transient expression verified that the U6 gene promoter proFmU6.3 of Mandshurica mandshurica can be highly expressed on Mandshurica mandshurica Transformation studies in related plants have provided highly efficient promoter sequences.

Description of drawings

Figure 1 is the sequence alignment of the U6 gene promoter proFmU6.3 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 proFmU6.3, it can be seen that the 1442bp fragment of Mandshurica mandshurica U6 gene promoter proFmU6.3 has been obtained through PCR amplification;

Figure 3 shows the GUS staining of the aseptic vaccine of Mandshurica mandshurica. Control is the control group transiently infected with Agrobacterium GV3101.

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 proFmU6.3 of Mandshurica Fraxinus U6 gene, the specific operation is as follows:

(1) According to the conservation of U6 snRNA sequence among different species, use Arabidopsis AtU6 snRNA sequence (GTCCCTTCGGGGACATCCGATAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCATAAATCGAGAAATGGTCCAAAATTTT) and soybean GmU6 snRNA sequence (GTCCCTTCGGGGACATCCGATAAAATTGGAAC GATACAGAGAAGATTAGCATGGCCCCTGCGCAAGGATGACACGCACAAATCGAGAAATGGTCCAAAATTTT) was compared with the genome sequence of Fraxinus ash (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 proFmU6.3.

(2) Using Genomic DNA of Mandshurica bacterium as template, design specific primers on the upstream and downstream of Mandshurica U6 gene promoter proFmU6.3:

proFmU6.3-F:ACCACCGTCGTCTCCTCCA

proFmU6.3-R:CATCCAGAAGTTCACCTAAAGCA

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 1442bp promoter proFmU6.3 of Fraxinus 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 proFmU6.3 sequence of the U6 gene promoter of Mandshurica mandshurica contains the USE element and the key site TATA-like Box for U6 snRNA transcription, as shown in Figure 1, and the two The position of the element relative to the transcription start site is consistent with that of Arabidopsis AtU6-1, AtU6-26, AtU6-29 and soybean GmU6-16g-1, GmU6-16g-2, GmU6-19g-2 promoter sequence positions, This is of great significance to the exercise of its functions.

Example 2:

The activity detection of Mandshurica U6 gene promoter proFmU6.3, the specific operation is as follows:

(1) Construction of the detection vector for proFmU6.3 activity of Mandshurica U6 gene promoter:

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-proFmU6.3-F: CAGTGGTCTCTGTCCAGTCCT ACCACCGTCGTCTCCTCCA

pNC-proFmU6.3-R: CGGTCTCAGCAGACCACAAGT CATCCAGAAGTTCACCTAAAAGCA

(The underlined sequence is homologous to the pNC-121-pro vector)

Using the proFmU6.3 positive recombinant monoclonal plasmid of Mandshurica mandshurica U6 gene promoter as a template, the proFmU6.3 DNA fragment containing homology arms at both ends was amplified by PCR, and the above fragment was combined with pNC-121- The pro plasmids were mixed, pipetted and mixed 10-20 times, treated with a PCR instrument at 50°C for 45 minutes, and kept at 4°C to obtain the pNC-121-pro:FmU6.3 promoter activity detection vector.

(2) Agrobacterium transformation verification of Mandshurica U6 gene promoter proFmU6.3:

The constructed promoter activity detection vector pNC-121-pro:FmU6.3 was transformed into Agrobacterium strain GV3101, and the seedlings of Mandshurica mandshurica inoculated for 15 days were used as explant materials for transient transformation, and Agrobacterium was transiently infected and co-cultured for 3 Two days later, the Mandshurica mandshurica seedlings were taken out and observed by GUS staining to evaluate the starting ability and expression activity of the Mandshurica mandshurica U6 gene promoter proFmU6.3.

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 no-load GV3101 Agrobacterium were used as a control, and the staining conditions are shown in Figure 3.

It can be seen that the present invention obtains the RNA polymerase type III promoter of the Fraxinus Fraxinus U6 snRNA gene in Fraxinus Fraxinus U6 gene promoter, proFmU6.3, and has been verified to have a promoter activity. Therefore, the present invention provides a high-efficiency promoter sequence for the transformation research of Mandshurica mandshurica and related plants.

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 proFmU6.3 and its cloning and application

<130> proFmU6.3

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1442

<212>DNA

<213> Fraxinus mandshurica

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accaccgtcg tctcctccac caacaccaac accaacacta cagaaacgca ctacaatggt 60

ttgatgtcac atgttaataa tgagtctcaa tgaaccaaaa tgtcggttac atccatcaaa 120

cacctcacac ccatcccgat tctccaccaa aatttggtca caaatcaacc ctcgagaccc 180

ctaccaccac cattattatc gccgcagcca gacccaccgc cactgcagct agaaaaccca 240

aaacccccca acagcaccaa aaccaccact accaccaaca taagcaccac catctccacc 300

acaaatccac catatccaac acattcaaaa ccaaaacgcg aaacacatct tagatctaaa 360

cacccaaatc acaccgcatc accgctacaa caacgaccac cacccaaaca ccaccactac 420

gagcactgca ccatagcagt gattgcccat gaacaataga tcttctcaat aagagagact 480

tcgttgcgga tcctgtggtg ccgctgcacc aacacaacga catctctctt ggtgaagctt 540

gaaatggttc cgtttcaaac accacggcc tcgggcgggg caaaaagtga agccccgatt 600

ttgaaaccaa ggaccgttgg gtcgaccacg aagattatca aatggaagtc tcatgggaaa 660

ggtgcaacaa cgacgagaga gagaacggaa atagaaatga attagggttt aatttataat 720

atttaaatat ggattattaa ttaaaacctg acttgattct tgattatga aaatggaaaa 780

ttaaaatcca ttatagctgt aatttcctca gtaaacggtt ctaaacaagt ttaaggtaga 840

caaaatgaat aaaatgcaaa tcaaatactt tgtaggctca ccaacaaggc ctggcccgta 900

attgaaacta gaagtgaagc ctaagaaccc gagcccaaaa acttaccagt cctattggaa 960

ctgaatctca tatattctag tcccacatcg acaaaaggca gaattccttt gcaatttata 1020

ttgcgattgc tggcaataga gtttgtccct tcggggacat ccgataaaat tggaacgata 1080

cagagaagat tagcatggcc cctgcgcaag gatgacacgc acaaatcgag aaatggtcca 1140

aatttttttt gttcttgaat tcatgtagtt ttccgtagaa aaatcatcct ttggtttttt 1200

tatttttttt gtcgatttgc agtttgtcta tatttttgag gactttggtg agtggttctt 1260

atggacctta atttgatgag gtttttgtca aaaaaccatc tatattgtta gggtttcagg 1320

caagctttat ttattattaa ggtttctatc gcgaaatgac gatttttttc ttttttttcg 1380

gtatgcagtt tcatcgaaat tgttgaggta tttgtcgaat gctttaggtg aacttctgga 1440

tg 1442

Claims (6)

1. The promoter proFMU6.3 of the fraxinus mandshurica U6 gene is characterized in that the DNA nucleotide sequence of the promoter proFMU6.3 is shown as SEQ ID NO. 1.

2. A method for cloning a gene promoter, characterized in that a method for cloning the fraxinus mandshurica U6 gene promoter profmu6.3 according to claim 1 comprises the steps of:

(1) Designing a specific primer proFMU6.3-F: ACCACCGTCGTCTCCTCCA, proFmU6.3-R: CATCCAGAAGTTCACCTAAAGCA by taking the fraxinus mandshurica DNA as a template;

(2) PCR amplification was performed in a 50. Mu.L system using LA Taq enzyme;

(3) Cloning the amplified product onto pCloneEZ-TOPO vector, converting colibacillus DH5 alpha, picking up recombinant monoclonal sequencing to obtain the promoter proFMU6.3 of fraxinus mandshurica U6 gene with length of 1442 bp.

3. The method of cloning the fraxinus mandshurica U6 gene promoter profmu6.3 according to claim 2, wherein in the step (2), the PCR amplification reaction procedure is as follows: 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.

4. The method of cloning the fraxinus mandshurica U6 gene promoter fmu6.3 as claimed in claim 2, wherein the downstream primer is located 300bp downstream of fraxinus mandshurica snRNA in the genomic sequence to ensure that the cloned sequence contains the complete 102bp U6 snRNA.

5. 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 proFMU6.3 in claim 1, and the promoter activity detection vectors pNC-121-pro: FMU6.3 are respectively and instantly introduced into fraxinus mandshurica seedlings.

6. The application of the mangrove Liu Qi promoter is characterized in that the application of the mangrove U6 gene promoter proFMU6.3 in the technical field of molecular breeding of the mangrove is disclosed in claim 1.

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