CN103667288B - A kind of suppress wheat aphid cytochrome C oxidase VIIc subunit gene to be expressed dsRNA and application - Google Patents
A kind of suppress wheat aphid cytochrome C oxidase VIIc subunit gene to be expressed dsRNA and application Download PDFInfo
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Abstract
The invention discloses a kind of suppress wheat aphid cytochrome C oxidase VIIc subunit gene to be expressed dsRNA and application.The invention provides a kind of double stranded rna molecule, as shown in the sequence 2 of sequence table.The present invention also protects the DNA molecular of described RNA molecule of encoding.The present invention also protects the application of described RNA molecule or described DNA molecular, for following (1) is at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.The present invention has significant application value for the control of aphid in agriculture production.
Description
Technical field
The present invention relates to a kind of suppress wheat aphid cytochrome C oxidase VIIc subunit gene to be expressed dsRNA and application.
Background technology
Wheat aphid (especially grain aphid) is one of primary pest of harm Wheat in China production, and according to statistics, the annual wheat aphid hazard area of China up to 0.17 hundred million hectare, can account for 62% of the total cultivated area of wheat, cause underproduction 15%-30%, can up to 50% time serious.In recent years, due to factors such as global warming, cropping system changes, the fecundity of aphid and adaptability are significantly strengthened, and its harm is on the rise.At present, control of aphids is to spray insecticide, and a large amount of agricultural chemicals that uses not only is harmful to people and animals, and causes serious environmental pollution.Cultivating aphids resistance wheat breed is the most effective way preventing aphid damage, but owing to lacking effective aphid-resistant gene in existing germ plasm resource, resistance mechanism is still not clear, and conventional breeding is difficult to prove effective.Excavate and utilize novel aphid-resistant gene and to cultivate wheat anti-aphid new germ plasm by genetically engineered significant.
The RNAi technology of mediated plant has become one of focus of farm crop Insect resistant gene engineer, is expressed the dsRNA of corresponding insect specific gene by host plant, its corresponding gene reticent after insect's food-taking plant thus reach the object of Control pests harm.The process of RNAi is that double-stranded RNA (dsRNA) enters in organism, is cut into the siRNA of 21-23nt by Dicer enzyme, and siRNA and RNA induces silencing complex to combine, and is combined, is identified by Dicer, cause the decline of expression of target gene amount with the said target mrna of complementary sequence.In recent years, utilize that dsRNA is external to feed or inject to screen RNA target gene, cause target gene to be expressed and reticent, be widely used in qualification and functional analysis that insect growth grows key gene.
Summary of the invention
The object of this invention is to provide a kind of suppress wheat aphid cytochrome C oxidase VIIc subunit gene to be expressed dsRNA and application.
The invention provides a kind of double stranded rna molecule, as shown in the sequence 2 of sequence table.
The present invention also protects the DNA molecular of described RNA molecule of encoding.The DNA molecular shown in sequence 1 that described DNA molecular is sequence table.
Recombinant expression vector containing described RNA molecule, transgenic cell line, recombinant bacterium or expression cassette all belong to protection scope of the present invention.
Recombinant expression vector containing described DNA molecular, transgenic cell line, recombinant bacterium or expression cassette all belong to protection scope of the present invention.
The present invention also protects described RNA molecule or the application of described DNA molecular in preparing product; The purposes of described product be following (1) at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.Described aphid specifically can be wheat aphid, more specifically can be grain aphid.Described aphid can be the aphid with cytochrome C oxidase VIIc subunit gene.Described cytochrome C oxidase VIIc subunit gene specifically can be there is sequence table sequence 5 shown in DNA fragmentation DNA molecular or there is the DNA molecular with the DNA fragmentation shown in the sequence 5 of sequence table with the DNA fragmentation of more than 80% homology.
The present invention also protects a kind of product, and its activeconstituents is described RNA molecule or described DNA molecular; The purposes of described product be following (1) at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.Described aphid specifically can be wheat aphid, more specifically can be grain aphid.Described aphid can be the aphid with cytochrome C oxidase VIIc subunit gene.Described cytochrome C oxidase VIIc subunit gene specifically can be there is sequence table sequence 5 shown in DNA fragmentation DNA molecular or there is the DNA molecular with the DNA fragmentation shown in the sequence 5 of sequence table with the DNA fragmentation of more than 80% homology.
The present invention also protects the application of described RNA molecule or described DNA molecular, for following (1) is at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.Described aphid specifically can be wheat aphid, more specifically can be grain aphid.Described aphid can be the aphid with cytochrome C oxidase VIIc subunit gene.Described cytochrome C oxidase VIIc subunit gene specifically can be there is sequence table sequence 5 shown in DNA fragmentation DNA molecular or there is the DNA molecular with the DNA fragmentation shown in the sequence 5 of sequence table with the DNA fragmentation of more than 80% homology.
The present invention also protects the application of material in preparing product of the expression for suppressing aphid cells in vivo cytochrome C oxidase VIIc subunit gene; The purposes of described product is following (a) and/or (b) and/or (c): (a) is anti-to eliminate aphis; B () promotes that aphid is dead; C () suppresses aphid growth.Described aphid specifically can be wheat aphid, more specifically can be grain aphid.Described aphid can be the aphid with cytochrome C oxidase VIIc subunit gene.Described cytochrome C oxidase VIIc subunit gene specifically can be there is sequence table sequence 5 shown in DNA fragmentation DNA molecular or there is the DNA molecular with the DNA fragmentation shown in the sequence 5 of sequence table with the DNA fragmentation of more than 80% homology.
The present invention also protects a kind of product, and its activeconstituents is the material of the expression suppressing aphid cells in vivo cytochrome C oxidase VIIc subunit gene; The purposes of described product is following (a) and/or (b) and/or (c): (a) is anti-to eliminate aphis; B () promotes that aphid is dead; C () suppresses aphid growth.Described aphid specifically can be wheat aphid, more specifically can be grain aphid.Described aphid can be the aphid with cytochrome C oxidase VIIc subunit gene.Described cytochrome C oxidase VIIc subunit gene specifically can be there is sequence table sequence 5 shown in DNA fragmentation DNA molecular or there is the DNA molecular with the DNA fragmentation shown in the sequence 5 of sequence table with the DNA fragmentation of more than 80% homology.
The present invention is based on the conserved sequence of the cDNA of the cytochrome C oxidase VIIc subunit of grain aphid, provide a kind of dsRNA.The external dsRNA provided by the invention that feeds, can by RNAi reticent grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene, and then produce lethal effect to grain aphid, and along with the increase of dsRNA concentration and feeding time prolongation, the mortality ratio of grain aphid increases all gradually.The present invention has significant application value for the control of aphid in agriculture production.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of the pcr amplification product in the process of preparation dsRNA-1 and dsRNA-2.
Fig. 2 is the agarose gel electrophoresis figure of dsRNA-1 and dsRNA-2.
Fig. 3 is amplification curve and the typical curve of identifying reference gene with the primer pair of P3-F and P3-R composition.
Fig. 4 is the amplification curve of primer pair identification of cell cytochrome C oxidase VIIc subunit gene, typical curve and solubility curve with P2-F and P2-R composition.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.T7 in-vitro transcription test kit: NEB company.
Grain aphid: reference: Qian Youting, Zhou Guanghe, Zhang Shuxiang, Zhang Xiangcai. the research of grain aphid sexual generation. plant protection, 1982,01,14-15.; The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science; Grain aphid is inoculated on the seedling of wheat breed section agriculture 199, puts into growth cabinet (20 ± 2 DEG C, 60%-80% humidity, 16 h light/8 h dark) and breed.
Embodiment 1, preparation for the dsRNA of silenced cell cytochrome C oxidase VIIc subunit gene
1, extract grain aphid total serum IgE and reverse transcription is cDNA.
2, the cDNA obtained with step 1 is for template, and carry out pcr amplification with the primer pair of P1-F and P1-R composition, obtain pcr amplification product, the agarose gel electrophoresis figure of pcr amplification product is shown in Figure 1A.
P1-F(upstream primer):
tAATACGACTCACTATAGGGaGTGGTTCAAAAGCCGTACAAGCGT;
P1-R(downstream primer):
tAATACGACTCACTATAGGGaGGGAAGACCAAAACCGGTGCTAAAGA.
The region of underscore mark is T7 promoter sequence.
PCR reaction system: 10 × PCRBuffer5 μ L, dNTP(2.5mmolL
-1) 4 μ L, rTaq0.5 μ L, upstream primer (20 μm of olL
-1) 1 μ L, downstream primer (20 μm of olL
-1) 1 μ L, template 1 μ L, use ddH
2o complements to 50 μ L.
PCR reaction conditions: 94 DEG C of 4min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 39 circulations; 72 DEG C of 10min; 4 DEG C of preservations.
3, the preparation of dsRNA-1
The pcr amplification product that recycling step 2 obtains also as template, adopts T7 in-vitro transcription test kit to carry out in-vitro transcription (hatching 16 hours for 42 DEG C), digests residual template DNA and single stranded RNA, obtain dsRNA-1 with DNaseI and RNaseA.
The agarose gel electrophoresis figure of dsRNA-1 is shown in Fig. 2 A.Checked order by dsRNA-1, sequencing result shows, the double-stranded RNA shown in sequence 2 that dsRNA-1 is sequence table.With without RNase water dissolution dsRNA-1, carry out quantitatively, being then placed in-20 DEG C of refrigerators and preserving with spectrophotometer (wavelength 260nm).
Embodiment 2, preparation for the dsRNA of reticent GFP gene
1, the double chain DNA molecule shown in sequence 3 of composition sequence table.
2, the double chain DNA molecule obtained with step 1 is for template, and carry out pcr amplification with the primer pair of P4-F and P4-R composition, obtain pcr amplification product, the agarose gel electrophoresis figure of pcr amplification product is shown in Figure 1B.
P4-F(upstream primer):
tAATACGACTCACTATAGGGaCGGGAACTACAAGACACG;
P4-R(downstream primer):
tAATACGACTCACTATAGGGcTTTGGAAAGGGCAGATT.
PCR reaction system and PCR reaction conditions are with the PCR reaction system in the step 2 of embodiment 1 and PCR reaction conditions.
3, the preparation of dsRNA-2
The pcr amplification product that recycling step 2 obtains also as template, adopts T7 in-vitro transcription test kit to carry out in-vitro transcription (hatching 16 hours for 42 DEG C), digests residual template DNA and single stranded RNA, obtain dsRNA-2 with DNaseI and RNaseA.
The agarose gel electrophoresis figure of dsRNA-2 is shown in Fig. 2 B.Checked order by dsRNA-2, sequencing result shows, the double-stranded RNA shown in sequence 4 that dsRNA-2 is sequence table.With without RNase water dissolution dsRNA-2, carry out quantitatively, being then placed in-20 DEG C of refrigerators and preserving with spectrophotometer (wavelength 260nm).
Embodiment 3, dsRNA are suppressing the application in aphid growth
One, aphid artificial diet preparation and raise the preparation of device
Artificial diet compound method and the structure of raising device see reference document (Li Caixia, Koryo cutting edge of a knife or a sword, high tinkling of pieces of jades, Li Runzhi. hjolomorphism artificial nutrient liquid raises the research of aphid. Agricultural University Of Shanxi's journal, 1997,17 (3): 225-228.LiCX, GaoLF, GaoLL, LiRZ.Studyontherearingofaphidsonaartificiallyholidicdiet s.JournalofShanxiAgriculturalUniversity, 1997,17 (3): 225-228. (inChinese)).With the bacterial filter artificial diet in 0.2 μm of aperture, be dispensed in the centrifuge tube of 2.0mL sterilizing, be stored in the refrigerator of-20 DEG C, avoid multigelation.
Two, dsRNA is suppressing the application in aphid growth
The feeding method of grain aphid sees reference document: entangle quick, Liu Shusheng. utilize artificial diet to raise aphid technology. East China insect journal, 2004,13 (2): 102-109.JiuM, LiuSS.Aphidrearingwithartificialdiets.EntomologicalJourn alofEastChina, 2004,13 (2): 102-109.
1, packet transaction
DSR8 group: eachly raise in device the grain aphid of putting into 15 3 ages, adopt mixed fodder first (preparation method of mixed fodder first: add 750ngdsRNA-1 in every 100 μ L artificial diet) aphid of feeding, the mixed fodder first more renewed for every two days, every two days statistics mortality ratio the grain aphid of getting survival detect the relative expression quantity of cytochrome C oxidase VIIc subunit gene.
GFP group: eachly raise in device the grain aphid of putting into 15 3 ages, adopt mixed fodder second (preparation method of mixed fodder second: add 750ngdsRNA-2 in every 100 μ L artificial diet) aphid of feeding, the mixed fodder second more renewed for every two days, every two days statistics mortality ratio the grain aphid of getting survival detect the relative expression quantity of cytochrome C oxidase VIIc subunit gene.
Control group: each aphid raises in device the grain aphid of putting into 15 3 ages, adopt artificial diet to feed aphid, every artificial diet, every two days statistics mortality ratio grain aphid of getting survival more renewed for two days detects the relative expression quantity of cytochrome C oxidase VIIc subunit gene.
Often group arranges 5 re-treatments (namely raising device for 5).
Cultivating condition: 20 ± 2 DEG C, humidity 60%-80%, 16 h light/8 h dark.
2, mortality statistics result
Use Excel2003 software to carry out statistical analysis to mortality ratio, calculating mean value and variance, and carry out the analysis (t-test, P<0.01 or 0.05) of significant difference.The results are shown in Table 1.
The mortality statistics analytical results that table 1 is respectively organized, mean+SD (%)
| 2d | 4d | 6d | 8d | |
| DSR8 group | 65±3 ＊＊ | 89±10 ＊＊ | 96±3.7 ＊＊ | 100±2.2 ＊＊ |
| GFP group | 8.89±3.85 | 11.11±7.69 | 15.56±3.85 | 17.78±3.85 |
| Control group | 6.00±5.96 | 12.33±5.58 | 17.67±3.65 | 18.33±5.58 |
*represent compared with control group, result difference significantly (P<0.05);
*represent compared with control group, result difference extremely significantly (P<0.01).
For DSR8 group, feeding dsRNA-1 time longer grain aphid mortality ratio higher, after feeding 6 days, the average mortality of grain aphid is 96%, and after feeding 8 days, the average mortality of grain aphid is 100%, all there is significant difference compared with control group.For GFP group, the mortality ratio of each time point of feeding dsRNA-2 and the mortality ratio of control group do not have significant difference.
3, the relative expression quantity result of cytochrome C oxidase VIIc subunit gene
Get the grain aphid of survival, extract total serum IgE and reverse transcription is cDNA(reverse transcription system is 10 μ L), be diluted to as template after 20 times of volumes with TE damping fluid, adopt the relative expression quantity of real-time fluorescence quantitative PCR identification of cell cytochrome C oxidase VIIc subunit gene.Primer pair for the identification of cytochrome C oxidase VIIc subunit gene is made up of P2-F and P2-R.Primer pair (ACTIN gene) for the identification of reference gene is made up of P3-F and P3-R.
P2-F(upstream primer): CGGTGCTAAAGAAAACTGCT;
P2-R(downstream primer): TGTCTAACGCCCGTGAGA.
P3-F(upstream primer): AGCCAAGGCGGTGATT;
P3-R(downstream primer): TCCGTTGCCCAGAAGC.
Real-time fluorescence quantitative PCR system: upstream primer (10 μm of olL
-1) 0.5 μ L, downstream primer (10 μm of olL
-1) 0.5 μ L, 2 × TransStart
tMgreenqPCRSuperMix12.5 μ L, PassiveReferenceDye0.5 μ L, template 1 μ L, use ddH
2o complements to 25 μ L.
Real-time fluorescence quantitative PCR program: 95 DEG C of 30s, 95 DEG C of 5s, 57 DEG C of 15s, 72 DEG C of 10s, 40 circulations.
2-△ △ Ct method (Ct represents cycle number) is adopted to calculate, i.e. △ △ Ct=(Ct
cytochrome C oxidase VIIc subunit gene-Ct
aCTIN gene)
test group-(Ct
cytochrome C oxidase VIIc subunit gene-Ct
aCTIN gene)
control group, use Excel2003 software to carry out statistical analysis, calculating mean value and variance, and carry out the analysis (t-test, P<0.01 or 0.05) of significant difference.
The relative expression quantity of grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene is in table 2.
The relative expression quantity of table 2 grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene, mean+SD (%)
| 0d | 2d | 4d | 6d | 8d | |
| DSR8 group | 100±6 | 63±28 ＊ | 42±8 ＊＊ | 33±7.8 ＊＊ | 18±3.2 ＊＊ |
| GFP group | 101±4 | 93±4 | 114±6 | 98±8 | 96±7 |
*represent compared with control group, result difference significantly (P<0.05);
*represent compared with control group, result difference extremely significantly (P<0.01); Before namely 0d feeds.
The experimental group of feeding dsRNA-1, along with the lengthening of feeding time, the relative expression quantity of grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene significantly reduces.Compared with control group, the relative expression quantity of feeding dsRNA grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene after-1 two day is significantly lower than control group.Compared with control group, the relative expression quantity of each time point grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene of feeding dsRNA-2 does not have significant difference.Result shows, can be caused the RNAi effect of grain aphid cells in vivo cytochrome C oxidase VIIc subunit gene by feeding dsRNA-1, causes the expression amount of cytochrome C oxidase VIIc subunit gene obviously to reduce, thus causes aphid dead.
4, the primer pair that foundation P3-F and P3-R forms identifies the typical curve equation of reference gene
Get grain aphid, extract total serum IgE and reverse transcription is cDNA(reverse transcription system is 10 μ L), with after TE damping fluid 10 times of gradient dilutions as template, adopt real-time fluorescence quantitative PCR, adopt P3-F and P3-R composition primer pair qualification reference gene (ACTIN gene).
Amplification curve is shown in Fig. 3 B, and typical curve is shown in Fig. 3 A.
Typical curve equation is: the corresponding Ct value of y=2.536x+13.074(y, x corresponding extension rate denary logarithm value), R
2=0.9644.
5, the typical curve equation of the primer pair identification of cell cytochrome C oxidase VIIc subunit gene of foundation P2-F and P2-R composition
Get grain aphid, extract total serum IgE and reverse transcription is cDNA(reverse transcription system is 10 μ L), with after TE damping fluid 10 times of gradient dilutions as template, adopt real-time fluorescence quantitative PCR, adopt P2-F and P2-R composition primer pair identification of cell cytochrome C oxidase VIIc subunit gene.
Amplification curve is shown in Fig. 4 B, and typical curve is shown in Fig. 4 A, and melting curve is shown in Fig. 4 C.
Typical curve equation is: the corresponding Ct value of y=-3.44x+9.1(y, the negative of x corresponding extension rate denary logarithm value), R
2=0.99.
The result of step 4 and step 5 shows, the primer pair (P2-F and P2-R) for the identification of cytochrome C oxidase VIIc subunit gene and the primer pair (P3-F and P3-R) for the identification of reference gene all specificity are good, and amplification efficiency is relatively consistent.
Claims (7)
1. a double stranded rna molecule, as shown in the sequence 2 of sequence table.
2. the DNA molecular of RNA molecule described in claim 1 of encoding.
3. the recombinant expression vector containing DNA molecular described in RNA molecule described in claim 1 or claim 2, transgenic cell line, recombinant bacterium or expression cassette.
4. the application of DNA molecular in preparing product described in RNA molecule described in claim 1 or claim 2; The purposes of described product be following (1) at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.
5. a product, its activeconstituents is DNA molecular described in RNA molecule described in claim 1 or claim 2; The purposes of described product be following (1) at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.
6. the application of DNA molecular described in RNA molecule described in claim 1 or claim 2, for following (1) is at least one in (4): (1) is anti-to eliminate aphis; (2) promote that aphid is dead; (3) aphid growth is suppressed; (4) expression of aphid cells in vivo cytochrome C oxidase VIIc subunit gene is suppressed.
7. apply as claimed in claim 4, product as claimed in claim 5, or, apply as claimed in claim 6, it is characterized in that: described aphid is wheat aphid.
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