Use of rice HDT701 gene in breeding plant varieties with high tolerance or high sensitivity to arsenic stress

Abstract

The application discloses application of a rice HDT701 gene in cultivating plant arsenic stress high tolerance or high sensitivity varieties, and belongs to the technical field of genetic engineering. The nucleotide sequence of the HDT701 gene is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence for coding the amino acid sequence shown in SEQ ID NO. 2. The application can improve the ability of plants to resist arsenic stress by reducing the expression of HDT701, or can improve the sensitivity of plants to arsenic stress by increasing the expression of HDT701, and can be applied to plant genetic engineering breeding, and provides a new method for creating or improving new germplasm of plants resisting arsenic stress or indicating plants in arsenic pollution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of rice HDT701 gene in regulating plant arsenic stress response and / or breeding of plant varieties resistant to arsenic stress or sensitive to arsenic stress. BACKGROUND

[0002] Arsenic is a kind of metalloid substance widely existing in the natural environment, and arsenic is toxic to humans, animals and plants. Because rice is cultivated by flooding, it has a very high absorption efficiency of arsenic. As the staple food of half of the world's population, the arsenic accumulated in rice will enter the human body through the food chain and thus threaten human health. It is of great significance to improve rice quality by mining and applying gene resources related to arsenic stress resistance in plants. In addition, sensitive indicator plants can quickly respond to environmental pollution. For example, water hyacinth can sensitively indicate arsenic pollution. If wastewater contains trace amounts of arsenic, leaf symptoms such as spots, yellowing and wilting will appear within 2 hours. However, different indicator plants have different growth environments, and it is impossible to use one plant to detect the pollution of all ecological environments. If a specific gene of a specific plant can be regulated to make it more sensitive to a specific pollutant, the plant can be used specifically for pollution detection in a specific growth environment. Therefore, it is of great significance to mine and clone genes that regulate plant response to environmental pollution and develop new methods for breeding plants sensitive to environmental pollution, especially crops such as rice.

[0003] However, the genetic regulatory network of plants responding to arsenic stress is complex, and the molecular regulation mechanisms of plants with different genetic backgrounds differ greatly. Although some genes of plants responding to arsenic stress have been reported, it is still a technical problem to be solved to mine and clone more new genes with the function of regulating plant resistance to arsenic stress, and to develop new methods for breeding plant varieties resistant to arsenic stress or breeding environmental indicator plants sensitive to environmental pollution. SUMMARY

[0004] The purpose of the present application is to provide application of rice HDT701 gene or its orthologous gene in regulating plant response to arsenic stress and / or breeding of plant varieties resistant to arsenic stress or sensitive to arsenic stress.

[0005] The present application clones rice HDT701 gene, constructs and compares phenotypes and survival rates of HDT701 overexpression lines and RNAi interference lines under arsenic stress. Compared with the control material, the HDT701 RNAi interference lines show higher survival rate under arsenic stress than the control, while the HDT701 overexpression transgenic plants show lower survival rate under arsenic stress. The HDT701 gene nucleotide sequence is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2. The gene is expected to be applied to plant genetic engineering breeding, and provides a theoretical basis for creating or improving plants sensitive to arsenic stress. Considering the degeneracy of codons, modifying the bases of the above-mentioned nucleotide sequence without changing the amino acid sequence also belongs to the protection scope of the present application.

[0006] In one aspect, the present application provides a rice HDT701 gene or its orthologous gene in regulating plant resistance to arsenic stress, and the HDT701 gene nucleotide sequence is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

[0007] Further, the regulation of plant resistance to arsenic stress is to improve the ability of plants to resist arsenic stress by reducing the expression of HDT701 gene or its orthologous gene, or to improve the sensitivity of plants to arsenic stress by overexpressing HDT701 gene or its orthologous gene.

[0008] In a preferred embodiment, the plant is a monocotyledon.

[0009] Further, the plant is a plant of the family Poaceae.

[0010] Further, the plant is a plant of the genus Oryza.

[0011] Further, the plant is rice.

[0012] In another aspect, the present application provides a protein encoded by a rice HDT701 gene or its orthologous gene in regulating plant resistance to arsenic stress, and the HDT701 gene nucleotide sequence is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

[0013] Further, the method for improving the ability of the plant to resist arsenic stress is by reducing the expression of the HDT701 gene or its ortholog gene, or by over-expressing the HDT701 gene or its ortholog gene.

[0014] In preferred embodiments, the plant is a monocotyledon.

[0015] Further, the plant is a plant of the family Poaceae.

[0016] Further, the plant is a plant of the genus Oryza.

[0017] Further, the plant is rice.

[0018] In preferred embodiments, the plant is a monocotyledon.

[0019] Further, the plant is a plant of the family Poaceae.

[0020] Further, the plant is a plant of the genus Oryza.

[0021] Further, the plant is rice.

[0022] In preferred embodiments, the plant is a monocotyledon.

[0023] Further, the plant is a plant of the family Poaceae.

[0024] Further, the plant is rice.

[0025] Further, the plant species with high resistance to arsenic stress can be bred by reducing the expression of the HDT701 gene or the protein encoded by the orthologous gene thereof, and / or the plant species with sensitivity to arsenic stress can be bred by overexpressing the HDT701 gene or the protein encoded by the orthologous gene thereof.

[0026] In a preferred embodiment, the plant is a monocotyledon.

[0027] Further, the plant is a plant of the family Poaceae.

[0028] Further, the plant is a plant of the genus Oryza.

[0029] Further, the plant is rice.

[0030] Compared with the prior art, the present application has the following advantages:

[0031] 1) The present application provides a new method for regulating the resistance of plants to arsenic stress, i.e. increasing the resistance of plants to arsenic stress by reducing the expression of the HDT701 gene or the protein encoded thereby, or increasing the sensitivity of plants to arsenic stress by overexpressing the HDT701 gene or the protein encoded thereby.

[0032] 2) The present application provides a new method for breeding plant species with resistance to arsenic stress, i.e. breeding the plant species with high resistance to arsenic stress by reducing the expression of the HDT701 gene or the protein encoded thereby, and / or breeding the plant species with sensitivity to arsenic stress by overexpressing the HDT701 gene or the protein encoded thereby.

[0033] 3) The present application can also regulate the resistance of plants to arsenic stress or breed plant species with resistance or sensitivity to arsenic stress by regulating the protein encoded by the HDT701 gene or the orthologous gene thereof.

[0034] 4) The new method provided by the present application can increase the resistance of plants to arsenic stress or increase the sensitivity of plants to arsenic stress. BRIEF DESCRIPTION OF DRAWINGS

[0035] The method for regulating the resistance of plants to arsenic stress and / or breeding plant species with resistance to arsenic stress of the present application and the beneficial effects thereof will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Figure 1 The identification results of the rice HDT701 overexpression line (A) and the RNAi line (B) are shown.

[0037] Figure 2Phenotype analysis of HDT701 overexpression lines under arsenic stress; WT, wild type; HDT701-OE1 and HDT701-OE7, overexpression lines; Control, control; 100 μΜ As(III) treatment, 100 μΜ NaAsO2[As(III)] treatment for 3 days.

[0038] Figure 3 Sensitivity analysis of HDT701-RNAi interference lines to arsenic stress; WT, wild type; HDT701-R2 and HDT701-R6, RNAi interference lines; Control, control; 100 μΜ As(III) treatment, 100 μΜ NaAsO2[As(III)] treatment for 3 days.

[0039] Figure 4 Survival rate analysis of HDT701 transgenic overexpression lines (A) and interference lines (B) under arsenic stress. Student's t-test analysis, * indicates p < 0.05 (n = 3) DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0041] Unless otherwise defined, all technical and scientific terms used in the present application have the same meanings as commonly understood by those of ordinary skill in the art to which the present application belongs.

[0042] The nucleotide sequence of the rice HDT701 gene in the embodiments is shown in SEQ ID NO. 1:

[0043] ATGGAGTTCTGGGGTCTTGAAGTCAAGCCTGGACAGACTGTCAAATGTGAGCCTGAAGATGAACGCTTTTTGCACCTTTCTCAGGCTGCTCTTGGGGAATCAAAGAAAGGATCTGACAATGCAGTAATGTATGTTAAAACTGATGATCAAAAGCTAGTCATTGGAACCCTCTCAGCTGACAAGTTCCCTCAAATCCAGTTTGATTTGGTCTTTGACAAAGAGTTTGAGCTGTCACACACTTCAAAGACTGCTAGTGTGTTCTTTTCTGGCTACAAAGTTTCCCAGCCGGCTGAGGAAGATGAAATGGATTTTGATTCTGAAGAAGTTGAAGATGAAGAGGAGGAAGAAAAGATCATTCCAGCTCCCAGGGCAAATGGCAAAGTTGAAGGGAAGGAAAATGAGCAGAAAAAACAAGGCAAGACAGATTCTTCAGCTTCAAAATCAAAGGCTGCAGTGAATGACGATGATGATGATGATGACAGTGATGAGGATGATTCTGAGGACGAAGATCTTTCTCCTGAGGATGATGATGATGATTCTTCTGAGGATGATTCCAGCGAAGATGATGAGGATGAGAGTGACGAGGAAGATACTCCCAAGAAGCCAGAGACTGGAAAGAGGAAAGTAGCTGAAATTGTGTTGAAGACACCTTCGTCTGATAAGAAAGCAAAGATTGCTACACCGTCAGGCCAGAAGACAGGTGACAAGAAGGGTGTCCATGTAGCAACTCCACATCCGGCAAAGCAGGCTAGCAAGACCCCCGTGAATGACAAGTCAAAGGAGAAGTCCCCAAAATCCGGTGGTGGGTCAATTTCTTGCAAGTCATGCAGCAAGACGTTCAACAGTGAAATGGCTCTGCAATCTCACTCGAAGGCCAAGCACCCCGCCAAGTGA

[0044] The amino acid sequence of the rice HDT701 protein is shown in SEQ ID NO. 2:

[0045] MEFWGLEVKPGQTVKCEPEDERFLHLSQAALGESKKGSDNAVMYVKTDDQKLVIGTLSADKFPQIQFDLVFDKEFELSHTSKTASVFFSGYKVSQPAEEDEMDFDSEEVEDEEEEEKIIPAPRANGKVEGKENEQKKQGKTDSSASKSKAAVNDDDDDDDSDEDDSEDEDLSPEDDDDDSSEDDSSEDDEDESDEEDTPKKPETGKRKVAEIVLKTPSSDKKAKIATPSGQKTGDKKGVHVATPHPAKQASKTPVNDKSKEKSPKSGGGSISCKSCSKTFNSEMALQSHSKAKHPAK

[0046] Example 1

[0047] Construction and identification of HDT701 gene overexpression and RNAi interference lines

[0048] Take 0.5 g of seedlings of rice variety "Zhonghua 11" germinated and grown on 1 / 2MS solid medium for 14 days, extract total RNA using TriZol reagent (Invitrogen Corporation), perform reverse transcription, then amplify the target gene, after obtaining the target gene, connect it to a plant binary expression vector, and transfer it into rice through Agrobacterium, the specific experiment is as follows:

[0049] RNA reverse transcription: take 1 μg of high-quality (OD 260 / OD 280 : 1.8-2.0; OD 260 / OD 230 ≈2.0) RNA for reverse transcription (Promega reverse transcription kit) to obtain first-strand cDNA.

[0050] HDT701 gene overexpression vector construction: cDNA as template, using KOD FX high fidelity enzyme (ToYoBo company) PCR amplification. Reaction system is: 2x PCR buffer 10 μL, 2 mM dNTPs 2 μL, F primer (5'-CGGGATCCATGGAGTTCTGGGGTCTTGA-3') 0.5 μL, R primer (5'-CGGGATCCTCACTTGGCGGGGTGCTTGG-3') 0.5 μL, cDNA template 1 μL, KOD FX (1 U / μl) 0.4 μL, add water to 20 μL. Reaction conditions: 98 °C for 5 min; 98 °C for 15 sec, 56 °C for 30 sec, 68 °C for 30 sec, 35 cycles; 68 °C for 5 min. After the reaction, the gel recovery kit (Qiagen company) recovers the PCR product. The pCU1301 vector (modified from pCAMBIA1301 vector, containing maize ubiquitin gene promoter) is linearized by restriction endonuclease BamH I (NEB company), and the enzyme digestion system is: vector 2 μg, BamH I endonuclease 2 μL, 10x buffer 10 μL, make up water to 100 μL. Enzyme digestion conditions: 25 °C, 2 hours. After the vector enzyme digestion product and the PCR product are purified and recovered, the DNA concentration is measured using NanoDrop 2000, and the recombination kit (Invitrogen company) is used for recombination, and the recombination system is: target fragment: 14 ng, vector fragment: 130 ng, 5x buffer 2 μL, Exnase II 1 μL, add water to 10 μL. Recombination conditions: 37 °C, 30 min. Take all the products and add to 100 μL of E. coli DH5α competent cells, and the transformation product is coated on LB solid medium (containing kanamycin resistance, the concentration of kanamycin is 50 mg / L). 37 °C culture overnight, and 4 single colonies are selected for colony PCR identification. Two positive clones are selected for sequencing, and a positive clone containing the HDT701 gene sequence is obtained, and finally an overexpression vector containing the HDT701 target gene is obtained.

[0051] HDT701 gene RNAi interference vector construction: the cDNA obtained above was used as a template for PCR amplification using KOD FX high-fidelity enzyme (ToYoBo Company). The reaction system was as follows: 2x PCR buffer 10 μL, 2 mM dNTPs 2 μL, F primer (5'-CGGGATCGAGCTCAAGGAAAATGAGCAGAAAAA-3') 0.5 μL, R primer (5'-GGGGTACCACTAGTTTGGCGGGGTGCTTGGCCTT-3') 0.5 μL, cDNA template 1 μL, KOD FX (1 U / μl) 0.4 μL, and water was added to 20 μL. The reaction conditions were as follows: 98°C for 5 min; 98°C for 15 sec, 56°C for 30 sec, 68°C for 30 sec, 35 cycles; 68°C for 5 min. After the reaction, the PCR product was recovered by a gel recovery kit (Qiagen Company). The RNAi interference vector pTCK303 was linearized by restriction enzymes Spe I and Sac I, and the enzyme digestion system was as follows: 2 μg of vector, 2 μL of Spe I and Sac I endonuclease, 10x buffer 10 μL, and water was added to 100 μL. The enzyme digestion conditions were as follows: 25°C, 2 hours. After the vector enzyme digestion product and the PCR product were purified and recovered, the DNA concentration was determined using NanoDrop 2000, and recombination was performed using a recombination kit (Invitrogen Company). The recombination system was as follows: target fragment: 14 ng, vector fragment: 130 ng, 5x buffer 2 μL, Exnase II 1 μL, and water was added to 10 μL. The recombination conditions were as follows: 37°C, 30 min. The entire product was added to 100 μL of E. coli DH5α competent cells, and the transformed product was plated on LB solid medium (containing kanamycin resistance, and the concentration of kanamycin was 50 mg / L). It was cultured at 37°C overnight, and 4 single colonies were selected for colony PCR identification. Two positive clones were selected for sequencing, and finally the RNAi interference vector of the HDT701 gene was obtained.

[0052] Genetic transformation: the overexpression vector and the RNAi interference vector constructed in Example 1 were introduced into rice variety Zhonghua 11 by the flower dipping method using Agrobacterium GV3101-mediated genetic transformation method. The experimental method was referred to the literature: Agrobacterium-mediated transgenic technology method exploration; Zhang Zhao-yi-chun; Nongjia Keji, 2017, No. 9. The rice seeds after flower dipping were harvested, surface sterilized with 2% sodium hypochlorite, and sown on 1 / 2MS solid medium containing 25 μg / L hygromycin. The resistant seedlings (root length and true leaves) were selected as transgenic positive seedlings. Ten positive T1 generation transgenic seedlings were propagated to obtain T2 generation. The T2 generation seeds were germinated using a medium containing hygromycin. If the seeds can grow normally, it proves that the strain is a homozygous strain.

[0053] We selected transgenic lines for qRT-PCR detection, and finally identified 2 HDT701 overexpression and interference transgenic lines each Figure 1 ).

[0054] Example 2

[0055] Phenotype analysis of HDT701 overexpression transgenic lines under arsenic stress

[0056] Wild type WT and HDT701 overexpression lines OE1 and OE7 rice seeds were sown on filter paper after 5% NaClO disinfection for germination, and at the 10th day, the seedlings with consistent growth were selected, and then treated with 100 μM NaAsO2[As(III)] for 3 days to observe the phenotype Figure 2 ). The results showed that under arsenic stress, compared with wild type, the leaves of HDT701 overexpression lines were severely curled, and some plants were severely wilted and yellow. The above results showed that overexpression of HDT701 reduced the tolerance of rice to arsenic stress, making rice more sensitive to arsenic stress.

[0057] Example 3: Sensitivity phenotype analysis of HDT701-RNAi interference lines to arsenic stress

[0058] Wild type WT and HDT701 RNAi interference line seeds were sown on filter paper after 5% NaClO disinfection for germination, and at the 10th day, the seedlings with consistent growth were selected, and then treated with 100 μM NaAsO2[As(III)] for 3 days to observe the phenotype. The results showed that WT and HDT701 RNAi interference lines grew healthily under normal growth conditions, and there was no obvious phenotype difference Figure 3 ); while under arsenic stress, most of the seedlings in wild type showed severe curling and yellowing of leaves, while HDT701 RNAi interference lines (HDT701-R2 and HDT701-R6) were less stressed, and most of the seedlings still showed green leaves Figure 3 ).

[0059] Example 4: Survival rate analysis of HDT701 overexpression and interference lines after arsenic stress

[0060] The survival rate of HDT701 overexpression line seedlings after arsenic stress treatment was further detected. Wild type and HDT701 overexpression lines that grew normally and consistently for 10 days were transferred to 100 uM NaAsO2[As(III)] for treatment. After 3 days of treatment, they were transferred to normal growth conditions for 14 days. The results showed that the survival rate of HDT701 overexpression lines was significantly lower than that of the control Figure 4 A).

[0061] At the same time, we also analyzed the survival rate of HDT701 interference strain seedlings after arsenic stress treatment. Wild type and HDT701 interference strain that grew normally and consistently for 10 days were transferred to 200uM NaAsO2[As(III)] for treatment. After 3 days of treatment, they were transferred to normal growth conditions for 14 days. The results showed that the survival rate of HDT701 interference strain was significantly higher than that of the control (P<0.05) (Figure 6B). Figure 4 B).

[0062] In summary, the above results confirm that HDT701 gene is involved in the response to arsenic stress, and thus can be mutated using gene engineering techniques such as CRISPR-Cas9 gene editing technology to reduce its expression, thereby improving the tolerance of plants to arsenic stress. Or by overexpressing HDT701 to improve the sensitivity of plants to arsenic. Therefore, HD5701 can be used in genetic breeding to improve the tolerance and sensitivity of plants to arsenic stress.

[0063] >SEQ ID NO. 1

[0064] ATGGAGTTCTGGGGTCTTGAAGTCAAGCCTGGACAGACTGTCAAATGTGAGCCTGAAGATGAACGCTTTTTGCACCTTTCTCAGGCTGCTCTTGGGGAATCAAAGAAAGGATCTGACAATGCAGTAATGTATGTTAAAACTGATGATCAAAAGCTAGTCATTGGAACCCTCTCAGCTGACAAGTTCCCTCAAATCCAGTTTGATTTGGTCTTTGACAAAGAGTTTGAGCTGTCACACACTTCAAAGACTGCTAGTGTGTTCTTTTCTGGCTACAAAGTTTCCCAGCCGGCTGAGGAAGATGAAATGGATTTTGATTCTGAAGAAGTTGAAGATGAAGAGGAGGAAGAAAAGATCATTCCAGCTCCCAGGGCAAATGGCAAAGTTGAAGGGAAGGAAAATGAGCAGAAAAAACAAGGCAAGACAGATTCTTCAGCTTCAAAATCAAAGGCTGCAGTGAATGACGATGATGATGATGATGACAGTGATGAGGATGATTCTGAGGACGAAGATCTTTCTCCTGAGGATGATGATGATGATTCTTCTGAGGATGATTCCAGCGAAGATGATGAGGATGAGAGTGACGAGGAAGATACTCCCAAGAAGCCAGAGACTGGAAAGAGGAAAGTAGCTGAAATTGTGTTGAAGACACCTTCGTCTGATAAGAAAGCAAAGATTGCTACACCGTCAGGCCAGAAGACAGGTGACAAGAAGGGTGTCCATGTAGCAACTCCACATCCGGCAAAGCAGGCTAGCAAGACCCCCGTGAATGACAAGTCAAAGGAGAAGTCCCCAAAATCCGGTGGTGGGTCAATTTCTTGCAAGTCATGCAGCAAGACGTTCAACAGTGAAATGGCTCTGCAATCTCACTCGAAGGCCAAGCACCCCGCCAAGTGA

[0065] >SEQ ID NO. 2

[0066] MEFWGLEVKPGQTVKCEPEDERFLHLSQAALGESKKGSDNAVMYVKTDDQKLVIGTLSADKFPQIQFDLVFDKEFELSHTSKTASVFFSGYKVSQPAEEDEMDFDSEEVEDEEEEEKIIPAPRANGKVEGKENEQKKQGKTDSSASKSKAAVNDDDDDDDSDEDDSEDEDLSPEDDDDDSSEDDSSEDDEDESDEEDTPKKPETGKRKVAEIVLKTPSSDKKAKIATPSGQKTGDKKGVHVATPHPAKQASKTPVNDKSKEKSPKSGGGSISCKSCSKTFNSEMALQSHSKAKHPAK

[0067] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Numerous modifications to the embodiments described above will be apparent to persons skilled in the art and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of rice HDT701 gene in regulating the ability of plants to resist arsenic stress, characterized in that, The amino acid sequence encoded by the HDT701 gene is shown as SEQ ID NO. 2, and the plant is rice.

2. The use according to claim 1, wherein, the ability of the plant to resist arsenic stress is the ability of the plant to resist arsenic stress by reducing the expression of the HDT701 gene, or the sensitivity of the plant to arsenic stress by overexpressing the HDT701 gene.

3. The use of the protein encoded by the rice HDT701 gene in regulating the ability of a plant to resist arsenic stress, characterized in that, The amino acid sequence encoded by the HDT701 gene is shown as SEQ ID NO. 2, and the plant is rice.

4. The use according to claim 3, wherein, the ability of the plant to resist arsenic stress is the ability of the plant to resist arsenic stress by reducing the expression of the protein encoded by the HDT701 gene, or the sensitivity of the plant to arsenic stress by overexpressing the protein encoded by the HDT701 gene.

5. Use of the rice HDT701 gene in the breeding of plant varieties with high resistance to arsenic stress and / or sensitivity to arsenic stress, characterized in that, The amino acid sequence encoded by the HDT701 gene is shown as SEQ ID NO. 2, and the plant is rice.

6. Use of the protein encoded by the rice HDT701 gene in breeding plant varieties having high resistance to arsenic stress and / or being sensitive to arsenic stress, characterized in that, The amino acid sequence encoded by the HDT701 gene is shown as SEQ ID NO. 2, and the plant is rice.

Images