WO2011050716A1

WO2011050716A1 - Paraquat-resistant gene and use thereof

Info

Publication number: WO2011050716A1
Application number: PCT/CN2010/078137
Authority: WO
Inventors: 夏勉; 乔琳; 王丽英; 孔祥凤; 刘军华; 李毅
Original assignee: 北京未名凯拓作物设计中心有限公司
Priority date: 2009-10-30
Filing date: 2010-10-27
Publication date: 2011-05-05
Also published as: CN102548388A

Abstract

The present invention provides a protein having the amino acid sequence as shown in SEQ ID NO: 2. A transgenic plant expressing the protein is resistant to at least 800 mg/l paraquat. The present invention further provides a gene encoding the protein, as well as a method of screening, testing and using of the gene. The present invention also provides a method of culturing the transgenic plant.

Description

Paraquat resistance gene and its application

The invention belongs to the technical field of transgenic plants, and in particular, the invention relates to the application of a high paraquat resistance gene in the cultivation of transgenic plants. Background technique

Paraquat (1,1-dimethyl-4,4'-bipyridyl, molecular weight 257.2) is the second largest herbicide after glyphosate. It is a fast-acting contact herbicide with both Suction, mainly used for weeds in orchards, mulberry gardens, tea gardens, forest belts, etc. It is estimated that future applications will increase and increase. Paraquat has been widely used in agricultural cultivation for nearly 50 years and has been widely accepted and welcomed in more than 100 countries around the world. Since the 1990s, with the demand for agricultural production, several pesticide companies in China have started to produce paraquat raw drugs on a large scale, and have continuously improved the production process. The products have been promoted and applied in various places and played a certain role in production.

Paraquat is a non-selective herbicide. Plants absorb it very quickly. Even if it is rained shortly after spraying, it does not affect the effect of the drug. The absorbed agent is transmitted through the reverse flow in the xylem. Under suitable conditions, a large amount of the drug is The leaves absorb and conduct to other parts, and this conduction is only a non-protoplast (xylem) conduction, so that the foliar treated paraquat usually stagnates within the treated leaves. As a typical photosynthetic system I inhibitor, the activity of the white grass is determined by light. Toxicological studies have shown that the biotoxicity and mutagenicity of paraquat is caused by the reduction and re-oxidation of paraquat when a dioxygen compound is present, during which a negative radical 02_ is produced during the reaction. Cell accumulation in the body. Ju-Fang Ma et al. cloned the 6-phosphate glucose dehydrogenase gene zwf from a strain of Pseudomonas aeruginosa, which has an effective detoxification effect on the negative free radical 0 ₂ _ produced by paraquat.

U.S. Patent Nos. 6,451,543 B1 and US Pat. No. 7,482,425 B2 disclose in SEQ ID No: 27, respectively, a protein identical to SEQ ID No: 2 of the present invention, but they are all used for integration into a lipid matrix, thereby being suitable for FRET analysis. The polypeptide on the membrane does not suggest the use of the invention.

Jinki Jo et al. (Biochem. Biophys. Res. Commun. 285 (4), 885-890 (2001)) found a gene greatly different from the SEQ ID No: 2 structure involved in the present invention from Achromobacter (ie pqrA, GenBank: AAF86626.1), the encoded protein has some resistance to paraquat. Jinki Jo et al. transferred the pqrA gene into tobacco, and when it was well expressed, it had some resistance to paraquat compared to wild-type transgenic tobacco. When the transgenic tobacco plants with pqrA gene are compared with wild-type tobacco, the wild type grows in semi-solid medium and when the concentration of paraquat is ΙμΜ, it will wither. When paraquat reaches 20μΜ (about 5mg/L), the plant will die. And genetically modified tobacco Still normal growth; however, when the concentration of paraquat reaches 50 μΜ (about 12.5 mg / L), the loss of chlorophyll content in the leaves of transgenic tobacco will also reach about 15%, so the gene can tolerate paraquat The concentration in the medium is substantially maintained at about 5 mg/L.

At present, there are few studies on transgenic plants that are resistant to high paraquat, and there is still no commercialization. The research mainly stays in the laboratory stage. According to the experience of the present inventors, the amount of 20% paraquat water in the field weeding is 100-200 ml/mu, and is diluted to 25 L water according to the conventional usage. Generally, 800-1600 mg/L is a commonly used field spraying concentration, and in actual use, It is used by some farmers in over-dosage, so one of the reasons for restricting its commercialization is that the tolerance of transgenic plants to P. humilis is not high, and the resistance far removed from actual use has affected the practical application. In addition, it is not clear that breeding transgenics The suitability of genes selected for plants for different plants (eg, tobacco, cotton, rice, maize, soybeans, etc., common crops that differ greatly from their genetic background); moreover, multidrug resistance protein There are many genes in it, and there are nearly 20,000 articles on the public database PubMed that introduce thousands of multi-drug resistance proteins. From there, there is no clear direction to obtain high paraquat resistance protein and its coding gene. By luck and choice, verification, because the protein originally selected cannot be expected to be highly resistant, so work Amazing, actually difficult to complete.

The inventors have surprisingly discovered, through long and arduous research, that the transgenic plants can be rendered tolerant to paraquat (ie, resistant) after introduction of the protein and its encoding gene as shown in SEQ ID No: 2 into plants. It reaches a high level of 3200mg/L, and at the same time makes the plant tolerance to the concentration of H. purpurea in the medium reach a high level of 50mg/L, and the high resistance is still used for a variety of plants, and the application prospect is broad. Summary of the invention

The technical problem to be solved by the present invention is to satisfy the requirement of practical application of paraquat in the process of preventing weeds when planting plants or weeds after weeding, and the high concentration of paraquat in the plants to be planted. The growth under the conditions is not affected, thereby specifically killing weeds. The technical problem to be solved by the present invention is also a method for screening, detecting and applying genes for high paraquat resistance to plants.

Surprisingly, the high paraquat-resistant pqrK2 gene of the present invention and the protein encoded thereby can not only bring the corresponding transgenic plants a paraquat concentration capable of withstanding conventional field application, but are more tolerant to 200 mg/L. The concentration of paraquat on the left and right makes the tolerance of redundancy in actual use more suitable for practical application; in addition, the plant adaptation of the high paraquat resistant pqrK2 gene and its encoded protein of the present invention It has a wide range of high paraquat resistance in a large number of transgenic plants that are significantly different from the tobacco genetic background.

Specifically, in a first aspect, the present invention provides a method of growing a plant comprising the steps of planting a high paraquat resistant transgenic plant and applying a high concentration of paraquat, wherein the transgenic plant is introduced with a code such as SEQ ID No: 2 The gene of the protein (designated herein as pqrK2). Due to the high paraquat resistance pqrK2 gene of the present invention and its The protein of the code can bring high paraquat resistance to the corresponding transgenic plants, and thus can tolerate high concentrations of paraquat, so that when high concentration of paraquat is applied, the growth of the corresponding transgenic plants is not affected, but specifically removed or prevented. Weeds are removed sexually. Thus, the method of planting plants of the first aspect of the invention also includes a method of removing weeds, or a method of removing weeds when planting plants. Preferably, in the first aspect of the invention, the plant is tobacco, corn, rice, cotton, rape or soybean, and particularly preferably corn, rice, cotton or soybean.

As used herein, "administering" refers to field application, such as field spraying. Suitably, the concentration of paraquat in this context refers to the concentration applied in the field, which is more close to the practical application than the concentration of paraquat in the medium, unless otherwise specified. Of course, the inventors have also found that the high paraquat-resistant pqrK2 gene of the present invention and the protein encoded thereby have a plant tolerance of at least 50 mg/L in the medium, which is much higher than the prior art. The wild pqrA gene confers a concentration tolerance of 5 mg/L in the medium. This confirms from another aspect that the pqrK2 gene of the present invention and the protein encoded thereby are highly paraquat resistant.

In this context, "high concentration" of paraquat refers to a concentration of paraquat greater than 800 mg/L unless otherwise specified. Preferably, the concentration of paraquat is 100 (T3200 mg/L, more preferably 1600~3200 mg/L, such as 1600 mg/L commonly used in the field, or 1700 mg/L, 1800 mg/L, 2000 mg/L, 2300 which may be excessively applied. Mg/L, 2600 mg/L, 2900 mg/L, or 3200 mg/L. In a specific embodiment of the present invention, the high paraquat resistant pqrK2 gene of the present invention and the encoded protein thereof can be brought to the corresponding transgenic plants. Paraquat resistance up to 3200 mg/L. Accordingly, "high paraquat resistance" as used herein refers to the property of the corresponding plants that is tolerant to the high concentration of paraquat.

Herein, the "PqrK2 protein" refers to a protein having an amino acid sequence as shown in SEQ ID No: 2 or a functionally equivalent mutant protein thereof, preferably a protein having the amino acid sequence of SEQ ID No: 2. Accordingly, "pqrK2 gene" herein refers to a gene encoding the above PqrK2 protein. The gene of the present invention is a nucleic acid molecule which may be in the form of DNA or in the form of RNA, preferably in the form of DNA. The DNA form includes natural cDNA and synthetic cDNA, and the DNA may be a coding strand or a template strand. The nucleic acid molecule encoding the protein of SEQ ID No: 2 of the present invention can be easily obtained by a person skilled in the art by a conventional technique such as a PCR method, a recombinant method or a synthetic method, knowing the specific sequence or Its fragment. Once obtained, these sequences can be cloned into vectors, transformed or transfected into corresponding cells, and then propagated by conventional host cells, from which a large number of nucleic acid molecules are isolated. Of course, those skilled in the art can search for encoding under stringent conditions by deleting, substituting and/or adding one or several amino acids in the protein represented by SEQ ID No: 2, or by hybridization, according to the teachings herein. The nucleic acid which hybridizes with the nucleic acid of the protein represented by SEQ ID No: 2, and the mutant protein and its gene which are functionally equivalent to the PqrK2 protein of the present invention and its pqrK2 gene are selected, which are also included in the scope of the present invention. A nucleic acid encoding a protein deleted, substituted and/or added with the protein of SEQ ID No: 2 or a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoding the protein of SEQ ID No: 2 can be introduced into paraquat-free resistance. In the bacterium, the protein equivalent to the amino acid sequence as shown in SEQ ID No: 2 can be screened. Mutant protein and its coding gene. Further, it is also preferred to introduce a nucleic acid encoding a deletion, substitution and/or addition of the protein represented by SEQ ID No: 2 or a nucleic acid which hybridizes under stringent conditions with a nucleic acid encoding the protein represented by SEQ ID No: 2, into a plant. The paraquat resistant transgenic plant can also be screened for a mutant protein having the same amino acid sequence as the protein represented by SEQ ID No: 2 and a gene encoding the same. For example, a gene encoding a mutant protein functionally equivalent to the candidate PqrK2 protein can be introduced into a plant to observe whether or not the protein having the amino acid sequence as shown in SEQ ID No: 2 is functional, thereby selecting a functionally equivalent mutant. Protein and its coding gene.

The expressions of the proteins and amino acids, genes and nucleotides used herein are all recognized methods in the art, such as the amino acid or amino acid residue symbols shown in Table 1, and those skilled in the art can reverse the corresponding Trinucleotide codon. In a specific embodiment of the present invention, the gene sequence encoding the protein represented by SEQ ID No: 2 is shown in SEQ ID NO: 1.

Table 1 Amino acid abbreviations

Amino acid three letter abbreviation one letter abbreviation amino acid three letter abbreviation one letter abbreviation alanine Ala A leucine Leu L arginine Arg R lysine Lys K asparagine Asn N methionine Met M aspartate Asp D styrene Ph F Cysteine Cys C Proline Pro P Glutamine Gin Q Serine Ser S Glutamate Glu E Threon Thr T Glycine Gly G Tryptophan Trp w Histidine His H Tyrosine Tyr Y isoleucine lie I valine Val V

Preferably, in the first aspect of the invention, the gene encoding the protein as set forth in SEQ ID No: 2 is introduced into the transgenic plant by a plant transformation vector, preferably the plant transformation vector is pCAMBIA1303-pqrK2. The term "vector" as used herein refers to bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors commonly used in the art. Depending on the purpose of the application, "vector" can be divided into "cloning vector", "expression vector" and "transformation vector" in this context, which means that the purpose of use is to clone and verify the gene, express the corresponding gene and Corresponding gene conversion. Vectors suitable for use in the present invention include, but are not limited to, a vector for expression in bacteria (prokaryotic expression vector), a vector for expression in yeast (e.g., Pichia vector, Hansenula vector, etc.), expressed in insect cells. Baculovirus vector, vector for expression in mammalian cells (vaccinia virus vector, retroviral vector, adenoviral vector, gland) A viral vector for expression in a plant, a plant viral vector for expression in plants, and various vectors for expression in mammalian mammary glands. In addition to the cloning vectors necessary in the cloning process, it is preferred that the vector of the fourth aspect of the invention is a transformation vector, in particular a plant transformation vector. In a specific embodiment of the invention, the plant transformation vector is pCAMBIA1303-pqrK2, the map of which is shown in FIG. pCAMBIA1303-pqrK2 was constructed by first excising the approximately 2.5 kb gus-gfp fusion gene of pCAMBIA1303, retaining the 35S promoter portion, and inserting the pjrK2 gene into the target gene using Ncol and Bstpl.

Preferably, in the first aspect of the invention, the gene encoding the protein as shown in SEQ ID No: 2 is derived from a high paraquat resistant bacterial cell. The term "cell" as used herein may be a prokaryotic cell or a eukaryotic cell, such as a bacterial cell, a yeast cell, a plant cell, an insect cell, a mammalian cell or the like. Preferred high paraquat resistant bacterial cells are E. coli cells. Such high paraquat-resistant Escherichia coli can be obtained by screening methods in the following aspects.

Application is usually carried out according to the instructions of the operator of paraquat, as indicated by the operator's stated start time, application concentration, application range and duration. Usually, the amount of 20% paraquat water in the field weeding is 100-200ml/mu, which is calculated according to the conventional method of dilution to 25L water. Generally, 800-1600mg/L is the commonly used field spray concentration. Preferably in the first aspect of the invention, wherein the start time of administration, i.e., the time of starting the application, may be when weeds are found to grow. Also preferred in the first aspect of the invention, wherein the start time of application is from 3 to 60 days after the start of growth of the plant, preferably from 5 to 50 days after the start of growth of the plant, more preferably from 7 to 30 days after the start of growth of the plant. The time at which the plant begins to grow refers to the time at which the plant seeds are grown under conditions suitable for plant seed growth. The inventors have found that, after too late application, weeds also grow before application and have a certain effect on the growth of the plants.

In a second aspect, the invention provides a method of screening for high paraquat resistant bacteria, comprising

1) The soil contaminated with paraquat is inoculated into a liquid selective medium containing paraquat and shaken at 35 ° C for one week;

2) applying the culture solution obtained by the liquid culture to a solid selective medium, and incubating at 35 ° C until a clearly visible colony grows;

3) taking a single colony of solid culture, inoculated in a liquid selective medium, shaking culture at 35 ° C until the cell concentration reaches 10 ⁷ ~ 10 ⁸ cfu / mL; optionally, repeat steps 2) -3);

Wherein, the liquid selective medium is a liquid BS inorganic salt medium containing paraquat, preferably having a content of paraquat of 200 mg/L; the solid selective medium is a solid BS inorganic salt medium containing paraquat, preferably a herb The dry content is 200 mg/L _; the formulation of the liquid BS inorganic salt medium is: per 100 mL of the medium containing: Ν3⁄4ΗΡ0 ₄ · 23⁄40 7. 0g, KH ₂ P04 3. 0g, NaCl 0· 25g, MgS0 ₄ · 7H ₂ 0 0· 3g, CaCl ₂ · 2H ₂ 0 0 · 02g, FeCl ₃ · 6H ₂ 0 0. 045g, MnS0 ₄ ·4Η ₂ 0 0. 01g, ZnS0 ₄ ·7Η ₂ 0 0. 01g, CuS0 ₄ ·5Η ₂ 0 0. 002g, CoCl ₂ ·6Η ₂ 0 0. 003g, NiCl ₂ ·6Η ₂ 0 0. 003g, and N3⁄4Mo0 ₄ · 2H ₂ 0 0. 002g, pH 7.5, the rest is water; solid BS inorganic salt culture The formulation of the base is a formulation of a liquid BS inorganic salt medium further containing 13. 5 g of agar powder in the formulation. Preferably in the second aspect of the invention, wherein the bacterium is Escherichia coli.

In a third aspect, the present invention provides a selective medium for screening high paraquat resistant bacteria, which is a BS inorganic salt medium containing paraquat, wherein the BS inorganic salt medium contains a formulation of: Each lOOOOmL of medium contains: Ν3⁄4ΗΡ0 ₄ · 23⁄40 7. 0g, KH ₂ P04 3. 0g, NaCl 0. 25g, MgS0 ₄ · 7H ₂ 0 0. 3g, CaCl ₂ · 2H ₂ 0 0. 02g, FeCl ₃ · 6Η ₂ 0 0. 045g, MnS0 ₄ ·4Η ₂ 0 0. 01g, ZnS0 ₄ ·7Η ₂ 0 0. 01g, CuS0 ₄ ·5Η ₂ 0 0. 002g, CoCl ₂ «6H ₂ 0 0. 003g, NiCl ₂ · 6H ₂ 0 0. 003g, and _{_{N¾Mo0 4 · 2H 2 0 0. 002g}} , pH 7. 5, the remainder being water, and optionally the BS formulation containing an inorganic salt medium further contains agar powder 13. 5g. The BS inorganic salt medium is a liquid BS inorganic salt medium, and the BS inorganic salt medium contains a formulation containing no 13. 5 g of agar powder; and the BS inorganic salt medium is a solid BS inorganic salt. 5克琼粉。 The medium, the composition of the solution containing 13. 5g agar powder.

In a separate aspect of the invention, the invention provides a high paraquat resistant transgenic plant or a progeny thereof, a seed into which a gene encoding a protein as set forth in SEQ ID No: 2 is introduced, preferably the plant is tobacco, Corn, rice, cotton, canola or soy. Such plants or their progeny, seeds can be obtained by the planting method of the first aspect of the invention. In addition, the present invention also provides a non-reproductive agricultural product processed from the above-mentioned transgenic plants or their progeny and the usable parts of the seeds, such as food or textiles, such as tobacco, rice (rice-free rice after dehydration) Seed embryos, cotton wool, vegetable oil, etc. Where the processing method is well known to those skilled in the art, it is entirely possible to carry out the method of processing the corresponding non-transgenic plants into corresponding agricultural products.

In a fourth aspect, the present invention provides a method for identifying a high paraquat resistant transgenic plant or a progeny thereof, a seed into which a gene encoding a protein as set forth in SEQ ID No: 2 is introduced, comprising from the transgenic plant or Amplification of the sequence encoding the protein as shown in SEQ ID No: 2 was carried out in the progeny and the seed, and the amplified sequence was subjected to sequence analysis and compared with the gene encoding the protein as shown in SEQ ID No: 2. Methods for amplification therein are well known, such as PCR amplification. Since the gene encoding the protein represented by SEQ ID No: 2 of the present invention is isolated from bacteria, the natural plant or its seed does not contain the gene if the sequence can be amplified from the plant or its seed. The sequence is identical to the gene encoding the protein as set forth in SEQ ID No: 2, then the plant or its seed is a high paraquat resistant transgenic plant into which a gene encoding the protein as set forth in SEQ ID No: 2 is introduced or Its offspring, seeds.

In a fifth aspect, the invention provides the use of a gene encoding a protein as set forth in SEQ ID No: 2 for the cultivation of high paraquat resistant transgenic plants. Under the condition of known genes, methods for cultivating transgenic plants are precedent. For example, genes can be introduced into plants or tissues by means of Agrobacterium transformation or microprojectile bombardment or electroporation, and then the plants or tissues thereof can be cultured. And screened in the presence of paraquat. Preferably, in the sixth aspect of the invention, the plant is tobacco, corn, rice, cotton, rape or soybean, and particularly preferably corn, rice, cotton or soybean. For ease of understanding, the present invention will be described in detail below through the specific drawings and embodiments. It is to be understood that the specific examples and drawings are not to be construed as limiting. It is apparent that those skilled in the art can make various modifications and changes to the present invention within the scope of the present invention, and such modifications and changes are also included in the scope of the present invention. In addition, the present invention is hereby incorporated by reference in its entirety to the extent of the disclosure of the disclosure of the disclosure of the disclosure of the of DRAWINGS

Fig. 1 is a schematic diagram showing the construction process of the pCAMBIA1303-pqrK2 vector obtained by inserting the pqrK2 gene into the vector pCAMBIA1303.

Figure 2 is a photograph showing the performance of transgenic tobacco plants and wild-type plants on a medium with a concentration of 50 mg/L of paraquat. The resistance of the transgenic tobacco plants transformed with pCAMBIA1303-pqrK2 in the left panel is significantly better than that in the wild. Plant type.

Figure 3 is an electrophoresis photograph of PCR amplification of genomic DNA of pQrK2 transgenic tobacco T _Q plants. The lanes 1 to 10 are transgenic tobacco, the lane M is the molecular weight marker, the lane + is the positive control, and the - is the negative control.

Fig. 4 is a comparative photograph of 2 days after spraying 3200 mg/L of paraquat after 30 days of growth of tobacco seedlings, in which the area indicated by the arrow marked with "wild" is planted with wild-type plants, and other well-developed areas are planted with the high of the present invention. Paraquat resistant plants.

Fig. 5 is a comparative photograph of before and after spraying 3200 mg/L of paraquat after 7 days of growth of cotton seedlings, Fig. 5a is a photograph before spraying, and Fig. 5b is a photograph of 120 hours after spraying, wherein B of Fig. 5b is wild type cotton which has been dried. And A in Figure 5b is the unaffected high paraquat resistant cotton of the present invention.

Fig. 6 is a photograph showing the results of the test of the tolerance of paraquat in the leaves of rice seedlings, wherein the area A is the high paraquat resistance rice seedling of the invention, and the B area is the wild type rice seedling.

Fig. 7 is a comparative photograph of 120 hours after spraying 3200 mg/L of paraquat after 12 days of growth of the corn seedling, in which the wild type corn of the B area was dried, and the high paraquat resistant corn of the present invention of the A area was not affected.

Fig. 8 is a comparative photograph of 120 hours after spraying 3200 mg/L of paraquat after soybean seed growth for 18 days, in which the wild type corn of the B area was dried, and the high paraquat resistant corn of the present invention of the A area was not affected. detailed description

The methods used in the following examples are the methods described in commonly used molecular biology, tissue culture techniques, and agronomic manuals unless otherwise specified. Example 1. Acquisition of the pqrK2 gene

According to the environmental survey, it was found that the activated sludge in the coking wastewater plant of Wuhan Iron and Steel Group Co., Ltd. was contaminated with paraquat. Soil samples from the site were collected and inoculated into a liquid selective medium containing paraquat (ie, in BS inorganic salt medium (per 100 mL of medium containing: Ν3⁄4ΗΡ0 ₄ ·2Η ₂ 0 7. 0g, KH ₂ P04 3. 0g, NaCl 0. 25g, MgS0 ₄ ·7Η ₂ 0 0. 3g, CaCl ₂ ·2Η ₂ 0 0. 02g, FeCl ₃ ·6Η ₂ 0 0. 045g, MnS0 ₄ ·4Η ₂ 0 0. 01g, ZnS0 ₄ ·7Η ₂ 0 0. 01g, CuS0 ₄ ·5Η ₂ 0 0. 002g, CoCl ₂ · 6H ₂ 0 0. 003g, NiCl ₂ · 6H ₂ 0 0. 003g, and N3⁄4Mo0 ₄ · 2H ₂ 0 0. 002g, pH 7. 5 The remaining water was added to a shake flask of 200 mg/L paraquat, and cultured at 35 ° C with shaking at 120 rpm for one week.

Then, the culture solution obtained by the culture was applied to a solid selective medium (1. 5 g of agar powder was added per 100 mL of the medium), and cultured at 35 ° C under constant temperature until colonies which were clearly visible were grown. Then, a single colony was picked, transferred to a liquid selective medium, and cultured at 35 ° C with shaking at 120 rpm until the cell concentration reached 10 ^{7 to} 10 ⁸ cfu/mL. Then, the above steps of this paragraph were repeated, and the screening was repeated in a solid selective medium and a liquid selective medium to finally obtain 6 strains which were more resistant to paraquat. One strain of Escherichia coli E. _C0 li was named KT-q5366. It was sequenced by gene and found a gene designated pqrK2, the sequence of which is shown as Seq ID No: 1. The protein encoded by PqrK2 is Seq ID No:

2 is shown. Example 2. Construction of plant expression vector

The genomic DNA of KT-q5366 was extracted as a template, and primers pqrK2-F (5'-CATGCCATGGCAATGAACCCTTATATTTATC-3') and pqrK2-Ra2 (5'-) which respectively introduce Nco I and Bstp I restriction sites were used.

GGGTcACCCTTAATGTGGTGTGCTTCGT -3' ) was amplified by PCR and annealed at 55 °C to amplify the 333 bp PCR fragment pqrK2_333. Then, pqrK2_333 was digested with Ncol and Bstp I, and the product was recovered as a ligation fragment, and pCAMBIA1303 (available from Invitrogen) was double-digested with Ncol and Bstp I as a vector, a T4 DNA ligase ligation fragment and a vector. Finally, the clones with kanamycin resistance were screened and verified as correct plasmids. pCAMBIA1303-pqrK2o Example 3, Plant transformation and seedling screening

Tobacco, cotton, rice, corn, and soybean are transformed using conventional Agrobacterium transformation. Specifically, the constructed plant expression vector pCAMBIA1303-pqrK2 was transformed into Agrobacterium, and then the above plants were separately transformed. After transformation, the T _Q generation (transformed contemporary) transgenic plant seedlings were screened with hygromycin (50 mg/L) resistance and a certain concentration (25-50 mg/L) of paraquat, and then obtained from the screening. Genomic DNA extracted from leaves of transgenic plants T _Q generation, by PCR The pqrKl gene was positive. Then, the T _Q plants were selfed, and one generation of plants were obtained: 30 tobacco, 23 rice, 19 maize, 21 cotton, and 22 soybean. The first-generation seeds were planted separately according to 50-100 grains per plant. When the plants were grown to the 4-5-leaf stage, the leaves of the plants were taken to test the resistance of the leaves to paraquat and/or to spray the plants. Among them, the process of leaf resistance to paraquat is as follows: Each single plant takes about 2 cm long in the middle of new leaves, soaked in lml (5μΜ paraquat, 0.025% Tween) paraquat solution for 24 hours to observe the leaves; the process of spraying experiments As follows: spray the seedlings with paraquat at concentrations of 5mg/L, 15mg/L, 100mg/L, 150mg/L., 200mg/L, and 300mg/L, and add wild type as a negative control to observe and count normal growth. Resistant seedlings. The specific results are as follows:

1, tobacco

A total of 26 tobacco T _Q plants with positive resistance to paraquat were screened. Figure 2 shows the performance of T _Q transgenic tobacco plants and wild-type plants on a medium with a paraquat concentration of 50 mg/L, indicating that certain transgenic tobacco plants have certain resistance to paraquat, and these T _Q positive-positive transgenic tobacco plants The genomic DNA extracted from the leaves can amplify a pqrK2 gene fragment of about 330 bp in size (see Figure 3).

After self-crossing, 26 plants were obtained, named K2-1 26, and 100 seeds were planted in each 1st generation plant. The wild type tobacco was used as a control spray experiment. The results showed that the wild type all died after 5 days of treatment under 800 mg/L paraquat, and no significant damage was observed in the positive transgenic tobacco plants. Under the condition of 1600 mg/L paraquat, the wild type was completely dead after 2 days of treatment, and large. Some positive transgenic tobacco plants grew well. Even when sprayed at a concentration of 3200 mg/L paraquat, as shown in Table 2, the seedlings of a large number of first-generation plants grew well and showed good resistance to paraquat.

Table 2 Screening of T1 Generation Transgenic Tobacco Seeds

K2-1 46 K2-14 49

Κ2-2 38 K2-15 34

Κ2-3 43 K2-16 48

Κ2-4 51 K2-17 30

Κ2-5 54 K2-18 43

Κ2-6 34 K2-19 49

Κ2-7 42 Κ2-20 34

Κ2-8 46 K2-21 34

Κ2-9 33 Κ2-22 42

K2-10 45 Κ2-23 38

K2-11 30 Κ2-24 45

K2-12 25 Κ2-25 55

K2-13 51 Κ2-26 52 2, cotton

After the selected paraquat-resistant cotton T _Q plants were self-crossed, 23 plants of the first generation were obtained, and 50 seeds were planted for each of the first-generation plants. The wild-type cotton was used as a control spray test. When the concentration of paraquat was 800 mg/L, the wild type cotton was withered and the transgenic cotton grew well. When the concentration of paraquat reached 3200 mg/L, the seedlings of 8 cotton plants showed good resistance to paraquat, and the growth status was as shown in Fig. 5.

3, rice

After self-crossing the selected paraquat-resistant rice T _Q plants, 21 plants of the first generation were obtained, named ΚΤ00493-Γ21, and 100 seeds were planted in each 1st generation plant, and wild type rice was used as control. Leaf resistance to paraquat ability experiments and spraying experiments.

The results of the leaf resistance to paraquat test are shown in Fig. 6. The leaf of the rice seedlings with positive paraquat resistance in zone A is obviously superior to the leaves of wild type plants which are conventionally planted in B zone (without adding paraquat). .

After the spraying experiment, when the concentration of paraquat was 800 mg/L, the wild type rice was withered, and the transgenic rice grew well. Even when sprayed at a concentration of 200 mg/L of paraquat, as shown in Table 3, the seedlings of a large number of first-generation plants also grew well and showed excellent resistance to paraquat. Table 3 Screening of T1 Generation Transgenic Rice Seedlings

Plant number Number of resistant seedlings: Plant number Number of resistant seedlings

KT00493-1 35 KT00493- 12 55

KT00493-2 43 KT00493-13 35

KT00493-3 43 KT00493-14 43

KT00493-4 37 KT00493-15 43

KT00493-5 51 KT00493-16 49

KT00493-6 34 KT00493-17 34

KT00493-7 37 KT00493-18 34

KT00493-8 33 KT00493-19 32

KT00493-9 29 KT00493-20 51

KT00493-10 39 KT00493- 21 37

KT00493-11 47 4, corn

After the selected paraquat-resistant maize T _Q plants were self-crossed, 28 plants of the first generation were obtained, and 50 seeds were planted for each of the first-generation plants, and the wild-type maize was used as a control spray test. When the concentration of paraquat was 800 mg/L, the wild type cotton was withered and the transgenic corn grew well. When the concentration of paraquat reached 3200 mg/L, the seedlings of 11 corn plants showed good resistance to paraquat, and the growth status was as shown in Fig. 7.

5, soybean

After the selected paraquat-resistant soybean T _Q plants were self-crossed, 30 plants of the first generation were obtained, and 50 seeds were planted for each of the first-generation plants, and the wild-type maize was used as a control spray test. When the concentration of paraquat was 800 mg/L, the wild type cotton was withered and the transgenic soybean grew well. When the concentration of paraquat reached 3200 mg/L, the seedlings of 13 soybean plants showed good resistance to paraquat, and the growth status was as shown in Fig. 8.

Claims

Claim

A method of growing a plant comprising the steps of planting a high paraquat resistant transgenic plant and applying a high concentration of paraquat, wherein the transgenic plant is introduced into a gene encoding a PqrK2 protein.

The method of growing plants according to claim 1, wherein the concentration of paraquat is 100 (T3200 mg/L, preferably 1600 to 3200 mg/L, such as 1600 mg/L or 3200 mg/L.

The method of planting a plant according to claim 1 or 2, wherein the transgenic plant is introduced with a gene encoding a protein as shown in SEQ ID No: 2, preferably wherein the protein represented by SEQ ID No: 2 is encoded. The gene sequence is SEQ ID NO:

1 is shown.

The method of planting a plant according to claim 1 or 2, wherein the gene encoding the protein represented by SEQ ID No: 2 is introduced into the transgenic plant by a plant transformation vector, preferably the plant transformation vector is pCAMBIA1303-pqrK2, It is also preferred that the gene encoding the protein as shown in SEQ ID No: 2 is derived from a high paraquat resistant bacterial cell.

5. The method of growing a plant according to claim 1, wherein the start time of the application is from 3 to 60 days after the start of growth of the plant. The method of growing a plant according to claim 1 or 2, wherein the plant is tobacco, corn, rice, cotton, canola or soybean.

7. A method of screening for high paraquat resistant bacteria, comprising

Wherein, the liquid selective medium is a liquid BS inorganic salt medium containing paraquat, preferably having a content of paraquat of 200 mg/L; the solid selective medium is a solid BS inorganic salt medium containing paraquat, preferably a herb The dry content is 200 mg/L _; the formulation of the liquid BS inorganic salt medium is: per 100 mL of the medium containing: Ν3⁄4ΗΡ0 ₄ · 23⁄40 7. 0g, KH ₂ P04 3. 0g, NaCl 0· 25g, MgS0 ₄ · 7H ₂ 0 0· 3g, CaCl ₂ · 2H ₂ 0 0 · 02g, FeCl ₃ · 6H ₂ 0 0. 045g, MnS0 ₄ ·4Η ₂ 0 0. 01g, ZnS0 ₄ ·7Η ₂ 0 0. 01g, CuS0 ₄ ·5Η ₂ 0 0. 002g, CoCl ₂ ·6Η ₂ 0 0. 003g, NiCl ₂ ·6Η ₂ 0 0. 003g, and N3⁄4Mo0 ₄ · 2H ₂ 0 0. 002g, pH 7.5, the rest is water; solid BS inorganic salt culture The formulation of the base is a formulation of a liquid BS inorganic salt medium further containing 13. 5 g of agar powder in the formulation.

A selective medium for screening high paraquat-resistant bacteria, which is a BS inorganic salt medium containing paraquat, wherein the BS inorganic salt medium contains a formula: per 100 mL of the medium contains: Na ₂ HP0 ₄ · 23⁄40 7. 0g, KH ₂ P04

3. 0g, NaCl 0. 25g, MgS0 ₄ ·7Η ₂ 0 0. 3g, CaCl ₂ ·2Η ₂ 0 0. 02g, FeCl ₃ ·6Η ₂ 0 0. 045g, MnS0 ₄ ·4Η ₂ 0 0. 01g, ZnS0 ₄ ·7Η ₂ 0 0. 01g, CuS0 ₄ ·5Η ₂ 0 0. 002g, CoCl ₂ ·6Η ₂ 0 0. 003g, NiCl ₂ ·6Η ₂ 0 0. 003g, and Na ₂ Mo0 ₄ · 2H ₂ 0 0. 5克琼粉粉。 The 002g, pH 7.5, further comprising water, and further comprising 13. 5g agar powder.

9. A method of identifying a high paraquat resistant transgenic plant or a progeny thereof or a seed thereof, which comprises a gene encoding a protein as set forth in SEQ ID No: 2, comprising encoding from the transgenic plant or its progeny, seed The amplified sequence was subjected to sequence analysis as compared with the gene encoding the protein shown in SEQ ID No: 2, as the gene sequence of the protein shown in SEQ ID No: 2 was amplified.

10. Use of a gene encoding a protein as set forth in SEQ ID No: 2 for cultivating a high paraquat resistant transgenic plant, preferably the plant is wheat, corn, rice, cotton, canola or soybean.