CN117025591A - Biological mosquito control method using microalgae - Google Patents
Biological mosquito control method using microalgae Download PDFInfo
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Abstract
The invention provides double-stranded RNA, which consists of a forward fragment and a reverse fragment, wherein the nucleotide sequence of the forward fragment is the sequence shown as SEQ ID NO.2 in a sequence table, and the nucleotide sequence of the reverse fragment is the sequence shown as SEQ ID NO.3 in the sequence table. The double-stranded RNA and the expression vector containing the double-stranded RNA provided by the invention transform chlamydomonas and chlorella to obtain engineering algae strains, and feed the aedes larva, so that the normal growth and development of the aedes larva can be retarded, the pupation and eclosion of the aedes larva can be inhibited, the aedes larva can be effectively killed, the method can be used for biological control of aedes, and meanwhile, the method has the advantages of environmental friendliness and biological ecological safety, and the pesticide consumption is reduced.
Description
Technical Field
The invention relates to the field of mosquito prevention and control, in particular to a biological mosquito control method by utilizing microalgae.
Background
Mosquito-borne infectious diseases are public health problems that are valued worldwide. Among the most common mosquito-borne viruses are derived from the flaviviridae family of flaviviruses, dengue virus (DENV), zika virus (Zika virus, ZIKV) and Yellow Fever Virus (YFV), and chikungunya virus (Chikungunya virus, CHIKV), of the alphaviridae genus, respectively. The transmission medium of the four diseases is Aedes albopictus.
The use of pyrethroid insecticides and organophosphate insecticides is the most common means of controlling mosquitoes, but the high cost and enhancement of mosquito resistance gradually expose their disadvantages, and thus a new type of mosquito-vector intervention is needed. Sterile Insect Technology (SIT) and Wolbachia infection male mosquito technology are relatively advanced biological mosquito control means, but radiation in the former experiment can cause low mating ability of partial male Aedes, and partial non-target arthropod insects in the latter experiment are also infected by Wolbachia strains, so that the method has hidden danger on biological and ecological security threat. Thus, both techniques are still in need.
RNA interference is one of the most widely used tools in cell biology, and can trigger the silencing effect of a target gene by double-stranded RNA (dsRNA). RNAi gene fragments are transmitted into organisms in a manner that is classified into non-microbial and microbial. Non-microbial mediated RNAi includes injection, infusion and nanoparticle mediated methods, while microbial mediated includes bacterial, yeast and microalgae mediated methods.
Microalgae are a multi-line group consisting of single-cell eukaryotes, living in aquatic and terrestrial environments, and can be photoautotrophic, heterotrophic, and mixotrophic. In nature, part of microalgae are used as food sources by Aedes larva due to their small size and rich nutrition.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and constructs an expression vector by taking CYP314A1 gene (GenBank accession number: LOC109413055, full length 3510bp of the gene, using ORF Finder to predict that the open reading frame of the gene is 1617bp and the length of the coded amino acid sequence is 538 aa) as an RNAi target, thereby being applicable to biological control of aedes and having the characteristics of environmental friendliness and higher commercial application value.
The first aspect of the invention provides a double-stranded RNA, which consists of a forward fragment and a reverse fragment, wherein the nucleotide sequence of the forward fragment is the sequence shown as SEQ ID NO.2 in the sequence table, and the nucleotide sequence of the reverse fragment is the sequence shown as SEQ ID NO.3 in the sequence table.
In a second aspect, the present invention provides an expression vector comprising a receiving vector, a supply vector and the double stranded RNA of claim 1.
The receiving vector is a vector for receiving and carrying exogenous gene fragments in the process of polygene assembly, and can be a commonly used receiving vector in the process of polygene assembly, and the invention is not particularly limited. In a specific embodiment of the present invention, the recipient vector employs pMaa7IR/XIR, but it is understood that the present invention may also employ other plasmids and the like.
The supply vector is a vector for transferring the target gene to the receiving vector in the process of multi-gene assembly, and a supply vector commonly used in the process of multi-gene assembly can be used, and the present invention is not particularly limited thereto. In a specific embodiment of the present invention, pT282 is used as the receiving vector, but it should be understood that other plasmids and the like may be used in the present invention.
Preferably, the receiving vector is pMaa7IR/XIR, the supply vector is pT282, the forward fragment is located between the HindIII and BamHI restriction sites of the pT282 vector, the reverse fragment is located between the SalI and XbaI restriction sites of the pT282 vector, and the receiving vector is ligated to the supply vector by EcoRI cleavage.
In a third aspect, the invention provides a transgenic microalgae prepared by transforming the edible microalgae of the aedes larva with the expression vector in the second aspect.
Preferably, the microalgae are chlamydomonas (e.g., chlamydomonas reinhardtii, etc.), chlorella (e.g., chlorella pyrenoidosa, chlorella vulgaris, etc.).
In a fourth aspect, the invention provides the use of double-stranded RNA according to the first aspect of the invention, or an expression vector according to the second aspect of the invention, or a transgenic microalgae according to the third aspect of the invention, in the preparation of an aedes preparation.
In a fifth aspect, the invention provides the use of a double-stranded RNA according to the first aspect of the invention, or an expression vector according to the second aspect of the invention, or a transgenic microalgae according to the third aspect of the invention, in the preparation of a formulation for retarding the normal growth and development of Aedes larvae.
In a sixth aspect, the invention provides a biological mosquito control method using microalgae, and the transgenic microalgae in the third aspect of the invention is used for feeding mosquito larvae.
The double-stranded RNA and the expression vector containing the double-stranded RNA provided by the invention transform chlamydomonas and chlorella to obtain engineering algae strains, and feed the aedes larva, so that the normal growth and development of the aedes larva can be retarded, the aedes larva can be killed, the method can be used for biological control of aedes, and simultaneously has the advantages of environmental protection and biological ecological safety, and the pesticide consumption is reduced.
Drawings
FIG. 1 shows the result of EcoRI cleavage after vector construction. M is DNA Maker-D10000,1: maa7IR/CYP314A1IR.
FIG. 2 shows the PCR amplification results of transgenic Chlamydomonas strains. Wherein M is DNA Maker-D10000, and 1-10 is Maa7IR/CYP314A1IR No. 1-10 transformant strain.
Fig. 3 shows aedes larval length (fig. 3A) and body width measurements (fig. 3B) for different foods, where a: aedes larval length data, B: aedes larva width data; water: clear water group, fodder: murine food group, CC425: non-transgenic chlamydomonas group, ma 7: pMaa7/IR (empty) group, CYP314: recombinant chlamydomonas CYP314A1-RNAi group.
Detailed Description
The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Test aedes: aedes albopictus eggs stored in the Biotechnology institute of Tropical agricultural sciences B402 laboratory are hatched in a culture environment of 26 (+ -1) deg.C, 75 (+ -2) percent humidity and a dark/12-hour light cycle of 12 hours including 1 hour dusk period at the beginning and end of each cycle.
Example 1 method for constructing Aedes albopictus CYP314RNAi expression vector
(1) Acquisition of Aedes albopictus CYP314RNAi Forward and reverse fragments
The coding region sequence of the CYP314A1 part of the gene of Aedes aegypti supplied by GenBank was used to construct an interference fragment (sequence shown in SEQ ID NO. 1) of the RNA vector by PCR amplification by the company of Biotechnology (Shanghai) Co., ltd. Ggtactgaagtatcctagca and ctttgactgaggtcgatga are used as primers. The reaction system was 1. Mu.L of cDNA template, 1. Mu.L of primer F, 1. Mu.L of primer R, 1 XMix 12.5. Mu.L, 0.5. Mu.L of Taq enzyme, 1. Mu.L of DMSO, and up to 25. Mu.L of dd H2O. The PCR reaction condition is that the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95℃for 1min, annealing at 58℃for 30s, extension at 72℃for 1min,35 cycles; extending at 72℃for 10min. The PCR products were detected by electrophoresis on a 1% agarose gel. And then connecting with pMD18-T, transforming E.coliDH5α competent cells by using a connection product, picking a monoclonal strain, culturing, and performing colony PCR, and obtaining pMD-18T-CYP314A1 by sequencing and verifying correctness. The forward fragment (sequence shown in SEQ ID NO. 2) obtained by double cleavage with HindIII and BamHI, and the reverse fragment (sequence shown in SEQ ID NO. 3) obtained by double cleavage with SalI and XbaI.
Double-digestion of pMD-18T-MAPK4 and an intermediate vector pT282 with HindIII and Bam HII, respectively, gel-cutting to recover the digested CYP314A1 forward fragment and linearization vector pT282, and ligating overnight at 16deg.C with T4 ligase to transform E.coli DH5a competent cells, picking up monoclonal colonies, extracting plasmids, and double-digestion with HindIII and Bam HII to identify recombinant plasmids to obtain pT282-CYP314A1 forward fragment plasmids. Then, the recombinant plasmids of the p MD-18T-CYP314A1 and p T282-CYP314A1 forward fragments are subjected to double digestion by using Xba I and Sal I, reverse fragments of the CYP314A1 and linearized pT282-CYP314A1 forward fragment vectors are recovered by cutting gel and connected, E.coli DH5a competent cells are transformed, monoclonal colonies are picked up, plasmids are extracted, and the plasmids are identified by double digestion by using Xba I and Sal I. Then EcoRI enzyme digestion identification is carried out, and the intermediate vector pT282-CYP314A1 inserted in the forward and backward directions from the CYP314A1 interference fragment is obtained.
The intermediate vector p T-282-CYP 314A1 and RNAi empty vector p Maa7IR/XIR are digested by EcoRI, the forward and reverse repeated fragment of CYP314A1 and the linearized pMaa7IR/XIR vector are recovered, the recovered vector is subjected to dephosphorylation treatment, then the ligation product is connected overnight at 16 ℃, the ligation product is transformed into escherichia coli, and PCR and EcoRI digestion identification are carried out after plasmid extraction, and the result shows that the CYP314RNAi expression vector is successfully constructed, and the result is shown in figure 1.
EXAMPLE 2CYP314A1 RNAi expression vector transformation of Chlamydomonas reinhardtii
The CYP314A1 RNAi expression vector was transformed into Chlamydomonas reinhardtii by shock transformation (shock conditions: MODE HV, VOLTAGE 0905V, PLENGTH 0.22MS, PULSES 90, INTERVAL 200ms, POLARITY UNIPLAR), and twice screening was performed using TAP culture with paromomycin and TAP medium with 5-Fluoroindole, L-trytophan. 113 transformed CYP314A1 RNAi Chlamydomonas reinhardtii strains are obtained.
The transgenic Chlamydomonas reinhardtii DNA was extracted, primers were designed for Maa7IR/XIR vector, 58 positive strains, each with a PCR amplified band meeting the expectations, were obtained, and the partial results are shown in FIG. 2.
It should be noted that other chlamydomonas reinhardtii, other edible microalgae such as chlorella and other aedes larva may be used in addition to chlamydomonas reinhardtii in the present embodiment, and the present invention is only exemplified herein by chlamydomonas reinhardtii and is not limited thereto. According to the embodiment, other algae can achieve the same effect as chlamydomonas reinhardtii, and the invention is not described herein.
Example 3 Small Scale larval feeding experiments
Experimental grouping: the murine diet group (murine diet feeding), the clear water group (clear water feeding), the non-transgenic chlamydomonas group (chlamydomonas CC425 feeding), the pMaa7/IR (empty vector chlamydomonas feeding), the recombinant chlamydomonas CYP314A1-RNAi group (CYP 314A1 RNAi chlamydomonas reinhardtii feeding).
The test method comprises the following steps: after centrifugation, 10mL of fresh transgenic algae liquid was taken daily and fed to aedes albopictus larvae (the same experiment was performed with aedes aegypti, with similar results).
The body length and body width of each group of larvae on the fourth day are measured under a microscope, and the result shows that the body size of the clear water group larvae is not greatly changed from the L1 larva period, and the body length and body width of the recombinant chlamydomonas CYP314A-RNAi group larvae are respectively reduced by 21.2 percent and 38.6 percent compared with those of the non-transgenic chlamydomonas group larvae, so that the recombinant chlamydomonas can slow the normal growth and development of the Aedes larvae (figure 3AB, table 1).
TABLE 1 measurement results of aedes larva body Length and body width
| Aedes larva length (mum) | Aedes larva width (mum) | |
| Clean water group | 1839 | 213 |
| Feed group | 5860 | 801 |
| Non-transgenic Chlamydomonas CC425 group | 4640 | 632 |
| pMaa7/IR (empty) group | 4050 | 475 |
| Recombinant chlamydomonas CYP314A1-RNAi group | 3657 | 388 |
The death number of each group of larvae is continuously detected for 11 days, and the statistical analysis result shows that: the death rate of the larvae in the non-transgenic chlamydomonas group and the feed group is 0%, which indicates that the chlamydomonas strain can be used as a nutrition source of the aedes larvae; the larval mortality of the transgenic chlamydomonas was 100%, with the larvae of the group 2 transformant all dying on the fifth day, indicating that the recombinant strain had significant lethality to the aedes larvae (table 2).
TABLE 2 aedes larva cumulative mortality (%)
| Day1 | Day2 | Day3 | Day4 | Day5 | Day6 | Day7 | Day8 | Day9 | Day10 | Day11 | |
| Water | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 3.33 | 10.00 | 10.00 | 13.33 | 16.67 | 16.67 |
| Fodder | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| CC425 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| Maa7 | 0.00 | 0.00 | 6.67 | 13.33 | 16.67 | 20.00 | 23.33 | 23.33 | 26.67 | 26.67 | 30.00 |
| CYP314-1 | 66.67 | 76.67 | 76.67 | 83.33 | 86.67 | 86.67 | 86.67 | 100.00 | 100.00 | 100.00 | 100.00 |
| CYP314-2 | 50.00 | 56.67 | 56.67 | 93.33 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 |
In table 2, water: clear water group, fodder: murine food group, CC425: non-transgenic chlamydomonas group, ma 7: pMaa7/IR (empty) group, CYP314: recombinant chlamydomonas CYP314A1-RNAi group.
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.
Claims (8)
1. A double-stranded RNA is characterized by comprising a forward fragment and a reverse fragment, wherein the nucleotide sequence of the forward fragment is shown as SEQ ID NO.2 in a sequence table, and the nucleotide sequence of the reverse fragment is shown as SEQ ID NO.3 in the sequence table.
2. An expression vector comprising a receiving vector, a supply vector, and the double-stranded RNA of claim 1.
3. The expression vector of claim 2, wherein the receiving vector is pMaa7IR/XIR, the supply vector is pT282, the forward fragment is located between the Hind iii and bamhi restriction enzyme sites of the pT282 vector, the reverse fragment is located between the sali and xbai restriction enzyme sites of the pT282 vector, and the receiving vector and the supply vector are joined by ecori cleavage.
4. A transgenic microalga prepared by transforming an aedes larva edible microalgae with the expression vector of claim 2 or 3.
5. The transgenic microalgae of claim 4 wherein the microalgae is chlamydomonas or chlorella.
6. Use of the double-stranded RNA of claim 1, or the expression vector of claim 2 or 3, or the transgenic microalgae of claim 4 or 5, for the preparation of an aedes preparation.
7. Use of the double-stranded RNA of claim 1, or the expression vector of claim 2 or 3, or the transgenic microalgae of claim 4 or 5, for the preparation of a formulation for retarding the normal growth and development of aedes larva.
8. A biological mosquito control method using microalgae, characterized in that the transgenic microalgae according to claim 4 or 5 are used for feeding mosquito larvae.
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