阅读说明：本技术 一种同时沉默小菜蛾精氨酸激酶基因PxAK和整合素β1亚基基因Pxβ的RNAi载体 (A kind of RNAi carrier of while silencing diamondback moth arginine kinase gene PxAK and 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,涉及一种可以在转基因植物中同时表达小菜蛾精氨酸激酶基因<I>PxAK</I>和整合素β<Sub>1</Sub>亚基基因<I>Pxβ</I>双链RNA(dsRNA)结构的RNAi载体,通过农杆菌介导的方法将该RNAi载体导入植物基因组,从而获得抗小菜蛾的转基因植株。本发明通过接虫试验,表明转基因植株可以同时沉默小菜蛾<I>PxAK</I>和<I>Pxβ</I>基因26.6%-30.9%的转录表达水平,且小菜蛾幼虫发育历期显著延长,化蛹率降低,从而导致死亡率升高了22.5%,为小菜蛾的防治提供了一种新方法,也为害虫防治提供了新策略。(The invention belongs to agricultural biological technical field, being related to one kind in transgenic plants while can express diamondback moth arginine kinase gene PxAK And integrin beta 1 Subunit gene Pxβ The RNAi carrier is imported Plant Genome by the method for mediated by agriculture bacillus, to obtain the transgenic plant of anti-diamondback moth by the RNAi carrier of double-stranded RNA (dsRNA) structure.The present invention by connect worm test, show transgenic plant can simultaneously silencing diamondback moth PxAK With Pxβ The transcriptional expression of gene 26.6%-30.9% is horizontal, and diamondback moth larvae development duration significantly extends, and percentage of pupation reduces, and increases 22.5% so as to cause the death rate, provides a kind of new method for the prevention and treatment of diamondback moth, also provide new strategy for control of insect.)

1. a kind of silencing diamondback moth arginine kinase gene simultaneouslyPxAKAnd integrin beta₁Subunit genePxβRNAi carrier, it is special Sign is: selecting diamondback moth arginine kinase gene respectivelyPxAK384 bp sequences and integrin beta₁Subunit genePxβ497 Bp sequence is target, and the seamless integration fusion sequence of the two target sequences is obtained using seamless integration technology rapid build, with The fusion sequence is that target constructs plant expression RNAi carrier, fusion target sequence core as shown in SEQ ID NO:1 Nucleotide sequence.

2. a kind of preparation method of RNAi carrier described in claim 1, which comprises the following steps:

(1) diamondback moth RNA, reverse transcription cDNA are extracted, using the cDNA as template, expands arginine kinase genePxAKAnd integration Plain β₁Subunit genePxβOverall length, and be inserted into cloning vector pEASY-blunt obtain recombinant cloning vector pEASY- respectivelyPxAK And pEASY-Pxβ；

(2) in diamondback moth arginine kinase genePxAKAnd integrin beta₁Subunit genePxβInside have chosen respectively a length of 384 bp and The conservative region of 497 bp obtains the fusion sequence of the two gene target sequences using seamless integration technology；

(3) by the PCR amplification fusion sequence, and sequence attB is added respectively at the both ends of sequence₁And attB₂For Gateway Homologous recombination；

(4) the target sequence segment is reacted with plant expression vector pHellsgate-4 using Gateway homologous recombination technique, It is inserted into fusion sequence target in carrier respectively with positive and negative both direction, includes one between two of them sequence fragmentPDKIt is interior Containing subsequence, byCaMV 35SPromoter starting expression, to obtain the plant table that can express target sequence segment dsRNA Up to RNAi carrier, it is named as pHells-dsAK-βCarrier.

3. a kind of application of RNAi carrier as described in claim 1, which is characterized in that utilize Agrobacterium-mediated Transformation method will pHells-dsAK-βCarrier arabidopsis thaliana transformation, acquisition can simultaneously expressing genePxAKWithPxβThe dsRNA of target sequence, and it is right The resistant transgenic arabidopsis of diamondback moth.

Technical field

The invention belongs to agricultural biological technical field, being related to one kind in transgenic plants while can express diamondback moth essence Histidine kinase genePxAKAnd integrin beta₁Subunit genePxβThe RNAi carrier of target sequence double-stranded RNA (dsRNA) structure is led to The RNAi carrier is imported Plant Genome to obtain the transgenic plant of anti-diamondback moth and is by the method for crossing mediated by agriculture bacillus The prevention and treatment of diamondback moth provides a kind of new strategy.

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.

Arginine kinase (Arginine kinase, AK) is a kind of phosphokinase, can be catalyzed the intracorporal ATP of insect and turn ADP is turned to, is that the mode (Zhou of energy is uniquely provided in insect bodies to discharge energy needed for growth and developmentet al., 2000).After having been reported that the dsRNA for showing feeding phyllotreta striolata arginine kinase gene, there is development and prolongs in phyllotreta striolata Late, the symptoms (Zhao such as the death rate increases and reproductive capacity declineset al., 2008).Integrin (Integrin) is one kind by α The transmembrane protein family for the heterodimer being made up of with β subunit non-covalent bond can form 24 by 18 α subunits and 8 β subunits The different integrin (Hynes, 2002) of kind.Wherein β₁Integrin is by β₁Subunit and α subunit composition, have accounted for integration fibroin The half of family's quantity is bio-tissue and the indispensable protein (Schnittert of allelotaxiset al., 2018).There is report Road shows feeding diamondback moth integrinβ ₁After the dsRNA of subunit gene, the death rate of diamondback moth can be improved significantly (Mohamed and Kim, 2011).These results show that arginine kinase gene and integrin beta₁Subunit gene, which has, to be made For the potentiality of the target gene of mediated plant insect RNAi pest control.Show to express multiple targets simultaneously in addition, also having been reported that RNAi efficiency (the Tzin to black peach aphid can be improved in the tobacco of gene dsRNAet al., 2015).Therefore, we are with arginine Kinase genePxAKAnd integrin beta₁Subunit genePxβFusion sequence as target construct plant expression RNAi carrier, with Phase provides new strategy for the prevention and treatment of diamondback moth.

Summary of the invention

In view of this, providing one kind can be in genetically modified plants it is an object of the invention to overcome the deficiencies of existing technologies In express diamondback moth arginine kinase gene simultaneouslyPxAKAnd integrin beta₁Subunit genePxβFusion sequence dsRNA structure The RNAi carrier is imported Plant Genome by the method for mediated by agriculture bacillus by RNAi carrier, to obtain turning for anti-diamondback moth Gene plant provides a kind of new strategy for the prevention and treatment of diamondback moth.

To achieve the above object, the invention adopts the following technical scheme:

Simultaneous selection of the present invention diamondback moth arginine kinase genePxAKIn one section of conserved sequence and integrin beta₁Subunit base CausePxβIn one section of conserved sequence, obtain the fusion sequence of the two conserved sequences, by seamless integration technology with the fusion sequence It is classified as target, which is inserted into positive and negative both direction by plant expression by Gateway homologous recombination technique and is carried In body pHellsgate-4, building can be expressedPxAKWithPxβThe plant of target fusion sequence dsRNA expresses RNAi carrier；It is logical The method for crossing agrobacterium mediation converted, by the genome of RNAi carrier insertion plant, acquisition can express diamondback moth simultaneously Target genePxAKWithPxβThe transgenic plant of target sequence dsRNA.By connecing worm feeding trial, the feeding transgenosis is analyzed The diamondback moth target gene of plantPxAKWithPxβTranscriptional expression it is horizontal, larvae development goes through phase, the percentage of pupation of polypide and the death rate Deng insect resistant effect of the evaluation transgenic plant to diamondback moth.

Applicant provide a kind of rapid buildsPxAKGene andPxβThe plant expression RNAi of gene target fusion sequence is carried Body, and provide application of the carrier in Genetic Transformation in Higher Plants, concrete application is shown in steps are as follows:

(1) extract diamondback moth RNA, reverse transcription cDNA, using the cDNA as template, respectively with PxAKF/PxAKR and Px β F/ Px β R is primer pair amplifies arginine kinase genePxAKAnd integrin beta₁Subunit genePxβOverall length, and insertion clone carries respectively Recombinant cloning vector pEASY- is obtained in body pEASY-bluntPxAKAnd pEASY-Pxβ.In diamondback moth arginine kinase genePxAKAnd integrin beta₁Subunit genePxβThe conservative region for inside having chosen a length of 384 bp and 497 bp respectively, utilizes seamless group Dress technology has been quickly obtained the fusion sequence of the two gene target sequences, then with PCR(Ploymerase Chain Reaction) method has carried out gene cloning to the fusion sequence, utilizes primer pair attB₁-AKF / attB₂- β R is in clone's sequence The both ends of column are added to sequence attB respectively₁And attB₂For Gateway homologous recombination；Utilize Gateway homologous recombination technique The target sequence segment is reacted with plant expression vector pHellsgate-4, makes fusion sequence target with positive and negative both direction point It Cha Ru not include one between two of them sequence fragment in carrierPDKIntron sequences, so that the target can be expressed by obtaining The plant of sequence fragment dsRNA expresses RNAi carrier, is named as pHells-dsAK-βCarrier.

PxAKF:5 '-ATG GTG GAC GCT GCA ACT CTT G -3 '；

PxAKR:5 '-TTA CAG GGA CTT CTC GAT CTT GAT GAG C -3 '；

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 '；

attB₁- AKF: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’。

(2) plant is expressed by RNAi carrier pHells-ds using freeze-thaw methodAK-βIt is thin to import Agrobacterium GV3101 competence It is intracellular, then imported in arabidopsis seed cdna group by the genetic transforming method of During Agrobacterium arabidopsis bud, acquisition turns base Because of arabidopsis seed；It collects these seeds to be seeded in the screening and culturing medium containing 50 mg/L kanamycins, germination growth After a week, the arabidopsis for still keeping green state is considered as transgenic arabidopsis.

(3) genome for extracting transgenic arabidopsis carries out PCR expansion with the primer of specificity using the genome as template Increase the target gene of diamondback mothPxAKWithPxβTarget sequence segment, to transgenic plant carry out PCR positive Molecular Identification, and And its target gene of transgenic plant transcriptional expression is analyzed by RT-PCR and RT-qPCR technologyPxAKWithPxβDsRNA energy Power.

(4) transgenic plant connects worm feeding trial: expressing diamondback moth genePxAKWithPxβTarget sequence dsRNA's The plant of transgenic plant and normal wild type is long to being tested after 4th week, respectively by diamondback moth newly hatched larvae choose to this two On plant blade, transparent plastic cup on cover, and cover the lid with strainer and prevent its escape, keep the growth of plant. Analyze diamondback moth target genePxAKWithPxβIt is silenced level, while comparing its development duration, percentage of pupation and the death rate, is assessed The ability of the anti-diamondback moth of transgenic plant.

The positive effect of the present invention is as follows: the expression diamondback moth arginine kinase gene of the inventionPxAKAnd integrin β₁Subunit genePxβThe transgenic plant of fusion sequence dsRNA, while inhibiting diamondback moth target genePxAKWithPxβTranscription Horizontal 23.3% -24.7%, extends larvae development and goes through the phase, reduce percentage of pupation, and improve the death of diamondback moth 22.5% Rate provides new strategy for the prevention and treatment of diamondback moth.

Detailed description of the invention

Fig. 1: contain diamondback moth arginine kinasePxAKGene and integrin beta₁Subunit genePxβThe plant table of fusion sequence Up to RNAi carrier pHells-dsAK-βT-DNA section map；LB and RB is the right boundary of T-DNA in figure；CaMV 35SFor Promoter,PDK intronFor introne,OCS terminatorFor terminator,NPTIIFor kalamycin resistance gene.

Fig. 2: the RNAi carrier pHells-ds of mediated by agriculture bacillusAK-βArabidopsis thaliana transformation；A figure is to be transformed intends in Fig. 2 Southern mustard；B figure is the seed collection of the arabidopsis of Agrobacterium-mediated Transformation in Fig. 2；C figure is that normal arabidopsis seed is seeded in not in Fig. 2 It is grown in the culture medium of the antibiotic containing kanamycins；D figure is that normal arabidopsis seed is seeded in that is mould containing 50 mg/L cards in Fig. 2 It is grown in the culture medium of plain antibiotic；In Fig. 2 E figure be arabidopsis after Agrobacterium-mediated Transformation seed be seeded in containing 50 mg/L cards that It is grown in the culture medium of mycin antibiotic, wherein arrow instruction is the transgenic arabidopsis with kalamycin resistance.

Fig. 3: expression diamondback moth arginine kinase genePxAKAnd integrin beta₁Subunit genePxβFusion sequence dsRNA's Transgenic arabidopsis PCR positive detection schematic diagram；In figure 1-15 be transgenic arabidopsis, C be normal control arabidopsis, P be containing There is the positive plasmid of target gene sequence,18s rDNAFor arabidopsis reference gene.

Fig. 4: while expressing diamondback moth arginine kinase genePxAKTarget sequence dsRNA and integrin beta₁Subunit genePx βThe transgenic arabidopsis RT-PCR detection schematic diagram of target sequence dsRNA；In figure number 301,302,303,305,310,31, 317,32,330,374,380,384,395 and 398 be different transgenic arabidopsis strains, and C is normal control arabidopsis, and P is Positive plasmid containing target gene sequence,18s rRNAFor arabidopsis reference gene.

Fig. 5: while expressing diamondback moth arginine kinase genePxAKTarget sequence dsRNA and integrin beta₁Subunit genePx βThe transgenic arabidopsis RT-qPCR detection schematic diagram of target sequence dsRNA；In figure number 302,303,317,32,380,384, 395 and 398 be different transgenic arabidopsis strains,PxAKGene relative expression quantity uses arabidopsisSandGene is internal reference.

Fig. 6: while expressing diamondback moth arginine kinase genePxAKTarget sequence dsRNA and integrin beta₁Subunit genePx βThe method of 398 strain of the transgenic arabidopsis feeding diamondback moth of target sequence dsRNA.

Fig. 7: while expressing diamondback moth arginine kinase genePxAKTarget sequence dsRNA and integrin beta₁Subunit genePx βEvaluation of resistance of 398 strain of transgenic arabidopsis of target sequence dsRNA to diamondback moth；A figure in Fig. 7 is the quasi- south of transgenosis Influence of 398 strain of mustard to diamondback moth larvae development duration；B figure in Fig. 7 is 398 strain of transgenic arabidopsis to diamondback mothization The influence of pupa rate；C figure in Fig. 7 is influence of 398 strain of transgenic arabidopsis to the diamondback moth death rate.WT is normal right in figure According to arabidopsis.

Fig. 8: while expressing diamondback moth arginine kinase genePxAKTarget sequence dsRNA and integrin beta₁Subunit genePx β398 strain of transgenic arabidopsis of target sequence dsRNA is to diamondback moth target genePxAKWithPxβMrna expression Inhibition；Diamondback moth four-age larva after A figure in Fig. 8 transgenic arabidopsis that has been feedingPxAKThe relative expression levels of gene； Diamondback moth pupa on the one after B figure in Fig. 8 transgenic arabidopsis that has been feedingPxAKThe relative expression levels of gene；C figure in Fig. 8 For diamondback moth four-age larva after feeding transgenic arabidopsisPxβThe relative expression levels of gene；D figure in Fig. 8 is feeding Diamondback moth pupa on the one after transgenic arabidopsisPxβThe relative expression levels of gene.

Specific embodiment

Following embodiment defines the present invention, and describes the present invention in building diamondback moth arginine kinase genePxAKWith Integrin beta₁Subunit genePxβThe plant of target fusion sequence expresses RNAi carrier, and creation being capable of expressing gene simultaneouslyPxAKWithPxβThe transgenic arabidopsis of fusion sequence dsRNA, and to the method that its anti-diamondback moth ability is evaluated.It is retouched according to below It states and embodiment, those skilled in the art can determine essential characteristic of the invention, and without departing from spirit of that invention and model In the case where enclosing, various changes and modifications can be made to the present invention, so that it is applicable in different purposes and condition.