CN108588072B - dsRNA of cotton bollworm CYP4L11 gene and application thereof - Google Patents

dsRNA of cotton bollworm CYP4L11 gene and application thereof Download PDF

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CN108588072B
CN108588072B CN201810385347.0A CN201810385347A CN108588072B CN 108588072 B CN108588072 B CN 108588072B CN 201810385347 A CN201810385347 A CN 201810385347A CN 108588072 B CN108588072 B CN 108588072B
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cotton bollworm
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靳明辉
萧玉涛
程英
廖重宇
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Agricultural Genomics Institute at Shenzhen of CAAS
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Abstract

The invention relates to the field of agricultural pest control, in particular to dsRNA of a cotton bollworm CYP4L11 gene and application thereof. The dsRNA of the cotton bollworm CYP4L11 gene has at least 80 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1. The invention also provides a coding gene of the dsRNA. The invention also provides application of the dsRNA or the coding gene thereof in preventing and treating cotton bollworms. The invention interferes cytochrome P450 enzyme by RNAi means, increases the sensitivity of the cotton bollworm to gossypol, and can realize specific and green prevention and control on the cotton bollworm.

Description

dsRNA of cotton bollworm CYP4L11 gene and application thereof
Technical Field
The invention relates to the field of agricultural pest control, in particular to dsRNA of a cotton bollworm CYP4L11 gene and application thereof.
Background
The cotton bollworm is a omnivorous pest worldwide and seriously harms the yield and quality of crops in China. At present, the cotton bollworm is controlled mainly by two means of planting transgenic cotton and applying pesticides, but along with the expansion of the planting range of the transgenic cotton, the cotton bollworm in many areas has already generated resistance to the transgenic cotton, and the control of the cotton bollworm still depends on chemical pesticides. However, the application of a large amount of chemical pesticides can cause the drug resistance of pests to be enhanced, and simultaneously, the application of the chemical pesticides can pollute the environment, destroy the ecology and even bring a series of food safety problems. The development of environmentally friendly, target-specific pest control strategies has been imminent.
Double-stranded RNA (dsrna) -mediated gene silencing, commonly referred to as RNA interference (RNAi), functions by preventing translation or transcription of homologous genes. RNAi technology has the characteristics of high specificity, convenient operation and the like, and has been applied to agricultural pest control. For example: baum et al (2007) express vacuolar ATPase gene dsRNA through transgenic maize, resulting in stunting development and increased mortality of corn rootworm eating the transgenic maize; the research of Mao et al (2007) finds that after cotton bollworms eat transgenic cotton expressing dsCYP6AE14, the larvae grow and develop slowly and finally die. Therefore, the RNAi technology has feasibility for preventing and controlling agricultural pests.
Gossypol is one of the plant secondary metabolites produced by cotton and is toxic to many organisms. Under the stress of gossypol, the bollworm carries out self-protection by inducing and highly expressing detoxification metabolic enzymes including cytochrome P450 and the like. The cytochrome P450 enzyme system is a multifunctional oxidase widely existing in organisms and can metabolize various endogenous and exogenous compounds. Therefore, the sensitivity of the cotton bollworm to the gossypol is increased by interfering the expression of the key CYP genes, and the purpose of preventing and controlling the cotton bollworm can be achieved.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide dsRNA of a cotton bollworm CYP4L11 gene.
The dsRNA of the cotton bollworm CYP4L11 gene is characterized in that the nucleotide sequence of one strand of the dsRNA has at least 80 percent of homology with the nucleotide sequence shown in SEQ ID NO. 1. Further preferably, the nucleotide sequence of one strand of said dsRNA has at least 95% homology with the nucleotide sequence shown in SEQ ID No. 1.
The invention also provides a coding gene of the dsRNA. For example, but not limited to, a coding gene with a nucleotide sequence shown in SEQ ID NO. 2.
The invention also provides products such as a recombinant expression vector containing the dsRNA or the coding gene thereof, a recombinant microorganism (such as a recombinant strain), a transgenic cell line, a transgenic plant or a kit (such as an expression kit containing the dsRNA or the coding gene thereof), and the like.
The invention also provides a preparation method of the dsRNA, which comprises the following steps:
1) taking total RNA of the cotton bollworm, and obtaining cDNA through RT-PCR;
2) amplifying the target gene fragment by PCR technology: designing a specific primer with a T7 sequence, and taking the cDNA obtained in the first step as a template to obtain a PCR product with a T7 sequence and a target gene fragment;
3) dsRNA is prepared by using a Kit T7High Yield RNA Transcription Kit.
Specifically, the transcriptome under different host treatments of the cotton bollworm is analyzed by utilizing the bioinformatics technology, the CYP4L11 gene is obtained, then the total RNA of the cotton bollworm is taken according to the following sequential entry (1), and the cDNA is obtained by RT-PCR; (2) designing a specific primer with a T7 sequence; (3) amplifying the target gene fragment by PCR technology: taking the cDNA obtained in the first step as a template, and amplifying the cDNA with the specific primer designed in the second step to obtain a PCR product with a T7 sequence and a target gene fragment; (4) preparing dsRNA by using a Kit T7High Yield RNA Transcription Kit; (5) performing microinjection experiments to deliver target dsRNA into the body; (6) preparing artificial feed; (7) the injected bollworm larvae are fed to artificial feed containing gossypol, and the weight change is measured.
The dsRNA is transferred into the bodies of cotton bollworm larvae by an injection method, Green Fluorescent Protein (GFP) is used as a control, the injected larvae are fed on artificial feed containing 0.1 percent of gossypol, and the weight growth change after 3 days is recorded. The result shows that the weight growth of the larvae of the experimental group is obviously inhibited, the fluorescence quantitative PCR result proves that the target gene is obviously silenced, and the experiment also proves that the silencing target gene does not influence the growth and development of the insects. The invention provides a candidate gene with good effect for a future cotton bollworm RNA interference (RNAi) mediated pest control strategy, and simultaneously provides a theoretical basis for future field application.
The invention also aims to provide the application of the dsRNA or the coding gene in the aspect of preventing and controlling cotton bollworms. The application is preferably achieved by injecting the dsRNA into the larvae of the cotton bollworm by injection. Preferably, the injection is metered to about 2.5-5 μ g per larva.
The application of the invention can be achieved in other ways than by injection. For example, but not limited to, introduction into the body of the worm by feeding, feeding includes direct dsRNA feeding, and also includes feeding a bacterial fluid expressing the dsRNA. Alternatively, the dsRNA can be introduced into transgenic cotton to make into plant-derived expressed dsRNA.
The invention also aims to provide the application of the recombinant expression vector, the recombinant microorganism, the transgenic cell line, the transgenic plant or the kit in the aspect of preventing and controlling the cotton bollworm.
In the prior art, cotton bollworms are mainly prevented and controlled by chemical pesticides, so that on one hand, the drug resistance of pests is easily caused, and on the other hand, the environment is polluted. The gossypol is used as plant secondary substance existing in nature, and is used for pest control by utilizing the characteristic of toxicity, so that the environment-friendly and pollution-free effect can be achieved. The invention interferes cytochrome P450 enzyme by RNAi means, increases the sensitivity of the cotton bollworm to gossypol, and can realize specific and green prevention and control on the cotton bollworm.
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FIG. 1 shows the change in body weight gain 3 days after the administration of dsRNA (dsCYP4L11) in accordance with the present invention, compared to dsGFP injection.
FIG. 2 shows the interference efficiency of dsRNA of cotton bollworm CYP4L11 gene on its target gene in the examples of the present invention.
FIG. 3 shows the change in body weight gain 3 days after the dsRNA of the invention (dsCYP4L11) was injected and fed to normal diet, with dsGFP injection as a control.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The molecular biological experiments, which are not specifically described in the examples, were carried out according to the methods specified in molecular cloning, A laboratory Manual (third edition) J. SammBruke, or according to the kit and product instructions.
Example 1 cloning of CYP4L11 Gene
1. The transcriptome of the cotton bollworm which is measured in the laboratory and feeds different host plants is combined with programs such as NCBI Blast and the like to determine the CYP4L11 gene of the cotton bollworm.
2. Test insects
The cotton bollworm indoor breeding strain (96S) was originally collected from the Xinxiang experimental base of the Chinese academy of agricultural sciences in 1996, and artificial feed breeding was carried out till now. The breeding conditions are that the temperature is 27 +/-2 ℃, the relative humidity is 75% +/-10%, the photoperiod is 14L: and 10D, feeding the prepared 10% sugar water after the imagoes are eclosized.
RNA extraction
Grinding collected larva, pupa and imago with liquid nitrogen, transferring 50-100mg of the powder into a 1.5mL RNase-free centrifuge tube, adding 1mL Trizol, and mixing well. The collected tissue was placed into a centrifuge tube, 200. mu.L Trizol was added, then ground with a plastic grinding rod, and 800. mu.L Trizol was added after grinding was complete.
Incubation was carried out at room temperature for 5min to facilitate complete separation of the nucleoprotein bodies in the homogenate. Add 200. mu.L chloroform, shake for 15s, and let stand at room temperature for 5 min. Then centrifuging for 15min at 12000rpm and 4 ℃; taking 400 mu L of the supernatant into a new centrifuge tube, adding isopropanol with the same volume, turning upside down to fully mix, standing at room temperature for 10min, and centrifuging at 12000rpm and 4 ℃ for 10 min; discarding the supernatant, adding 1mL of 75% ethanol, gently washing, and then centrifuging at 12000rpm and 4 ℃ for 5 min; the supernatant was removed, the precipitate was dried at room temperature, and 50 to 150. mu.L of ribozyme-free water was added to dissolve it sufficiently.
4. Synthesis of first Strand cDNA
First strand cDNA was synthesized from the extracted total RNA using the reverse transcription kit TransScript One-step gDNA Removal and cDNA Synthesis SuperMix according to the protocol. The specific operation is as follows:
the configuration system is shown in the following table 1:
TABLE 1
Figure BDA0001642022310000041
Mixing, and incubating at 42 deg.C for 15 min; the reverse transcriptase was inactivated by heating at 85 ℃ for 5 seconds. After the reaction is finished, the mixture is stored in a refrigerator at the ultralow temperature of minus 80 ℃.
Example 2 preparation of dsCYP4L11
5. Preparation of target gene dsRNA
The GFP gene (green fluorescent protein) was selected as a control gene, which is not present in insects, and a large number of studies have been conducted to synthesize dsRNA using GFP as a control gene.
1) Design of target gene primers:
the BLAST database was used to search for amino acid sequences of the same target gene from closely related species of Helicoverpa armigera, and the sequences of the selected genes were determined by local alignment of transcriptome data determined in the laboratory. The sequence of the selected CYP4L11 gene is shown in SEQ ID NO. 3.
SEQ ID NO.3：
Figure BDA0001642022310000042
Figure BDA0001642022310000051
According to the sequence of a target gene, Primer design software Primer Premier 5 is adopted to design a Primer with a T7 promoter, and the Primer is sent to Shanghai workers for Primer synthesis. The target gene primer sequences are shown in table 2 below:
TABLE 2
Figure BDA0001642022310000052
2) PCR amplification of target fragment and preparation of dsRNA
The gene was amplified using Fastpfu enzyme with Helicoverpa armigera cDNA as template. The reaction system is shown in table 3 below:
TABLE 3
Figure BDA0001642022310000061
After mixing the above reaction solutions, they were centrifuged briefly and then reacted in a PCR instrument according to the following procedure (see Table 4):
TABLE 4
Figure BDA0001642022310000062
After completion of PCR amplification, the PCR product was recovered according to Wizard SV Gel and PCR Clean-Up System kit instructions. Subsequently, referring to the T7High Yield RNA Transcription Kit of Nanjing Novozam company, using the instruction, dsRNA was synthesized, and the reaction system was as shown in the following Table 5:
TABLE 5
Figure BDA0001642022310000063
The components were gently mixed by pipette, centrifuged briefly, and incubated at 37 ℃ for 4 h. mu.L of DNaseI was added to the reaction system, incubated at 37 ℃ for 15min, and the transcribed DNA template was digested. And (3) carrying out electrophoretic analysis and purification on the synthesized dsRNA, and storing for later use. The synthesized RNA sequence is shown as SEQ ID NO. 1.
SEQ ID NO.1：
Figure BDA0001642022310000071
The DNA sequence for coding the polypeptide is shown as SEQ ID NO. 2.
SEQ ID NO.2：
Figure BDA0001642022310000072
Example 3 injection experiments with dsCYP4L11
dsRNA injection
And selecting 3-instar larvae with uniform body types on the same day, and placing on ice. 2.5. mu.g of dsRNA was injected into the larvae at the abdominal penultimate and third podic internodes through capillaries pulled out by a needle puller. 24 larvae were injected per treatment, triplicate, and dsGFP injected as a control.
6.1 measurement of weight gain of Cotton bollworm on gossypol feed after dsRNA injection
The injected larvae were placed singly in a 24-well plate containing normal feed for 24 hours and then weighed for recording M1, transferred to feed containing 0.1% gossypol, and after 72 hours M2 was weighed. As a result, it was found that the weight gain of larvae injected with dsCYP4L11 was significantly inhibited (p <0.05) compared to the control group (dsGFP) (fig. 1).
6.2 silencing efficiency assay
After 24 hours and 72 hours of dsRNA injection, samples were prepared and silencing efficiency of target genes was tested by qPCR technique, and the fluorescent quantitative primer design for CYP4L11 was q-CYP4L 11-F: CGCTAATATAACTGCTCTT, q-CYP4L 11-R: ACCTTCATCGTCTATCTT, reference gene selection β -actin: q- β -actin-F: CCTGGTATTGCTGACCGTATGC, q- β -actin-R: CTGTTGGAAGGTGGAGGGAA. all assays included 3 biological replicates, each replicate containing 5 samples, and differential significance analysis was performed by using independent sample t-test using SPSS software (FIG. 2).
The result shows that after the dsRNA is injected for 24 hours, the silencing efficiency of the target gene reaches 50 percent, and the silencing efficiency is good for lepidoptera. There was no significant silencing effect 72 hours after dsRNA injection.
6.3 Effect of the Gene of interest on insect growth
After the target gene dsRNA is injected, the dsRNA is fed to normal feed together with dsGFP, the weight change is observed, and no obvious difference is found between the weight increase of the two types of feed after 3 days, so that the possibility that the target gene participates in the regulation of the growth and development of cotton bollworm larvae is eliminated (figure 3). The results show that the cotton bollworm gene CYP4L11 is involved in the detoxification metabolic process of gossypol.
The present invention may be embodied in many different forms and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Shenzhen agricultural genome institute of Chinese agricultural science institute
<120> dsRNA of cotton bollworm CYP4L11 gene and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>448
<212>RNA
<213>Artificial
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gcauccaagg caacaauagg ggucauauca auugguuguc cguuggccag accccgcagu 60
uuaucuguca aauaucucug auucuugcaa aauacuggca agaaguuuug caguauauug 120
aaguggaagg cggguguuaa gaaucuccuu ugagucuucc acaucuggcc ugucgauguc 180
aguagaccua gaccaagcca guuuaauagg aagucguagg uccugccuuu ugugaugugc 240
uccgugcuug auauuagugc cucaacauau cuagaguggg uuacaaucac gacaggagug 300
gugaauaaau gcacucugaa cgcaccgcca uacuugcucg ccagucucgc uaacaagucc 360
aacauuucug uugauuuaac aauaaacaga ugugcauuuc ccaauauagg caaaggcgug 420
gguccauuca gguuaaaccg ucggcuuu 448
<210>2
<211>448
<212>DNA
<213>Artificial
<400>2
aaagccgacg gtttaacctg aatggaccca cgcctttgcc tatattggga aatgcacatc 60
tgtttattgt taaatcaaca gaaatgttgg acttgttagc gagactggcg agcaagtatg 120
gcggtgcgtt cagagtgcat ttattcacca ctcctgtcgt gattgtaacc cactctagat 180
atgttgaggc actaatatca agcacggagc acatcacaaa aggcaggacc tacgacttcc 240
tattaaactg gcttggtcta ggtctactga catcgacagg ccagatgtgg aagactcaaa 300
ggagattctt aacacccgcc ttccacttca atatactgca aaacttcttg ccagtatttt 360
gcaagaatca gagatatttg acagataaac tgcggggtct ggccaacgga caaccaattg 420
atatgacccc tattgttgcc ttggatgc 448
<210>3
<211>1497
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<213> Helicoverpa armigera (Helicoverpa armigera)
<400>3
aatttttatt ttttcaaaaa aatgattaca ttattaatat gtacggtttt agtgtttgtt 60
ttgttattat catggataaa tttggtgaag gaaagccgac ggtttaacct gaatggaccc 120
acgcctttgc ctatattggg aaatgcacat ctgtttattg ttaaatcaac agaaatgttg 180
gacttgttag cgagactggc gagcaagtat ggcggtgcgt tcagagtgca tttattcacc 240
actcctgtcg tgattgtaac ccactctaga tatgttgagg cactaatatc aagcacggag 300
cacatcacaa aaggcaggac ctacgacttc ctattaaact ggcttggtct aggtctactg 360
acatcgacag gccagatgtg gaagactcaa aggagattct taacacccgc cttccacttc 420
aatatactgc aaaacttctt gccagtattt tgcaagaatc agagatattt gacagataaa 480
ctgcggggtc tggccaacgg acaaccaatt gatatgaccc ctattgttgc cttggatgct 540
cttgataacg tgacagaatc cataatggga gtatgcgtgg atgcacagaa acatcagtct 600
gaatacgtga aaagtatcga agaactgtcc gcaatagtaa cgatgaggat gcaaaatcct 660
ttcatgggcc aagacgctat tttcaatttg ctgccttaca agaagaaaca agagaaagct 720
ttgaagattg tacatggaca gacccataaa gtaattgaag caagacgagc ggaactaaga 780
cgcgctaata taactgctct tcccgacagc aatgatattg gtattaagaa caagcacgca 840
ttcttggacc tgttgctatt agctgaaatg gacggcaaaa agatagacga tgaaggtgtg 900
agggaacagg tcgacacatt catgtttgag ggtcacgaca cgacgacttc aggcattgtg 960
tacacactct actgtctgtc gaaacacaga gatatccaag aaaaaatcta cgaagagctt 1020
caaacaatat tcggcagtga aatggaaaga gaccctacat ataccgaact caaccaaatg 1080
aagtatctgg aattggtgct caaggaatca atgcggctgt tcccaccagt gccactcatc 1140
gagaggaaaa ttttgaggga ttgtgagatt ggaggcttaa cactagtaaa aggcacatcg 1200
gtactgatca acatctacca gatccagagg cagccagagt tgtatgaaaa tcctttagag 1260
ttccggccgg agaggttcga agctccactg aagaacccct tcagttggct ggcatttagt 1320
gctggtccga gaaattgtat aggtcaaaag ttcgcgatga tggaactgaa aataactatc 1380
tctgaaatga tcaaaaactt ctacatactg ccagcacctc aagagcctga actgagcgca 1440
gacctcgtgc taagatctaa gaatggagtc cacatcaaac ttatgcctag gaaataa 1497

Claims (8)

1. The dsRNA of the cotton bollworm CYP4L11 gene is characterized in that the nucleotide sequence of one strand of the dsRNA is shown as SEQ ID NO. 1.
2. A gene encoding the dsRNA of claim 1.
3. The encoding gene as claimed in claim 2, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.
4. A recombinant expression vector, a recombinant microorganism, a transgenic cell line or a kit comprising the dsRNA of claim 1 or the encoding gene of claim 2 or 3.
5. A method of making the dsRNA of claim 1, comprising the steps of:
1) taking total RNA of the cotton bollworm, and obtaining cDNA through RT-PCR;
2) amplifying the target gene fragment by PCR technology: designing a specific primer with a T7 sequence according to the cotton bollworm CYP4L11 gene sequence, and taking the cDNA obtained in the first step as a template to obtain a PCR product with a T7 sequence and a target gene fragment;
3) dsRNA was prepared using Kit T7High Yield RNA Transcription Kit.
6. Use of the dsRNA of claim 1 or the coding gene of claim 2 or 3 for controlling Helicoverpa armigera.
7. The use according to claim 6, wherein the use is effected by injecting dsRNA into cotton bollworm larvae by injection or by feeding the cotton bollworm larvae.
8. The use of the recombinant expression vector, the recombinant microorganism, the transgenic cell line or the kit of claim 4 for the control of Helicoverpa armigera;
alternatively, the use of a transgenic plant comprising the dsRNA of claim 1 or the encoding gene of claim 2 or 3 for the control of Helicoverpa armigera.
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