The RNAi carrier and its application of silencing diamondback moth integrin β_1 subunit gene Px β
阅读说明：本技术 沉默小菜蛾整合素β1亚基基因Pxβ的RNAi载体及其应用 (The RNAi carrier and its application of silencing diamondback moth integrin β_1 subunit gene Px β ) 是由 杨广 傅淑 刘昭霞 陈金芝 孙庚晓 尤民生 于 2019-08-28 设计创作，主要内容包括：本发明属于农业生物技术领域,涉及沉默小菜蛾整合素β<Sub>1</Sub>亚基基因<I>Pxβ</I>的RNAi载体及其应用。选取小菜蛾整合素β<Sub>1</Sub>亚基基因<I>Pxβ</I>中497 bp的保守序列片段为靶标序列,利用Gateway技术将该靶标序列片段以正反两个方向插入植物表达载体pHellsgate-4中,构建表达靶标序列dsRNA结构的植物表达RNAi载体；通过农杆菌介导转化法,将该载体插入植物的基因组,获得表达靶标序列dsRNA结构的转基因植株。接虫试验表明转基因植株使小菜蛾靶标基因<I>Pxβ</I>的转录水平下调了44.7%-49.8%,同时也显著地降低小菜蛾化蛹率,减轻了蛹重,从而导致死亡率升高15.0%,为小菜蛾的防治提供了一种新方法。(The invention belongs to agricultural biological technical fields, are related to silencing diamondback moth integrin beta 1 Subunit gene Pxβ RNAi carrier and its application.Choose diamondback moth integrin beta 1 Subunit gene Pxβ In 497 bp conserved sequence segment be target sequence, using Gateway technology by the target sequence segment with positive and negative both direction insertion plant expression vector pHellsgate-4 in, building expression target sequence dsRNA structure plant express RNAi carrier；The genome of carrier insertion plant is obtained into the transgenic plant of expression target sequence dsRNA structure by Agrobacterium_mediated method.Worm is connect experiments have shown that transgenic plant makes diamondback moth target gene Pxβ Transcriptional level lowered 44.7%-49.8%, while it is also significant reduce diamondback moth percentage of pupation, alleviate pupa weight, increase 15.0% so as to cause the death rate, provide a kind of new method for the prevention and treatment of diamondback moth.)
1. a kind of silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier, it is characterised in that: select diamondback moth integrin β1Subunit genePxβ497 bp sequences as target sequence, the plant of building expression target gene sequence dsRNA expresses RNAi Carrier, the nucleotide sequence of the target sequence is as shown in SEQ ID NO:1.
2. silencing diamondback moth integrin beta according to claim 11Subunit genePxβRNAi carrier, it is characterised in that: it is logical It crosses Gateway homologous recombination technique target sequence is inserted into plant expression vector pHellsgate-4, building expression target base Because the plant of sequence dsRNA expresses RNAi recombinant vector pHells-dsβ, the diamondback moth gene in the carrierPxβTarget sequence piece Section is arranged in opposite directions with positive and negative both direction, and centre includes onePDKIntron sequences, withCaMV 35SPromoter starting expression,OCS terminatorFor terminator, there is eukaryon kalamycin resistance and protokaryon Spectinomycin resistance.
3. silencing diamondback moth integrin beta as described in claim 11Subunit genePxβRNAi carrier preparation method, feature It is, comprising the following steps:
(1) diamondback moth RNA, reverse transcription cDNA are extracted, using the cDNA as template, amplification obtains integrin beta1Subunit genePxβEntirely It is long, and it is inserted into acquisition recombinant cloning vector pEASY- in cloning vector pEASY-BluntPxβ；
(2) in diamondback moth integrin beta1Subunit genePxβThe conservative region of a length of 497 bp is inside selected, PCR clones the sequence Column, and the homology arm sequence attB for Gateway homologous recombination is added respectively at the both ends of cloned sequence1And attB2；
(3) utilize Gateway homologous recombination technique by the target sequence segment of clone and plant expression vector pHellsgate-4 Connection, is inserted into target sequence segment in carrier respectively with positive and negative both direction, includes one between two of them sequence fragmentPDKIntron sequences, withCaMV 35SPromoter starting expression,OCS terminatorFor terminator, having eukaryon card, that is mould Plain resistance and protokaryon Spectinomycin resistance, to be expressedPxβThe plant of target sequence dsRNA expresses RNAi carrier, name For pHells-dsβCarrier.
4. one kind contains silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier microbial transformant.
5. one kind according to claim 4 contains silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier micro- life Object transformant, it is characterised in that: the microbial transformant is to include silencing diamondback moth integrin beta1Subunit genePxβRNAi The Escherichia coli and Agrobacterium of carrier.
6. one kind contains silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier be applied to anti-diamondback moth genetically modified plants In preparation.
7. one kind according to claim 6 contains silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier be applied to In the preparation of anti-diamondback moth genetically modified plants, it is characterised in that: gained genetically modified plants successful expression diamondback moth integrin beta1 Subunit genePxβThe dsRNA of target sector sequence.
8. one kind according to claim 6 contains silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier be applied to In the preparation of anti-diamondback moth genetically modified plants, it is characterised in that: target gene is integrated after genetically modified plants obtained by diamondback moth feeding Plain β1SubunitPxβTranscriptional expression level significantly reduce, and pupa mitigates again, percentage of pupation decline, significantly risen so as to cause the death rate It is high.
Technical field
The invention belongs to agricultural biological technical fields, and in particular to silencing diamondback moth integrin beta1Subunit genePxβ's RNAi carrier and its application.One kind can express diamondback moth integrin beta in transgenic plants1Subunit genePxβDouble-stranded RNA (dsRNA) RNAi carrier is imported Plant Genome by the method for mediated by agriculture bacillus, to obtain by the RNAi carrier of structure The transgenic plant of anti-diamondback moth provides a kind of new strategy for the prevention and treatment of diamondback moth.
Background technique
Diamondback moth (Plutella xylostellaIt L. is) one of global important pests on brassicaceous vegetable, according to Estimate that diamondback moth is up to 7.7 hundred million dollars of (Li to the economic loss and expenses for prevention and control in China every yearet al., 2016).Currently, The means of prevention and treatment diamondback moth are still based on chemical pesticide, but are largely not only caused to other biology even mankind using pesticide Harm, and pollution will also result in environment, can more seriously drug resistance M8003 line be formed to diamondback moth.Table has been reported Bright diamondback moth has produced drug resistance to most pesticides, and is first and is reported in field to the production of Bt proteotoxin Insect (the Furlong of raw resistanceet al., 2013).Therefore, it has been extremely urgent for preventing and treating the new technology research and development of diamondback moth.
RNA interference (RNAi) refers to that external source or endogenous double-stranded RNA (double strand RNA, dsRNA) cause organism Inside the cracking of the mRNA (mRNA) of complementary pairing or inhibit the expression of its albumen therewith, this phenomenon is in 1998 in nematode In for the first time report (Fireet al., 1998).A kind of effective means of the RNAi technology as gene regulation is extensive at present The research (eating less recklessly, 2019) for being applied to insect functional genomics and control of insect etc..It is studied in the RNAi of early stage In, the effect of gene silencing is realized generally by modes such as injection, mixing foodstuff feedings, but these modes are in Field Pests In prevention and treatment using relatively difficult, first appeared using Expressed in Transgenic Plant pest gene dsRNA within 2007 and prevented and treated cotton The report of earworm, i.e. mediated plant insect RNAi pest control (Mao et al., 2007).Pass through Expressed in Transgenic Plant evil The mode of worm target gene dsRNA provides a kind of effective solution thinking for Field Pests improvement.
Integrin (Integrin) is the transmembrane protein of a kind of heterodimer being made up of α and β subunit non-covalent bond Family can form 24 kinds of different integrins (Hynes, 2002) by 18 α subunits and 8 β subunits.Wherein β1Integrin is By β1Subunit and α subunit composition, have accounted for the half of integrin protein family quantity, be bio-tissue and allelotaxis must Need albumen (Schnittert et al., 2018).It has been reported that and shows feeding diamondback moth integrinβ 1The dsRNA of subunit gene Afterwards, the death rate of diamondback moth can significantly improve (Mohamed and Kim, 2011).These results show that integrin beta1 Subunit gene has the potentiality of the target gene as mediated plant insect RNAi pest control.
Summary of the invention
In view of this, providing a kind of silencing diamondback moth integrin it is an object of the invention to overcome the deficiencies of existing technologies β1Subunit genePxβRNAi carrier and its application.Diamondback moth integrin beta is expressed in transgenic plant1SubunitPxβGene The RNAi carrier is imported Plant Genome by the method for mediated by agriculture bacillus, to be resisted by the RNAi carrier of dsRNA structure The transgenic plant of diamondback moth provides a kind of new strategy for the prevention and treatment of diamondback moth.
It is described that technical scheme is as follows:
A kind of silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier: selection diamondback moth integrin beta1Subunit genePxβ The conserved sequence of one section of 497 bp long in (the Genbank number of logining: GQ178290.1,2487 bp) is target, building expression target The plant for marking gene order dsRNA expresses RNAi carrier, the nucleotide sequence of the target such as SEQ ID NO:1.
A kind of silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier: pass through Gateway homologous recombination technique will Target sequence is inserted into plant expression vector pHellsgate-4, and the RNAi of building expression target sequence dsRNA structure, which is recombinated, to be carried Body pHells-dsβ；The diamondback moth gene in the carrierPxβTarget sequence segment is arranged in opposite directions with positive and negative both direction, tundish Containing onePDKIntron sequences, withCaMV 35SPromoter starting expression,OCS terminatorFor terminator, there is eukaryon Kalamycin resistance and protokaryon Spectinomycin resistance.
A kind of silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier preparation method, comprising the following steps:
(1) extract diamondback moth RNA, reverse transcription cDNA, using the cDNA as template, using Px β F/Px β R as primer pair (Px β F: 5'- ATG TCG GCG ACG AGG GGT CTA G -3'；Px β R:5 '-TTA CTT GCC AGC GTA CGT CGG GTT C -3 ') amplification acquisition integrin beta1Subunit genePxβOverall length, and be inserted into cloning vector pEASY-Blunt and obtain Recombinant cloning vector pEASY-Pxβ。
(2) in diamondback moth integrin beta1Subunit genePxβThe conservative region of a length of 497 bp has inside been selected, and has been utilized Primer pair attB1-βF / attB2- β R(attB1- β F:5 '-ggg gac aag ttt gta caa aaa agc agg ctc a - GCC AAC GCT TGC CGC TTC TGG -3'；attB2- β R:5 '-ggg gac cac ttt gta caa gaa Agc tgg gtc-GAT GTC GTA CAC GCC GCC CTC -3 ') from plasmid pEASY-PxβMiddle clone genePxβPhase 497 targeting regions bp RNAi (SEQ ID NO:1) answered, and the both ends of cloned sequence respectively add for Gateway it is same The homology arm sequence attB of source recombination1And attB2；
(3) utilize Gateway homologous recombination technique by the target sequence segment of clone and plant expression vector pHellsgate-4 Connection, is inserted into target sequence segment in carrier respectively with positive and negative both direction, includes one between two of them sequence fragmentPDKIntron sequences, withCaMV 35SPromoter starting expression,OCS terminatorFor terminator, having eukaryon card, that is mould Plain resistance and protokaryon Spectinomycin resistance, to be expressedPxβThe plant of target sequence dsRNA expresses RNAi carrier, name For pHells-dsβCarrier.
One kind containing silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier, i.e. pHells-dsβCarrier it is micro- Bioconversion body.
Further, a kind of to contain silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier, i.e. pHells-dsβ The Escherichia coli or Agrobacterium of carrier.
One kind containing silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier in anti-diamondback moth genetically modified plants system Application in standby.
One kind containing silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier applied to anti-diamondback moth transgenosis plant In the preparation of object, gained genetically modified plants successful expression diamondback moth integrin beta1Subunit genePxβDsRNA structure.
One kind containing silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier applied to anti-diamondback moth transgenosis plant Object, target gene integrin beta after diamondback moth feeding genetically modified plants1SubunitPxβTranscriptional expression level significantly reduce, and pupa weight Mitigate, percentage of pupation decline, is significantly increased so as to cause the death rate.
One kind containing silencing diamondback moth integrin beta1Subunit genePxβRNAi carrier applied to anti-diamondback moth transgenosis plant The preparation method of object the following steps are included:
(1) plant is expressed by RNAi carrier pHells-ds using freeze-thaw methodβIt imports in Agrobacterium GV3101 competent cell, then It is imported in arabidopsis seed cdna group by the genetic transforming method of During Agrobacterium arabidopsis bud, obtains transgenic arabidopsis Seed；It collects these seeds to be seeded in the screening and culturing medium containing 50 mg/L kanamycins, be grown one week to germination Afterwards, the arabidopsis for still keeping green state is considered as transgenic arabidopsis.
(2) genome for extracting transgenic arabidopsis carries out PCR expansion with the primer of specificity using the genome as template Increase the gene of diamondback mothPxβTarget sequence segment, PCR positive Molecular Identification is carried out to transgenic plant, and passes through RT- PCR and RT-qPCR technology analyzes transgenic plant transcriptional expression genePxβThe ability of target sequence dsRNA.
(3) transgenic plant connects worm feeding trial: expressing diamondback moth genePxβThe transgenosis of target sequence dsRNA It is tested after plant and the plant of normal wild type length to 4th week, is respectively chosen diamondback moth newly hatched larvae to both plant On blade, transparent plastic cup on cover, and cover the lid with strainer and prevent its escape, keep the growth of plant.It analyzes small Diamond-back moth target genePxβIt is silenced level, while comparing its pupa weight, percentage of pupation and the death rate, assesses the anti-pickles of transgenic plant The ability of moth.
The positive effect of the present invention is as follows: the present invention provides one kind to contain silencing diamondback moth integrin beta1Subunit genePx βRNAi carrier and its application.Select diamondback moth integrin beta1Subunit genePxβIn the conserved sequence of one section of 497 bp long be Target, building plant express RNAi carrier.The plant expresses RNAi carrier and is applied in the preparation of anti-diamondback moth genetically modified plants, Express diamondback moth integrin beta1Subunit genePxβThe transgenic plant of target sequence dsRNA inhibits diamondback moth target genePxβ 44.7% -49.8% transcriptional expression it is horizontal, and alleviate diamondback moth hero pupa weight, reduce percentage of pupation, and improve diamondback moth 15.0% death rate provides new strategy for the prevention and treatment of diamondback moth.
Detailed description of the invention
Fig. 1: contain diamondback moth integrin beta1Subunit genePxβThe plant of target sequence expresses RNAi carrier pHells-dsβ T-DNA section map.LB and RB is the right boundary of T-DNA in figure；CaMV 35SFor promoter,PDK intronTo include Son,OCS terminatorFor terminator,NPTIIFor kalamycin resistance gene,βFor diamondback moth integrin beta1SubunitPxβBase Because of target sequence.
Fig. 2: the RNAi carrier pHells-ds of mediated by agriculture bacillusβArabidopsis thaliana transformation.A is arabidopsis to be transformed；B is agriculture The seed collection of the arabidopsis of Agrobacterium-transformation；C is that normal arabidopsis seed is seeded in the culture medium without kanamycins antibiotic Middle growth；D is seeded in the culture medium containing 50 mg/L kanamycins antibiotic for normal arabidopsis seed to be grown；E is agriculture bar The seed of arabidopsis, which is seeded in the culture medium containing 50 mg/L kanamycins antibiotic, after bacterium conversion grows, and wherein arrow indicates Be the transgenic arabidopsis with kalamycin resistance.
Fig. 3: expression diamondback moth integrin beta1Subunit genePxβThe transgenic arabidopsis PCR positive of target sequence dsRNA is examined It measures and is intended to.1-15 is transgenic arabidopsis in figure, and C is normal control arabidopsis, and P is the positive matter containing target gene sequence Grain,18s rDNAFor arabidopsis reference gene.
Fig. 4: expression diamondback moth integrin beta1Subunit genePxβThe transgenic arabidopsis RT-PCR fine jade of target sequence dsRNA Sepharose electrophoresis detection schematic diagram.In figure number 201,202,203,205,208,209,21,214,218,22,23,24, 297,298 and 299 be different transgenic arabidopsis strains, and C is normal control arabidopsis, and P is to contain target gene sequence Positive plasmid,18s rRNAFor arabidopsis reference gene.
Fig. 5: expression diamondback moth integrin beta1Subunit genePxβThe transgenic arabidopsis RT-qPCR of target sequence dsRNA is examined It measures and is intended to.Number 202,205,208,209,218,23,24 and 297 is different transgenic arabidopsis strains in figure,PxβBase Because relative expression quantity uses arabidopsisSandGene is internal reference.
Fig. 6: expression diamondback moth integrin beta1Subunit genePxβ24 strain of transgenic arabidopsis of target sequence dsRNA is fed The method for raising diamondback moth.
Fig. 7: expression diamondback moth integrin beta1Subunit genePxβ24 strain pair of transgenic arabidopsis of target sequence dsRNA The evaluation of resistance of diamondback moth.A is influence of 24 strain of transgenic arabidopsis to diamondback moth percentage of pupation；B is transgenic arabidopsis 24 Influence of the strain to diamondback moth hero pupa weight；C is influence of 24 strain of transgenic arabidopsis to the diamondback moth death rate；WT is normal Compare arabidopsis.
Fig. 8: expression diamondback moth integrin beta1Subunit genePxβ24 strain pair of transgenic arabidopsis of target sequence dsRNA Diamondback mothPxβThe inhibition of mrna expression.A is diamondback moth four-age larva after feeding transgenic arabidopsisPxβGene Relative expression levels；B is diamondback moth pupa on the one after feeding transgenic arabidopsisPxβThe relative expression levels of gene.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention Technical solution is described further, but following examples is only present invention example therein, does not represent Ben Fa Rights protection scope defined by bright, the scope of the present invention are subject to claims.