CN110396125B

CN110396125B - Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants

Info

Publication number: CN110396125B
Application number: CN201810378111.4A
Authority: CN
Inventors: 叶健; 龚雨晴; 赵平芝
Original assignee: Institute of Microbiology of CAS; University of Chinese Academy of Sciences
Current assignee: Institute of Microbiology of CAS; University of Chinese Academy of Sciences
Priority date: 2018-04-25
Filing date: 2018-04-25
Publication date: 2021-08-13
Anticipated expiration: 2038-04-25
Also published as: CN110396125A

Abstract

The invention discloses application of an arabidopsis transcription factor gene PIF3 in insect stress resistance of plants. The invention provides an application of any one of the following substances 1) -3) in regulating and controlling insect resistance of plants: 1) protein PIF 3; 2) a DNA molecule encoding the protein PIF 3; 3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein PIF 3; experiments prove that after the PIF3 gene is overexpressed, arabidopsis shows a phenotype of resisting bemisia tabaci. In order to facilitate the identification and screening of transgenic Arabidopsis plants, the vectors used may be processed, for example, by adding plant selectable markers or antibiotic markers having resistance. The PIF3 gene provided by the invention provides gene resources for breeding new insect-resistant plant varieties and has good potential application value.

Description

Application of arabidopsis transcription factor gene PIF3 in insect stress resistance of plants

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of an arabidopsis transcription factor gene PIF3 in insect stress resistance of plants.

Background

Agricultural insect pests are one of main agricultural disasters in China, and have the characteristics of multiple types, large influence and frequent outbreak of disasters, the occurrence range and the severity of the agricultural insect pests cause great loss to national economy, particularly agricultural production in China, and besides directly endanger crops, the agricultural insect pests carry pathogens or parasites to cause epidemic outbreaks of diseases and double damages of the diseases and the pests to seriously influence the national civilian life. Common agricultural pests in China include whitefly, rice planthopper, corn borer, cotton bollworm, cotton aphid, wheat red spider, locust and the like. In recent years, frequent outbreaks of various crop pests and diseases and aggravation of a series of agricultural problems caused by abuse and unreasonable utilization of chemical fertilizers and pesticides urgently need a more environment-friendly and sustainable method for realizing green plant protection. Improving the insect resistance of the plant itself is the most fundamentally effective method. These current methods include the following: adopting biotechnology to culture resistant varieties, such as stably expressing toxin protein Bt of Bacillus thuringiensis, agglutinin of various plants, etc. for resisting chewing insects; stably expressing dsRNA of genes related to key development, physiology and metabolism of insects; the agricultural means is improved, the cultivation density is controlled, the use of nitrogen fertilizer is controlled, and the robust growth of field crops is ensured; according to the pest damage period of certain pests, the ecological pest resistance of the plant is improved by sowing the pest early or late; controlling population density of pests using natural enemies, and the like. In agricultural pests, piercing-sucking mouthpart pests such as whitefly, aphid, coccid, spider mite, thrips and the like are easy to spread and spread by various media, and most of the piercing-sucking mouthpart pests are carriers and intermediate media of pathogenic microorganisms, but an effective method for resisting the piercing-sucking insects is not found at present.

Bemisia tabaci (Bemisiatabaci) is a vector insect that transmits plant viruses, is an important pest of many crops worldwide, and viruses transmitted by bemisia tabaci are as many as 200, causing significant economic loss on food and economic crops such as wheat, tobacco, tomato, cotton, soybean. The bemisia tabaci not only can transmit plant viruses to cause diseases to host plants, but also can cause great threat to the host plants. The bemisia tabaci directly stabs and absorbs plant juice to cause plant weakness, nymphs and adults can also secrete honeydew to induce the generation of sooty mould, and photosynthesis is seriously influenced. At present, the tobacco whitefly is still prevented and controlled by a chemical insecticide with high toxicity and low selectivity in production, so that the problems of drug resistance, pollution, residue and the like of pests are caused, and the safety of the tongue tip of the common people is seriously influenced. The double hazards of the plant and the vector insect cause great difficulty in effective prevention and control in production, so that the improvement of the resistance level of the plant has important significance for effectively controlling bemisia tabaci and plant virus diseases.

PIFs (phytochrome interacting factors), which are phytochrome interacting factors, are a bHLH family of transcription factors that can interact with photoreceptor photopigment proteins. PIF3 as a transcription factor can act with photoreceptors phyA and phyB, and after seedlings growing in the dark are irradiated, PIF3 in vivo can be rapidly degraded under the control of phytochrome to negatively control a phyB signal pathway. The homologous genes of PIF3, PIF1, PIF4 and PIF5 also play an important role. PIF1 encodes a nuclear bHLH protein that can be induced by light and that can be involved in the photopigment signaling pathway; PIF4 encodes a nuclear localization bHLH protein, which can interact with phyB protein to negatively regulate phyB-mediated red light response pathway and participate in SAS process; PIF5 functions as a phyB negative regulator, with protein levels regulated by phyB. In addition, both the PIF6 and PIL1 genes are members of the PIF3 transcription factor gene family. PIF7 negatively regulates phyB-mediated seedling de-etiolation. The current research on PIFs focuses on the regulation of plant optical signal channels, and reports on the resistance of plants to biotic stress are not reported.

At present, the research of plant molecular biology is mostly carried out by taking Arabidopsis thaliana (Arabidopsis thaliana) as a model plant, and the Arabidopsis thaliana belongs to cruciferae plants, has the advantages of small genome, fast growth and the like, and is widely used for the research of plant genetics, cell biology, molecular biology and the like. Generally, by means of agrobacterium infection or other transgenic methods, a related gene cloning vector can be transferred into wild arabidopsis thaliana to obtain an over-expression or gene knockout strain of the gene, and then insect bioassay experimental detection is performed to research the function of the gene in insect resistance, so that insect-resistant gene resources are discovered. At present, some genes which can significantly improve the insect resistance of plants, such as protease inhibitor genes (PI genes), amylase inhibitor genes, other plant lectin genes and the like, are discovered.

Disclosure of Invention

An object of the present invention is to provide use of any one of the following 1) to 3).

The invention provides an application of any one of the following substances 1) -3) in regulating and controlling insect resistance of plants:

1) protein PIF 3;

2) a DNA molecule encoding the protein PIF 3;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein PIF 3;

the protein PIF3 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(2) and (b) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

It is understood that one skilled in the art can substitute, delete and/or add one or several amino acids based on the disclosed amino acid sequences without affecting their activity to obtain mutant sequences of the proteins.

It is understood that, considering the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

In the above application, the DNA molecule is any one of the following 1) to 3):

1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

2) DNA molecules which hybridize under stringent conditions with the DNA sequences defined in 1) and which code for proteins having the same function;

3) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in 1) and encoding a protein having the same function.

The above stringent conditions are hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS, and washing the membrane with the solution.

In the application, the insect resistance is the resistance to bemisia tabaci.

In the above application, the plant is a dicotyledonous plant or a monocotyledonous plant;

or the plant is a dicotyledon, and the dicotyledon is a cruciferae plant in particular; the cruciferous plant is specifically arabidopsis thaliana.

The application of any substance in 1) to 3) in breeding insect-resistant plants is also the protection scope of the invention;

or the application of any substance in the following 1) to 3) in serving or preparing pesticides is also the protection scope of the invention;

or the application of any substance of the following 1) to 3) in the disinsection is also the protection scope of the invention;

or, the application of any one of the following substances 1) to 3) in plant pest control and/or preparation of plant pest control products is also within the scope of the present invention;

1) protein PIF 3;

2) a DNA molecule encoding the protein PIF 3;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein PIF 3.

In the application, the insect resistance is the resistance to bemisia tabaci;

or, the plant is a dicotyledonous plant or a monocotyledonous plant;

Another object of the present invention is to provide a method for breeding insect-resistant transgenic plants.

The method provided by the invention comprises the following steps: improving the expression quantity and/or activity of a DNA molecule of the protein PIF3 in a target plant to obtain a transgenic plant, wherein the insect resistance of the transgenic plant is higher than that of the target plant;

the protein PIF3 is (1) or (2) as follows:

In the above method, the expression level and/or activity of the DNA molecule encoding the protein PIF3 in the target plant is increased by introducing the DNA molecule encoding the protein PIF3 into the target plant.

In the method, the disease resistance is the resistance to bemisia tabaci;

in the above method, the plant is a dicotyledonous plant or a monocotyledonous plant;

Experiments prove that after the PIF3 gene is overexpressed, the gene is transferred into arabidopsis thaliana to obtain transgenic arabidopsis thaliana, the transgenic arabidopsis thaliana is expressed as a bemisia tabaci resistant phenotype, the gene or protein expressed by the gene is proved to have the effect of improving the insect resistance of plants, gene resources are provided for breeding new insect-resistant plant varieties, and the transgenic arabidopsis thaliana has good potential application value.

Drawings

FIG. 1 shows Western Blot detection results of plant proteins of strains of Arabidopsis thaliana overexpressed by PIF3 gene.

FIG. 2 shows the oviposition and number of pseudo pupae of Bemisia tabaci in wild type and PIF3 gene overexpression Arabidopsis thaliana.

FIG. 3 shows the amount of eggs laid and the number of false pupae of Bemisia tabaci on wild type and pifQ mutant Arabidopsis thaliana.

FIG. 4 is a double selection experiment of Bemisia tabaci on wild type and pifQ mutant Arabidopsis thaliana under different lighting conditions.

FIG. 5 shows the expression levels of the terpene synthase genes TPS10, TPS14 and TPS21 in wild type and pifQ mutant Arabidopsis thaliana under different illumination conditions.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The pENTR-3C cloning vector in the following examples is a commonly used cloning vector, and is commercially available; the arabidopsis variety is Columbia ecotype; the pifQ mutant is constructed in the laboratory, and the materials of pif1, pif3, pif4 and pif5 arabidopsis thaliana single-mutant used in the construction process are purchased from a SALK mutant library; agrobacterium EHA105 strain was purchased from Bomaide (Biomed) Bio Inc.

The pH7-WG2Y vector was a vector obtained by inserting YFP tag (SEQ ID NO: 5) into the PstI and BamHI cleavage sites of pH7-WG2 vector (purchased from Invitrogen).

The main reagents in the following examples are:

reagents used for molecular cloning: high fidelity DNA polymerase Phusion was purchased from Thermo Fisher Scientific; restriction enzymes were purchased from NEB; t4DNA ligase was purchased from NEB; 1kb DNA marker and 2000bp DNA marker were purchased from Bomader (Biomed); gelsafe was purchased from Hao, formerly; SYBR qPCR Mix was purchased from TOYOBO.

Reagents used for protein-related experiments: 40% Acrylamide from Sigma; cocktail protease inhibitors were purchased from Roche; coomassie Brilliant blue was purchased from Sigma; IPTG was purchased from Inolco; antibodies were purchased from Beijing Quanji Biotech; prestained protein molecular weight marker was purchased from EasyBIO corporation; developer is available from GE Healthcare; lucifersae was purchased from Sigma.

The plasmid miniprep kit and the DNA purification recovery kit are purchased from AxyGen company; plant DNA and RNA miniprep kits were purchased from Qiagen; the agarose gel DNA recovery kit is purchased from Beijing Quanzijin Biotech company; reverse transcription II one step DNA Removal and DNA Synthesis SuperMix kits were purchased from Kyoto Seiko Biotech, Inc.

Spectinomycin (Spe), rifampicin (Rif) and hygromycin (Hyg) were purchased from Inolco; the various other chemicals used in the examples were all imported or home-made analytical grade reagents.

The primers used in the examples were synthesized by the EnxElite Achiette corporation and subjected to related sequencing.

Example 1 cloning of PIF3 Gene and construction of vector for expressing PIF3 Gene

Cloning of PIF3 Gene

The nucleotide sequence of the cDNA of the PIF3 gene is shown as sequence 1, and the amino acid sequence of the encoded PIF3 protein is shown as sequence 2.

A CDS (sequence coding for aminoacids in protein) protein coding region DNA sequence 1 of the PIF3 gene was artificially synthesized, and an upstream primer F and a downstream primer R were designed based on the coding region sequence analysis.

An upstream primer F: 5'-CAAGGGTACCATGCCTCTGTTTGAGCTTTT-3' (sequence 3)

A downstream primer R: 5'-CAAGCTCGAGGACGATCCACAAAACTGATC-3' (sequence 4)

Taking DNA molecules shown in the sequence 1 as a template, and carrying out PCR amplification by using an upstream primer F and a downstream primer R to obtain a PCR amplification product of 1575 bp.

Secondly, construction of a vector for expressing PIF3

And (3) a vector 35S for expressing PIF3, wherein PIF3-YFP is a vector obtained by recombining a PIF3 gene shown in a sequence 1 into an expression vector pH7-WG2Y through LR reaction.

The construction method of the vector comprises the following steps of firstly connecting the obtained 1575bp PCR amplification product to a pENTR-3C cloning vector to obtain an intermediate vector; and performing LR reaction on the intermediate vector and the expression vector pH7-WG2Y to obtain a vector 35S for expressing PIF3, namely PIF3-YFP, and connecting the PIF3 gene to the expression vector pH7-WG2Y through sequencing verification.

Example 2 application of PIF3 gene in regulation and control of plant insect resistance

Construction of PIF3 transgenic plant expressing PIF3 gene

The PIF 3-expressing vector 35S prepared in example 1, PIF3-YF, was transformed into Agrobacterium EHA105 strain to obtain a recombinant Agrobacterium expressing PIF 3. And then transferring the recombinant strain into a wild type arabidopsis col-0 plant to obtain a PIF 3-transferred arabidopsis seedling.

The transformation method comprises the following specific steps: the recombinant Agrobacterium expressing PIF3 was inoculated in 5mL LB liquid medium containing antibiotics (rifampicin 50. mu.g/m L, spectinomycin 50. mu.g/m L) and cultured overnight at 28 ℃ at 220 rpm; transferring 5mL of LB liquid culture medium containing the recombinant agrobacterium expressing PIF3 into 250mL of liquid culture medium, and culturing for 16h at 28 ℃ and 220 rpm; collecting the bacterial liquid into a 250mL centrifugal barrel, centrifuging at 4000rpm for 15min, and collecting thalli; the cells were suspended in 400m L transformation medium (1/2MS, 5% sucrose, 40. mu.L Silwet); taking newly-flowering and well-growing wild type arabidopsis thaliana col-0 for transformation, cutting off green fruit pods by using scissors before transformation, then reversely buckling the arabidopsis thaliana in the resuspended agrobacterium for 10min, immersing a flower container of a plant in a bacterial liquid, adopting a pollen tube soaking method for transformation, flatly placing the arabidopsis thaliana in a pot, wrapping the arabidopsis thaliana by using a preservative film, and moving the arabidopsis thaliana to a dark place for 24 h; removing the cover the next day, lifting the plant and transferring to normal conditions for continuous culture; after about 3 weeks, seeds were harvested for selection of transformants.

Second, detecting

1. Hygromycin resistance selection

The selection resistance gene carried by the pH7-WG2Y vector is hygromycin, the hygromycin resistance is used for screening transformants, the hygromycin resistance is positive, T1 generation is used for transferring PIF3 Arabidopsis seedlings, a single plant is harvested, the hygromycin resistance test is carried out on T2 generation seeds, a strain with resistance 3/4 and no resistance in the rest 1/4 is selected, and the over-expression vector connected with the target gene in the strain is inserted in a single copy mode. And (3) removing the plants with hygromycin resistance from the strains, harvesting the single plants, screening the hygromycin resistance by using the obtained seeds of the T3 generations, if the plants of the T4 generations are not separated, indicating that the transgenic strains are homozygotes which can be used for seed reproduction and bemisia tabaci bioassay experiments, wherein the homozygotes are transformed from the seeds of PIF3 Arabidopsis thaliana of the T4 generation, and sowing the seeds of the PIF3 Arabidopsis thaliana of the T4 generation.

2. Detection by western blotting method

1) Extraction of plant Total protein

Taking 0.1g of T4 to replace a leaf of a PIF3 Arabidopsis plant, putting the leaf into a 1.5mL centrifuge tube, covering the centrifuge tube tightly, putting the centrifuge tube into liquid nitrogen for precooling, then grinding the centrifuge tube into powder by using a grinding rod, adding 500 mu L of 2 xSDS buffer, swirling the powder until the powder is mixed evenly, boiling the powder in boiling water for 5min to denature protein, centrifuging the powder at 10000rpm for 10min, and taking the supernatant for electrophoresis. Wild type Arabidopsis thaliana was used as a control.

2) Protein content in over-expressed strains was determined by Western Blot

The same amount of wild type and plant protein of T4 generation transformed PIF3 Arabidopsis plant (35S: PIF3-YFP) are respectively taken, electrophoresis is carried out by using a voltage stabilizing mode, the electrophoresis is firstly carried out for about 20min by using 80V voltage until protein bands run out of separation gel, and the electrophoresis is carried out for about 1h by transforming 100V voltage. And finally, detecting the protein content in the over-expression strain by using an Anti-YFP antibody. To be provided with

Anti-Actin serves as an internal reference.

The results of the WesternBlot test are shown in FIG. 1, and it can be seen that the target protein is strongly expressed by the T4 generation PIF3 Arabidopsis plant (35S: PIF 3-YFP).

The empty vector pH7-WG2Y vector is transferred into wild arabidopsis thaliana by the same method to obtain T4 transfer empty vector arabidopsis thaliana.

Methods for overexpression of PIF3 gene in other plants can be performed with reference to this example.

Third, the insect resistance detection of the transgenic PIF3 Arabidopsis thaliana

Bemisia tabaci MeAM1(mtCOI, GenBank accession MF579701) was used as a Bemisia tabaci for experiments and was captured on tomato plants in the sunward region of Beijing. The bemisia tabaci is bred in an insect breeding cage, and the cotton is used as a host plant for growth and propagation. The indoor temperature is 25 ℃, the relative humidity is 65%, and the illumination period is 12h of illumination/12 h of darkness.

Firstly, wild type arabidopsis seedlings, T4 empty vector-transferred arabidopsis seedlings and T4 generation-transferred PIF3 arabidopsis plants (PIF3-OE) grow on a culture dish for 21 days, then the seedlings are transferred to soil to grow for 14 days, and a tobacco whitefly growth test experiment is carried out by adopting a leaf cage method: in the bemisia tabaci oviposition experiment, 3 female insects and 3 male insects are placed in a leaf cage (diameter 45 mm; height 30mm), and the number of eggs is counted after 10 days. In the experiment of the development of the bemisia tabaci pseudo pupae, 16 female insects are placed in a leaf cage, after the egg laying amount of the female insects reaches 2 days, all the adult bemisia tabaci are removed, and the eggs are left to continue to develop in the leaf cage; and counting the number of fake pupae after 20 days. 8 strains of each strain, the experiment was repeated 3 times, and the results were averaged.

As shown in FIG. 2, the oviposition amount (FIG. 2A) and the number of pseudo pupae (FIG. 2B) of Bemisia tabaci on T4 generation transgenic PIF3 Arabidopsis plants (PIF3-OE) were significantly reduced compared to wild type Arabidopsis thaliana. This shows that the insect resistance of the T4 generation PIF3 Arabidopsis plant (PIF3-OE) is obviously improved, and the PIF3 overexpression improves the insect resistance of the plant.

The results of the wild type arabidopsis seedlings and the T4 empty vector arabidopsis seedlings have no significant difference.

Example 3 PIF3 Gene regulating plant insect resistance

Construction of pifQ mutants (pif1, pif3, pif4, pif5)

The purchased pif1, pif3, pif4 and pif5 mutants arabidopsis thaliana (only the pif1, pif3, pif4 or pif5 genes are inactivated compared with the wild type) are sown, arabidopsis thaliana plants which just blossom and bolt and have good growth conditions are selected, pif1 and pif5 are hybridized, and pif3 and pif4 are hybridized. Carefully selecting the buds which do not bloom in the female parent plant, and completely removing stamens, petals and sepals by using a pointed-end forceps to leave the stigma. And (4) repeatedly smearing the stamens of the flowers of the male parent just blossomed on the stigma of the female parent, and filling the pores above the stigma with anthers. After 2-3 days, the stigma is observed to obviously extend and become thick, which represents successful hybridization, and if the change is not too large or the stigma slowly withers, the hybridization fails, and the stigma needs to be redone in time. After the seeds are mature, the hybrid seeds are carefully harvested. F1 plants of pif1/pif5 are continuously crossed with F1 plants of pif3/pif4, F2 generation seeds of pif1/pif3/pif4/pif5 are paved on an MS flat plate, plant markers with completely opened cotyledons are selected after 3 days of growth in the dark, the plants are moved into soil for continuous growth after light adaptation treatment, after the plants are mature, single plant genome DNA is taken, and four loci of fixed pif1/pif3/pif4/pif5 are homozygous, so that the pifQ four-mutant plants are obtained.

II, detection of insect resistance of pifQ mutant

Wild type Arabidopsis thaliana and pifQ mutant plants were tested for insect resistance according to the third example 2.

As a result, as shown in FIG. 3, the number of eggs laid by Bemisia tabaci (FIG. 3A) and the number of pseudo pupae (FIG. 3B) were significantly increased in the pifQ mutant plants as compared with that of wild type Arabidopsis thaliana. This indicates that the insect resistance of the pifQ mutant plants is reduced, and further verifies the insect resistance of the PIF3 overexpression plants.

Tri, PifQ mutant bemisia tabaci double selection experiment

Selecting wild arabidopsis thaliana and pifQ mutant arabidopsis thaliana with consistent plant size and leaf number, and selecting 5 illumination conditions: dark, natural light, red light (770-622 nm), far-red light (735-730 nm) and blue light (492-455 nm), treating for 2 hours under different illumination conditions, and placing into an insect cage (30 x 30 cm); freezing about 200 Bemisia tabaci on ice for 1min, then releasing between two plants, carrying out the selection process under the same illumination condition, and counting and recording the number of Bemisia tabaci on each plant after the Bemisia tabaci is selected for 25 min; 6 biological replicates were selected for each experiment.

The results are shown in fig. 4, and it can be seen that bemisia tabaci is more prone to select pifQ mutant plants than wild type arabidopsis under natural light and red light conditions, which indicates that the insect resistance of the pifQ mutant plants is reduced, and further verifies the insect resistance of the plants over-expressed by PIF 3.

Fourthly, the gene expression level of the PifQ mutant terpene synthase genes TPS10, TPS14 and TPS21

Treating the wild arabidopsis thaliana and the pifQ mutant arabidopsis thaliana under the illumination condition of the third step, extracting sample RNA by using an RNA extraction kit after sampling, inverting by using a reverse transcription kit to obtain cDNA, and detecting the gene expression amounts of the repellent terpene compounds TPS10, TPS14 and TPS21 of the wild arabidopsis thaliana and the pifQ mutant arabidopsis thaliana under different illumination.

As shown in fig. 5, under natural light and red light conditions, compared with wild arabidopsis thaliana, the gene expression levels of the repellent terpene compounds TPS10 (fig. 5A), TPS14 (fig. 5B) and TPS21 (fig. 5C) in the pifQ mutant arabidopsis thaliana were significantly reduced, and were more attractive to bemisia tabaci.

Therefore, after the Arabidopsis PIF3 gene is over-expressed, the resistance of the plant to bemisia tabaci can be remarkably improved, and after the PIF3 gene and homologous genes PIF1, PIF4 and PIF5 thereof are knocked out, the resistance of the plant to bemisia tabaci is remarkably reduced.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Glu Glu Ser Gly Asp Gly Arg Lys Glu Ala Gly Pro Ser Arg Thr Gly

325 330 335

Leu Gly Ser Lys Arg Ser Arg Ser Ala Glu Val His Asn Leu Ser Glu

340 345 350

Arg Arg Arg Arg Asp Arg Ile Asn Glu Lys Met Arg Ala Leu Gln Glu

355 360 365

Leu Ile Pro Asn Cys Asn Lys Val Asp Lys Ala Ser Met Leu Asp Glu

370 375 380

Ala Ile Glu Tyr Leu Lys Ser Leu Gln Leu Gln Val Gln Ile Met Ser

385 390 395 400

Met Ala Ser Gly Tyr Tyr Leu Pro Pro Ala Val Met Phe Pro Pro Gly

405 410 415

Met Gly His Tyr Pro Ala Ala Ala Ala Ala Met Ala Met Gly Met Gly

420 425 430

Met Pro Tyr Ala Met Gly Leu Pro Asp Leu Ser Arg Gly Gly Ser Ser

435 440 445

Val Asn His Gly Pro Gln Phe Gln Val Ser Gly Met Gln Gln Gln Pro

450 455 460

Val Ala Met Gly Ile Pro Arg Val Ser Gly Gly Gly Ile Phe Ala Gly

465 470 475 480

Ser Ser Thr Ile Gly Asn Gly Ser Thr Arg Asp Leu Ser Gly Ser Lys

485 490 495

Asp Gln Thr Thr Thr Asn Asn Asn Ser Asn Leu Lys Pro Ile Lys Arg

500 505 510

Lys Gln Gly Ser Ser Asp Gln Phe Cys Gly Ser Ser

515 520

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 3

caagggtacc atgcctctgt ttgagctttt 30

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 4

caagctcgag gacgatccac aaaactgatc 30

<210> 5

<211> 717

<212> DNA

<213> Artificial Sequence

<400>5

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

Claims

1. The application of any one of the following substances 1) to 3) in improving the resistance of plants to bemisia tabaci:

1) protein PIF 3;

2) a DNA molecule encoding the protein PIF 3;

the protein PIF3 is composed of an amino acid sequence shown in a sequence 2 in a sequence table.

2. Use according to claim 1, characterized in that: the DNA molecule is a DNA molecule with a coding region shown as a sequence 1 in a sequence table.

3. Use according to any one of claims 1-2, characterized in that: the plant is a dicotyledonous plant or a monocotyledonous plant.

4. Use according to claim 3, characterized in that: the dicotyledonous plant is a crucifer; the cruciferous plant is specifically arabidopsis thaliana.

5. The application of any one of the following substances 1) to 3) in cultivating the bemisia tabaci resistant plants;

or, the application of any one of the following substances 1) to 3) in serving as or preparing pesticide for killing bemisia tabaci;

or, any one of the following substances 1) to 3) is applied to killing the bemisia tabaci;

or, any substance in the following 1) to 3) is applied to the control of the bemisia tabaci pests of plants and/or the preparation of products for controlling the bemisia tabaci pests of plants;

1) protein PIF 3;

2) a DNA molecule encoding the protein PIF 3;

6. Use according to claim 5, characterized in that: the plant is a dicotyledonous plant or a monocotyledonous plant.

7. Use according to claim 6, characterized in that:

the plant is a dicotyledon, and the dicotyledon is a cruciferae plant; the cruciferous plant is specifically arabidopsis thaliana.

8. A method for cultivating transgenic plants with high resistance to Bemisia tabaci comprises the following steps: increasing the expression level of a DNA molecule of the protein PIF3 coded in a target plant to obtain a transgenic plant, wherein the resistance of the transgenic plant to bemisia tabaci is higher than that of the target plant;

the protein PIF3 is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table.

9. The method of claim 8, wherein:

the expression level of the DNA molecule coding for the protein PIF3 in the target plant is improved by introducing the DNA molecule coding for the protein PIF3 into the target plant.

10. The method according to any one of claims 8-9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant.

11. The method of claim 10, wherein: