CN110029108B - Endogenous whole-life-history constitutive promoter of seaweed and application thereof - Google Patents
Endogenous whole-life-history constitutive promoter of seaweed and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of seaweed genetic engineering, and relates to a seaweed endogenous whole-life-history constitutive promoter and application thereof. The promoter is a nucleotide sequence shown in SEQ ID NO. 1; 1 by substitution, deletion or addition of one or more nucleotides; or a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence limited by SEQ ID No. 1 in the sequence table and has transcription initiation function. The enteromorpha actin gene 2(actin2) promoter is obtained by adopting the technologies of chromosome walking, gene cloning, DNA sequencing and the like, and has a sequence shown as SEQ ID NO. 1; on the basis, the cloned actin gene 2 promoter is inserted into the upstream of a reporter gene GUS, a transformation vector is constructed and introduced into different life history stages of enteromorpha, and the GUS gene can be detected to be efficiently expressed in the vegetative growth period and the reproductive period. The technology can improve the transgenic efficiency and the biological safety of the enteromorpha and has important significance for variety improvement of algae and development of gene engineering products.
Description
Technical Field
The invention belongs to the technical field of seaweed genetic engineering, and relates to a seaweed endogenous whole-life-history constitutive promoter and application thereof.
Background
Large-sized seaweeds belong to lower plants and mainly comprise green algae, brown algae and red algae. Many of the economic species have been artificially cultivated on a large scale, such as Enteromorpha prolifera, Ulva lactuca and Monostroma nitidum of green algae, Laminaria japonica, Undaria pinnatifida and Hizikia fusiforme of brown algae, and Porphyra tenera, Gelidium amansii and Eucheuma muricatum of red algae. At present, the total cultivation area of the global macroalgae exceeds three million mu, the annual output exceeds 600 million tons fresh weight, and the macroalgae cultivation method is partially used as nutritional food, is also used for important chemical raw materials such as iodine, mannitol, phycocolloid and the like, and extraction raw materials of marine medicines and protein reagents, and has very high economic value.
In recent years, the cultivated seaweed also shows outstanding application potential in the fields of new energy sources (such as ethanol and methane produced by fermentation), new materials (such as seaweed polysaccharide textile fibers), novel expression systems (protein medicines prepared by recombination) and the like. In order to meet the requirement of rapid directional breeding of seaweed, the development of an efficient and safe seaweed genetic engineering technology and a complete set of methods are urgently needed. Among them, a promoter is an important vector element for efficient expression and directed inducible expression of an introduced gene.
A promoter is an integral part of a gene and is a DNA sequence that is specifically recognized and bound by RNA polymerase, controlling the initiation time and the extent of expression of the gene (transcription). Promoters, like "switches", do not control gene activity per se, but rather control transcription of genes by binding to proteins called transcription factors. At present, seaweed genetic transformation systems represented by kelp, undaria pinnatifida and laver are preliminarily established, and stable expression can be realized in seaweed by introducing exogenous functional genes. However, many of the promoter elements used are derived from terrestrial organisms, and thus have many disadvantages.
Firstly, the broad CaMV35S high-efficiency promoter (from cauliflower mosaic virus) is widely used in higher plants, and the efficiency of driving the expression of exogenous genes in seaweeds is generally low; in addition, the most widely used SV40 promoter line in seaweed is derived from a primate simian vacuolating virus, and therefore, the use of exogenous promoters brings two outstanding problems of inefficient expression of large-sized seaweed transgenes and potential safety hazards. Secondly, the life history of the seaweed has two stages of vegetative growth phase and reproductive phase, and the germ cells in the reproductive phase are often used as receptor materials for genetic transformation. After transformation, if the foreign gene is integrated at the single germ cell stage, all cells contain the introduced gene in the grown new plant; if the foreign gene is integrated at the multicellular stage after germ cells germinate, the grown new plant is often a chimera, i.e., only a portion of the cells in the new plant contain the introduced gene. Therefore, if screening is performed at the single germ cell stage after transformation, the generation of chimeras can be effectively avoided. However, the promoters currently used are only examined for promoter activity in the vegetative growth phase, and are not examined for promoter activity in the reproductive phase of seaweeds.
Disclosure of Invention
Aiming at the demand of seaweed genetic engineering on the endogenous full-life history constitutive promoter of the seaweed, the invention aims to provide the endogenous full-life history constitutive promoter of the seaweed and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
an endogenous full-life history constitutive promoter of seaweed, an enteromorpha actin gene 2(actin2) promoter is a nucleotide sequence shown as SEQ ID NO. 1;
1 by substitution, deletion or addition of one or more nucleotides;
or a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence limited by SEQ ID NO. 1 in the sequence table and has transcription initiation function.
Preferably, the enteromorpha actin gene 2 promoter is a nucleotide sequence shown in SEQ ID NO. 1.
The promoter is obtained from marine green algae.
The promoter is obtained from Ulva (Ulva) plant Enteromorpha (Ulva prolifera).
Further, an enteromorpha endogenous whole life history constitutive actin gene promoter pUPAtin 2 is cloned by utilizing a chromosome walking technology, a gene cloning technology and a DNA sequencing technology of molecular biology, and the sequence of the promoter is determined; on the basis, the cloned pUPAtin 2 promoter sequence is respectively inserted into the upstream of a beta-Glucuronidase (GUS) gene and a herbicide glufosinate-resistance gene (bar) by using a cloning technology, a series of enteromorpha conversion vectors are constructed, and the high-efficiency expression of the introduced foreign gene in the vegetative growth period and the reproductive period can be realized through conversion.
An algal expression system: the expression system contains the promoter.
The application of a seaweed endogenous whole-life-history constitutive promoter in driving high-efficiency expression of exogenous genes in the vegetative growth period of seaweed.
The promoter is applied to driving the high-efficiency expression of exogenous genes in the seaweed reproductive stage.
The promoter is applied to enteromorpha genetic breeding.
The promoter is applied to the improvement of the quality of the seaweed or the construction of a seaweed expression system. Preferably, the enteromorpha actin gene 2 promoter is a nucleotide sequence shown in SEQ ID NO. 1.
The Actin (Actin) gene provided by the invention is a constitutive expression gene commonly existing in the biological world, and the enteromorpha Actin gene 2(Actin2) promoter provided by the invention is named as pUPAtin 2 and is derived from large-scale economic green algae, namely enteromorpha (Ulva prolifera).
Compared with the prior art, the method has the following beneficial effects:
1. according to the enteromorpha actin gene 2 promoter, an enteromorpha actin gene 2 upstream segment is obtained through a chromosome Walking technology (Genome Walking), and a specific amplification primer is further designed after the promoter function verification, so that the enteromorpha actin gene 2 promoter can be effectively obtained.
2. The enteromorpha endogenous actin gene 2 promoter obtained by the invention can replace promoters from animal viruses (such as SV40 and CMV) and higher plant viruses (CaMV35S and AMT) used at present in seaweed transgenic research or application, completely avoids the introduction of virus nucleic acid sequences, has higher biological safety, and is beneficial to the application of transgenic seaweeds in the future.
3. Actin is a high-level constitutive expression protein, an actin gene promoter is a high-efficiency expression regulation element, and the expression efficiency of driving exogenous genes of the seaweed transgenic vector constructed by the enteromorpha actin gene 2 promoter can be obviously improved.
4. The enteromorpha actin gene 2 promoter is used for driving the expression of a selective marker gene (such as a herbicide glufosinate resistance gene bar), and the concentration of a screening reagent (such as glufosinate) can be increased by improving the expression level of the resistance gene, so that the generation of resistant clones (false positive) is reduced, the ratio of transgenic resistant clones (true positive) is increased, and the screening efficiency is effectively improved.
5. The life history of the seaweed has two stages of vegetative growth and reproductive stages, and reproductive cells in the reproductive stage are often used as receptor materials for genetic transformation. After transformation, if the foreign gene is integrated at the stage of a single germ cell, all cells in the grown new plant contain the introduced gene; therefore, if screening is performed at the single germ cell stage after transformation, the generation of chimeras can be effectively avoided. The enteromorpha actin gene 2 promoter has the characteristic of life history, can drive the gene to express in the reproductive stage and drive the resistance marker gene to express in the reproductive cell stage, and is used for realizing the purpose.
Drawings
Fig. 1 is a 5' upstream sequence bioinformatics analysis diagram of enteromorpha actin gene 2 provided by the embodiment of the invention.
FIG. 2 is a schematic diagram of enteromorpha transformation vectors pUPA2-GUS, pUPA2NI-GUS, pUPA2NH-GUS, pUPA1-GUS, pBI221 and pSV40-GUS provided by the embodiment of the invention.
FIG. 3 shows the quantitative determination results of the enteromorpha conversion of pUPA2-GUS, pUPA2NI-GUS, pUPA2NH-GUS, pUPA1-GUS, pBI221 and pSV40-GUS provided by the embodiment of the present invention.
FIG. 4 shows RT-PCR detection results of actin1 and actin2 genes of Enteromorpha prolifera in vegetative and reproductive stages in accordance with the present invention.
FIG. 5 is a schematic diagram of the construction of an enteromorpha conversion vector pUPA2-bar provided by an embodiment of the invention.
FIG. 6 shows the PCR detection result of the glufosinate-resistant strain transformed from Enteromorpha prolifera by pUPA2-bar according to the embodiment of the present invention. M is DNA marker Trans2K plus II; 1: blank control; 2-6: bombarding individual plants in the group of pUPA 2-bar; 7-9: and (4) a negative control group.
Detailed Description
The elements of the present invention and the effects achieved by the elements are described in further detail below with reference to specific embodiments.
The enteromorpha endogenous whole life history constitutive actin gene 2 promoter sequence and the application thereof in obtaining transgenic seaweed; can especially realize the expression and the function of the exogenous gene in the reproductive period, can effectively improve the transgenic efficiency, and has important significance for the variety improvement of the algae and the development of gene engineering products.
Example 1: cloning, sequencing and bioinformatics analysis of enteromorpha (U.prolifera) endogenous whole-life-history constitutive actin gene 2 promoter pUPAtin 2
1. Cloning and sequencing of 5' upstream sequence of enteromorpha actin gene 2
The enteromorpha total DNA template is prepared by utilizing a plant genome DNA extraction kit of Tiangen company according to a conventional mode, and the extracted enteromorpha genome DNA strip is clear and complete through 0.8% agarose electrophoresis detection, so that the PCR amplification requirement can be met. Three specific primers (SP1: 5'-CAAGACCGCACCATTGACCTGA-3'; SP2: 5'-GGAGCATCATCGCCAGCAAATC-3'; SP3: 5'-TCAACGAAAGGACGACGAGTGG-3') are designed by utilizing a Genome Walking Kit of TaKaRa company to aim at a 5 'upstream sequence of an enteromorpha endogenous whole-life history constitutive actin gene 2, chromosome Walking is carried out according to the Kit specification, amplified fragments are subjected to agarose gel recovery, TA cloning connection and nucleotide sequence determination, and the total 1739bp of the 5' upstream sequence of the actin gene is obtained.
2. Bioinformatics analysis of 5' upstream sequence of Enteromorpha actin gene 2
By utilizing an on-line analysis tool PLANTCARE (website http:// bioinformatics. psb. agent. be/webtools/plantacare/html) of plant cis-acting elements, bioinformatics analysis discovers that the 5 ' upstream sequence of enteromorpha actin gene 2 not only has some common promoter regulatory elements such as TATA-Box and CAAT-Box, but also has special elements such as a high-efficiency transcription element (5 ' UTR Py-rich stretch) and a 5 ' UTR intron (see figure 1), and the sequence can be preliminarily determined to have the function of a promoter.
Example 2: enteromorpha actin gene 2 promoter pUPAtin 2 driving reporter gene GUS to be efficiently and constitutively expressed in vegetative growth period of algae
1. Cloning of enteromorpha actin gene 2 promoter fragment and construction of pUPA2-GUS vector
According to the 5' upstream sequence of the enteromorpha actin gene 2 obtained in the example 1, aiming at the area with promoter activity predicted by bioinformatics, a primer (actin2-F14B-HindIII: 5-TGATTACGCCAAGCTTccacctatcaactatctgcg tctt-3', wherein the italic letters represent the recognition site for the restriction enzyme Hind III; actin2-R14B-SmaI: 5-AGGGACTGACCACCCGGGcttgtttcagcgcacctcc-3', wherein the italic letters indicate the recognition site for the restriction enzyme Sma I capital letters indicate subsequent use
Needed by the HD Cloning Kit seamless connection Cloning Kit for constructing the vector and the vectorEnd homologous fragment), PCR amplification is carried out, the amplified fragment is recovered by agarose gel, TA clone connection and nucleotide sequence determination are carried out, and the SEQ ID NO 1 sequence is obtained.
The commercial vector pBI221(CaMV35S promoter driving. beta. -glucuronidase GUS gene) from Clontech was used as a starting vector, which was digested simultaneously with restriction enzymes Hind III and Sma I, the original CaMV35S promoter upstream of the GUS gene was excised, subjected to 1% agarose gel electrophoresis, and used OMEGA
The Gel Extraction Kit recovers the enzyme-cleaved large fragment (carrying the GUS gene). Using TaKaRa high fidelity PCR enzyme
Max DNA Polymerase PCR amplification of single clone of the sequenced error-free primer pair actin2-F14B-Hind III/actin 2-R14B-SmaI amplified fragment was used
The HD Cloning Kit seamless connection Cloning Kit connects the obtained PCR product with HindIII and Sma I double digestion large fragments of pBI 221. The ligation products were then transformed into competent cells of the E.coli Top10 strain, and positive transformants were selected on LB plates containing ampicillin. And (3) carrying out amplification culture on the positive transformant, extracting plasmids, carrying out double enzyme digestion identification (Hind III and Sma I) on the plasmids, and sequencing the plasmids with correct enzyme digestion results by a sequencing company. Thus, the algal transformation vector pUPA2-GUS (see FIG. 2) was obtained, and transcription of the β -glucuronidase GUS gene was driven by the enteromorpha actin gene 2 promoter.
In addition, specific primers (actin2-F14: 5'-CCACCTATCAACTATCTGCGTCTT-3'; actin2-R24: 5'-CTTGTTTCAGCGCACCTCCGGACTAAACTCGGTTGATTCTGTTTCTT CCCTTACACCC-3') are designed according to the 5 'upstream sequence of the enteromorpha actin gene 2 obtained in the example 1 for PCR amplification, so that a truncated enteromorpha actin gene 2 upstream sequence is obtained, and the sequence lacks 213 bp (5' UTR intron) from the upstream-251 to-39 of the ATG (initiation codon) of the enteromorpha actin gene 2. This fragment was further amplified by PCR using the specific primers actin2-F14B-HindIII and actin2-R14B-SmaI as described previously, and the promoter-truncated vector pUPA2NI-GUS was constructed in the same manner as pUPA2-GUS (see FIG. 2).
In addition, specific primers (actin2-F14: 5'-CCACCTATCAACTATCTGCGTCTT-3'; actin2-R26: 5'-GATCCCGGCAGGGACGTGCCTGTACACCCAGCTTTCGTGTCG-3'; actin2-F26: 5'-CGACACGAAAGCTGGGTGTACAGGCACGTCCCTGCCGGGATC-3'; actin2-R14: 5'-CTTGTTTCAGCGCACCTCC-3') are designed according to the 5 'upstream sequence of the enteromorpha actin gene 2 obtained in example 1 and are subjected to recombinant PCR amplification, so that a truncated enteromorpha actin gene 2 upstream sequence is obtained, and the sequence lacks 10 bp (high efficiency transcription element 5' UTR Py-rich stretch) from the upstream-264 to-255 of the ATG initiation codon of the enteromorpha actin gene 2. This fragment was further PCR amplified using the aforementioned specific primers actin2-F14B-HindIII and actin2-R14B-SmaI, and the promoter-truncated vector pUPA2NH-GUS (see FIG. 2) was constructed in the same manner as pUPA 2-GUS.
SEQ ID NO.1pUpActin2
CCACCTATCAACTATCTGCGTCTTCTGAGCAACTTGATAATGATGGAGATGCACCTAGGTTGTCAGAGTGTTGGACACATGGTACATCACCCATGTTTTGGCACACCAGCTCCTCCTCATACACTTCATCAACAAAACCAAGCAGATTTTCAAAGCATCGAGTTTCGACAACAGCTGCATCATTATCGCCTAGGCGATGGAGGTTCATTGTACAGTGTCGTTGGCAGCGTACTGAATTTCATTGATCTTCTCTTCGCGGTCAAGTTGTCACAGACATTTCACAGCCTCTCCACGACTGTAGAGGGCTTCGCAGACATAATTTTTATGGCAGCCTCTCTCGGCAAACATTTCAACAACATCACGAGGCGCAACATCAGTCGCAACTGGCTTTCTGCGCATACTGGAGCCACCAACATCTGCCAAATCAGCCAGATTTGCCTCAACCCAACTTTCAATTCGGTCTTGTCTTGTGTCGAGCCTAGAAGTTGCTCACTGCCACATGACAGGTGCACACAGGCTACGGAAACTGACTCCCACAGCATGAGCAAGCGACACGGACTTAAGTACTAGGAGCCAGCAACAGGTGTGCTGCGAGTTACATAATGGCCACCGTGCTTTGCTAGTGATATTTGTCCTATGAGGTCGGTCAAAGCCATCTGAATACAGCCCTTTTTGGCCTCAACTTGGATTTTAAAGCACCCTTCACGTTAAAATGCGATAAAGCGAAACGGCCTCTTTAAAGCCTCGAAATCGTTTTAACGTTAAAACTGTCAAGTAAAATAGCATTTTAAAGTAGGTGAACATGGGCGTAACGTTCAATTTACAGCTCTACTCGCCGTTTGCTGCGATGGTGCTGAATTCTCTCATATCGACAGCTCGCATGCTGCTTCAAAGCGGATGAAAAGGCGTAGTGACGTCGGTGTGAAGATTTTCAAAAACATGTCATGTTGAATTTTTCTCGACCCGCACAGCGTGTGTCTGACGAACCACAAATACGGAGCAGCCCGGTTATTCCTGCCACGAAGAGGAAACTTTTGCCGGGTCCATTCCCTACGGAGCCCGCAACGACTCTGTGACGTTCGTACGGTAATGTCCCGCTATTGGTGGCTCAAATCAGTGCCTGTGCGGACGATCAGTGTGCTGCTAAAACACGACAGAGAAAGCTCGACATTGAAAACGTTACTGCGTCCTGTCTTATCCTGTGGAACCAATAAATAACTCATCGTTACATTATGCGCACGGTGCGCATCCAGGACCCAGGTGGTGGGGGCCTGGTGGCGACAGGGATTCGTGTCGCGACATGTAGCGAATGCAGCTGAATGGTTTTGGCAGGAGGGCAAGGCGAGCAATGCACGAGGCTTAGACCGGTCAACCTTCGATTCGAGGTAGCTCCACAAATATGAAGTCGACAGTGCACACGCAAGTTGCATTGGAAGCAGTCGTTTGTGACGTCAACGACACGAAAGCTGGGTGTAAGGGAAGAAACAGGCACGTCCCTGCCGGGATCATCCAGAAATGTCACATGATGTCGCCGCCTTTTGCGGAGTACATGGATTCATGACCGAGCTGCGTATGTGGTTGTGTACTCAAGTGTCGACTTGATGGTGGGTTGCGCACGTCGAGGCCTTGTTGCAGATGTGCCCGTGACCCGTGAGTGTGCTTTGCAACTTTTTCTGCCATTTGAATTGTCTCGAGGTGCAGAATCAACCGAGTTTAGTCCGGAGGTGCGCTGAAACAAG
(a) Sequence characteristics:
length: 1739 base pairs
Type: nucleotide, its preparation and use
Chain type: single strand
Topology structure: linearity
(b) Molecular type: DNA
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial sources were: enteromorpha (Ulva prolifera)
2. Gene gun transformed enteromorpha
1) Preparation of particle of gene gun
Weighing 60mg of gold powder (consumable part matched with a gene gun of American Bio-Rad company, the diameter of which is 1.0 μm), adding 1ml of absolute ethyl alcohol, and violently oscillating for 1 minute; centrifuging at 10000 rpm for 10 s, and removing supernatant; adding 1ml of sterile water for resuspension, centrifuging to separate out supernatant, repeating for 3 times, finally suspending the gold powder in 1ml of sterile water, subpackaging according to 50 mu l of each part, and storing at 4 ℃ for later use.
When in transformation, 1 part of gold powder suspension (50. mu.l of 1 part) is transferred into a 1.5ml centrifuge tube for oscillation, and 5. mu.l of DNA (namely the ready-made vector or the transformation vector obtained in the step 1, comprising 6 of pBI221, pSV40-GUS, pUPA1-GUS, pUPA2-GUS, pUPA2NI-GUS or pUPA2NH-GUS, 1. mu.g/. mu.l) and 50. mu.l of DNA are sequentially added in the continuous oscillation processl CaCl2(2.5mol/L), 20. mu.l spermidine (0.1mol/L), shaking for 3 minutes; centrifuging at 10000 rpm for 10 s, and removing the supernatant; rinsing with 250 μ l ethanol, centrifuging to remove supernatant, and repeating for 1 time; 60 μ l of absolute ethanol resuspended the gold powder.
2) Gene gun bombarded seaweed
Taking cultured enteromorpha (U.prolifera) thallus, shearing into 2 cm-long algae sections by aseptic operation, using round sterile bolting silk (400 meshes, diameter 4cm) as a bearing body of the algae sections during conversion, and uniformly spreading the algae sections in the center to form a round effective bombardment range with the diameter of about 2cm as a sample. One bombardment per sample, approximately 12. mu.l of gold powder suspension per bombardment. The transformation is carried out in a sterile room or a clean bench by using a high-pressure helium type gene gun (model: PDS1000/He) which is a product of American Bio-Rad company; wiping and disinfecting the surface and the inside of an operation table and a gene gun by using 70% ethanol, wherein consumable materials for bombardment comprise a fissile film (Rupture disk), a DNA slide glass (Macrocarrier) and a blocking net (Stopping screen), soaking in 70% absolute ethanol for 20 minutes in advance, and drying under an ultraviolet lamp for later use; the conversion parameters were: the recipient cells were 6.0cm from the barrier mesh and a vacuum of 28 inches of mercury was applied. The experiment set up was a blank control group bombarded with naked gold powder without plasmid DNA, and the procedure was the same.
Quantitative detection of GUS Gene
After transformation, transferring the seaweed materials of the transformation group and the control group into VSE culture solution at a culture temperature of 15.0 + -0.5 deg.C and a light-dark period of 12h/12h, wherein the light intensity is about 50 mmol.m-2·s-1. GUS enzyme can catalyze and decompose non-fluorescent 4-methyl umbelliferone-beta-D-glucuronide (4-MUG) into fluorescent 4-methyl umbelliferone (4-MU), and based on the principle and the standard method, the activity of the GUS enzyme which is recombined and expressed in the transgenic seaweed can be quantitatively measured, so that the promoter activities of different promoters are reflected.
The specific operation steps are as follows: after 48h of transformation, the transgenic seaweed fronds were frozen in liquid nitrogen and ground into powder, and 1ml of protein extraction buffer (50 mM Na as protein extraction buffer) was added2HPO4(pH 7.0),10mM Na2EDTA (pH 8.0), 0.1% Triton X-100,10 mM. beta. -mercaptoethanol),after mixing, centrifuging at 4 ℃ 10000g for 15min, taking supernatant, and storing in ice bath for later use. Determination of soluble protein concentration in supernatant Using Bradford protein quantitation kit of Tiangen Co., 60. mu.l of supernatant was added to 540. mu.l of a reaction solution (50 mM Na as a reaction solution) preheated at 37 ℃2HPO4(pH 7.0),10mM Na2EDTA (pH 8.0), 0.1% Triton X-100,10 mM. beta. -mercaptoethanol, 2mM 4-methylumbelliferyl-. beta. -D-glucuronide, 100. mu.l of the reaction mixture was taken out to 900. mu.l of the reaction termination solution (0.2M Na. beta. -D-glucuronide) at 0, 5, 15, 30, and 60min, respectively2CO3) Detecting the fluorescence value at 455nM under 365nM exciting light by using a microplate reader, and calculating the activity of GUS enzyme with the unit of nM MU min-1(mg protein)-1。
The detection result shows that both enteromorpha actin gene 2 promoter pUPAtin 2 and enteromorpha pUPAtin 1 promoter, high-efficiency promoter CaMV35S in higher plants and high-efficiency promoter SV40 in mammals can drive the high-efficiency constitutive expression of GUS gene in the vegetative growth period of enteromorpha, wherein the activity of pUPAtin 2 and SV40 promoter is equivalent to about 1.5 times that of CaMV35S promoter, and the pUPAtin 2 and the SV40 promoter are slightly lower than that of enteromorpha pUPA1 promoter (see figure 3). In addition, the results of transformation of the vector pUPA2NI-GUS or pUPA2NH-GUS after promoter truncation into Enteromorpha show that the promoter activities of the vector pUPA2NI-GUS and the vector pUPA2NH-GUS are both obviously reduced compared with the full-length promoter and are almost reduced to the background level, and the 5 'UTR intron and the high-efficiency transcription element 5' UTR Py-rich stretch in the Enteromorpha actin gene 2 promoter pUPAActin 2 are necessary elements for maintaining the high activity of the pUPAActin 2 promoter (see figure 3).
The results prove that the 5 ' upstream sequence of the enteromorpha endogenous constitutive actin gene 2 obtained by amplification in example 1 has a promoter function, the 5 ' UTR intron and the high-efficiency transcription element 5 ' UTR Py-rich stretch are essential elements, and the promoter can efficiently drive the exogenous gene to realize high-efficiency constitutive expression in the vegetative growth period of the enteromorpha.
Example 3: differential analysis of transcription levels of enteromorpha actin genes actin1 and actin2 in different stages of seaweed life history
1. RT-PCR primer design of enteromorpha actin genes actin1 and actin2
RT-PCR specific amplification primers for performing transcription level difference analysis are designed according to cDNA sequences of enteromorpha actin gene actin1 and actin2 genes, and are respectively (ACT1RT-F: 5'-CCGATGGGCAAGTAATCAC-3'; ACT1RT-R: 5'-TGAAGGTTGTATCATGAACTCC-3'; ACT2RT-F: 5'-AATCCCGCACTGTTAGGC-3'; ACT2RT-R: 5'-CAAACATGGTTGTCCCGC-3')
2. Preparation of materials in vegetative growth stage and reproduction stage in whole life history of enteromorpha
Culturing the seaweed material in VSE culture solution at 15.0 + -0.5 deg.C for 12h/12h with light intensity of 50 mmol.m-2·s-1. Then, taking out part of the seaweed and cutting into pieces of 1-5mm2Right and left size, induce reproduction. After about 60-72h, the cut algal bodies were observed by microscope, and algal fragments in which all vegetative cells were differentiated into germ cells were selected. Finally, the seaweed material in vegetative growth stage and reproductive stage is stored frozen in a refrigerator at-80 deg.C.
3. RT-PCR analysis of actin genes actin1 and actin2 in vegetative growth stage and reproductive stage in whole life history of enteromorpha
The Enteromorpha total RNA template is extracted and prepared by using a plant RNA extraction kit of the whole gold company according to the instruction, and the extracted Enteromorpha RNA strip is clear and complete through denaturing agarose electrophoresis detection, so that the requirement of reverse transcription PCR amplification can be met. Using PrimeScriptTMII 1st Strand cDNA Synthesis Kit to prepare Enteromorpha cDNA template. Using PrimeScriptTMThe RT reagent Kit with gDNA Eraser Kit performs fluorescent real-time quantitative PCR reaction. The reference gene is Enteromorpha prolifera 18S gene. The relative quantitative data analysis adopts a comparison threshold value method (2)-△△CtMethod), 3 replicates were set for each treatment.
RT-PCR analysis results show that the transcription level of the enteromorpha actin gene actin1 in vegetative growth cells is obviously higher than that of germ cells, and the transcription level of the enteromorpha actin gene actin2 is higher in both the vegetative stage and the reproductive stage and has no obvious difference (see figure 4). According to the experimental result of functional verification of the embodiment 2, the two promoters are both high-efficiency constitutive promoters in the vegetative growth stage; however, when enteromorpha cells are in a germ cell stage, the activity of the promoter pUPAtin 1 is almost reduced to 0, and the promoter pUPAtin 2 still has high transcription activity and is an endogenous constitutive promoter with whole life history.
Example 4: enteromorpha actin gene 2 promoter pUPAtin 2 drives selection marker gene bar to express in enteromorpha germ cells to realize screening of transformants
1. Cloning of enteromorpha actin gene promoter fragment and construction of pUPA2-bar vector
Specific primers for pUpActin 2(actin 2-2 FB-PstI: 5'-TGATTACGCCCTGCAGccacctatcaactatctgcg-3', wherein italics letters represent recognition sites for restriction enzyme Pst I; actin2 RB-SmaI: 5'-TTCTGGGCTCATCCCGGGcttgtttcagcgcacctccgg-3', wherein italics letters represent recognition sites for restriction enzyme SmaI; capital letters represent recognition sites for subsequent use) were designed for the bioinformatically predicted region having promoter activity according to the 5 ' upstream sequence of Enteromorpha actin gene 2 obtained in example 1
The HD Cloning Kit is seamlessly connected with the Cloning Kit to construct a fragment which is required by the vector and is homologous with the end of the vector), PCR amplification is carried out, the amplified fragment is recovered by agarose gel, TA Cloning connection is carried out, and nucleotide sequence determination is carried out, so as to obtain the fragment with the same sequence as SEQ ID NO. 1.
The method comprises the following steps of taking a carrier p35S-bar (a CaMV35S promoter drives a herbicide glufosinate resistance gene bar) constructed by a laboratory as an initial carrier, carrying out double digestion on the carrier by using restriction enzymes Pst I and Sma I, cutting off a bar gene front CaMV35S promoter, carrying out electrophoresis on the cut promoter by using 1% agarose gel, and then using OMEGA
The Gel Extraction Kit recovers the digested large fragment. Using TaKaRa high fidelity PCR enzyme
Max DNA Polymerase for the above sequencing-free actin2FB-PCR amplification of PstI and actin2RB-SmaI fragments monoclonal using
The HD Cloning Kit seamless connection Cloning Kit connects the obtained PCR product with Pst I and Sma I double-enzyme large fragments of p 35S-bar. The ligation products were then transformed into competent cells of the E.coli Top10 strain, and positive transformants were selected on LB plates containing ampicillin. And (3) carrying out amplification culture on the positive transformant, extracting plasmids, carrying out double enzyme digestion identification (Pst I and Sma I) on the plasmids, and sequencing the plasmids with correct enzyme digestion results by a sequencing company. Thus, the algal transformation vector pUPA2-bar (see FIG. 5) was obtained, and transcription of the bar gene was driven by the enteromorpha actin gene 2 promoter.
2. Gene gun transformed enteromorpha prolifera germ cell
1) Preparation of particle of gene gun
Referring to step 1 of example 2, gold powders carrying two plasmids, pUPA1-bar and pUPA2-bar, respectively, were co-prepared.
2) Bombardment of germ cells of seaweed by gene gun
Reference example 3 step 2 seaweed was first subjected to reproductive induction and biolistic transformation at the sporangial stage, the transformation process being referred to example 2 step 2.
Detection of the Bar Gene
After transformation, the alga body is recovered for 8 hours in a dark place, then herbicide glufosinate-P is added into the culture solution to enable the final concentration of the effective component PPT to be 40 mu g/ml, the screening time is one week, germ cells which are scattered in the period are diluted and transferred into VSE culture solution for amplification culture. The results showed that the bombardment of the group pUPA1-bar did not result in resistant plants, whereas the bombardment of the group pUPA2-bar resulted in resistant plants. When enough biomass is available, a total DNA template of the genome is prepared, and PCR detection is carried out by designing specific primers (bar 1-F: 5'-TCTGCACCATCGTCAACCACTACA-3'; bar 1-R: 5'-TCAAATCTCGGTGACGGGCAGGAC-3') according to a bar gene region, the PCR result of the wild type strain is negative, and the resistant strain is positive (see figure 6).
The results prove that under the high dose selection pressure of the herbicide glufosinate, the enteromorpha actin gene 2 promoter can successfully drive the bar gene to be transcribed and expressed at the germ cell stage in the life history of the enteromorpha, so that the positive transformed algae successfully obtains the resistance to the glufosinate, and the method can be used for constructing a stable expression system and obtaining the homozygous algae.
Sequence listing
<110> oceanic research institute of Chinese academy of sciences
<120> endogenous whole life history constitutive promoter of seaweed and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1739
<212> DNA
<213> Enteromorpha (Ulva prolifera)
<400> 1
ccacctatca actatctgcg tcttctgagc aacttgataa tgatggagat gcacctaggt 60
tgtcagagtg ttggacacat ggtacatcac ccatgttttg gcacaccagc tcctcctcat 120
acacttcatc aacaaaacca agcagatttt caaagcatcg agtttcgaca acagctgcat 180
cattatcgcc taggcgatgg aggttcattg tacagtgtcg ttggcagcgt actgaatttc 240
attgatcttc tcttcgcggt caagttgtca cagacatttc acagcctctc cacgactgta 300
gagggcttcg cagacataat ttttatggca gcctctctcg gcaaacattt caacaacatc 360
acgaggcgca acatcagtcg caactggctt tctgcgcata ctggagccac caacatctgc 420
caaatcagcc agatttgcct caacccaact ttcaattcgg tcttgtcttg tgtcgagcct 480
agaagttgct cactgccaca tgacaggtgc acacaggcta cggaaactga ctcccacagc 540
atgagcaagc gacacggact taagtactag gagccagcaa caggtgtgct gcgagttaca 600
taatggccac cgtgctttgc tagtgatatt tgtcctatga ggtcggtcaa agccatctga 660
atacagccct ttttggcctc aacttggatt ttaaagcacc cttcacgtta aaatgcgata 720
aagcgaaacg gcctctttaa agcctcgaaa tcgttttaac gttaaaactg tcaagtaaaa 780
tagcatttta aagtaggtga acatgggcgt aacgttcaat ttacagctct actcgccgtt 840
tgctgcgatg gtgctgaatt ctctcatatc gacagctcgc atgctgcttc aaagcggatg 900
aaaaggcgta gtgacgtcgg tgtgaagatt ttcaaaaaca tgtcatgttg aatttttctc 960
gacccgcaca gcgtgtgtct gacgaaccac aaatacggag cagcccggtt attcctgcca 1020
cgaagaggaa acttttgccg ggtccattcc ctacggagcc cgcaacgact ctgtgacgtt 1080
cgtacggtaa tgtcccgcta ttggtggctc aaatcagtgc ctgtgcggac gatcagtgtg 1140
ctgctaaaac acgacagaga aagctcgaca ttgaaaacgt tactgcgtcc tgtcttatcc 1200
tgtggaacca ataaataact catcgttaca ttatgcgcac ggtgcgcatc caggacccag 1260
gtggtggggg cctggtggcg acagggattc gtgtcgcgac atgtagcgaa tgcagctgaa 1320
tggttttggc aggagggcaa ggcgagcaat gcacgaggct tagaccggtc aaccttcgat 1380
tcgaggtagc tccacaaata tgaagtcgac agtgcacacg caagttgcat tggaagcagt 1440
cgtttgtgac gtcaacgaca cgaaagctgg gtgtaaggga agaaacaggc acgtccctgc 1500
cgggatcatc cagaaatgtc acatgatgtc gccgcctttt gcggagtaca tggattcatg 1560
accgagctgc gtatgtggtt gtgtactcaa gtgtcgactt gatggtgggt tgcgcacgtc 1620
gaggccttgt tgcagatgtg cccgtgaccc gtgagtgtgc tttgcaactt tttctgccat 1680
ttgaattgtc tcgaggtgca gaatcaaccg agtttagtcc ggaggtgcgc tgaaacaag 1739
Claims (7)
1. An endogenous whole-life-history constitutive promoter of seaweed, characterized in that: enteromorpha actin gene 2(actin2) The promoter is the nucleotide sequence shown in SEQ ID NO. 1.
2. An endogenous full-life history constitutive promoter of seaweed as claimed in claim 1 wherein: the promoter is obtained from marine green algae.
3. An algal expression system, characterized by: an expression system comprising the promoter of claim 1.
4. Use of the endogenous full-life history constitutive promoter of seaweed as claimed in claim 1, wherein: the promoter is applied to driving the high-efficiency expression of exogenous genes in the vegetative growth period of the seaweed.
5. Use of an endogenous full-life history constitutive promoter of seaweed as claimed in claim 4 wherein: the promoter is applied to driving the high-efficiency expression of exogenous genes in the seaweed reproductive stage.
6. Use of an endogenous whole-life history constitutive promoter of seaweed according to claim 4 or 5, characterized in that: the promoter is applied to enteromorpha genetic breeding.
7. Use of an endogenous whole-life history constitutive promoter of seaweed according to claim 4 or 5, characterized in that: the promoter is applied to the improvement of the quality of the seaweed or the construction of a seaweed expression system.
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| CN103497951A (en) * | 2013-09-05 | 2014-01-08 | 上海海洋大学 | Kelp gametophyte lhcf 6 gene promoter and application thereof |
| CN105907761A (en) * | 2016-01-04 | 2016-08-31 | 云南农业大学 | Paddy metallothionein gene OsMT2bL promoter and application thereof |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
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| CN103497951A (en) * | 2013-09-05 | 2014-01-08 | 上海海洋大学 | Kelp gametophyte lhcf 6 gene promoter and application thereof |
| CN105907761A (en) * | 2016-01-04 | 2016-08-31 | 云南农业大学 | Paddy metallothionein gene OsMT2bL promoter and application thereof |
| CN106520766A (en) * | 2016-05-31 | 2017-03-22 | 中国科学院海洋研究所 | Seaweed endogenesis constructive promoter and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Molecular Cloning and Analysis of a Cytosolic Hsp70 Gene from Enteromorpha prolifera (Ulvophyceae, Chlorophyta);Wandong Fu等;《Plant Mol Biol Rep》;20101231;第430-437页 * |
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