Plasma membrane intrinsic aquaporin for absorbing and transporting neonicotinoid insecticides, and coding gene and use therefor

Abstract

The disclosure discloses a Chinese cabbage plasma membrane intrinsic aquaporin, having an amino acid sequence as set forth in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene BraPIP2;1 thereof is as set forth in SEQ ID NO. 1. The plasma membrane intrinsic aquaporin has the characteristic of sensitively responding to neonicotinoid insecticides (thiamethoxam, imidacloprid, etc.) in the external environment. At the same time, it has the function of mediating transmembrane transport of the neonicotinoid insecticides, promoting accumulation of the neonicotinoid insecticides in plant roots and leaves, which has important application value in guiding efficient and simple use of pesticides, development of new systemic pesticides, etc.

Claims

1. A method for promoting uptake and transport of neonicotinoid insecticides in a plant, the method comprising introducing a binary overexpression vector containing a nucleotide sequence as set forth in SEQ ID NO: 1 into the plant for expression; applying neonicotinoid insecticides to the plant or its environment; and measuring a concentration of neonicotinoid insecticides in root and above ground portion of the plant, wherein the plant has an increased concentration of the neonicotinoid insecticides as compared to that of a control plant that has not been transformed with the binary overexpression vector containing the nucleotide sequence as set forth in SEQ ID NO: 1.

2. The method for promoting uptake and transport of neonicotinoid insecticides in plants of claim 1, wherein the plants comprise at least one of vegetables, rice, Zea mays, wheat, rape and Arabidopsis thaliana.

3. The method for promoting uptake and transportation of neonicotinoid insecticides in plants of claim 1, wherein the neonicotinoid insecticides comprise at least one of thiamethoxam, imidacloprid, acetamiprid, nitenpyram and dinotefuran.

4. The method of claim 1, wherein the increase in concentration of neonicotinoid insecticide is at least 50% in roots and 80% in above ground portion of the plants.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of subcellular localization of the BraPIP2;1 protein;

(2) tobacco leaf cells instantaneously transformed for 48 hours under confocal microscope observation. Scale=40 μm.

(3) FIG. 2 shows the effect of water channel inhibitors on the uptake of different neonicotinoid insecticides and nicotine in Chinese cabbage (FIG. 2A: thiamethoxam; FIG. 2B: imidacloprid; FIG. 2C: acetamiprid; FIG. 2D: nitenpyram; FIG. 2E: dinotefuran; and FIG. 2F: nicotine).

(4) FIG. 3 shows the molecular docking analysis of BraPIP2;1 with different neonicotinoid insecticides and nicotine.

(5) FIG. 4 is a schematic diagram of the expression characteristics of BraPIP2;1 and response characteristics thereof to thiamethoxam,

(6) where FIG. 4A shows the response characteristics of the root tissue BraPIP2;1 to external thiamethoxam; and FIG. 4B shows the response characteristics of the aboveground tissue BraPIP2;1 to external thiamethoxam.

(7) FIG. 5 is a schematic diagram of the growth and the thiamethoxam content of a BraPIP2;1 recombinant yeast strain under thiamethoxam stress,

(8) where FIG. 5A shows the growth of the recombinant yeast under different concentrations of thiamethoxam; and FIG. 5B is a schematic diagram of the thiamethoxam content in the recombinant yeast strain.

(9) FIG. 6 is a schematic diagram of the growth and the thiamethoxam content of BraPIP2;1 transgenic Arabidopsis thaliana plants under thiamethoxam stress,

(10) where FIG. 6A shows the growth of the transgenic Arabidopsis thaliana plants under different concentrations of thiamethoxam; FIG. 6B is a schematic diagram of the main root length of the transgenic Arabidopsis thaliana plants under different concentrations of thiamethoxam; FIG. 6C is a schematic diagram of the thiamethoxam content of the transgenic Arabidopsis thaliana plants; and BraPIP2;1#3 and BraPIP2;1#5 are overexpression Arabidopsis thaliana lines.

DETAILED DESCRIPTION OF THE INVENTION

(11) The technical solutions of the disclosure will be further described below in conjunction with examples.

Example 1 Cloning and Analysis of the Full-Length Encoding Region of the BraPIP2;1 Gene

(12) 1. ORF Amplification of BraPIP2;1

(13) The total RNA was extracted from a root sample of Chinese cabbage according to operation steps of using an RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China). Using 2 g of total RNA as a template and oligo (dT)18 as an anchor primer, and referring to instructions of a Primescript 1st Strand cDNA Synthesis Kit (Invirtrogen), 1st strand cDNA was synthesized. Primers were designed to perform PCR amplification to obtain a single cDNA fragment, and the primer sequences are as follows: upstream primer sequence (SEQ ID NO.3): ATGGCGAAGGACGTGGAAGC; and downstream primer sequence (SEQ ID NO.4): TTAAACGTTGGCAGCACTTCTG.

(14) An amplification system is 50.0 μL in total volume, including 20 ng of cDNA, 10.0 μL of 5×Prime STAR buffer, 4.0 μL of 2.5 mmol.Math.L-1 dNTPs, 2.0 μL of 10 mmol.Math.L-1 forward primer and reverse primer each, 1.25 U of PrimeSTAR HS DNA Polymerase, and the balance of redistilled water.

(15) An amplification program is: pre-denaturation at 95° C. for 5 min, denaturation at 98° C. for 10 s, renaturation at 55° C. for 15 s, and extension at 72V for 60 s, 35 cycles in total.

(16) The PCR product was sent to Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing result shows that the full-length cDNA sequence of BraPIP2;1 is 2550 bp. The cDNA of BraPIP2;1 and the amino acid sequence of a protein encoded by BraPIP2;1 are as set forth in SEQ ID No. 1 and SEQ ID No. 2, respectively.

Example 2 Subcellular Localization of BraPIP2;1

(17) Referring to Liu et al. (Liu T, et al. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nat Commun 5, 4686. 2014), BraPIP2;1 was constructed into a subcellular localization vector pBinGFP4, and transformed into an Agrobacterium strain GV3101 (deposited in the laboratory of Jiangsu Academy of Agricultural Sciences) by a freeze-thaw method. Tobacco was transformed instantaneously, and fluorescence was observed under a fluorescence microscope after 48 h-60 h. It was found that the green fluorescent signal was mainly distributed on the cell membrane or nuclear membrane (see FIG. 1), indicating that BraPIP2;1 is mainly located on the cell membrane and nuclear membrane related to transmembrane transport.

Example 3 Inhibiting the Activity of Aquaporins can Reduce the Uptake and Accumulation of Neonicotinoid Pesticides in Vegetables

(18) Referring to the paper (Wenfeng W, Wan Q, Li Y, et al. Uptake, translocation and subcellular distribution of pesticides in Chinese cabbage (Brassica rapa var. chinensis)[J]. Ecotoxicology and Environmental Safety, 2019.), water channel inhibitors, mercuric chloride and glycerol, of different concentrations were added to equal volumes of Hoagland nutrient solutions containing 2 mg/L nicotine compounds (thiamethoxam, imidacloprid, acetamiprid, nitenpyram, dinotefuran and nicotine). After 48 h, the concentrations of the nicotine compounds in the Chinese cabbage plants (with three leaves and one bud) were determined respectively. Each treatment was repeated five times.

(19) To determine whether aquaporins can transport the neonicotinoid insecticide molecules and nicotine, the water channel inhibitors mercuric chloride (HgCl2) and glycerol of different concentrations were added to equal volumes of nutrient solutions containing 2 mg/L of different neonicotinoid insecticides (thiamethoxam, imidacloprid, acetamiprid, nitenpyram and dinotefuran) and nicotine, and the concentrations of the nicotine compounds in the plants were determined respectively.

(20) The test results are as shown in FIG. 2. After the water channel inhibitors were added, the content of the neonicotinoids in the plants was significantly reduced, while the content of nicotine in the plants did not change significantly. It can be seen that, the water channel inhibitors can significantly inhibit the uptake of the neonicotinoid insecticides in Chinese cabbage, but not the uptake of nicotine, indicating that the aquaporins are involved in the transmembrane transport of the neonicotinoid insecticides.

Example 4 Molecular Docking Analysis of BraPIP2;1 Protein with Different Neonicotinoids and Nicotine

(21) A 3D structure of BraPIP2;1 was obtained by homology modeling of the BraPIP2;1 amino acid sequence, and whether the neonicotinoid insecticide molecules (thiamethoxam, imidacloprid, acetamiprid, nitenpyram and dinotefuran) and nicotine molecules can pass through the pores in the middle of the BaPIP2;1 crystal structure was verified with the molecular docking technology. The docking range includes the whole protein conformation, so the docking results of the neonicotinoid insecticide molecules and nicotine molecules entering the central pores of the protein can be screened from many conformations generated to analyze whether the insecticide-like molecules can pass through the protein pores.

(22) The software used for docking is AutoDockTools-1.5.6, the size of a docking box is set to 100 Å*126 Å*100 Å, semi-flexible docking is adopted, the algorithm uses the Lamarckian genetic algorithm, and the docking generates 200 results. The docking result is shown in FIG. 3: The neonicotinoid insecticide molecules (thiamethoxam, imidacloprid, acetamiprid, nitenpyram and dinotefuran) bind to the center of the protein channel, which proves that thiamethoxam and other neonicotinoid insecticide molecules do not produce steric hindrance when entering the channel. While the nicotine molecule cannot bind to the center of the protein channel, which proves that the nicotine molecule will produce steric hindrance when entering the channel.

(23) The result above indicates that the BraPIP2;1 protein can transport neonicotinoid insecticides, but not nicotine.

Example 5 Response of the BraPIP2;1 Gene to Environmental Thiamethoxam Stress

(24) Seedlings of Chinese cabbage with the same growth condition were put in a Hoagland's nutrient solution containing 10 mg/L thiamethoxam (the Hoagland's nutrient solution was purchased from Beijing Coolaber Technology Co., Ltd.), and treated in the Hoagland's nutrient solution for 6 and 24 hours (treatment group).

(25) At the same time, a Hoagland's nutrient solution without thiamethoxam was used as the control group.

(26) Treated root and aboveground tissue RNA of two groups was extracted respectively, the 1st strand cDNA was obtained by reverse transcription as a template, specific expression primers were designed based on the cDNA sequence of BraPIP2;1, the Chinese cabbage Tublin was used as an internal reference, and the expression of the gene transcription level was detected by quantitative RT-PCR.

(27) Quantitative PCR primer design:

(28) BraPIP2;1-F(SEQ ID NO 0.5): TCACTGGAACTGGAATCAACCC;

(29) BraPIP2;1-R(SEQ ID NO 0.6): CCTGAAGCCCTCAAAACGAAC:

(30) Tublin-F(SEQ ID NO. 7): ACTGGGTGTTTTGGGTTGGG;

(31) Tublin-R(SEQ ID NO. 8): TGAAGGGGATTGCTCTGATGAC.

(32) The quantification instrument is 7500 Real Time PCR System (Applied Biosystem), and a qPCRT system was configured according to the instructions of a kit 2×TSINGKE Master qPCR Mix (Tsingke Biotechnology Co., Ltd.). The PCR program is: pre-denaturation at 95° C. for 10 min, at 95° C. for 15s, and at 60° C. for 20 s, for 40 cycles.

(33) The result of qRT-PCR showed that BraPIP2;1 was expressed both in roots and aboveground parts. Compared with the control, under the condition of adding thiamethoxam, the expression abundance of BraPIP2;1 in the roots and aboveground parts was significantly increased after 6 hours and 24 hours of treatment (p<0.001) (as shown in FIG. 4), indicating that the transport and utilization of thiamethoxam is related to the BraPIP2;1 aquaporin.

Example 6 Overexpression of BraPIP2;1 Gene Saccharomyces cerevisiae can Improve Stress Tolerance to Thiamethoxam

(34) The BraPIP2;1 gene cloned in Example 1 was constructed into the Saccharomyces cerevisiae overexpression vector pYES2 (purchased from Clotech) referring to (He Lin, Jiang Lili, Wang Yucheng, Analysis of the stress tolerance of Tamarix thioredoxin peroxidase (ThPrx1) gene transformed into yeast, Journal of Northeast Forestry University, 2011, Vol. 39. No. 4, 101-104), and the constructed recombinant plasmid was named pYES2-BraPIP2;1. The pYES2-BraPIP2;1 and an empty vector pYES2 were transformed into Saccharomyces cerevisiae INVSc1 by lithium acetate precipitation. The recombinant yeasts were named INVSc1 (pYES2-BraPIP2;1) and INVSc1 (pYES2), respectively.

(35) Induction of recombinant strains: Single colonies of the control yeast INvsc1 (pYES2) and the recombinant yeast INvsc1 (pYES2-BraPIP2;1) were picked, inoculated in an SC-U liquid medium (with glucose of a final concentration of 2%) respectively, and incubated at 30° C. for 24 h on a shaker. The OD600 value is measured, and the amount of bacterial solution required is calculated such that the OD600 of bacteria in 10 ml of induction medium (SC-U+2% galactose) is 0.4. Induce expression was carried out at 30° C. for 24 h. The OD600 value was measured again, and the amount of bacterial solution required was calculated.

(36) Growth experiment of the recombinant strains under thiamethoxam stress: Referring to the method disclosed in (He Lin, Jiang Lili, Wang Yucheng, Analysis of the stress tolerance of Tamarix thioredoxin peroxidase (ThPrx1) gene transformed into yeast, Journal of Northeast Forestry University, 2011, Vol. 39. No. 4, 101-104), the OD600 value of the induced bacterial solution is measured, and the amount of bacterial solution required is calculated such that the OD600 of bacteria in 200 μL of bacterial solution is 2. The bacterial solution was centrifuged at 8500 r/min for 1 min, and the supernatant was discarded.

(37) Thiamethoxam stress treatment: The bacteria were resuspended in 200 μL of thiamethoxam solutions of 0, 10, 100, 400, 800 and 1600 mg/L respectively, and subjected to stress at 30° C. for 72 h. The bacterial solutions were diluted by 10, 100, 1000, 10000 and 100000 times, and then 3 μL of bacterial solutions were spread on a solid medium of SC-U (with glucose of a final concentration of 2%), and incubated at 30° C. for 48 h. The result is as shown in FIG. 5A: The transgenic strain (pYES2-BraPIP2;1) grows the same as the wild strain (pYES2) without the thiamethoxam, and grows significantly weaker than the wild strain with the thiamethoxam.

(38) The thiamethoxam content experiment of the recombinant strains: The OD600 value of the induced bacterial solution was measured and the amount of bacterial solution required was calculated such that the OD600 of bacteria in 500 ml of bacterial solution is 0.1. The bacterial solution was centrifuged at 8500 r/min for 1 min, and the supernatant was discarded. The bacteria were resuspended in 500 ml of SC-U liquid media containing 10 and 100 mg/L thiamethoxam (with glucose of a final concentration of 2%) respectively, and subjected to stress at 30° C. for 6 h, and the OD600 value was determined. After centrifugation at 6000 rpm for 10 min, the bacteria were washed three times with ultrapure water, and disrupted with glass beads combined with ultrasound, and thiamethoxam was extracted to measure the amount. Under the stress of 10 mg/L thiamethoxam, the thiamethoxam content of the recombinant strain was 0.0071 ng/OD, which was significantly higher than that of the control strain (0.0038 ng/OD). Under the stress of 100 mg/L thiamethoxam, the thiamethoxam content of the recombinant strain (pYES2-BraPIP2;1) was 0.028 ng/OD, which was significantly higher than that of the control strain (pYES2, 0.011 ng/OD) (see FIG. 5B).

Example 7 Overexpression of BraPIP2;1 Gene in Arabidopsis thaliana can Increase the Uptake and Accumulation of Thiamethoxam in Arabidopsis thaliana

(39) The BraPIP2;1 gene cloned in Example 1 was constructed into the plant binary overexpression vector pCAMBIA2301 (purchased from Clotech). By the Agrobacterium tumefaciens-mediated method (Valvekens, D., van Montagu, M., and Lijsebettens, M. V. (1988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536-5540.), the inflorescence of wild-type Arabidopsis thaliana Col-0 (purchased from ATCC, USA) was infected with the constructed overexpression vector. Through screening (resistance pure line seedlings were screened on a 1/2MS plate containing 50 μg/mL kanamycin), BraPIP2;1 overexpression Arabidopsis thaliana pure line materials BraPIP2;1#3 and BraPIP2;1#5 were identified and obtained.

(40) The pure line seeds of the BraPIP2;1#3 and BraPIP2;1#5 obtained in the previous transgenic experiment and a wild-type Col-0 material (WT) were germinated on a 1/2MS medium respectively, and then transferred to 1/2MS media (Qingdao Hopebio Co., Ltd., Item No. HB8469-12, PH=5.6) containing 10 and 50 mg/L thiamethoxam respectively. After 20 days, the growth of the seedlings was observed and the main root length was measured. The result is as shown in FIGS. 6A and 6B respectively. The main root length of the transgenic strain is shorter than that of the wild type.

(41) In addition, germinated seedlings of the same size were transplanted into soil containing 10 mg/L thiamethoxam, and after 20 days of culture, the root and aboveground samples of the transgenic Arabidopsis thaliana and the control were harvested respectively, washed with deionized water, and weighed. The thiamethoxam content of each sample was measured and calculated with reference to (Wenfeng W, Wan Q, Li Y, et al. Uptake, translocation and subcellular distribution of pesticides in Chinese cabbage (Brassica rapa var. chinensis)[J]. Ecotoxicology and Environmental Safety, 2019.). The result is as shown in FIG. 6C. The thiamethoxam content of the roots and aboveground parts of the transgenic plant is higher than that of the wild plant.

(42) The examples above illustrate that the aquaporin BraPIP2;1 gene resource has the characteristic of sensitively responding to neonicotinoid insecticides in the external environment. At the same time, it has the function of rapidly mediating the uptake and transport of neonicotinoid pesticides, promoting the accumulation of neonicotinoid pesticides in plants, which has important application value in improving (agricultural) crop utilization of pesticides and pesticide development, and lays a foundation for further improvement of pesticide structure and development of systemic pesticides to improve pesticide utilization.