CN103215299B - Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application - Google Patents
Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application Download PDFInfo
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Abstract
The invention relates to a plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and an application, belonging to the field of gene engineering. The plasmid pHT305AKR is a recombinant plasmid obtained by inserting deoxyribonucleic acid (DNA) shown by SEQ ID NO.1 between BamHI and PstI enzyme cutting sites of the plasmid pHT305a. The plasmid pHT305AKR not only realizes the research of expressing dsRNA in bacillus thuringiensis, but also provides a powerful means for applying RNAi (RNA interference) technique to identifying the function of bacillus thuringiensis genes and researching a functional genome of the bacillus thuringiensis, and can also be used for synthesizing dsRNA by utilizing host bacterium, so that the cost for obtaining the dsRNA is reduced. The invention also provides a direction for exogenous transformation of the bacillus thuringiensis and research of bacillus thuringiensis biopesticide, so that the plasmid has a promising application prospect. Meanwhile, a novel way is provided for controlling the plutella xylostella, and a new field is created for controlling the pests.
Description
Technical field
The present invention relates to a kind of plasmid and the application of expressing small cabbage moth arginine kinase gene dsRNA, belong to genetically engineered field.
Background technology
Small cabbage moth belongs to lepidopteran diamond-back moth section, English name: Diamondback moth; Formal name used at school:
plutella xylostella(L.), be the worldwide insect that migrates, the cresss such as wild cabbage, broccoli, Chinese cabbage, rape of mainly causing harm.From nineteen fifty-three reported first small cabbage moth DTT has been produced since resistance, up to the present, small cabbage moth creates resistance in various degree to more than 50 kinds of sterilants, almost relates to all control medications.
Arginine kinase (Arginine Kinase, AK) (ATP:Argine N-phosphotransferase EC 2.7.3.3) belongs to phosphagen kinase families, extensively be present in invertebrates and mollusk, be similar to creatine kinase (the Creatine Kinase in vertebrates, CK), be one operate with intracellular energy, Muscle contraction, ATP regenerate have direct relation important kinases (Zhang Yuanchen etc. the clone of tobacco budworm arginine kinase gene and mrna expression analysis [J], insect journal, 54 (7): 754-761.).
Bacillus thuringiensis (
bacillus thuringiensisbe called for short Bt) be a kind of widely distributed ground Gram-positive genus bacillus, the parasporal crystal with insecticidal activity can be produced in the sporulation phase, and parasporal crystal (parent toxin) has active (the Wight A P of virus killing to the various insects such as lepidopteran or Diptera, Davis M E. Design and preparation of organic-inorganic hybrid catalysts [J]. Chem Rev, 2002,102 (10): 3589-3614.), be current most widely used general, study the microbial pesticide the most deep, turnout is maximum.Although Bt sterilant has harmless to person poultry safety, free from environmental pollution, have certain specificity and the optionally advantage such as insecticidal action, the problems such as also existence and stability is poor, the longevity of residure is short, insecticidal spectrum is narrow, have had a strong impact on the practical application of Bt.This makes transformation Bt become a kind of inevitable.Some foreign genes, due to the successful structure of shuttle vector, integrative vector, Bt swivel base-Resolution vector etc., to be successfully transferred in bacillus thuringiensis by present stage.But these transformations all concentrate on bacillus thuringiensis transformation itself, particularly to the transformation of its toxalbumin.
RNA interference (i.e. RNAi) utilizes double-stranded RNA (double strain RNA, dsRNA) silence (Geley of corresponding gene is mediated, S., Muller C. RNAi:ancient mechanism with a promising future. Exp Gerontol, 2004,39 (7): 985-998).Its interference mechanism be the member Dicer enzyme spcificity of RNaseIII ribozyme family be combined with double-stranded RNA and cut into the microRNA fragment of 21 ~ 23 nt sizes.Silencing complex (the RNA-induced silencing complex that the microRNA fragment of double-strand causes with RNA subsequently, RISC) combine, untwist into strand, and be combined with the mRNA with complementary sequence under the mediation of RISC, thus cause the degraded of mRNA.By injection and the functional gene of feeding dsRNA experimental study insect, find the grown significant impact of the silence of some genes on insect.Therefore, utilize RNAi effect to suppress the function of indispensable gene in insect and then to cause it dead, with the harm of protective plant from insect, this is feasible in theory.
Summary of the invention
The object of the present invention is to provide a kind of plasmid can expressing small cabbage moth arginine kinase gene dsRNA in bacillus thuringiensis, for the control of small cabbage moth provides new way.
Plasmid of the present invention, at plasmid pHT305a
bamHi and
pstthe recombinant plasmid that the DNA shown in SEQ ID NO.1 obtains is inserted, called after plasmid pHT305AKR between I restriction enzyme site.
The present invention also protects the engineering bacteria that described plasmid pHT305AKR builds.
The present invention also protects the bacillus thuringiensis engineering bacteria that described plasmid pHT305AKR builds.
The present invention also protects the application of described bacillus thuringiensis engineering bacteria in preparation Bt biotic pesticide.
Details are as follows:
In described plasmid pHT305AKR, foreign gene is small cabbage moth arginine kinase portion gene, and nucleotide sequence is specifically as shown in SEQ ID NO. 4.
Described plasmid pHT305AKR can copy in a large number in intestinal bacteria, can express in bacillus thuringiensis bacterial strain.
Also protection scope of the present invention is belonged to as the engineering bacteria of plasmid construction with plasmid pHT305AKR.
The plasmid of the expressing gene dsRNA obtained as the plasmid construction that sets out with plasmid pHT305AKR also belongs to protection scope of the present invention; Containing forward promotor pro3 α (+) and the sub-pro3 α (-) of reverse starting in described plasmid pHT305AKR; Described forward promotor pro3 α (+) and the sub-pro3 α (-) of reverse starting are bacillus thuringiensiss
cry III Athe promotor of gene, the two is reverse complementary sequence; Described forward promotor pro3 α (+) sequence is as shown in SEQ ID NO. 2, and described reverse starting pro3 α (-) sequence is as shown in SEQ ID NO. 3.
With plasmid pHT305AKR as set out plasmid obtain new plasmid and the engineering bacteria utilizing this novel plasmid to obtain also belongs to protection scope of the present invention.
The present invention also protects a kind of method utilizing described plasmid to carry out RNA interference, is the target gene of interference is inserted shuttle plasmid pHT305AKR's as foreign gene
sali and
sphbetween I restriction enzyme site, obtain the dsRNA expressing target gene, make corresponding target gene reticent.
The present invention also protects and a kind ofly utilizes described shuttle plasmid pHT305AKR to transform bacillus thuringiensis and the method for Bt biotic pesticide that obtains.
tool of the present invention has the following advantages:
1, the present invention utilizes smooth end to connect at random and digestion with restriction enzyme obtains the plasmid of the dsRNA that can express arginine kinase gene.
2, plasmid provided by the invention not only achieves the research expressing dsRNA bacillus thuringiensis, for the research of the Function Identification and bacillus thuringiensis functional genome that RNAi technology are applied to Bacillus thuringiensis Genes provides strong means, also host bacteria can be utilized to synthesize dsRNA, reduce the cost obtaining dsRNA.
3, the present invention is that the transformation of bacillus thuringiensis provides new thinking, is the insecticidal effect improving B. thuringiensis bio sterilant, and a new direction opened up by development B. thuringiensis bio agricultural chemicals.
4, the present invention is by studying RNAi method and the effect of small cabbage moth arginine kinase gene, for the control of small cabbage moth provides new way, also offers reference at injurious insect control for RNAi theory and technology, starts the frontier of injurious insect control.
Accompanying drawing explanation
Fig. 1 is the structural representation of pHT305a.
Fig. 2 is the structural representation of pHT305AKR.
Fig. 3 is the electrophoresis schematic diagram of forward promotor, reverse starting and goal gene fragment; Swimming lane 1:
pro3a(+), swimming lane 2:AK fragment, swimming lane 3:
pro3a(-).
Fig. 4 is RNA electrophorogram; Wherein swimming lane 1: engineering bacteria, swimming lane 2: original bacteria.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following concrete enforcement, if no special instructions, is ordinary method.Test materials used in following specific embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Enzyme cuts restriction enzyme used, enzyme connects T4 ligase enzyme used, PCR Taq enzyme used is all purchased from TAKARA company, glue reclaims test kit, high-purity Plasmid Miniprep Kit and PUM-T support agent box are all purchased from Bio Teke company, total RNA from animal tissues extraction test kit and culturing cell/bacterium total RNA extraction reagent box are all purchased from Tian Gen biochemical technology company limited, the M-MLV ThermoScript II of reverse transcription is purchased from invitrogen company, the carrier pHT305a(structural representation that sets out is shown in Fig. 1) close male teacher by key lab of University Of Agriculture and Forestry In Fujian and given, penbritin (hereinafter abbreviated as Amp) is purchased from Sheng Gong biotechnology company limited, erythromycin (hereinafter abbreviated as Erm) is purchased from Solarbio company.
Embodiment 1, plasmid pHT305AKR build
One, bacillus thuringiensis
cry III Athe preparation of the promoter fragment of gene
1.1 extract plasmid pHT305a
Get the single bacterium colony incubated overnight in LB liquid nutrient medium (containing 50ng/ml Amp and 50ng/ml Erm) containing plasmid pHT305a bacterial strain, extract plasmid pHT305a with high-purity Plasmid Miniprep Kit (products catalogue: DP1002).
1.2 pcr amplification bacillus thuringiensiss
cry III Apro3 α (+) promoter fragment of gene
With plasmid pHT305a for template, design pair of primers (introduce respectively by two primer 5 ＇ ends
bamHi and
sphi restriction endonuclease recognition sequence and protection base).
Upstream primer 01F:5 ＇-GTCT
gGATCC(underscore is GAAATTAGTTATACAAGCATT-3 ＇
bamHi recognition sequence);
Downstream primer 01R:5 ＇-ACAT
gCATGC(underscore is TTTTCTTCCTCCCTTTCTT-3 ＇
sphi recognition sequence).
Carry out PCR with above-mentioned primer, obtain the PCR primer of 1072bp.
PCR primer carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue.
Reclaim product to check order, sequencing result shows that the nucleotide sequence of PCR primer is as shown in SEQ ID NO. 2.
1.3
cry III Athe enzyme of pro3 α (+) promoter fragment of gene is cut
Pro3 α (+) promoter fragment is used
bamHi and
sphi restriction endonuclease carries out double digestion reaction (37 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue, be enzyme cut after
cry III Apro3 α (+) promoter fragment of gene.
1.4 pcr amplification bacillus thuringiensiss
cry III Apro3 α (-) promoter fragment of gene
With plasmid pHT305a for template, design pair of primers (introduce respectively by two primer 5 ＇ ends
psti and
sali restriction endonuclease recognition sequence and protection base).
Upstream primer 02F:5 ＇-TGCA
cTGCAG(underscore is GAAATTAGTTATACAAGCATT-3 ＇
psti recognition sequence);
Downstream primer 02R:5 ＇-AGAT
gTCGAC(underscore is TTTTCTTCCTCCCTTTCTT-3 ＇
sali recognition sequence).
Carry out PCR with above-mentioned primer, obtain the PCR primer of 1072bp.
PCR primer carries out agarose gel electrophoresis, reclaims reagent (products catalogue: DP1602) carry out purifying recovery with glue.
Reclaim product to check order, sequencing result shows that the nucleotide sequence of PCR primer is as shown in SEQ ID NO. 3.
1.5
cry III Athe enzyme of pro3 α (-) promoter fragment of gene is cut
Pro3 α (-) promoter fragment is used
psti and
sali restriction endonuclease carries out double digestion reaction (37 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue, be enzyme cut after
cry III Apro3 α (-) promoter fragment of gene.
Two, the preparation of small cabbage moth arginine kinase gene fragment
2.1 extract small cabbage moth total serum IgE
Get diamondback moth larvae (strain from this experiment is raised), use total RNA from animal tissues to extract test kit (products catalogue: DP431) and extract total serum IgE.
2.2 reverse transcriptions obtain cDNA
Use M-MLV reversed transcriptive enzyme, the total serum IgE reverse transcription obtained in step 2.1 is become cDNA.
2.3 pcr amplification goal gene fragments
According to small cabbage moth AK complete genome sequence (GenBank:HQ327310.1), determine that size is the gene fragment of 325bp.Utilize the following primer of Primer Premier 5.0 software design, and one end of a primer adds wherein
sphi restriction enzyme site and protection base.
Upstream primer 03F:5 ＇-ACAT
gCATG(underscore is CCCGCCAACGCTTGCCGCTT-3 ＇
sphi recognition sequence);
Downstream primer 03R:5 ＇-TGGTACTTGGACGCCACCT-3 ＇.
Carry out PCR with above-mentioned primer, obtain the PCR primer of 325bp.
PCR primer carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue.
Reclaim product to check order, sequencing result shows that the nucleotide sequence of PCR primer is as shown in SEQ ID NO. 4.
2.4 goal gene fragments are in the connection of PUM-T carrier
Object fragment is connected with PUM-T support agent box (products catalogue: DP6701).
The enzyme of 2.5 goal gene fragments is cut
Use in above-mentioned steps 2.4
sphi restriction endonuclease carries out endonuclease reaction (37 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue, be enzyme cut after goal gene fragment.
Three, the preparation of carrier framework
3.1 extract plasmid pHT305a
Get the single bacterium colony incubated overnight in LB liquid nutrient medium (containing 50ng/ml Amp and 50ng/ml Erm) containing plasmid pHT305a bacterial strain, extract plasmid pHT305a with high-purity Plasmid Miniprep Kit (products catalogue: DP1002).
Plasmid pHT305a uses by 3.2
bamHi restriction endonuclease carries out endonuclease reaction (30 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, with Bio Teke glue reclaim test kit (products catalogue: DP1602) carry out purifying recovery, be enzyme cut after carrier framework.
Four, plasmid pHT305AKR builds
4.1 the enzyme that above-mentioned steps one obtains is cut after forward promotor pro3 α (+) fragment cut with the enzyme that above-mentioned steps two obtains after goal gene fragment carry out being connected (16 DEG C 12 hours) with T4 ligase enzyme.
Enzyme connects product and carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) and carries out purifying recovery, obtain the connection product of forward promotor and goal gene fragment with Bio Teke glue.
The connection product obtained in above-mentioned steps 4.1 is used by 4.2
sphi restriction endonuclease carries out endonuclease reaction (37 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with glue.
4.3 connection product above-mentioned steps 4.2 obtained and carrier framework T4 ligase enzyme carries out being connected (16 DEG C 12 hours).
Enzyme connects product agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) and carries out purifying recovery, obtain the connection product containing forward promotor and goal gene fragment with Bio Teke glue.
4.4 by the connection product conversion bacillus coli DH 5 alpha competence in above-mentioned steps 4.3, coated plate, 37 DEG C of incubated overnight.
4.5 picking positive colony bacterial plaques shake bacterium amplification, plasmid is extracted with high-purity Plasmid Miniprep Kit (products catalogue: DP1002) of Bio Teke, with the plasmid extracted for template, carry out pcr amplification with the primer that 01 primer pair and 03 primer pair are combined into, obtain the PCR primer of 2027bp.
4.6 plasmids extracted in above-mentioned steps 4.5 are used
psti and
sali restriction endonuclease carries out double digestion reaction (37 DEG C 6 hours).
Digestion products carries out agarose gel electrophoresis, reclaims test kit (products catalogue: DP1602) carry out purifying recovery with Bio Teke glue, be follow-up ligase enzyme cut after the carrier framework of reverse starting pro3 α (-) fragment.
4.7 the enzyme that above-mentioned steps one obtains is cut after reverse starting pro3 α (-) fragment cut with the enzyme that above-mentioned steps 4.6 obtains after carrier framework carry out being connected (16 DEG C 12 hours) with T4 ligase enzyme.
The 4.8 connection product conversion bacillus coli DH 5 alpha competence that above-mentioned steps 4.7 is obtained, coated plate, 37 DEG C of incubated overnight.
4.9 picking positive colony bacterial plaques shake bacterium amplification, plasmid is extracted with high-purity Plasmid Miniprep Kit (products catalogue: DP1002) of Bio Teke, with the plasmid extracted for template, carry out pcr amplification with the primer pair of 01F and 02R composition, obtain the PCR primer of about 2.5kb; PCR primer checked order, sequencing result shows in PCR primer containing, for example the nucleotide sequence shown in SEQ ID NO.1.
The structural representation of plasmid pHT305AKR is shown in Fig. 2.
The structure of embodiment 2, bacillus thuringiensis engineering bacteria and application
One, plasmid extraction
Get the single bacterium colony incubated overnight in LB liquid nutrient medium (containing 50ng/ml Amp and 50ng/ml Erm) containing plasmid pHT305AKR bacterial strain, extract plasmid pHT305AKR with high-purity Plasmid Miniprep Kit (products catalogue: DP1002).
Two, bacillus thuringiensis BMB171 competence preparation
2.1 by BMB171 in 30 DEG C, 200r/min incubator overnight cultivate.
2.2 proceed in LB substratum by 1% inoculum size, and 30 DEG C, 220r/min is cultured to OD
600till 1.2-1.5.
2.3 by the nutrient solution in step 2.2 in placing 10min on ice, proceed to the large centrifuge tube of ice precooling, 6000r/min, 4 DEG C of centrifugal 10min.
2.4 supernatant discarded, add the SG damping fluid of 10ml ice precooling, and 6000r/min, 4 DEG C of centrifugal 10min, remove supernatant.Repeat 3 times.
2.5 use SG damping fluid from outstanding thalline, are sub-packed in Ep pipe ,-80 DEG C of preservations.
Three, engineering bacteria builds
5ul plasmid adds in 200ul BMB171 competent cell by 3.1, is transferred in electric shock cup after mixing, places 30min on ice and allows it thoroughly cool.
3.2 electricity turn: it is 2.5kv, 25uF that electricity turns condition, 200 Ω, ice bath 5min after electric shock.
3.3 recoveries: the bacterium liquid in electric shock cup is gone in EP pipe, and adds SOC substratum, 30 DEG C of shaking culture 2h.
3.4 coated plate: reclaim thalline, coat on LB flat board (containing Erm and Amp), be inverted overnight incubation for 30 DEG C.
3.5 picking positive colony bacterial plaques shake bacterium amplification, take bacterium colony as template, carry out bacterium colony PCR checking with the primer pair that primer pair 01 and 02 forms.
Carry out PCR with above-mentioned primer, obtain the PCR primer of 2497bp.
PCR primer carries out agarose gel electrophoresis, reclaims reagent (products catalogue: DP1602) carry out purifying recovery with glue.
Reclaim product to check order, sequencing result shows that the nucleotide sequence of PCR primer is as shown in SEQ ID NO.1.
Four, the application of bacillus thuringiensis engineering bacteria
The total serum IgE of 4.1 extraction recombinant bacterial strains and original strain.
The positive single bacterium colony of picking, in liquid LB nutrient solution, incubator overnight is cultivated, and utilizes foster cell/bacterium total RNA extraction reagent box (products catalogue: DP430) to extract engineering bacteria total serum IgE.
4.2 will extract the total serum IgE that obtains respectively with s1 nuclease process 30min in above-mentioned steps 4.1.
The ferment treatment product obtained in above-mentioned steps 4.2 is carried out agarose gel electrophoresis by 4.3.
Electrophoresis result as shown in Figure 4, demonstrates plasmid provided by the invention and can realize in bacillus thuringiensis, express small cabbage moth arginine kinase gene dsRNA.
4.4 feeding tests
Folium Raphani is immersed in the bacterium liquid after 4.1 process, is then placed in culturing bottle, raise diamondback moth larvae.Observe its death condition.
Statistics small cabbage moth, in the mortality ratio of the 2nd day, the 4th day and the 6th day, is respectively 70 %, 82.22 %, 97.78 %.Illustrate that improved engineering bacteria has obvious insecticidal effect, for the control of small cabbage moth provides new way.
<110> University Of Agriculture and Forestry In Fujian
<120> expresses plasmid and the application of small cabbage moth arginine kinase gene dsRNA
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 2497
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<213> artificial sequence
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<213> bacillus thuringiensis (Bacillus thuringiensis)
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atttaacaag aataataaat aaacacaatt tttattcttg ttagttcata aaaatatgaa 720
aaacttaaat ttctttaaat tttaggacta aatttattaa atttcaatca atatgttcct 780
cgcaaaacgt attgtaatta agaatattct cttgattggg tattttttta attttggtca 840
aaacttctac tgctgaggta cttttatggc cagggctatt ttgaatccgc ttattctaat 900
aaatttctta aaatagtata gatagtattt tttcttatga ggtttatgta aaattcataa 960
atattttaaa tcgaatagat aaaaaatttt tcacaatatc atcctactaa tctcgtaata 1020
gcaggattta tctttttaat aattcttaca aaatgcttgt ataactaatt tc 1072
<210> 4
<211> 325
<212> DNA
<213> small cabbage moth (Plutella xylostella)
<400> 4
tggtacttgg acgccacctc ctccagcttg gtcttgtcgg ccgccagctt gggcagcttg 60
atgtgcacgg aggcgcgcac ggtggtgccc aggttggtgg ggcagaaggt caggaaaccg 120
agacggtcgt ggtgcgagaa cggcacgcgc ttctcaatgt cgttgacggc ggccaccagc 180
ctggcgtaca cggccttcag gtcgccaccc atctgcatgg agatcaggcg gaggtggtcc 240
tcctcgttgc accacaccag gaaggtcttg ttctcgttgt ggaagatacc acgtccagag 300
ggccagaagc ggcaagcgtt ggcgg 325
Claims (4)
1. express a plasmid of small cabbage moth arginine kinase gene dsRNA, it is characterized in that: the plasmid of described expression small cabbage moth arginine kinase gene dsRNA is at plasmid pHT305a
bamHi and
pstinsert the recombinant plasmid that the DNA shown in SEQ ID NO.1 obtains between I restriction enzyme site, called after plasmid pHT305AKR, structure is as shown in Figure of description 2.
2. the engineering bacteria of plasmid pHT305AKR structure as claimed in claim 1.
3. the bacillus thuringiensis engineering bacteria of plasmid pHT305AKR structure as claimed in claim 1.
4. the application of bacillus thuringiensis engineering bacteria as claimed in claim 3 in preparation Bt biotic pesticide.
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| CN105296516A (en) * | 2014-07-15 | 2016-02-03 | 中国农业科学院棉花研究所 | DsRNA expression vector and application |
| CN104694566B (en) * | 2014-11-12 | 2018-11-02 | 石河子大学 | RNAi carrier and its application of HaAK genes dsRNA are expressed in transfer-gen plant |
| CN110438151B (en) * | 2019-08-28 | 2021-08-31 | 福建农林大学 | RNAi vector for plant-mediated silencing of plutella xylostella arginine kinase gene PxAK and application thereof |
| CN110511958B (en) * | 2019-08-28 | 2020-09-08 | 福建农林大学 | RNAi vector for simultaneously silencing plutella xylostella arginine kinase gene PxAK and integrin beta 1 subunit gene Px beta |
| CN111793639B (en) * | 2020-07-02 | 2022-05-24 | 福建农林大学 | Method for improving insecticidal activity of Bt by mixing with RNAi engineering bacteria |
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Non-Patent Citations (7)
| Title |
|---|
| Expression vector Pht3ag, complete sequence;Li Q., 等;《GenBank: GU046701.1》;20091109;第1页 * |
| Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription;M T de Souza, 等;《Journal of Bacteriology》;19930531;第175卷(第10期);第2953页第1栏第4段 * |
| Yu W, You M.Plutella xylostella arginine kinase mRNA, complete cds.《GenBank: HQ327310.1》.2010, * |
| 外源蛋白表达载体在重组短芽孢杆菌中的遗传稳定性分析;李强, 等;《中国科技论文在线》;20100521;第2页第2段 * |
| 小菜蛾精氨酸激酶基因dsRNA在大肠杆菌中的表达及其效应;徐秀凤;《中国优秀硕士学位论文全文数据库》;20121115;摘要第iii页，正文第53页附录1 * |
| 小菜蛾精氨酸激酶基因的RNA干扰及其效应;杨广, 徐秀凤, 等;《首届"全国青年昆虫学科技工作者创新论坛"论文摘要集》;20120723;第23页第1段 * |
| 带cry3 Aa启动子的aiiA基因在苏云金芽孢杆菌中的表达;朱晨光, 喻子牛, 等;《生物工程学报》;20030731;第19卷(第4期);第397页最后1段 * |
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