CN104694548B

CN104694548B - Insect thoratropic hormone gene and its application in terms of plant anti-insect

Info

Publication number: CN104694548B
Application number: CN201310651573.6A
Authority: CN
Inventors: 朱祯; 王晓芳; 魏晓丽; 戴艳
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2013-12-05
Filing date: 2013-12-05
Publication date: 2017-12-15
Anticipated expiration: 2033-12-05
Also published as: CN104694548A

Abstract

The invention discloses a kind of application of insect thoratropic hormone gene in terms of plant anti-insect.The invention provides a kind of DNA molecular for being used to express the hairpin RNA that can suppress insect thoratropic hormone.The structure of DNA molecular provided by the present invention is SEQ_{It is positive}‑X‑SEQ_Reversely；The SEQ_{It is positive}Sequence be the 1st 659 of sequence 1；The SEQ_ReverselySequence and the SEQ_{It is positive}Sequence reverse complemental；The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive}And the SEQ_ReverselyIt is not complementary.It is demonstrated experimentally that by formula provided by the present invention（I）Shown DNA fragmentation great expression in plant under the driving of cotton curve leaf disease virus PRP promoters, the insect resistance capacity of transfer-gen plant can be effectively improved.Biological control of the present invention for plant-feed insect, engineering of insect-resistant plant, biology field play important application value.

Description

Insect thoratropic hormone gene and its application in terms of plant anti-insect

Technical field

The invention belongs to biological technical field, is related to a kind of insect thoratropic hormone gene and its in terms of plant anti-insect Application.

Background technology

The physiology courses and behavior reaction etc. such as the growing, cast off a skin of insect, metamorphosis, diapause, reproduction, polytypism are all It is unable to do without the participation of hormone.Moulting hormone（ecdysone）It is that animal can be adjusted by shirtfront glandular secretion（Mainly arthropod Door Insecta, Crustacea animal）The hormone of husking.Shrimp, the young of crab and adult have husking phenomenon, Apterigota animal Larva and adult have husking phenomenon, and Pterigota animal only has larva to have husking phenomenon.Most insects larva has periodically Husking phenomenon.Neuropeptide is a kind of secretion peptide, passes through post-translational cleavage by macromolecule, inactive preprohormone.God Various aspects through peptide hormone in insect life cycle play an important role, such as common thoratropic hormone （Prothoracicotropic Hormone, PTTH）There is important facilitation to the prothoracic gland synthesis moulting hormone of insect.

Efficient utilization of the raising of for many years crop yield dependent on chemical synthesis insecticide.But as insecticide exists Extensive application in agriculture, forestry, some have the problem of to be solved gradually to cause concern, such as environmental pollution, and edge effect is to non-target Mark and endangered caused by insect, caused by natural equilibrium multilated the problems such as the increase of Pest rampancy and pest resistance to insecticide.To understand Certainly these problem scientists have done many trials in the pest-resistant technical elements for finding more effectively alternative chemical insecticide, wrap The utilization of natural enemy is included, the use, the research and development of anti-pest GM crop etc. of the insecticide based on natural products.In above strategy, It is most widely used with expressing the genetically modified crops of bacillus thuringiensis toxalbumin.It is main that Bt toxalbumin can kill some Pest population using crop as food, and it is harmless to vertebrate and other biologies.What Bt protein insecticidal agents were most widely used at present is Cry1A crystalline proteins family, the Cry1Ab in particular for transgenic corns and the Cry1Ac for transgene cotton.In State, transgenosis Bt cottons have played very big effect since 1997 are commercialized during to bollworm resisting.In recent years, Big Tanaka in laboratory or plantation Bt genetically modified crops has been found that the insect that resistance is produced to Bt albumen, at least isolates 3 The microspecies of 7 generation resistances of kind insect.On the other hand, due to long-term plantation Bt genetically modified crops, the pest population knot in farmland Structure generates change, accounts for leading population structure by single insect and is developing progressively multiple insects and accounts for leading population structure, this More difficulties are brought for control insect.In summary, it would be highly desirable to which a kind of environmentally safe, group specificity is strong, to crop character There is no dysgenic new pest-resistant method to occur.Insect hormone has turned into a major fields of insecticide research and development, no It is same as acting on the conventional pesticides of nervous system in the past, it is acted on, and toxicity is low, pollutes less, natural enemy and beneficial organism are influenceed It is small, contribute to the development of sustainable agriculture, be advantageous to pollution-free green food production, be beneficial to man health.

RNA is disturbed（RNA interference, RNAi）A kind of newfound mechanisms of gene regulation, be by with target gene The double-stranded RNA of sequence homology（dsRNA）A kind of silencing specific genes phenomenon induced.There are some researches show dsRNA is direct The ovum, haemocoele or local organization of insect are injected into, the specific silence of remote target gene can be triggered.In addition, except directly noting DsRNA is penetrated, by the method for feeding dsRNA insect can also be induced to produce RNAi phenomenons.There are some researches show feeding phase at present Answer the dsRNA of target gene successfully reduces the shallow brown volume moth of apple（Epiphyas postvittana）Larva carboxy-lesterase expresses water Flat, honeybee（Apis mellifera）Rapamycin target protein（Targetof rapamycin, amTOR）Expression and Bollworm（Helicoverpa armigera）The expression of P450 genes.Thus, developed newly based on RNAi technology Insect pest control method, high specific and the new strategy of Environmental security are provided for agricultural insect management.

Insect hormone plays an important role in the growing of insect.The insect hormone of insect growth is adjusted, It is synthesized, involved in transport and metabolic pathway key gene is most to have very strong species specificity, i.e., with other species Genetic homology it is low, almost there is no homology especially compared with human gene, this largely reduces RNAi skills " effect of missing the target " of art, so as to provide guarantee for application security aspect.Therefore, insect hormone dependency basis is expressed in plant The dsRNA of cause, it the advantage is that only there is inhibitory action to the insect of phytophagous, while can also be according to insect hormone related gene Species specificity, a certain or a certain growing for insect of category is had an impact by expressing not homotactic dsRNA, from And reach the purpose of accurate, safe control insect pest.

The content of the invention

It is an object of the invention to provide a kind of application of insect thoratropic hormone gene in terms of plant anti-insect.

The invention provides it is a kind of can be with the DNA molecular of the hairpin RNA of expression inhibiting insect thoratropic hormone.

DNA molecular provided by the present invention is specially such as formula（I）Shown DNA fragmentation：

SEQ_{It is positive}-X-SEQ_Reversely（I）

The SEQ_{It is positive}Sequence be sequence 1 1-659 positions；

The SEQ_ReverselySequence and the SEQ_{It is positive}Sequence reverse complemental（The 879-1537 positions of sequence 1）；

The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive}And The SEQ_ReverselyIt is not complementary.

In the present invention, formula（I）Described in X sequence as shown in the 669-869 positions of sequence 1 in sequence table.

More specifically, formula（I）The nucleotide sequence of shown DNA fragmentation is as shown in sequence 1 in sequence table.

DNA molecular provided by the present invention is alternatively following DNA fragmentation first and DNA fragmentation second：The DNA fragmentation first is sequence The 10-667 positions of sequence 4 in list；The DNA fragmentation second is the reverse complemental sequence of the 10-667 positions of sequence 4 in sequence table Row.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the DNA fragmentation fall within the guarantor of the present invention Protect scope.

The recombinant vector can be recombinant expression carrier, or recombinant cloning vector.

The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture Bacillus carrier and carrier available for plant micropellet bombardment etc., as pGreen0029, pCAMBIA3301, pCAMBIA1300, The derivative plant expression vector of pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.The plant expression Carrier can also include 3 ' end untranslated regions of foreign gene, i.e., processed comprising polyadenylation signals and any other participation mRNA Or the DNA fragmentation of gene expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Using institute When stating gene constructed recombinant expression carrier, any enhanced, composing type, tissue can be added before its transcription initiation nucleotides Idiotype or inducible promoter, such as cauliflower mosaic virus（CAMV）35S promoter, ubiquitin gene Ubiquitin start Son（pUbi）, stress induced promoter rd29A etc., they can be used alone or are used in combination with other plant promoters； In addition, when using the gene constructed recombinant expression carrier of the present invention, enhancer, including translational enhancer or transcription also can be used to increase Hadron, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but required and coded sequence Reading frame it is identical, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon is wide General, can be natural or synthesis.Translation initiation region can come from transcription initiation region or structural gene. For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be processed, such as The coding that adding can express in plant can produce the enzyme of color change or the gene of luminophor, resistant antibiotic Label or anti-chemical reagent marker gene etc..Also any selected marker can be not added with, is directly screened and converted with adverse circumstance Plant.

In the present invention, the promoter for starting the genetic transcription in the recombinant expression carrier is specially PRP promoters Or T7 promoters.

Further, the sequence of the PRP promoters is as shown in sequence 2 in sequence table.

In one embodiment of the invention, the recombinant expression carrier is specially as follows（a）Or（b）：

（a）The restructuring matter obtained after DNA fragmentation shown in forward direction insetion sequence 1 at the restriction enzyme site of pC2300PN carriers Grain；The restriction enzyme site is specially Pst I；

The pC2300PN carriers are the DNA fragmentation shown in by sequence in sequence table 2（PRP promoters）It is inserted into Between Pst I and the EcoR I of pCAMBIA2300 carriers, while by DNA fragmentation shown in sequence 3（T-NOS terminators）It is inserted into Obtained recombinant plasmid after between Pst I and Hind III.

（b）The 10-667 positions nucleotides of insetion sequence 4 between two reverse T7 promoters of L4440 carriers（Or its Reverse complementary sequence）The recombinant plasmid obtained afterwards；The restriction enzyme site is specially Bgl II and Xho I.In the recombinant plasmid, The 10-667 positions nucleotides of sequence 4（Or its reverse complementary sequence）By two reverse T7 promoter two-way startups.

By above formula（I）The RNA molecule that shown DNA fragmentation encodes to obtain falls within protection scope of the present invention.

Above formula（I）Shown DNA fragmentation, or the recombinant expression carrier, expression cassette or recombinant bacterium, or the RNA molecule In following a1）Or a2）In application fall within protection scope of the present invention：

a1）Pest control；

a2）Cultivate pest-resistant genetically modified plants.

It is a further object to provide a kind of method for cultivating pest-resistant genetically modified plants.

The method provided by the present invention for cultivating pest-resistant genetically modified plants, specifically may include following steps：By the DNA Fragment is imported in purpose plant, obtains pest-resistant genetically modified plants.

In above-mentioned application or method, the pest-resistant death for being presented as promotion worm, and/or suppress the worm survived Body weight increase.

In the above-mentioned methods, the DNA fragmentation is imported in the purpose plant particular by will carry the DNA The recombinant expression carrier described above of fragment imports in the purpose plant what is realized.

In the above-mentioned methods, the recombinant expression carrier for carrying the DNA fragmentation is imported into the purpose plant, tool Body can be：It is situated between by using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, Agrobacterium Conventional biology methods conversion plant cell or the tissue such as lead, and the plant tissue of conversion is cultivated into plant.

It is also another object of the present invention to provide a kind of insecticide.

The active component of insecticide provided by the present invention is above formula（I）Shown DNA fragmentation, or the recombination expression carry Body, expression cassette or recombinant bacterium, or the RNA molecule.

Above formula（I）Shown DNA fragmentation, or the recombinant expression carrier, expression cassette or recombinant bacterium, or the RNA molecule Application in insecticide is prepared falls within protection scope of the present invention.

The above all of insecticide is the insecticide that may act on the insects such as bollworm.

In above-described application or method, the plant can be dicotyledon, or monocotyledon.At this In invention, the plant is dicotyledon, specially cotton（Gossypium hirsutum L.）, such as upland cotton （Gossypium hirsutum）Kind nasal mucus cotton No. 3, or tobacco（Such as Nicotiana tabacum cv.Xanthi）.

In the present invention, the worm in above-described application or method is bollworm, specially 2 age bollworms or 3 Age bollworm.

It is demonstrated experimentally that by formula provided by the present invention（I）Drive of the shown DNA fragmentation in cotton curve leaf disease virus PRP promoters Under dynamic in plant great expression, the insect resistance capacity of transfer-gen plant can be effectively improved.Life of the present invention for plant-feed insect Thing is prevented and treated, engineering of insect-resistant plant, and biology field plays important application value.

Brief description of the drawings

Fig. 1 is recombinant plasmid pLPTTH structural representations.Wherein, PTTH5 ' is the sequence 4 of sequence table from 5 ' ends 10-667 positions nucleotides.

Fig. 2 be embodiment 1 feeding experiment in two groups of processing bollworms mortality analysis block diagram.

Fig. 3 is interference carrier PRP:DsPTTH structural representation.Wherein, PTTH5 is that the sequence 4 of sequence table is last from 5 ' Hold 6-664 positions nucleotides.

Fig. 4 is the PCR detection figures of transgene tobacco.Wherein, M is DL2000plus Marker；CK is the open country of non-transgenosis Raw type tobacco；Swimming lane 1-10 is transgene tobacco, amplify about 521bp purpose bands for positive plant（At arrow）.

Fig. 5 is the death rate of cotton bollworm larvae in embodiment 3.Wherein, CK is to turn empty carrier tobacco control group.

Fig. 6 is transgenic tobacco leaf and the extent of injury for turning empty carrier tobacco leaf in embodiment 3.Wherein, A is to turn base Because of tobacco leaf；B is to turn empty carrier tobacco leaf.

Fig. 7 is feeding transgene tobacco and turns the body weight analysis of empty carrier tobacco survival bollworm.Wherein, CK is to turn sky Carrier tobacco control group.* the significant difference compared with CK group larval weights is represented（P<0.05）.

Fig. 8 is the PCR detection figures of transgene cotton.Wherein, M is DL2000plus Marker；CK is the open country of non-transgenosis Raw type cotton；Swimming lane 1-12 is transgene cotton, amplify about 264bp purpose bands for positive plant（At arrow）.

Fig. 9 is transgenosis cotton leaf and the extent of injury for turning empty carrier cotton leaf in embodiment 4.Wherein, A is a turn sky Carrier cotton leaf；B is transgene cotton blade.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

Quantitative test in following examples, it is respectively provided with and repeats to test three times, results averaged.

Bacterial expression vector L4440：The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research；With reference to Document：Timmons,L.and Fire,A.(1998)Specific interference by ingested dsRNA.Nature395:854–854.）；The details of the carrier can inquire on the internet, and specific network address is http://www.addgene.org/pgvec1?cmd=findpl&identifier=1654。

Escherichia coli HT115（DE3）：The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research；Ginseng Examine document：Timmons,L.,Court,D.L.and Fire,A.(2001)Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.Gene263:103–112.）.

Agrobacterium tumefaciems EHA105：The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research；With reference to Document：Hood EE,Gelvin SB,Melchers S,Hoekema A(1993)New Agrobacterium helper plasmids for gene transfer to plants(EHA105).Trans Res2:208-218。

Tobacco bred Nicotiana tabacum cv.Xanthi：The public can give birth to from Chinese Academy of Sciences's heredity with development Wu Xue research institutes obtain；Bibliography：A.NATO,S.BAZETOUX,Y.MATHIEU Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum(cv.Xanthi)Cell Suspension Cultures Physiologia Plantarum Volume41,Issue2,pages116–123,October1977。

Upland cotton（Gossypium hirsutum）Kind nasal mucus cotton No. 3：The public can be from Chinese Academy of Sciences's genetic development life Wu Xue research institutes obtain；Bibliography：Shen-Jie Wu,Hai-Hai Wang,Fei-Fei Li,Tian-Zi Chen,Jie Zhang, Yan-Jie Jiang, Yezhang Ding, Wang-Zhen Guo, Tian-Zhen Zhang.Enhanced Agrobacterium-mediated Transformation of Embryogenic Calli of Upland Cotton via Efficient Selection and Timely Subculture of Somatic Embryos.Plant Molecular Biology Reporter.2008Sep26(3):174-185。

Bollworm（Helicoverpa armigera）：The public can be from Chinese Academy of Sciences's heredity and Developmental Biology research Obtained；Bibliography：Wu KM,Lu YH,Feng HQ,Jiang YY,Zhao JZ.Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton.Science.2008Sep19;321(5896):1676-8。

LB culture mediums：Solvent is water, and solute and its concentration are as follows：Yeast extract 5g/L, peptone 10g/L, NaCl10g/L, PH to 7.0 is adjusted with the 10M NaOH aqueous solution.

YEB culture mediums：Solvent is water, and solute and its concentration are as follows：Beef extract 5g/L, yeast extract 1g/L, peptone 5g/ L, sucrose 5g/L, MgSO47H₂O0.04g/L, agar 15g/L（Fluid nutrient medium is then free of the component）, with 10M NaOH water Solution adjusts pH to 7.2.

Co-culture culture medium：The culture medium based on MS culture mediums, separately add final concentration of 0.1mg/L IAA（Indoles second Acid）, final concentration of 1mg/L 6-BA（6-benzyl aminopurine）, final concentration of 100 μM of AS（Acetosyringone）.

Screening and culturing medium：The culture medium based on MS culture mediums, separately add final concentration of 0.1mg/L IAA（Indoles second Acid）, final concentration of 1mg/L 6-BA（6-benzyl aminopurine）, final concentration of 400mg/L Cef（Cephalosporin）, it is final concentration of 100mg/L Kan（Kanamycins）.

Regeneration culture medium：The culture medium based on MS culture mediums, separately add final concentration of 1mg/L 6-BA（6- benzyl amino Purine）, final concentration of 400mg/L Cef（Cephalosporin）, final concentration of 100mg/L Kan（Kanamycins）.

Root media：The culture medium based on MS culture mediums, separately add final concentration of 200mg/L Cef（Cephalo is mould Element）, final concentration of 100mg/L Kan（Kanamycins）.

MS culture mediums：It is 5.8 to adjust PH, and formula is as follows：

Culture medium forms	Content mg/l
		NH₄NO₃	1650
KNO₃	1900
		KH₂PO₄	170
MgSO₄·7H₂O	370
		CaCl₂·2H₂O	440
MnSO₄·4H₂O	22.3
		ZnSO₄·7H₂O	8.6
H₃BO₃	6.2
		KI	0.83
Na₂MoO₄·2H₂O	0.25
		CuSO₄·5H₂O	0.025

CoCl₂·6H₂O	0.025
		Na₂-EDTA	37.25
FeSO₄·7H₂O	27.85
		Glycine	2
Vitamin B1	0.4
		Vitamin B6	0.5
Nicotinic acid	0.5
		Inositol	100
Sucrose	30000
		Agar	8000

B5 medium：It is 5.5 to adjust PH, and formula is as follows：

Culture medium forms	Content mg/l
		NaH₂PO₄·H₂O	150mg/L
KNO₃	3000mg/L
		(NH₄)₂SO₄	134mg/L
MgSO₄·7H₂O	500mg/L
		Na₂-EDTA	37.3mg/L
FeSO₄·7H₂O	27.8mg/L
		MnSO₄·H₂O	10mg/L
ZnSO₄·7H₂O	2mg/L
		H₃BO₃	3mg/L
Na₂MoO·2H₂O	0.25m g/L
		CuSO4·5H₂O	0.025mg/L
CoCl₂·6H₂O	0.025mg/L
		KI	10mg/L
Vitamin B1	10mg/L
		Vitamin B6	1mg/L
Glycine	2.0mg/L
		Folic acid	0.5mg/L
Inositol	100.0mg/L
		Sucrose	50g/L

MSB culture mediums：Organic matter+30g/L glucose+the 2.5g/ of inorganic salts+B5 medium of MS culture mediums Lphytalgel+1.1g/L MgCl₂。

Embodiment 1, cotton bollworm larvae feeding experiment

The encoding gene of the thoratropic hormone of bollworm（HaPTTH genes）CDNA as shown in the sequence 4 of sequence table, The sequence 2 of its ORFs such as sequence table forms from the nucleotides of 5 ' end the 30th to 710.

First, recombinant plasmid pLPTTH structure

1st, with the encoding gene of the thoratropic hormone of the bollworm shown in sequence 4（HaPTTH genes）For template, with drawing Thing P1-BglII/P2-XhoI enters performing PCR amplification, reclaims pcr amplification product.

P1-BglII：5’-GAAGATCTAC ACTCACGGGATAGTTTTCAG-3’（Underscore part is Bgl II knowledge Other sequence, sequence thereafter for the 646-667 positions of sequence 4 reverse complementary sequence）；

P2-XhoI：5’-CCGCTCGAGGCAACTGGAGAAAGCTGTAC-3’（Underscore part is Xho I identification sequence Row, sequence thereafter are the 10-29 positions of sequence 4）.

2nd, with the pcr amplification product of restriction enzyme Bgl II and Xho I double digestion steps 1, digestion products are reclaimed.

3rd, with restriction enzyme Bgl II and Xho I double digestion bacterial expression vector L4440, carrier framework is reclaimed.

4th, the digestion products of step 2 are connected with the carrier framework of step 3, obtains recombinant plasmid pLPTTH.Recombinant plasmid PLPTTH is that the sequence 4 of sequence table is inserted between Bgl II and Xho the I restriction enzyme sites for the carrier L4440 that sets out from 5 ' ends The recombinant plasmid obtained after the nucleotides of 10-667 positions.Recombinant plasmid pLPTTH structural representations are shown in Fig. 1.Two reverse T7 are opened The 10-667 positions of mover two-way startup sequence 4, can forms double-stranded RNA so on rna level, triggers RNAi, so as to Available for suppressing target gene（HaPTTH）Expression.

2nd, the preparation of recombinant bacterium and control bacterium

By the recombinant plasmid pLPTTH conversion Escherichia coli HT115 (DE3) obtained by step 1, recombinant bacterium is obtained.Using drawing Thing P1-BglII/P2-XhoI enters performing PCR checking to gained recombinant bacterium, as a result shows, gained recombinant bacterium expands available through PCR The purpose band that size is about 660bp, it is consistent with expected results.

Using as the bacterial expression vector L4440 conversion Escherichia coli HT115 (DE3) for the carrier that sets out, obtain compareing bacterium.

3rd, cotton bollworm larvae feeding experiment

1st, RNA samples are prepared

Recombinant bacterium prepared by step 2 is 37 DEG C in LB culture mediums, 200rpm shaken cultivations 8-10h；Then 2% is pressed（Body Product percentage composition）Ratio is transferred in fresh LB, shaken cultivation to OD₆₀₀For 0.6 or so；It is dense eventually to it to add IPTG Spend for 0.4mmol/L, shaken cultivation 4 hours, collection thalline；Total serum IgE is extracted from thalline（RNA sample first）.

Control bacterium prepared by step 2 is 37 DEG C in LB culture mediums, 200rpm shaken cultivations 8-10h；Then 2% is pressed（Body Product percentage composition）Ratio is transferred in fresh LB, shaken cultivation to OD₆₀₀For 0.6 or so；It is dense eventually to it to add IPTG Spend for 0.4mmol/L, shaken cultivation 4 hours, collection thalline；Total serum IgE is extracted from thalline（RNA sample second）.

2nd, the preparation of bollworm man-made feeds

Bollworm raises man-made feeds formula（1L）：Analysis for soybean powder 80g, wheat flour 80g, dusty yeast 16g, agar 14g, dimension life Plain C20 pieces, pangamic acid piece, sorbic acid 1g, sodium benzoate 1g, glacial acetic acid 5ml, Nepal gold ethyl ester 2g, benomyl （Benlate）0.05-0.1g, it is 1L to add water to volume；It is solid to boil after cooling down, and carries out feeding experiment.

3rd, cotton bollworm larvae feeding experiment

2 instar bollworm grubs are divided into two groups, every group sets three repetitions, each repeats 24 larvas, is fed respectively Experiment, it is specific as follows：

First group：The RNA samples first that concentration is 100ng/ μ l is uniformly sprayed on to the surface of man-made feeds（Every gram of feed spray Apply 10 μ l）；2 instar bollworm grubs are placed on the man-made feeds after spraying, change the man-made feeds of fresh warp thread processing daily；

Second group：The RNA samples second that concentration is 100ng/ μ l is uniformly sprayed on to the surface of man-made feeds（Every gram of feed spray Apply 10 μ l）；2 instar bollworm grubs are placed on the man-made feeds after spraying, change the man-made feeds of fresh warp thread processing daily；

After 10 days, the death rate of bollworm in two groups of statistics.

As a result as shown in fig. 2, it can be seen that the death rate of first group of cotton bollworm larvae is significantly higher than second group.

Embodiment 2, interference carrier PRP:DsPTTH structure

First, recombinant plasmid pC2300PN structure

In plant expression vector pCAMBIA2300（Purchased from CAMBIA companies；Detailed sequence is shown in GenBank: AF234315.1, network address：http://www.ncbi.nlm.nih.gov/nuccore/7638145）Upper structure connection cotton is bent Mosaic virus（CLCuV）Complementary strand promoter PRP and T-NOS terminator, obtain recombinant plasmid pC2300PN.Comprise the following steps that：

1st, synthetic DNA fragment "GAATTC+ sequence 2+CTGCAG", wherein sequence 2 is PRP promoter sequences.

2nd, the DNA fragmentation synthesized with restriction enzyme Pst I and EcoR I double digestions step 1, limited after recovery with passing through Property restriction endonuclease Pst I processed are connected with the pCAMBIA2300 carrier framework large fragments of EcoR I double digestions, and gained recombinant plasmid is ordered Entitled pCAMBIA2300-PRP.Recombinant plasmid pCAMBIA2300-PRP structure is described as：In the enzyme of pCAMBIA2300 carriers The recombinant plasmid obtained between enzyme site Pst I and EcoR I in insetion sequence table after DNA fragmentation shown in sequence 2.

3rd, synthetic DNA fragment "CTGCAG+ sequence 3+AAGCTT", wherein sequence 3 is T-NOS terminator sequences.

4th, with restriction enzyme Pst I and Hind III double digestions step 3 synthesize DNA fragmentation, after recovery with process Restriction enzyme Pst I are connected with the pCAMBIA2300-PRP carrier framework large fragments of Hind III double digestions, that is, obtain weight Group plasmid pC2300PN.Recombinant plasmid pC2300PN structure is described as：In the restriction enzyme site of pCAMBIA2300-PRP carriers The recombinant plasmid obtained between Pst I and Hind III in insetion sequence table after DNA fragmentation shown in sequence 3.

2nd, interference carrier PRP:DsPTTH structure

1st, synthetic DNA fragment "CTGCAG+ sequence 1+CTGCAG", wherein sequence 1 is interference fragment, altogether by 1537 nucleosides Acid composition, 1-659 positions are the positive sequence of HaPTTH genetic fragments（The 6-664 positions of sequence 4）；669-869 positions are interior Nucleotide sequence containing daughter nucleus；879-1537 positions are the reverse sequence of the HaPTTH genetic fragments（The 6-664 positions of sequence 4 Reverse complementary sequence）.By one section of introne between positive sequence and reverse sequence（intron）Sequence is separated to maintain carrier Stability；The DNA fragmentation transcribes generation band hairpin structure in plant cell（hairpin）DsRNA, trigger RNAi, so as to Available for the expression for suppressing target gene.

2nd, the DNA fragmentation synthesized with restriction enzyme Pst I digestions step 1, with passing through Pst I digestions after recovery The skeleton large fragment of pC2300PN carriers is connected, and obtains interference carrier PRP:dsPTTH.Interference carrier PRP:DsPTTH structure Schematic diagram is shown in Fig. 3, and its structure is described as：Disturbed at the restriction enzyme site Pst I of pC2300PN carriers shown in positive insetion sequence 1 The recombinant plasmid obtained after fragment.

Embodiment 3, the acquisition of transgene tobacco and Resistance Identification

First, the acquisition of transgene tobacco

1st, the interference carrier PRP for building embodiment 2:DsPTTH electric shocks are transferred to Agrobacterium tumefaciems EHA105, are recombinated Agrobacterium.

2nd, recombinational agrobacterium is turned to draw YEB solid mediums（50mg/L containing kanamycins, rifampin 50mg/L）, lived Change；Picking monoclonal bacterial plaque is in YEB fluid nutrient mediums（50mg/L containing kanamycins, rifampin 50mg/L）, 28 DEG C of 200rpm trainings Support 16~20 hours；Then 4% is pressed（Volumn concentration）Ratio be inoculated in YEB fluid nutrient mediums（Containing the μ of acetosyringone 100 M）, 28 DEG C, 200rpm shaken cultivations to OD₆₀₀Value reaches 0.5~0.6（About 4 hours）；4 DEG C, 4000rpm centrifugations, collect thalline； By thalline MS fluid nutrient mediums（Containing 200 μM of acetosyringone）Suspend, regulation OD values are 0.15~0.20, as recombinant bacterium bacterium Suspension.

3rd, by sterile tobacco（Nicotiana tabacum cv.Xanthi）Blade is cut into small leaf dish, is put into recombinant bacterium bacterium In suspension, infect 10 minutes.

4th, the small leaf dish after infecting is taken out, filter paper somewhat sucks unnecessary bacterium solution, and the leaf dish back side is laid on co-cultivation culture upward On base, 25 DEG C of dark condition cultures 72 hours；Leaf dish through co-cultivation is transferred on screening and culturing medium, 28 DEG C of 16h illumination/8h Cultivated 15~20 days under dark photoperiod；The leaf dish that has callus will be grown regeneration culture medium is transferred to by screening and culturing medium On, cultivated under 28 DEG C of 16/8h photoperiods to there is small regeneration bud to occur；Cut budlet and move on to strong plantlets and rootage in root media, Moved into after long root in vermiculite nutrition earth mixtures.

5th, PCR is identified

The seedling that will take root enters performing PCR identification（P3/P4）, the plant for showing 521bp or so band is PCR positive plants.

P3：5’-TGCTGACGTGCTTGACTACA-3’（The 1023-1042 positions of sequence 1）；

P4：5’-CTGCAGGCAACTGGAGAAAG-3’（Whether should be Pst I recognition sequence CTGCAG at underscore It is just right, sequence thereafter for the 1524-1537 positions of sequence 1 reverse complementary sequence）.

PCR reaction conditions：95 DEG C, 5 minutes；95 DEG C 40 seconds, 56 DEG C 40 seconds, 72 DEG C 40 seconds, 35 circulation；72 DEG C of extensions 10 Minute.

The electrophoretogram of PCR product is shown in Fig. 4, and it is positive plant that amplification, which obtains the plant that size is about 521bp purpose bands,.

2nd, the acquisition of tobacco is compareed

PC2300PN carriers are replaced into interference carrier PRP:DsPTTH, operated using step 1 identical, obtain turning zero load Body tobacco, the control as transgene tobacco.

3rd, insect resistance identification

What the transgene tobacco and step 2 that step 1 is obtained respectively obtained turns empty carrier tobacco progress feeding experiment, tool Body is as follows：Plant is placed in hot-house culture（Growth temperature is 25 DEG C, and periodicity of illumination is 16h illumination/8h dark）, plant strain growth is extremely During 7~10 leaf age, 3 instar bollworm grubs are inoculated with, every plant of tobacco is inoculated with 10 larvas；The death rate of larva is counted daily and is deposited The body weight of larva living, and observe tobacco leaf form.

Experiment sets the tobacco of non-transgenosis as wild type control simultaneously.

Test in triplicate, quantitative data results averaged.

The death rate of cotton bollworm larvae is shown in Fig. 5, it can be seen that on the 6th, 8 day transgenic tobacco plant after inoculation by The death rate of test worm is obviously higher than turning empty carrier tobacco.After inoculation 48 hours, transgenic tobacco leaf and turn empty carrier cigarette The extent of injury of blade of grass piece is shown in Fig. 6, it can be seen that compared to turning empty carrier tobacco leaf, transgenic tobacco leaf by Evil degree is significantly lighter.

In addition, the 2nd, 4,6, the 8 day bollworm for taking the surviving on plant after inoculation, weighs and enters worm tuple evidence Row variance analysis.As a result Fig. 7 is seen, it can be seen that the larval weight of feeding transgene tobacco is significantly less than feeding and turns empty carrier cigarette The larval weight of grass, P<0.05.

As the tobacco group of the non-transgenosis of wild type control, the death rate of larva, the body weight for the larva that survives, and tobacco The undermined degree of blade with basically identical, the no difference of science of statistics that turns empty carrier tobacco group.

Embodiment 4, the acquisition of transgene cotton and insect resistace identification

First, the acquisition of transgene cotton

Cotton Transformation regenerates genetic transformation using embryo, concretely comprises the following steps：The interference carrier PRP that embodiment 2 is built: DsPTTH electric shocks are transferred to Agrobacterium tumefaciems EHA105, obtain recombinational agrobacterium.Recombinational agrobacterium is activated into switching culture to OD600 =0.5 or so.4 DEG C, 5000r/min centrifugation 5min, collect thalline.Then with MSB0 Liquid Cultures of the 5ml containing 100 μm of ol/L AS Base（Formula：The inorganic salts of MS culture mediums, the organic matter of B5 medium, 30g/L glucose and 0.75g/L MgCl₂）Again suspend Precipitation, then MSB0 fluid nutrient mediums of the 15~45ml containing 100 μm of ol/L AS is transferred to, culture is left to OD600=0.5 on 28 DEG C of shaking tables The right side, obtain Agrobacterium when diluting nutrient solution OD600 value 0.3~0.7 and infect liquid, it is standby.Dip-dye is carried out on super-clean bench, uses operation Knife cuts upland cotton（Gossypium hirsutum）Stem section 7cm of kind nasal mucus cotton No. 3 or so, hypocotyl stem section is soaked in agriculture Bacillus infects 5~8min in liquid, blots plumular axis section bacterium solution with sterilizing filter paper, is placed on the co-cultivation culture for being covered with one layer of sterilizing filter paper On base, sealed with sealed membrane, 22 DEG C~25 DEG C co-cultivation 2d.Treat that the callus diameter that hypocotyl segment both ends otch is grown is big Cut independent subculture during about 0.5~1.0cm, be transferred to embryonic callus induction training during the diameter about 2cm of callus lines Support base（MSB2 culture mediums）On, in MSB2 culture mediums（Formula：Final concentration of 3.8g/L KNO is added in MSB₃With it is final concentration of 1.5g/L curing agent）Upper 1~3 appearance for having embryo callus subculture of subculture.When callus grows to diameter about 2~3cm, It is cut off from plumular axis section, inducing culture propagation is transferred to, can effectively remove Agrobacterium and prevent callus browning from showing As.Embryo callus subculture inducing culture is put into when callus lines diameter reaches 7~8mm（MSB2 culture mediums）In, between culture often Cultivated under the conditions of rule, every other month embryo callus subculture inducing culture of left/right rotation（MSB2 culture mediums）, 2~6 times, to appearance Yellow green or celadon, untill the embryo callus subculture of rice-shaped.The callus of doubtful differentiation is disperseed into subculture to differential medium（Match somebody with somebody Side：MSB adds 1.9g/L KNO₃, Sucrose30g/L, Phytogel4g/L, pH6.5）On, it is viable on differential medium Usually embryo callus.Screened by 10d or so resistance embryo, the embryo callus subculture survived is chosen into bottleneck bag tampon, made Cultivated with Ventilative bottle plug blake bottle, promote the formation and sprouting of embryoid.The plantlet tiling of sprouting is inoculated into paving filter paper Differential medium（Formula：MSB adds 1.9g/L KNO₃, Sucrose30g/L, Phytogel4g/L, pH6.5）On, induce into Seedling.Plantlet has been transferred to callus proliferation medium again（Formula：MSB adds 1.9g/L KNO₃, Sucrose30g/L, Phytogel4g/L, pH6.5）Big triangular flask in, take 2~3 leaves of plantlet under aseptic condition, a small amount of methods extract DNA, carry out The PCR amplifications of target gene, by positive plantlet grafting greenhouse.Ventilated after 3~4d of grafting, that is, outward winding bottle cap and cover bottleneck 2/3, Mouldy to prevent scion, 7d removes bottle cap, and 10~12d takes off bottle, 15d or so Xie Sheng and survived.

Wherein, PCR identifications are specific as follows：

The seedling that will take root enters performing PCR identification（P5/P6）, the plant for showing 264bp or so band is PCR positive plants.

P5：5’-TGTAACGTGGGTAGGTGTTTTC-3’（The 1057-1078 positions of sequence 1）；

P6：5’-GTAACGTAGGAACAAATTACGA-3’（The reverse complementary sequence of the 1299-1320 positions of sequence 1）.

PCR reaction conditions：95 DEG C, 5 minutes；95 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72 DEG C of extensions 10 Minute.

The electrophoretogram of PCR product is shown in Fig. 8, and it is positive plant that amplification, which obtains the plant that size is about 264bp purpose bands,.

2nd, the acquisition of transgene cotton is compareed

PC2300PN carriers are replaced into interference carrier PRP:DsPTTH, operated using step 1 identical, obtain turning zero load Body cotton, the control as transgene cotton.

3rd, the insect resistace detection of cotton

What the transgene cotton and step 2 that step 1 is obtained respectively obtained turns empty carrier cotton progress feeding experiment, tool Body is as follows：Plant is placed in hot-house culture（Growth temperature is 25 DEG C, and periodicity of illumination is 16h illumination/8h dark）.Put per culture dish 2 tender leafs, with absorbent cotton moisturizing, put neat 2 instar bollworm grub 6 of the growth of man-made feeds nursing simultaneously again per ware, add Paper covers, and is escaped with pest control.Its dead borer population, borer population living and blade were observed after the 3rd day and is killed degree etc..

Experiment sets the cotton of non-transgenosis as wild type control simultaneously.

Test in triplicate, quantitative data results averaged.

The death rate of cotton bollworm larvae is shown in Table 1, it can be seen that the death rate on transgene cotton blade by test worm is obvious Higher than turning on empty carrier cotton leaf by the death rate of test worm.The 7th day after worm is connect, transgene cotton blade and turn empty carrier cotton The extent of injury of floral leaf piece is shown in Fig. 9, it can be seen that compared to turning empty carrier cotton leaf, transgene cotton blade by Evil degree is significantly lighter.

Table 1 connects the 3rd day bollworm mortality statistics after worm（Unit：%）

Group	3rd day
		Transgene cotton	60.6
Turn empty carrier cotton	28.6

In addition, in the 10th day bollworm for taking the surviving on blade after connecing worm, weigh and by worm tuple according to progress side Difference is analysed.It the results are shown in Table 2, it can be seen that the larval weight of feeding transgene cotton blade is significantly less than feeding and turns empty carrier cotton The larval weight of floral leaf piece, P<0.05.

Table 2 connects the 10th day bollworm body weight statistics after worm

As the cotton group of the non-transgenosis of wild type control, the death rate of larva, the body weight for the larva that survives, and cotton The undermined degree of blade with basically identical, the no difference of science of statistics that turns empty carrier cotton group.

Claims

1.DNA fragments or recombinant vector or expression cassette or recombinant bacterium or RNA molecule are in following a1) or a2) in application：

A1 bollworm) is prevented and treated；

A2 the genetically modified plants of bollworm resisting) are cultivated；

Shown in the DNA fragmentation such as formula (I)：

SEQ_{It is positive}-X-SEQ_Reversely (I)

The SEQ_{It is positive}Sequence be sequence 1 1-659 positions；

The SEQ_ReverselySequence and the SEQ_{It is positive}Sequence reverse complemental；

The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive}It is and described SEQ_ReverselyIt is not complementary；

The recombinant vector is the recombinant vector containing the DNA fragmentation；

The expression cassette is the expression cassette containing the DNA fragmentation；

The recombinant bacterium is the recombinant bacterium containing the DNA fragmentation；

The RNA molecule is the RNA molecule that the DNA fragmentation encodes to obtain.

2. application according to claim 1, it is characterised in that：The 669- of sequence 1 in the sequence of the X such as sequence table Shown in 869.

3. application according to claim 2, it is characterised in that：The nucleotides sequence of the DNA fragmentation is classified as sequence in sequence table Row 1.

4. application according to claim 1, it is characterised in that：Start the DNA fragmentation transcription in the recombinant vector Promoter is PRP promoters or T7 promoters.

5. according to any described application in claim 1-4, it is characterised in that：The plant is dicotyledon or unifacial leaf Plant.

6. application according to claim 5, it is characterised in that：The dicotyledon is cotton or tobacco.

7. a kind of method for the genetically modified plants for cultivating bollworm resisting, comprises the following steps：DNA fragmentation is imported into purpose plant In, obtain the genetically modified plants of bollworm resisting；

Shown in the DNA fragmentation such as formula (I)：

SEQ_{It is positive}-X-SEQ_Reversely (I)

The SEQ_{It is positive}Sequence be sequence 1 1-659 positions；

The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive}It is and described SEQ_ReverselyIt is not complementary.

8. according to the method for claim 7, it is characterised in that：The 669- of sequence 1 in the sequence of the X such as sequence table Shown in 869.

9. according to the method for claim 8, it is characterised in that：The nucleotides sequence of the DNA fragmentation is classified as sequence in sequence table Row 1.

10. according to any described method in claim 7-9, it is characterised in that：The plant is dicotyledon or list Leaf plant.

11. according to the method for claim 10, it is characterised in that：The dicotyledon is cotton or tobacco.

12. a kind of insecticide, its active component is DNA fragmentation or recombinant vector or expression cassette or recombinant bacterium or RNA molecule；

The worm is bollworm；

Shown in the DNA fragmentation such as formula (I)：

SEQ_{It is positive}-X-SEQ_Reversely (I)

The SEQ_{It is positive}Sequence be sequence 1 1-659 positions；

13. insecticide according to claim 12, it is characterised in that：The of sequence 1 in the sequence of the X such as sequence table Shown in 669-869 positions.

14. insecticide according to claim 13, it is characterised in that：The nucleotides sequence of the DNA fragmentation is classified as sequence table Middle sequence 1.

15. insecticide according to claim 12, it is characterised in that：Start the DNA fragmentation in the recombinant vector to turn The promoter of record is PRP promoters or T7 promoters.