Application of nitrogen response gene LAO1 in regulation and control of plant growth

Abstract

The invention discloses application of a nitrogen response gene LAO1 in regulation and control of plant growth. And the amino acid sequence of the protein LAO1 is as shown in SEQ ID No.3. Experiments prove that the deletion of the LAO1 gene in the chlamydomonas genome can enable the chlamydomonas to be more suitable for a nitrogen-deficient environment, so that the growth rate and biomass of the chlamydomonas are improved, and the LAO1 gene can be used for improving the yield of products such as starch and triacylglycerol of the chlamydomonas under the nitrogen-deficient condition in practical application. The method has an important application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the nitrogen-responsive gene LAO1 in regulating plant growth. Background Technology

[0002] Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii As a single-celled eukaryotic microalga, *Chlamydomonas reinhardtii* has become a standard model organism in basic biology fields such as photosynthesis research, cell cycle regulation, and flagella assembly due to its highly annotated genome, mature genetic manipulation system, and conserved photosynthetic mechanism. *Chlamydomonas reinhardtii* not only plays an indispensable role in basic research but also demonstrates enormous potential in the field of biotechnology. For example, it can be used to produce biofuels (such as lipids, starch, and hydrogen) and high-value bioproducts (such as recombinant proteins, pigments, and antioxidants).

[0003] When subjected to environmental stress, *Chlamydomonas reinhardtii* undergoes metabolic rearrangement, resulting in inhibited vegetative reproduction, reduced photosynthetic capacity, and the targeted accumulation of energy storage compounds (such as starch and triglycerides) to maintain intracellular energy homeostasis. Currently, nitrogen deficiency induction is a routine technique to enhance lipid and starch accumulation in *Chlamydomonas reinhardtii*, but this comes at the cost of reduced cell growth and slower basal metabolism, severely limiting actual production efficiency. Existing improvement techniques, such as nitrogen supplementation, can partially alleviate growth inhibition, but this increases the risk of contamination and operating costs. Mutagenesis screening or genetic modification targeting known nitrogen metabolism-related genes (such as nitrate assimilation pathway genes) has limitations such as low modification efficiency and localized regulatory scope. Currently, no regulatory target has been found that can systematically enhance the basal growth capacity of *Chlamydomonas reinhardtii* under nitrogen deficiency conditions. Finding a strategy to significantly improve the growth rate and biomass production of *Chlamydomonas reinhardtii* without continuous exogenous nitrogen supplementation would provide an innovative solution to the problem of low culture efficiency caused by nitrogen deficiency stress. Summary of the Invention

[0004] The purpose of this invention is to improve the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions.

[0005] This invention first protects the application of the protein LAO1 or the nucleic acid molecule encoding said protein LAO1, which can be S1) or S2). S1) Regulates the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions; S2) Cultivating transgenic Chlamydomonas with altered growth capacity under nitrogen-deficient culture conditions; The application is achieved by inhibiting the expression level and / or activity of the protein LAO1.

[0006] In the above applications, the protein LAO1 can be a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3; a3) Proteins related to Chlamydomonas growth capacity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the proteins shown in a1) or a2).

[0007] The proteins in a2) above are labeled as shown in Table 1.

[0008] Table 1. Sequence of Labels

[0009] The protein in a3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0010] The proteins mentioned in a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The gene encoding the protein in a3) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0012] In the above applications, the nucleic acid molecule encoding the protein LAO1 can be a DNA molecule as follows (b1) or (b2) or (b3) or (b4) or (b5): (b1) A DNA molecule with a coding region as shown in SEQ ID No. 1; (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1; (b3) A DNA molecule with a nucleotide sequence as shown in SEQ ID No. 2; (b4) Hybridizes under stringent conditions to a DNA molecule defined by (b1) or (b2) or (b3), which is derived from Chlamydomonas and encodes any of the proteins described above, LAO1; (b5) A DNA molecule derived from Chlamydomonas and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the DNA molecule defined in (b1), (b2), or (b3) and encoding any of the proteins described above, LAO1.

[0013] The stringent conditions were: hybridization in a 2×SSC, 0.1% SDS solution at 68°C with two washes of 5 min each, followed by hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C with two washes of 15 min each.

[0014] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0015] Of these, SEQ ID No. 1 consists of 1683 nucleotides. SEQ ID No. 2 consists of 5393 nucleotides. The nucleotides shown in SEQ ID No. 1 or SEQ ID No. 2 encode the amino acid sequence shown in SEQ ID No. 3.

[0016] Those skilled in the art can readily use known methods, such as directed evolution and point mutation, to mutate the nucleotide sequence encoding any of the aforementioned proteins LAO1. Any artificially modified nucleotides that have 70% or higher identity with the nucleotide sequence of any of the aforementioned proteins LAO1 obtained by means of this invention, as long as they encode any of the aforementioned proteins LAO1, are derived from and equivalent to the nucleotide sequence of this invention.

[0017] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 70% or higher, 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence of the protein LAO1 as shown in SEQ ID No. 3 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0018] In the above applications, regulating the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions can be used to improve the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions.

[0019] In the above applications, the transgenic Chlamydomonas with altered growth capacity under nitrogen-deficient culture conditions can be transgenic Chlamydomonas with enhanced growth capacity under nitrogen-deficient culture conditions.

[0020] In any of the above-described applications, the inhibition of the expression level and / or activity of the protein LAO1 can be achieved by inserting a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1 described above.

[0021] In any of the above-described applications, the insertion of a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1 can be specifically achieved by inserting an resistance gene into the nucleic acid molecule encoding the protein LAO1.

[0022] Preferably, the nucleotide sequence of any of the above-described nucleic acid molecules encoding the protein LAO1 may be as shown in SEQ ID No. 2.

[0023] Preferably, the nucleotide sequence of any of the above-described resistance genes may be as shown in SEQ ID No. 4, positions 2046-4267 from the 5' end.

[0024] Preferably, the nucleotide sequence after inserting the resistance gene into the nucleic acid molecule encoding the protein LAO1 can be as shown in SEQ ID No. 4.

[0025] In any of the applications described above, the *Chlamydomonas* can be *Chlamydomonas reinhardtii*. Preferably, the *Chlamydomonas reinhardtii* can be *Chlamydomonas reinhardtii* CC5325.

[0026] In any of the above-described applications, the growth capacity may be the growth rate and / or biomass.

[0027] The present invention also protects a method for cultivating transgenic Chlamydomonas, which may include the following steps: inhibiting the expression level and / or activity of any of the proteins LAO1 described above in the original Chlamydomonas to obtain transgenic Chlamydomonas; compared with the original Chlamydomonas, the growth capacity of the transgenic Chlamydomonas is improved under nitrogen-deficient culture conditions.

[0028] In the above method, preferably, the growth capacity can be the growth rate and / or biomass.

[0029] In the above method, the *Chlamydomonas reinhardtii* can be *Chlamydomonas reinhardtii*. Preferably, the *Chlamydomonas reinhardtii* can be *Chlamydomonas reinhardtii* CC5325.

[0030] In any of the methods described above, the inhibition of the expression level and / or activity of any of the proteins LAO1 in Chlamydomonas aeruginosa can be achieved by inserting a foreign DNA molecule into the nucleic acid molecule encoding any of the proteins LAO1.

[0031] In the above method, the insertion of a foreign DNA molecule into the nucleic acid molecule encoding any of the aforementioned proteins LAO1 can be specifically achieved by inserting an resistance gene into the nucleic acid molecule encoding the protein LAO1.

[0032] In any of the methods described above, preferably, the nucleotide sequence of the nucleic acid molecule encoding the protein LAO1 described above may be as shown in SEQ ID No. 2.

[0033] In any of the methods described above, preferably, the nucleotide sequence of the resistance gene may be as shown in SEQ ID No. 4, positions 2046-4267 from the 5' end.

[0034] In any of the methods described above, preferably, the nucleotide sequence after inserting the resistance gene into the nucleic acid molecule encoding the protein LAO1 described above can be as shown in SEQ ID No. 4.

[0035] Experiments have shown that the *Chlamydomonas reinhardtii* cc5325 LAO1 gene knockout mutant (identified as LMJ.RY0402.044073 on the website) purchased from the Chlamydomonas Resource Center (https: / / www.chlamycollection.org / ) – the *lao1* mutant – is more adapted to nitrogen-deficient environments and can improve the growth capacity (e.g., growth rate and / or biomass) of *Chlamydomonas reinhardtii* under nitrogen-deficient conditions. Therefore, the deletion of the LAO1 gene from *Chlamydomonas reinhardtii* (i.e., inhibiting the protein LAO1 encoded by the LAO1 gene) allows *Chlamydomonas reinhardtii* to better adapt to nitrogen-deficient environments, thereby increasing its growth rate and biomass. In practical applications, this can be used to increase the yield of starch, triglycerides, and other products from *Chlamydomonas reinhardtii* under nitrogen-deficient conditions. This invention has significant application prospects. Attached Figure Description

[0036] Figure 1 This is a schematic diagram showing the insertion sites of the LAO1 gene and the resistance gene in the lao1 mutant.

[0037] Figure 2 The color change of the lao1 mutant in Example 3.

[0038] Figure 3 This is a line graph showing the density of lao1 mutant cells transferred to TAP-N liquid culture medium in Example 3.

[0039] Figure 4 The lao1 mutant OD transferred to TAP-N liquid medium in Example 3 750nm A line graph of the values. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] The solutes and their concentrations in TAP liquid medium were: 2.42 g / L Tris-base, 375.0 mg / L NH4Cl, 100 mg / L MgSO4·7H2O, 50 mg / L CaCl2·2H2O, 14.34 mg / L K2HPO4, 7.26 mg / L KH2PO4, 11.4 mg / L H3BO3, 22 mg / L ZnSO4·7H2O, 5.06 mg / L MnCl2·4H2O, 4.99 mg / L FeSO4·7H2O, 1.61 mg / L CoCl2·6H2O, 1.57 mg / L CuSO4·5H2O, and 1.10 mg / L (NH4)6Mo7O. 24 • 4H2O, 50 mg / L EDTA-Na and 1 mL / L glacial acetic acid, with water as the solvent, and the pH adjusted to 7.0 using HCl.

[0043] The solutes and their concentrations in TAP-N liquid medium were: 2.42 g / L Tris-base, 100 mg / L MgSO4·7H2O, 50 mg / L CaCl2·2H2O, 14.34 mg / L K2HPO4, 7.26 mg / L KH2PO4, 11.4 mg / L H3BO3, 22 mg / L ZnSO4·7H2O, 5.06 mg / L MnCl2·4H2O, 4.99 mg / L FeSO4·7H2O, 1.61 mg / L CoCl2·6H2O, 1.57 mg / L CuSO4·5H2O, and 1.10 mg / L (NH4)6Mo7O. 24 • 4H₂O, 50 mg / L EDTA-Na, and 1 mL / L glacial acetic acid, in water as solvent, with pH adjusted to 7.0 using HCl. Compared to TAP liquid medium, each 1 L of TAP-N liquid medium lacks 375.0 mg of NH₄Cl.

[0044] The solutes and their concentrations in TAP solid medium were: 2.42 g / L Tris-base, 375.0 mg / L NH4Cl, 100 mg / L MgSO4·7H2O, 50 mg / L CaCl2·2H2O, 14.34 mg / L K2HPO4, 7.26 mg / L KH2PO4, 11.4 mg / L H3BO3, 22 mg / L ZnSO4·7H2O, 5.06 mg / L MnCl2·4H2O, 4.99 mg / L FeSO4·7H2O, 1.61 mg / L CoCl2·6H2O, 1.57 mg / L CuSO4·5H2O, and 1.10 mg / L (NH4)6Mo7O. 24 • 4H2O, 50 mg / L EDTA-Na, 1 mL / L glacial acetic acid and 1.5% (m / v) Agar, in water as solvent, with pH adjusted to 7.0 using HCl.

[0045] Example 1: Discovery of protein LAO1 and its encoding gene The inventors of this application, through extensive experiments, discovered the LAO1 gene in *Chlamydomonas reinhardtii* cc5325. The nucleotide sequence of the LAO1 gene in the cDNA of *Chlamydomonas reinhardtii* cc5325 is shown in SEQ ID No. 1. The nucleotide sequence of the LAO1 gene in the genome of *Chlamydomonas reinhardtii* is shown in SEQ ID No. 2.

[0046] The LAO1 gene encodes the protein LAO1, the amino acid sequence of which is shown in SEQ ID No. 3.

[0047] Example 2: Obtaining and Genotyping the lao1 Mutant 1. Obtaining the lao1 mutant The inventors of this application purchased a knockout mutant of the LAO1 gene of *Chlamydomonas reinhardtii* cc5325 from the online website of the Chlamydomonas Resource Center in the United States (https: / / www.chlamycollection.org / ). The mutant's identification number on the website is LMJ.RY0402.044073. In the following text, the knockout mutant of the LAO1 gene of *Chlamydomonas reinhardtii* cc5325 will be referred to as the lao1 mutant.

[0048] 2. Genotyping of the lao1 mutant Total DNA was extracted from the lao1 mutant and used as a template. PCR amplification was performed using primer pairs lao1-check-F: 5′-GAGACGACCTGGGTGAGTGGATAAT-3′ and lao1-check-R: 5′-GCGTGTTGAAGATGACCTGTGGG-3′, yielding a 2967 bp DNA sequence. This DNA sequence was then sequenced and analyzed.

[0049] The results showed that in the lao1 mutant, the inserted sequence was a resistance gene, specifically inserted into the 6th exon region of the LAO1 gene. SEQ ID No. 4 is the genomic sequence of the LAO1 gene after the insertion of the resistance gene. The nucleotide sequence of the resistance gene is shown in SEQ ID No. 4 from positions 2046-4267 starting from the 5' end. The insertion of the resistance gene into the 6th exon of the LAO1 gene caused premature termination of the LAO1 protein. Ultimately, the LAO1 gene after the insertion of the resistance gene translated into a 175-amino acid protein, the amino acid sequence of which is shown in SEQ ID No. 5.

[0050] The insertion sites of the LAO1 gene and resistance gene, and the protein translation details in the lao1 mutant are shown in [reference needed]. Figure 1 (lao1 is a lao1 mutant, WT is Chlamydomonas reinhardtii cc5325).

[0051] Example 3: Phenotypic Identification of the lao1 Mutant 1. The Chlamydomonas reinhardtii (Chlamydomonas reinhardtii cc5325 or lao1 mutant) to be tested was cultured on TAP solid medium containing 20 µg / ml paromomycin. Then, a single colony was picked and inoculated into TAP liquid medium for pre-culture (in order not to affect the growth of Chlamydomonas, TAP liquid medium without any antibiotics was used for culture).

[0052] 2. Once the Chlamydomonas reinhardtii cell line turns dark green (approximately 5 days of pre-culture), wash the cells three times with TAP-N medium and then transfer them to 300 mL of TAP-N liquid medium, setting the initial cell count to 1 × 10⁻⁶ cells / mL. 5 The cells were cultured and the culture continued. During the culture period, the OD (oxidative stress) of *Chlamydomonas* was measured every 12 hours using a Nucleic Acid / Protein Analyzer DU730 (BECKMAN CULTER). 750nm The cell density of Chlamydomonas was obtained by counting cells using an upright phase-contrast microscope (Olympus BX53) and a cell counting chamber.

[0053] The color change of Chlamydomonas after 48 hours of cultivation is shown in the figure. Figure 2(cc5325-N is Chlamydomonas reinhardtii cc5325, lao1-N is lao1 mutant, Volume: 300ml is the transfer to 300 mL of TAP-N liquid medium, Volume: 1 ml is 1 mL taken from the culture of Chlamydomonas reinhardtii 48 h after inoculation with TAP-N liquid medium, 0 h is the initial inoculation of the test Chlamydomonas reinhardtii with TAP-N liquid medium, 48h is the 48 h after inoculation of the test Chlamydomonas reinhardtii with TAP-N liquid medium).

[0054] A line graph showing the cell density of Chlamydomonas aeruginosa transferred to 300 mL TAP-N liquid medium is shown below. Figure 3 (cc5325-N is Chlamydomonas reinhardtii cc5325, lao1-N is lao1 mutant).

[0055] Chlamydomonas OD transferred to 300 mL TAP-N liquid medium 750nm See the line chart of the values. Figure 4 (cc5325-N is Chlamydomonas reinhardtii cc5325, lao1-N is lao1 mutant).

[0056] The results showed that under nitrogen-deficient conditions, the lao1 mutant was greener than Chlamydomonas reinhardtii cc5325; under nitrogen-deficient conditions, the cell density and OD of the lao1 mutant were... 750nm The values ​​were all higher than those of *Chlamydomonas reinhardtii* cc5325, indicating that the cell mass and biomass of the lao1 mutant were significantly higher than those of *Chlamydomonas reinhardtii* cc5325 after nitrogen deficiency treatment. This shows that the LAO1 gene is involved in the regulation of nitrogen response; the lao1 mutant is more adapted to nitrogen-deficient environments. Under nitrogen-deficient conditions, compared with *Chlamydomonas reinhardtii* cc5325, the lao1 mutant showed significantly improved growth ability, manifested in a significantly faster growth rate and significantly increased biomass. In conclusion, the lao1 mutant can increase the yield of *Chlamydomonas* under nitrogen-deficient conditions.

[0057] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of protein LAO1 or the nucleic acid molecule encoding said protein LAO1, as S1) or S2). S1) Regulates the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions; S2) Cultivating transgenic Chlamydomonas with altered growth capacity under nitrogen-deficient culture conditions; The protein LAO1 is either a1), a2), or a3). a1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3; a3) Proteins related to Chlamydomonas growth capacity obtained by substituting and / or deleting and / or adding one or more amino acid residues of the proteins shown in a1) or a2). The application is achieved by inhibiting the expression level and / or activity of the protein LAO1.

2. The application according to claim 1, characterized in that: The nucleic acid molecule encoding the protein LAO1 is a DNA molecule of the following type (b1) or (b2) or (b3) or (b4) or (b5): (b1) A DNA molecule with a coding region as shown in SEQ ID No. 1; (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1; (b3) A DNA molecule with a nucleotide sequence as shown in SEQ ID No. 2; (b4) Hybridizes under stringent conditions with a DNA molecule defined in (b1) or (b2) or (b3) that is derived from Chlamydomonas and encodes the protein LAO1 described in claim 1; (b5) A DNA molecule derived from Chlamydomonas and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the DNA molecule defined in (b1), (b2), or (b3) and encoding the protein LAO1 of claim 1.

3. The application according to claim 1, characterized in that: The regulation of Chlamydomonas growth capacity under nitrogen-deficient culture conditions is to improve the growth capacity of Chlamydomonas under nitrogen-deficient culture conditions. The transgenic Chlamydomonas species whose growth capacity is altered under nitrogen-deficient culture conditions are transgenic Chlamydomonas species whose growth capacity is enhanced under nitrogen-deficient culture conditions.

4. The application according to claim 1, characterized in that: The inhibition of the expression level and / or activity of the protein LAO1 is achieved by inserting a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1.

5. The application according to claim 4, characterized in that: The insertion of a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1 is specifically achieved by inserting an resistance gene into the nucleic acid molecule encoding the protein LAO1. Preferably, the nucleotide sequence of the nucleic acid molecule encoding the protein LAO1 is shown in SEQ ID No. 2; Preferably, the nucleotide sequence of the resistance gene is shown in SEQ ID No. 4, positions 2046-4267 from the 5' end; Preferably, the nucleotide sequence after inserting the resistance gene into the nucleic acid molecule encoding the protein LAO1 is shown in SEQ ID No.

4.

6. The application according to any one of claims 1 to 5, characterized in that: The chlamydomonas mentioned is Chlamydomonas reinhardtii; Preferably, the *Chlamydomonas reinhardtii* is *Chlamydomonas reinhardtii* CC5325.

7. The application according to any one of claims 1 to 6, characterized in that: The growth capacity refers to the growth rate and / or biomass.

8. A method for cultivating transgenic Chlamydomonas, comprising the following steps: inhibiting the expression level and / or activity of the protein LAO1 described in claim 1 in the original Chlamydomonas to obtain transgenic Chlamydomonas; compared with the original Chlamydomonas, the growth capacity of the transgenic Chlamydomonas is improved under nitrogen-deficient culture conditions; Preferably, the growth capacity is the growth rate and / or biomass; Preferably, the Chlamydomonas is Chlamydomonas reinhardtii; Preferably, the *Chlamydomonas reinhardtii* is *Chlamydomonas reinhardtii* CC5325.

9. The method according to claim 8, characterized in that: The inhibition of the expression level and / or activity of the protein LAO1 in claim 1 in Chlamydomonas aeruginosa is achieved by inserting a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1.

10. The method according to claim 9, characterized in that: The insertion of a foreign DNA molecule into the nucleic acid molecule encoding the protein LAO1 is specifically achieved by inserting an resistance gene into the nucleic acid molecule encoding the protein LAO1. Preferably, the nucleotide sequence of the nucleic acid molecule encoding the protein LAO1 is shown in SEQ ID No. 2; Preferably, the nucleotide sequence of the resistance gene is shown in SEQ ID No. 4, positions 2046-4267 from the 5' end; Preferably, the nucleotide sequence after inserting the resistance gene into the nucleic acid molecule encoding the protein LAO1 is shown in SEQ ID No. 4.

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