CN110818784B

CN110818784B - Application of rice gene OsATL15 in regulating pesticide absorption and transport

Info

Publication number: CN110818784B
Application number: CN201911165317.XA
Authority: CN
Inventors: 徐汉虹; 肖钰艳; 林菲; 张汉林; 李志伟
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2021-06-25
Anticipated expiration: 2039-11-25
Also published as: WO2021104542A1; CN110818784A

Abstract

The invention discloses the application of rice gene OsATL15 in regulating the absorption and transport of pesticides. In the present invention, the gene OsATL15, which regulates the absorption and transport of pesticides in rice, is isolated and cloned from the rice variety Zhonghua 11. The nucleotide sequence, the encoded protein, and the recombinant vector, expression cassette, transgenic cell line and recombinant bacteria containing the gene OsATL15 can be obtained. Improve the absorption and transfer of pesticides by rice, so that the pesticides can reach the pest-damaging parts, and reduce the use of pesticides in paddy fields. Moreover, OsATL15 gene can be involved in regulating rice plant height, and has the effect of improving rice germplasm resources.

Description

Application of rice gene OsATL15 in regulation of absorption and transportation of pesticides

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a rice gene OsATL15 in regulation of absorption and transportation of pesticides.

Background

The rice is a main food crop in China and all over the world, about 22 hundred million people all over the world eat the rice, and 60 percent of people in China eat the rice. However, the excessive use of pesticides in the rice production process places a great strain on the ecological environment. It is estimated that in current pesticide use, only 2% of the pesticide applied will actually stay on the surface of the plant, and more so only 0.1% of the pesticide actually reaches the target of action (Wang et al, 2007). This means that more than 99.9% of the pesticide enters the environment, the chemical fertilizers and pesticides are inefficient to use, most of them run off into the soil and rivers, causing a decrease in soil quality, water pollution and a decrease in soil ecological diversity (Pimentel et al, 1992). Therefore, the enhancement of the pesticide targeting and the improvement of the effective utilization rate of the pesticide are the main targets of the current pesticide research and are the necessary ways to realize the reduction and the synergism of the pesticide.

The pesticide transport gene can realize the targeted accumulation of pesticide and improve the effective utilization of the pesticide. Therefore, by utilizing the separation and cloning of the pesticide transport gene and protein of the rice, the targeted accumulation regulation and control of the systemic delivery conductive pesticide can be hopefully realized, the environmental release is reduced, and the method is beneficial to rice breeding and has important economic value.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the rice gene OsATL15 in regulating the absorption and transportation of pesticides.

The invention also aims to provide a molecular marker detection primer of the rice gene OsATL 15.

The purpose of the invention is realized by the following technical scheme: the application of the rice gene OsATL15 in regulation of pesticide absorption and transportation is based on the discovery by the inventor of the invention that the OsATL15 gene can improve the pesticide absorption and transportation of rice, increase the pesticide absorption and transportation of rice, reach pest-damaged parts and reduce the pesticide usage amount in a paddy field; can also improve the pesticide transmission and guide performance of rice varieties in heredity and select rice varieties with pesticide high-efficiency utilization.

The pesticide is preferably systemic pesticide; further preferred are systemic insecticides; further preferably one of neonicotinoid insecticide, amide insecticide, organophosphorus insecticide, nereistoxin insecticide or carbamate insecticide; most preferably one of thiamethoxam, chlorantraniliprole, omethoate, dimehypo or methomyl.

The protein coded by the rice gene OsATL15 has the amino acid sequence of A) or B):

A. as shown in SEQ ID NO: 1;

B. the amino acid sequence of the derivative protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the A.

The nucleotide sequence of the rice gene OsATL15 is any one of the following A) to E):

A. as shown in SEQ ID NO: 2;

B. as shown in SEQ ID NO: 3;

C. hybridizes under stringent conditions to a nucleotide as defined by A or B and encodes a nucleotide sequence as defined in SEQ ID NO: 1;

D. has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence defined by a and encodes a polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO: 1;

E. has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence defined by B and encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a nucleotide sequence of an amino acid shown in the specification.

The application of the recombinant vector, the expression cassette, the transgenic cell line and the recombinant bacterium containing the rice gene OsATL15 in regulation of pesticide absorption and transportation also belongs to the protection scope of the invention.

A molecular marker detection primer of a rice gene OsATL15 is composed of a primer shown as SEQ ID NO: 4 and SEQ ID NO: 5 comprises an upstream primer and a downstream primer which are shown in the specification:

F:5’-TACTCGCCGCCGCCGTCATA-3’；

R:5’-CGAGGTTGAGCGTGTTGCTGTTCC-3’。

the application of the molecular marker detection primer of the rice gene OsATL15 in identifying rice varieties which efficiently utilize pesticides comprises the following steps: the molecular marker detection primer is used for amplifying the germplasm genome DNA or RNA of the rice variety to be detected, and the sensitivity of the target gene responding to pesticide treatment is judged through the change of gene expression.

A molecular marker detection kit of a rice gene OsATL15 comprises the molecular marker detection primer.

The molecular marker detection kit of the rice gene OsATL15 further comprises at least one of an enzyme for PCR, water for PCR and a buffer solution for PCR.

The application of the rice gene OsATL15 in cultivating transgenic rice with efficient pesticide utilization comprises the following steps: the rice gene OsATL15 is constructed on a plant expression vector, and the obtained recombinant expression vector is transferred into rice for expression to obtain a rice variety which efficiently utilizes pesticides.

The plant expression vector is preferably pCAMBIA 2300-35S.

The obtained recombinant expression vector is transferred into rice for expression, and the obtained recombinant plant expression vector can be transferred into plant cells or tissues by conventional biological methods such as agrobacterium mediation, plant virus vectors, direct DNA transfer, conductance transfer and the like.

The rice gene OsATL15 is applied to the regulation of rice plant height.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention utilizes Polymerase Chain Reaction (PCR) to separate and clone a gene OsATL15 for controlling pesticide absorption and transportation from a rice variety flower 11 into rice, and the application of the gene can increase the absorption and transportation of the rice to the pesticide, so that the pesticide reaches the pest position, and the usage amount of the pesticide in the rice field is reduced.

2. The expression of the gene OsATL15 in rice can be improved genetically to improve the pesticide conductance of rice varieties, select varieties with high pesticide utilization efficiency, and realize the reduction and the increase of the pesticide in rice fields.

3. The invention confirms the effect of the gene OsATL15 in pesticide absorption and transportation, and provides a new clue for researching the utilization mechanism of the rice to the pesticide.

4. The molecular marker detection primer of the gene OsATL15 provided by the invention can rapidly judge the sensitivity of the target gene in response to pesticide treatment through the change of gene expression level, and breed a new variety with high efficiency utilization and output.

Drawings

FIG. 1 is a diagram showing the results of real-time quantitative fluorescent PCR detection of OsATL15 gene expression level in OsATL15 over-expressed rice lines.

Fig. 2 is a diagram of the alignment result of the OsATL15 gene sequences of a CRISPR-knocked-out OsATL15 gene mutant plant and a wild-type plant.

FIG. 3 is a graph showing the results of measuring the thiamethoxam content in the roots and upper parts of the mutant rice plants having the OsATL15 gene.

FIG. 4 is a graph showing the results of measuring the chlorantraniliprole content in the roots and upper parts of the mutant rice plants of the OsATL15 gene.

FIG. 5 is a graph showing the results of measuring the omethoate content in the roots and upper parts of roots of mutant rice of OsATL15 gene.

FIG. 6 is a graph showing the results of examining the bisultap content in the roots and upper parts of roots of mutant rice having OsATL15 gene.

FIG. 7 is a graph showing the results of examining the content of methomyl in the roots and upper parts of roots of mutant rice of OsATL15 gene.

FIG. 8 is a graph showing the results of examining the transfer coefficient of thiamethoxam in mutant rice lines of OsATL15 gene.

FIG. 9 is a graph showing the results of examining the transfer coefficient of chlorantraniliprole in a mutant rice line of OsATL15 gene.

FIG. 10 is a graph showing the results of examining the transfer coefficient of omethoate in a mutant rice line of OsATL15 gene.

FIG. 11 is a graph showing the results of examining the transfer coefficient of bisultap in a mutant rice line of OsATL15 gene.

FIG. 12 is a graph showing the results of examining the transfer coefficient of methomyl in a mutant rice line of OsATL15 gene.

FIG. 13 is a graph showing the result of subcellular localization of OsATL15 protein-GFP in rice protoplasts; wherein FIGS. A, B, C and D are graphs showing the results of subcellular localization of protein GFP; FIGS. E, F, G and H are graphs showing the results of subcellular localization of OsATL15-GFP fusion protein; graphs a and E are results in bright field; panels B and F are green fluorescence in the dark field; panels C and G are membrane localization protein mCherry-1008 red fluorescence; views D and H are overlapping views; scale bar 10 μm.

FIG. 14 is a diagram showing the result of real-time fluorescent quantitative PCR detection of OsATL15 gene under the condition of 100. mu. mol/L Thiamethoxam (THX) treatment for 24 hours.

FIG. 15 shows the phenotype of mutant rice lines (10 cm on scale) of OsATL15 gene; wherein, A is a wild type rice middle flower 11, B is a mutant Crispr-10, C is a mutant Crispr-9, and D is a mutant Crispr-17.

FIG. 16 is a statistical chart of plant heights of OsATL15 gene mutant rice.

FIG. 17 is a statistical chart of root length of rice mutants of OsATL15 gene.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

In the embodiment, Escherichia coli DH5 alpha and Agrobacterium EHA105 are commonly used strains, and most molecular biology laboratories have preservation; the rice variety is wild type medium flower 11 (publicly used rice variety, commercially available).

The chemical reagents used in the examples were all imported or domestic analytical grade.

The primers used in the examples were synthesized by Shenzhen Huamao Gene Co, and the sequencing was performed by Shenzhen Huamao Gene Co.

Example 1 cloning of OsATL15 Gene and protein encoded thereby

RNA (OMEGA R6827-01) of 11 seedlings of wild japonica rice was extracted, and reverse transcription (Takara cat #6210A) was performed to obtain gDNA-removed cDNA (having nucleotides shown in SEQ ID NO: 1), which was used as a template for PCR amplification using a forward primer F1 and a reverse primer R1. A PCR product of 1896bp was obtained.

After sequencing, the 1896bp PCR product has the nucleotide sequence shown in SEQ ID NO: 2. The amino acid sequence of OsATL15 was obtained by amino acid translation of the coding sequence CDS using (http:// web. expasy. org/translate /) and encoded 631 amino acids. The coded protein is named as OsATL15, and the amino acid sequence of the protein is shown as SEQ ID NO: 3, respectively.

F1：5’-ATGGCAGATCAGAAGGTG-3’；

R1：5’-TCATAGGTGACGAGACTGAGGTG-3’。

Example 2 construction of plants overexpressing OsATL15

One, overexpression OsATL15 gene

1. Construction of recombinant expression vector pCAMBIA1300-35S-OsATL15

RNA of wild type Zhonghua 11 seedling was extracted, and cDNA was obtained by reverse transcription as a template (the method for extraction and reverse transcription was the same as in example 1), and PCR amplification was performed using primer 1(ACCCGGGGATCCTCTAGAGTCGAATGGCAGATCAGAAGGTG) and primer 2(ATGATACGAACGAAAGCTCTGCATCATAGGTGACGAGACTGAGGTG) (Takara cat # R045) under the following reaction conditions: 1 cycle: heating at 98 deg.C for 3 min; 32 cycles of: 98 deg.C, 30s, 58 deg.C, 30s, 72 deg.C, 1 min; 1 cycle: 72 ℃ for 5 min; the OsATL15 gene with 15 base homologous recombination arms was obtained at 16 ℃.

The PCR product was recovered by gel, and subjected to homologous recombination with a pCAMBIA2300-35S vector backbone (provided by Wuhan Boehringer) digested with both Sal I and Pst I by an In-fusion kit (Takara cat #639648) to obtain a recombinant plasmid. The recombinant plasmid was subjected to sample-feeding sequencing and named pCAMBIA1300-35S-OsATL15 as a recombinant expression vector.

2. Preparation of transgenic rice overexpressing OsATL15

1) The recombinant expression vector pCAMBIA1300-35S-OsATL15 is transferred into agrobacterium EHA105 (Olivia et al, 2019) by electric shock to obtain recombinant bacteria AGL1/pCAMBIA1300-35S-OsATL15 (plasmid is extracted from positive clone, and sequencing verification is carried out).

2) The recombinant strain AGL1/pCAMBIA1300-35S-OsATL15 is used for transforming medium flower 11 rice callus by an agrobacterium-mediated method, and the specific steps are as follows:

a single colony of AGL1/pCAMBIA1300-35S-OsATL15 was picked, inoculated into 10mL of Agrobacterium culture medium (containing 50mg/L kanamycin and 50mg/L rifampicin), and shake-cultured at 28 ℃ and 180rpm for 2-3 days. Centrifuging 4mL of bacterial solution at 4000rpm for 3min, pouring out the supernatant, adding a small amount of AAM culture medium to reconstitute the suspended cells, adding 20mL of AAM culture medium (containing 0.1mM acetosyringone As), performing shake cultivation at 28 deg.C and 150rpm in the dark for 1-2h, and culturing to OD₆₀₀About 0.4. Selecting rice callus of granular Zhonghua 11 (also called wild rice below) with good growth state, immersing the rice callus in the agrobacterium culture solution, shaking at 28 ℃ and 200rpm for 20min at 150 ℃, pouring out the callus, sucking excess bacteria solution with sterile filter paper, spreading the callus in a sterile plate containing multiple layers of filter paper, drying the callus on an ultra-clean bench (the callus is dispersed and does not cake), transferring the callus to a co-culture medium, and culturing for 2-3 days under dark condition. The calli were transferred to NB minimal medium containing 100mg/L hygromycin and 400mg/L cefamycin for 3-4 weeks (one screen). The surviving calli were transferred to a secondary screening medium (NB minimal medium containing 100mg/L hygromycin and 200mg/L cefuroxime) for 3 weeks. Transferring the resistant callus to a differentiation culture medium (containing 100mg/L hygromycin) for differentiation, transferring a regenerated plant to a greenhouse after the regenerated plant takes root on a strong seedling culture medium (about 3-4 weeks) containing 100mg/L hygromycin to obtain T₀Transgenic OsATL15 rice.

The media used in the above transformation were as follows:

co-culture medium: the callus and subculture medium was induced + As (0.1mM/L) + glucose (10g/L), pH 5.2.

The culture medium of the rice callus infected by agrobacterium comprises: AA macroelement, AA microelement, AA amino acid, MS vitamin, hydrolyzed casein (500mg/L), sucrose (68.5g/L), glucose (36g/L), As (0.1mM) and pH 5.2.

NB minimal medium: macroelement N6 + trace element B5 + organic component B5 + iron salt + hydrolyzed casein (300mg/L) + proline (500mg/L) + sucrose (30g/L) + agar (8g/L), pH 5.8.

Induction of callus and subculture medium: NB minimal medium +2, 4-D (2 mg/L).

Differentiation medium: NB minimal medium +6-BA (3mg/L) + NAA (1 mg/L).

Strong seedling culture medium: 1/2MS inorganic salt + NAA (0.5mg/L) + MET (0.25 mg/L).

Agrobacterium culture medium (YEP): 10g/L tryptone +10g/L yeast extract +5g/L sodium chloride +15g/L agar.

3. Molecular identification of OsATL15 transgenic rice

1) Preliminary identification by PCR

Extraction of T₀Transgenic OsATL15 rice genome DNA (OMEGA cat # D2485-02), and PCR identification with primers F2 and R2 (primer sequences are as follows) under the following reaction conditions: 1 cycle: heating at 98 deg.C for 3 min; 32 cycles of: 98 deg.C, 30s, 60 deg.C, 30s, 72 deg.C, 30 s; 1 cycle: 72 ℃ for 5 min; 16 ℃ is adopted. Screening out PCR positive (340bp) T₀Transgenic OsATL15 rice plants #1, #2 and # 3.

F2：5’-ACGGTGTCGTCCATCACAGTTTGCC-3’；

R2：5’-TTCCGGAAGTGCTTGACATTGGGGA-3’。

2) Analysis of transcript levels

Extraction of PCR-Positive T₀Total RNA of OsATL15 rice lines #1, #2 and #3 was transferred by passage, and the cDNA obtained by reverse transcription was used as a template, and real-time quantitative fluorescent PCR was performed using the following primers to detect the expression level of OsATL15 gene in each material at the transcription level. Fruit of Chinese wolfberryThe experiment was repeated 3 times and the results averaged. Wild type rice was used as a control.

Real-time quantitative fluorescent PCR was performed using Bio-Rad CFX 96. The PCR reaction system (20. mu.L) was carried out according to the product instruction SYBR Green Real-Time PCR Master Mix reagent (Takara) as follows: 10 μ L SYBR Green Real-Time PCR Master Mix, 2 μ L upstream and downstream primer Mix (both 10 μ M upstream and downstream primer concentration), 7 μ L RNase-free water, 1 μ L cDNA template. The specific reaction procedure is as follows: enzymatic heat activation at 95 deg.C for 30s for 1 cycle; denaturation at 95 ℃ for 5s, extension at 60 ℃ for 30s for 40 cycles.

Wherein, the primer sequence for amplifying the OsATL15 gene is as follows:

OsATL15 upstream primer F: 5'-TACTCGCCGCCGCCGTCATA-3', respectively;

OsATL15 downstream primer R: 5'-CGAGGTTGAGCGTGTTGCTGTTCC-3' are provided.

The primer sequence for amplifying the internal reference UBQ2 by using UBQ2 as an internal reference gene is as follows:

UBQ2 upstream primer: 5'-GCATCTCTCAGCACATTCCA-3', respectively;

UBQ2 downstream primer: 5'-ACCACAGGTAGCAATAGGTA-3' are provided.

The data are processed by using a complementary Ct method, i.e., the Ct value is the number of cycles that the fluorescence signal in the PCR tube passes through when reaching a set threshold value, and the delta Ct (OsATL15) -Ct (ACTIN1) is calculated by 2^－△△CtThe gene transcription level was measured, and the expression of OsATL15 gene in each material was analyzed and compared.

The real-time quantitative fluorescent PCR detection result of OsATL15 gene expression level in each experimental material is shown in FIG. 1, and the expression of OsATL15 gene is relative value, which shows that: compared with the non-transgenic wild rice japonica rice middle flower 11(WT), the PCR positive T₀The expression level of OsATL15 gene in the transgenic OsATL15 rice lines #1, #2 and #3 is obviously improved on the transcription level. PCR positive T₀The generation-OsATL 15 rice lines #1, #2 and #3 are positive T0 generation-OsATL 15 rice and are named OsATL15-OX1, OsATL15-OX2 and OsATL15-OX 3.

Example 3: construction of OsATL15 mutant plant by CRISPR knockout

1. Selection of target sequences based on exon sequences of OsATL15 using CRISPR/Cas9 system

Selecting a specific target sequence according to an OsATL15 exon sequence by utilizing a simple and efficient CRISPR/Cas9 system, wherein the target sequence comprises the following steps: GAGGCACGTCCTGGAGAAGGAGG are provided. The target sequence is specific to OsATL15 gene and inactivates OsATL15 protein.

2. Construction of pCRISPR/Cas9 recombinant vector containing the target sequence fragment

1) Design of adaptor primer with cohesive end according to target sequence

The designed target sequence was added to the specific sticky end linker (F: GGCA; R: AAAC) of the pCRISPR/Cas9 system and the complete linker primer was synthesized.

F3：5’-GGCA-GAGGCACGTCCTGGAGAAGG-3’；

R3：5’-AAAC-CCTTCTCCAGGACGTGCCTC-3’。

2) Annealing and complementing the adaptor primer with the cohesive end to form a double-stranded small fragment with the cohesive end

Diluting the F3 primer and the R3 primer into 10 mu M solutions, uniformly mixing 10 mu L of each solution, carrying out annealing reaction in a PCR instrument, reducing the temperature from 98 ℃ to 22 ℃, and enabling the F3 primer and the R3 primer to be complementary to form a double-stranded small fragment with a sticky end.

3) Cleavage of the original vector pOs-sgRNA containing sg-RNA (TAKARA Cat #632640)

The original vector pOs-sgRNA containing sg-RNA was digested with the restriction enzyme Bsa I, resulting in cohesive ends which could be complementary to the cohesive ends of the target sequence. The pOs-sgRNA original vector system was digested with Bsa I: 10 XBuffer Bsa I2. mu. L, Bsa I enzyme 1. mu. L, pOs-sgRNA vector 4. mu.g, ddH₂The amount of O is up to 20. mu.L, and the enzyme is cleaved at 37 ℃ for 12 h. Checking the size of the cut enzyme product with 1% agarose gel electrophoresis, purifying the cut enzyme product with a column chromatography kit (OMEGA Cat # D2500-02) to obtain pOs-sgRNA vector, adding sterilized ddH₂Dissolving O, and measuring the concentration for later use.

4) Ligating double-stranded small fragments with cohesive ends to the digested pOs-sgRNA vector to form a recombinant vector comprising the target sequence and sg-RNA

Connecting the double-stranded small fragment in the step 2) with the pOs-sgRNA vector cut in the step 3) by using T4 ligase to form a complete recombinant vector containing a target sequence aiming at the OsATL15 protein and sg-RNA. The 15 μ L linker is: 10 XT 4 ligation buffer 1.5 uL, double-stranded small fragment 4 uL, enzyme-cleaved pOs-sgRNA vector 3 u L, T4 DNA ligation buffer 1 u L, ddH₂O to 15. mu.L, and ligation was performed at 16 ℃ for 12 hours. And transforming the connecting product into escherichia coli DH5 alpha, culturing the kanamycin-resistant LB plate overnight, selecting a positive strain, and sequencing to obtain a recombinant vector which is correctly sequenced and contains a target sequence and sg-RNA.

5) LR-reactive recombination of a recombinant vector comprising a target sequence and sg-RNA and a vector pH-Ubicas9-7 comprising Cas9 with LRmix to form a complete recombinant vector comprising the target sequence-sg-RNA + Cas9

LR mix LR reaction recombination of the recombinant vector obtained in step 4) and the vector pH-Ubi-Cas9-7 (supplied by Wuhanbo Co., Ltd.) containing Cas9, LR reaction system: 25-50ng of recombinant vector containing target sequence and sg-RNA, 75ng of pH-Ubi-cas9-7 vector, 1. mu.L of 5 xlr clone TM Buffer, TE Buffer (pH8.0) were supplemented to 4.5. mu.L of LR clone TM 0.5 uL. Incubating the system at 25 ℃ for 2h, adding 2 mu L of 2 mu g/mu L of protease K after reaction, treating at 37 ℃ for 10min, transferring 2 mu L of reaction product into escherichia coli DH5 alpha, overnight culturing gentamicin resistant LB plate at 37 ℃, selecting positive strains for sequencing, and obtaining the complete pCRISPR/Cas9 recombinant vector containing OsATL15 protein target sequence-sg-RNA + Cas9 with correct sequencing.

3. The obtained pCRISPR/Cas9 recombinant vector is introduced into rice callus to obtain transgenic plant

According to the method for transferring the recombinant vector to OsATL15 rice in example 2, the complete recombinant vector containing the OsATL15 protein target sequence-sg-RNA + Cas9 obtained in step 5) is introduced into rice callus to prepare transgenic rice, and a transgenic plant with the OsATL15 protein completely inactivated can be obtained in a T0 generation plant.

4. Screening transgenic positive plants in transgenic plants

Transgenic plants (T) to be transplanted₀Generation) extracting DNA, proceeding with target sequence positionAnd (4) point detection, wherein 32 positive plants are detected in total.

5. Obtaining mutant plants using transgenic positive plants

1) Identification of the site of mutation

And (3) extracting DNA from the transplanted positive plant, designing specific primers F4 and R4 aiming at the DNA fragment containing the target site and within 500bp, amplifying the DNA fragment containing the target site, purifying and then sending the amplified 360bp PCR product to a company for sequencing, and comparing the sequencing result with the sequence of the wild plant to screen out a mutant plant. The results of the partial mutation analysis are shown in FIG. 2.

F4:5’-ATGGCAGATCAGAAGGTGATT-3’；

R4:5’-TGGAGCTGGTAGCCAAGAATCT-3’。

2) And (3) breeding the mutant plants, detecting that the plants without the transgenic elements such as hygromycin, Cas9 and the like in a T1 generation transgenic segregation population and harvesting seeds of the individual plants to obtain the function-deleted mutants without transgenic components, which are named as Crispr-10, Crispr-9 and Crispr-17 respectively.

Example 4: influence of OsATL15 mutant plant on absorption and transportation of pesticide

The following pesticides were selected for the experiments: neonicotinoid insecticides such as thiamethoxam, amide insecticide chlorantraniliprole, organophosphorus insecticide omethoate, nereistoxin insecticide such as bisultap and carbamate insecticide methomyl. The contents of the agricultural chemicals in the rice rhizome and leaves of the rice lines of Crispr-10, Crispr-9 and Crispr-17 and the rice mid-flower 11 obtained in example 3 were examined.

(1) The thiamethoxam is dissolved in DMSO to prepare a 40mM mother solution, and the mother solution is stored at 4 ℃ for later use. Picking out rice seedlings growing in a rice incubator for 14 days, carefully washing out root nutrient solution, and grouping 10 rice seedlings. Then placing the roots of the rice seedlings in 0.5mM calcium chloride buffer solution for pre-culture for 2 h; diluting the mother liquor to 100 μm with 0.5mM calcium chloride buffer solution, adjusting pH to 5.8, adding into 50mL centrifuge tube, 40mL each tube, transferring rice seedling into the tube, fixing with fixed value cup to ensure that the stem part of rice contacts the liquid surface, and standing in artificial climate incubator for 24 h. After 24h, the rice was removed and the roots were washed 4 times with 0.5mM (pH 5.8) calcium chloride buffer to ensure complete removal of the drug attached to the root surface. Drying surface water with filter paper, cutting off 1cm below and 1cm above the rhizome junction with a blade, and dividing the rice into root, stem and leaf. Respectively weighing and recording, adding liquid nitrogen into a mortar for grinding, adding 10mL of chromatographic grade acetonitrile for extraction, carrying out ultrasonic treatment for 30min, carrying out 14000g centrifugation for 10min, sucking 1mL of supernatant, passing through a 0.22 mu m needle microporous filter membrane to remove impurities, filling the filtrate into a liquid phase bottle, and placing a sample at the low temperature of-80 ℃ for preservation and detection. Each treatment was repeated for 3 groups. The same treatments were carried out on rice seedlings with chlorantraniliprole, omethoate, dimehypo and methomyl, 3 treatments each being repeated.

The results showed that the content of Thiamethoxam (THX), chlorantraniliprole, omethoate, dimehypo and methomyl in the upper part of the roots of the criprpr-10, criprpr-9 and criprpr-17 rice lines was higher than that of the wild type, while the content in the roots was not significantly different from that of the wild type (fig. 3, fig. 4, fig. 5, fig. 6, fig. 7); the transfer coefficient (RCF) was significantly reduced in Crispr-10, Crispr-9 and Crispr-17 rice lines compared to the wild type (FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12).

Example 5: subcellular localization of OsATL15 protein

Extracting total RNA of rice seedlings with 15-day-sized medium flowers 11, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid) serving as a template, carrying out PCR (polymerase chain reaction) amplification, and amplifying the full-length ORF (removing a stop codon) of OsATL15 by using primers as follows:

F5：5’-TCAGATCTCGAGCTCAAGCTTC ATGGCAGATCAGAAGGTG-3’；

R5：5’-CCTTGCTCACCATCAGGATCCCTAGGTGACGAGACTGAGGTG-3’。

and (3) carrying out enzyme digestion and recovery on the amplified target fragment, connecting the target fragment with an empty vector 322-d1-eGFPn (Beijing Huayun), fusing OsATL15 with GFP, transferring the fused vector 322-d1-eGFPn-OsATL15 and the empty vector into the rice protoplast after the sequencing identification is correct, normally culturing for 16h at room temperature, and observing the subcellular localization of the fused protein OsATL15-GFP and the protein GFP in the rice protoplast under a laser confocal microscope. As shown in FIG. 13, it was confirmed that the OsATL15 protein was specifically localized in rice cell membranes.

Example 6: development of OsATL15 molecular probe

The neonicotinoid insecticide thiamethoxam is taken as an example.

Seedlings of flower 11 of the 14-day-old variety were treated with 100. mu. mol/L thiamethoxam, and treated with solvent water as a Control (Control). After treatment, RNA was extracted, and after reverse transcription, expression of OsATL15 gene was detected by quantitative PCR technique in the same manner as in example 2, and the experiment was repeated three times.

As a result, as shown in FIG. 14, the specific expression of OsATL15 gene in rice roots, stems and leaves was strongly induced under the thiamethoxam-treated condition. Therefore, the primer is used as a molecular probe to perform expression analysis on OsATL15 gene in rice germplasm, can quickly judge the response processing capacity of target genes to thiamethoxam, and can be used for breeding new varieties with high thiamethoxam absorption.

Example 7 Effect of OsATL15 Gene mutation on plant height

The OsATL15 rice Crispr-10, Crispr-9 and Crispr-17 mutants obtained in example 3 and the wild type rice middle flower 11 were planted in a rice incubator and the phenotype was observed after two weeks. 15 strains of each strain, the experiment was repeated 3 times, and the results were averaged.

The phenotype was observed, and the plant heights of the rice lines Crispr-10, Crispr-9 and Crispr-17 were reduced compared to the wild-type rice mid-flower 11, with no significant change in root length (FIG. 15). When the plant heights and root lengths of rice plants were measured, the plant heights of the rice lines of Crispr-10, Crispr-9 and Crispr-17 were all reduced compared to the wild type rice mid-flower 11 (FIG. 16), while the root lengths were unchanged (FIG. 17).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> rice gene OsATL15 in regulation of absorption and transportation of pesticides

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 631

<212> PRT

<213> Rice (Oryza sativa)

<400> 1

Met Ala Asp Gln Lys Val Ile Leu Ala Glu Pro Leu Leu Pro Gly Lys

1 5 10 15

Glu Ala Asp Phe Ala Asp Asp Asp Asp Val Glu Ala Gln Leu Thr Ser

20 25 30

Tyr His Thr Gly Ala Ser Phe Ser Arg Thr Cys Leu Asn Leu Thr Asn

35 40 45

Ala Val Ser Gly Ile Gly Val Leu Ser Met Pro Tyr Ala Val Ser Gln

50 55 60

Gly Gly Trp Leu Ser Leu Leu Leu Phe Val Leu Val Gly Ala Val Cys

65 70 75 80

Tyr Tyr Thr Gly Thr Leu Ile Glu Arg Cys Met Arg Ala Asp Gly Ser

85 90 95

Ile Ala Ser Tyr Pro Asp Ile Gly Gln Tyr Ala Phe Gly Ala Thr Gly

100 105 110

Arg Arg Ala Val Ala Phe Phe Met Tyr Val Glu Leu Tyr Leu Val Ala

115 120 125

Ile Ser Phe Leu Val Leu Glu Gly Asp Asn Leu Asp Lys Leu Phe Pro

130 135 140

Gly Ala Thr Met Glu Ile Leu Gly Tyr Gln Leu His Gly Lys Gln Leu

145 150 155 160

Phe Ile Val Leu Ala Ala Ala Val Ile Leu Pro Thr Thr Trp Leu Lys

165 170 175

Asn Leu Gly Met Leu Ala Tyr Val Ser Ala Ala Gly Leu Ile Ala Ser

180 185 190

Val Ala Leu Thr Ala Ser Leu Ile Trp Ala Gly Val Ala Glu Thr Gly

195 200 205

Phe His Arg Asn Ser Asn Thr Leu Asn Leu Ala Gly Ile Pro Thr Ser

210 215 220

Leu Gly Leu Tyr Phe Val Cys Phe Thr Gly His Ala Val Phe Pro Thr

225 230 235 240

Ile Tyr Ser Ser Met Lys Asn Ser Lys His Phe Ser Lys Val Leu Leu

245 250 255

Ile Ser Ser Val Leu Cys Ser Leu Asn Tyr Gly Leu Thr Ala Val Leu

260 265 270

Gly Tyr Met Ile Tyr Gly Asp Asp Val Gln Ser Gln Val Thr Leu Asn

275 280 285

Leu Pro Ser Gly Lys Leu Tyr Thr Lys Ile Ala Ile Val Met Thr Leu

290 295 300

Val Asn Pro Leu Ala Lys Tyr Ala Leu Leu Val Ala Pro Ile Thr Ala

305 310 315 320

Ala Val Glu Glu Arg Leu Ser Leu Thr Arg Gly Ser Ala Pro Ala Arg

325 330 335

Val Ala Ile Ser Thr Ala Ile Leu Ala Ser Thr Val Val Val Ala Ser

340 345 350

Thr Val Pro Phe Phe Gly Tyr Leu Met Ser Phe Ile Gly Ser Phe Leu

355 360 365

Ser Val Met Ala Thr Val Leu Phe Pro Cys Leu Cys Tyr Leu Lys Ile

370 375 380

Tyr Lys Ala Asp Gly Ile His Arg Thr Glu Met Val Ala Ile Ala Gly

385 390 395 400

Ile Leu Leu Leu Gly Val Phe Val Ala Val Thr Gly Thr Lys Arg Gly

405 410 415

Ser His Lys Arg Ser Gly Val Thr Gly Phe Ser Ser Val Glu Thr Thr

420 425 430

Ser Thr Ala Ala Val Ala Met Ala Gly Ser Gln Thr Gln Ser Gln Arg

435 440 445

Leu Pro Thr Pro Thr Arg Gln Val Ala Gly Glu Arg Gly Trp Ser Arg

450 455 460

Trp Arg Gly Gly Gly Gln Trp Glu Ser Ala Val Ala Ala Ala Gln Ala

465 470 475 480

Ala Gly Glu Arg Gln Gln Ala Ala Val Arg Gly Arg Ser Arg Trp Lys

485 490 495

Gly Gly Val Gly Gly Cys Arg Arg Arg Lys Ala Ala Val Trp Ser Arg

500 505 510

Gly Leu Glu Glu Lys Glu Thr Arg Ala Pro Gly Pro Arg Arg Val Gly

515 520 525

Ser Trp Ser Arg Asn Ala Ala Thr Ser Ser Gly Leu Lys Arg Asn Leu

530 535 540

Arg Trp Cys Thr Ser Ser Ser Pro Gly Arg Ser Leu Ser Leu Ala Ser

545 550 555 560

Ser Asp Ala Gly Ser Tyr Ser Gly Trp Ser Tyr Ala Gly Arg Gly Gly

565 570 575

Trp Arg Ala Ser Arg Gly Ser Leu Ala His Thr Gly Gln Thr Leu Cys

580 585 590

Ala Phe Arg Glu Leu Thr Asp Gly Leu Gln Glu Leu Arg Asp Pro Arg

595 600 605

Arg Val Asp Glu Val Val Ala Asp Leu Gly Ala Glu His Glu Ala Ala

610 615 620

Thr Pro Gln Ser Arg His Leu

625 630

<210> 2

<211> 3676

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggcagatc agaaggtgat tctcgccgag ccgctgcttc cggggaagga agctgacttc 60

gccgacgacg acgacgtgga ggcgcagctc acttcgtacc acaccggcgc ctccttctcc 120

aggacgtgcc tcaacctcac caatgccgtc tccggcatcg gggtgctgtc catgccgtac 180

gcggtgtcgc agggcgggtg gctcagcctg ctgctcttcg tcctcgtcgg cgccgtgtgc 240

tactacaccg gcacgctcat cgagcgctgc atgcgcgccg acggctccat cgccagctac 300

ccggacatcg gccagtacgc cttcggcgcc accggcagga gagccgtcgc attcttcatg 360

tacgtcgagc tctacctcgt cgccataagc ttcctcgtcc tcgagggcga caatctcgac 420

aagctcttcc ccggcgcaac catggagatt cttggctacc agctccacgg caagcagcta 480

ttcattgtac tcgccgccgc cgtcatactg cccactacct ggctcaagaa tcttggcatg 540

ctcgcttacg tgtcagcggc cgggctcatc gcctcggtgg ccttgacggc gtcactgatc 600

tgggccggtg tcgccgagac cgggttccac cggaacagca acacgctcaa cctcgccgga 660

atccccacct ccctcggtct ctacttcgtc tgcttcaccg gccacgccgt cttcccgacg 720

atatactcct ccatgaagaa cagcaaacac ttctccaaag ttctcttgat ctcgtcggtg 780

ctgtgcagcc tcaactacgg gctcacggcg gtgctgggct acatgatcta cggtgacgat 840

gtgcagtcgc aggtgacgct gaacctgccc tccggcaagc tctacaccaa gatcgccatc 900

gtgatgacgc tggtgaaccc gttggccaag tacgcgctgc tcgtggcgcc gatcacggcg 960

gccgtcgagg agaggctgtc tctgacgcgg ggcagcgcgc cggcgagggt ggccatcagc 1020

accgccatcc tcgccagcac ggtggtggtg gcgtcgacgg tgcccttctt cggctacctc 1080

atgtcattca tcggctcgtt cctcagcgtc atggcgaccg tgctgttccc ttgcctctgc 1140

tacctcaaga tctacaaggc cgatggcatc caccgcacgg agatggtggc catcgccgga 1200

attctgctgc taggagtgtt cgtcgccgtc accggcacgt acacttcttt gctgcagatt 1260

atagccactt tctgaggtgg ttggttgctg ttggtctctg atagtagaca gtagtactta 1320

cagacaatag tgtgaccaca gtgtccttaa aataaagggc gcttgaaaaa attttcggag 1380

ttagtttgta tctaagataa agctgaatca ataatattga cttaggtgaa aaaattgtat 1440

atattattgg tgccaacaat gtcaatgttg tatttaataa atatcagata tgagacacta 1500

atttaggttt atttgtaaaa aaaaaaatgc caagtgacta atttgtttaa ggaacaaatc 1560

cataagagat gttgtcgaca atgtcaaggg acaaatttgt taaagtaaca aagccataag 1620

gggaggatcc actaaaaaaa aaggaatgga cagcgatacc tgtgttttgc tatttgaatg 1680

acggccgatg cttccaaaat ccaaacctac ccacctaggt actacagacc tgaagcaacg 1740

aagaacgaag gttttttcct ctgctatcga gaggtctttc gttgcatgtc attcaaatat 1800

ttatcaaaaa tcttaaaaga acaacaataa attgaaattc aacctattca agttgaggaa 1860

aaataataaa ttaaaatcta atgttttgtt atttttgttt taataaccta tatagattaa 1920

atttgaaatt gcataattgt taaatatgca cgtttgtgaa acgatatata atatattaag 1980

ctatattttt taacttttca aaaaaaaagc tatttaaata gcatggaaaa aaggaagagc 2040

attttactct ctaatgggaa tagttaactc acatatatag atgctaaata tgcttcacat 2100

gcatttgggg atagttaact cacatatata gatgcttgat taatcgccag tatgtttact 2160

gagaaagaga agaaaaggaa ctgataagtc aaaggaactg ataagtcatc gccccctaca 2220

agcccaaaca ggcaaaggtg tacgatgtgg gtcggagtga catggtcgtg gcttcgccgc 2280

tttgggtgga ggcgtggagc gatgcgggca gtggagcagc atgctttggg ccacccgacg 2340

caagaagaga aggctgacga acatagggtg gaataatttg tatgagtagc aaaggagggg 2400

cgtggctctg ggtgcccgga cgagacgctg gagcggagct gctctggcac tcggctcagc 2460

agcggaggag cgagctgttt ctaatccggc aacctctctg ctagatcacc cagctcttcg 2520

cctctactct aggcctccgt tgtcagattc agtagattcc acctctctac ctcattcccc 2580

acctctattc cttctacctc tattcttcca ctacttctaa tcctacacct ggggagccga 2640

tcccgatcgt ggcacgagtt cgatccagag gcggtcaaat tttaccgttc ctccaaccca 2700

aacatccatt tcatacttac ggttggattt tgctggcgca ggggatgcga accctatgga 2760

ataaggtcac catagggccc tcaactgaac agcgagattt tctaagatcc tttaactaga 2820

aaaccagaaa tgtgtacctc taaactctct caaaccattc accgaaggtc ccttggcagt 2880

atttttgcct ggttttactg acgtggcatc ctagtcagaa aaaaaataaa aaattacgtg 2940

cggcccacat gtcagctgca cttttttccc ttttcttttc ttctccttct gtactctctt 3000

ctctgattct ctcttcagca agcgcggcag ccacaagcgg agcggcgtga cggggttctc 3060

ctccgtggag acgacgagca cggccgcggt ggcgatggca ggttcccaga cccagagcca 3120

gcggttgccg acgccgacgc ggcaagtagc gggcgagcgc ggctggagca ggtggagggg 3180

cggcggtcag tgggagagcg cagttgcagc ggctcaagcg gcgggcgagc ggcagcaggc 3240

ggcggtgcgc ggccggagca ggtggaaggg cggcgttggc ggctgtaggc ggcggaaagc 3300

ggcggtctgg tcccggggac tcgaggagaa ggaaacgcga gcaccggggc cgaggcgcgt 3360

ggggagctgg tcgcgcaacg cggcgacgtc gtcggggctg aagaggaacc tgcggtggtg 3420

cacgagctcg tcgcctggac ggagcttgtc gctagcctcg tctgatgccg gctcgtactc 3480

ggggtggtca tacgccggac gcggcgggtg gcgcgcgtcg aggggctccc tcgctcacac 3540

cggccagacg ctctgcgcct tccgcgagct caccgacggc ctgcaagaac tgcgtgatcc 3600

ccgccgcgtc gacgaggttg tggcagatct gggtgctgaa cacgaagccg ccacacctca 3660

gtctcgtcac ctatga 3676

<210> 3

<211> 1896

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

atggcagatc agaaggtgat tctcgccgag ccgctgcttc cggggaagga agctgacttc 60

gccgacgacg acgacgtgga ggcgcagctc acttcgtacc acaccggcgc ctccttctcc 120

aggacgtgcc tcaacctcac caatgccgtc tccggcatcg gggtgctgtc catgccgtac 180

gcggtgtcgc agggcgggtg gctcagcctg ctgctcttcg tcctcgtcgg cgccgtgtgc 240

tactacaccg gcacgctcat cgagcgctgc atgcgcgccg acggctccat cgccagctac 300

ccggacatcg gccagtacgc cttcggcgcc accggcagga gagccgtcgc attcttcatg 360

tacgtcgagc tctacctcgt cgccataagc ttcctcgtcc tcgagggcga caatctcgac 420

aagctcttcc ccggcgcaac catggagatt cttggctacc agctccacgg caagcagcta 480

ttcattgtac tcgccgccgc cgtcatactg cccactacct ggctcaagaa tcttggcatg 540

ctcgcttacg tgtcagcggc cgggctcatc gcctcggtgg ccttgacggc gtcactgatc 600

tgggccggtg tcgccgagac cgggttccac cggaacagca acacgctcaa cctcgccgga 660

atccccacct ccctcggtct ctacttcgtc tgcttcaccg gccacgccgt cttcccgacg 720

atatactcct ccatgaagaa cagcaaacac ttctccaaag ttctcttgat ctcgtcggtg 780

ctgtgcagcc tcaactacgg gctcacggcg gtgctgggct acatgatcta cggtgacgat 840

gtgcagtcgc aggtgacgct gaacctgccc tccggcaagc tctacaccaa gatcgccatc 900

gtgatgacgc tggtgaaccc gttggccaag tacgcgctgc tcgtggcgcc gatcacggcg 960

gccgtcgagg agaggctgtc tctgacgcgg ggcagcgcgc cggcgagggt ggccatcagc 1020

accgccatcc tcgccagcac ggtggtggtg gcgtcgacgg tgcccttctt cggctacctc 1080

atgtcattca tcggctcgtt cctcagcgtc atggcgaccg tgctgttccc ttgcctctgc 1140

tacctcaaga tctacaaggc cgatggcatc caccgcacgg agatggtggc catcgccgga 1200

attctgctgc taggagtgtt cgtcgccgtc accggcacca agcgcggcag ccacaagcgg 1260

agcggcgtga cggggttctc ctccgtggag acgacgagca cggccgcggt ggcgatggca 1320

ggttcccaga cccagagcca gcggttgccg acgccgacgc ggcaagtagc gggcgagcgc 1380

ggctggagca ggtggagggg cggcggtcag tgggagagcg cagttgcagc ggctcaagcg 1440

gcgggcgagc ggcagcaggc ggcggtgcgc ggccggagca ggtggaaggg cggcgttggc 1500

ggctgtaggc ggcggaaagc ggcggtctgg tcccggggac tcgaggagaa ggaaacgcga 1560

gcaccggggc cgaggcgcgt ggggagctgg tcgcgcaacg cggcgacgtc gtcggggctg 1620

aagaggaacc tgcggtggtg cacgagctcg tcgcctggac ggagcttgtc gctagcctcg 1680

tctgatgccg gctcgtactc ggggtggtca tacgccggac gcggcgggtg gcgcgcgtcg 1740

aggggctccc tcgctcacac cggccagacg ctctgcgcct tccgcgagct caccgacggc 1800

ctgcaagaac tgcgtgatcc ccgccgcgtc gacgaggttg tggcagatct gggtgctgaa 1860

cacgaagccg ccacacctca gtctcgtcac ctatga 1896

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> molecular marker detection primer F of OsATL15

<400> 4

tactcgccgc cgccgtcata 20

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> molecular marker detection primer R of OsATL15

<400> 5

cgaggttgag cgtgttgctg ttcc 24

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F1

<400> 6

atggcagatc agaaggtg 18

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R1

<400> 7

tcataggtga cgagactgag gtg 23

<210> 8

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer 1

<400> 8

acccggggat cctctagagt cgaatggcag atcagaaggt g 41

<210> 9

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer 2

<400> 9

atgatacgaa cgaaagctct gcatcatagg tgacgagact gaggtg 46

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F2

<400> 10

acggtgtcgt ccatcacagt ttgcc 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R2

<400> 11

ttccggaagt gcttgacatt gggga 25

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer F of internal reference UBQ2

<400> 12

gcatctctca gcacattcca 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer R of internal reference UBQ2

<400> 13

accacaggta gcaataggta 20

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> target sequence

<400> 14

gaggcacgtc ctggagaagg agg 23

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F3

<400> 15

ggcagaggca cgtcctggag aagg 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R3

<400> 16

aaacccttct ccaggacgtg cctc 24

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F4

<400> 17

atggcagatc agaaggtgat t 21

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R4

<400> 18

tggagctggt agccaagaat ct 22

<210> 19

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F5

<400> 19

tcagatctcg agctcaagct tcatggcaga tcagaaggtg 40

<210> 20

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R5

<400> 20

ccttgctcac catcaggatc cctaggtgac gagactgagg tg 42

Claims

1. the application of rice gene OsATL15 in regulating the absorption and transport of pesticides, it is characterized in that, the protein encoded by described rice gene OsATL15 , its amino acid sequence is as shown in SEQ ID NO: 1;

The pesticide is one of neonicotinoid pesticides, amide pesticides, organophosphorus pesticides, necrotoxin pesticides or carbamate pesticides.

2. the application of rice gene OsATL15 according to claim 1 in regulating the absorption and transport of pesticide, it is characterized in that, described rice gene OsATL15 , its nucleotide sequence is any one in following A to C:

A. The nucleotide sequence as shown in SEQ ID NO:2;

B. The nucleotide sequence shown in SEQ ID NO: 3;

C. A nucleotide sequence that hybridizes to the nucleotides defined by A or B under stringent conditions and encodes the amino acid set forth in SEQ ID NO:1.

3. the application of the recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the described rice gene OsATL15 of claim 1 or 2 in regulating the absorption and transport of pesticides;

4. the application of a molecular marker detection primer of a rice gene OsATL15 in the identification of the rice variety of efficient utilization of pesticides, it is characterized in that comprising the steps: utilize the described molecular marker detection primer of the germplasm genomic DNA of the rice variety to be detected or RNA is amplified, and the sensitivity of the target gene in response to pesticide treatment is judged by the change in gene expression;

The pesticide is one of neonicotinoid insecticides, amide insecticides, organophosphorus insecticides, necrotoxin insecticides or carbamate insecticides;

The primers consist of upstream primers and downstream primers as shown in SEQ ID NO: 4 and SEQ ID NO: 5:

F: 5'-TACTCGCCGCCGCCGTCATA-3';

R: 5'-CGAGGTTGAGCGTGTTGCTGTTCC-3'.

5. the application of the rice gene OsATL15 described in claim 1 or 2 in cultivating the transgenic rice of efficient utilization of pesticides, it is characterized in that comprising the steps: described rice gene OsATL15 is constructed on the plant expression vector, the recombination obtained The expression vector is transferred into rice for expression, and a rice variety that efficiently utilizes pesticides is obtained;

6. the application of rice gene OsATL15 according to claim 5 in cultivating the transgenic rice of efficient utilization of pesticide, it is characterized in that:

The pesticide is a systemic pesticide;

The plant expression vector is pCAMBIA2300-35S;

The transformation of the obtained recombinant expression vector into rice for expression is to transform the obtained recombinant plant expression vector into plant cells or tissues by biological methods such as Agrobacterium-mediated, plant virus vector, direct DNA transformation, and electrical conduction transformation. .

7. The application of the rice gene OsATL15 of claim 1 or 2 in reducing the high and high plant height of rice plants.