Application of LRK6 gene in improving high temperature resistance of rice

Abstract

The invention provides the following technical scheme: the invention provides application of an LRK6 gene or a recombinant vector containing the LRK6 in improving the heat resistance of rice. The nucleotide sequence of the LRK6 gene is as shown in SEQ ID NO.1, the amino acid sequence of the expression protein of the LRK6 gene is as shown in SEQ ID NO.2, and the recombinant vector containing the LRK6 gene is an expression vector obtained by inserting the LRK6 gene into the expression vector. Experiments show that a transgenic rice strain is obtained by constructing a recombinant vector containing the LRK6 gene and introducing the expression vector into Zhonghua 11 rice by using an agrobacterium-mediated method. Compared with wild-type Zhonghua 11 rice, the transgenic rice strain has higher heat resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a... LRK6 Application of genes in improving the ability of rice to withstand high temperatures. Background Technology

[0002] Rice, as one of the three major food crops, is the primary food source for more than half of the world's population, and increasing its yield is of paramount importance in ensuring food security. However, rice is susceptible to various biotic and abiotic stresses during its growth, leading to reduced yields. Currently, with global warming and the frequent occurrence of extreme high-temperature weather, the normal growth and development of rice are adversely affected, threatening food security. Therefore, exploring the mechanisms of high-temperature tolerance in rice and creating heat-resistant varieties is an important research topic.

[0003] The growth and development of rice can be roughly divided into three stages: the vegetative stage, the reproductive stage, and the grain-filling stage. Each stage has extremely high temperature requirements, and high temperatures can affect each stage of rice growth and development to varying degrees.

[53] The rice's nutritional period mainly includes three stages: germination, seedling growth, and tillering. During seed germination and seedling growth, energy is primarily derived from starch. High temperatures can affect starch synthesis within the seed, leading to a decrease in germination rate and inhibited growth. [54-55] Rice seedlings are highly susceptible to environmental influences. The optimal temperature for seedling growth is 25℃-28℃. High temperatures can cause dehydration, yellowing of leaves, and root damage. Studies have shown that within the suitable temperature range for rice growth, the number of tillers increases with rising temperature. However, when the temperature exceeds the optimal growth temperature, tillering ceases, leading to a decrease in yield. [56-57] During the reproductive period, rice undergoes processes such as flower development, development of male and female ligands, pollination, and fertilization. During flower development, the nutrients for microspores and pollen growth mainly come from the tapetum. High temperatures can induce mutations in tapetum cells, preventing the tapetum from providing nutrients. This results in most microspores and pollen failing to develop, leading to pollen abortion and affecting fertilization.

[58] The development of male and female ligands and fertilization in rice are closely related to the meiotic division stage. Studies have found that the rice meiotic division stage is most sensitive to high temperature stress.

[59] The degree to which heat stress affects the meiotic division stage of rice varieties with different heat tolerance varies. For example, after high-temperature treatment during the meiotic division stage of the heat-tolerant variety Huang Huazhan and the heat-sensitive variety Shuanggui No. 1, it was found that Huang Huazhan had higher pollen fertility, yield, and antioxidant enzyme activity than the heat-sensitive variety Shuanggui No. 1.

[60] The grain-filling stage of rice is a critical period that determines rice yield and quality. High temperatures can negatively impact seed embryo development by increasing the grain-filling rate and reducing the grain-filling time.

[61] In addition, high temperatures can alter seed size, leading to a decrease in thousand-grain weight, reduced yield, increased chalkiness (the opaque portion of the white endosperm in rice), and a decline in rice quality. [62-64] . Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide LRK6 Genes or containing LRK6 New applications for gene recombination vectors.

[0005] To solve the above-mentioned technical problems, the present invention provides a specific solution through the following technical solution: This invention provides LRK6 Genes or containing LRK6 Application of gene recombination vectors in improving the drought resistance of rice.

[0006] The aforementioned LRK6 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] The aforementioned LRK6 The amino acid sequence of the expressed protein of the gene is shown in SEQ ID NO.2.

[0008] The aforementioned containing LRK6 The gene recombination vector is used to carry out the above-mentioned gene recombination. LRK6 The gene is inserted into the expression vector to obtain the expression vector.

[0009] In this invention, the application involves introducing the LRK6 gene into a target plant to obtain a transgenic plant with greater heat resistance than the target plant. The LRK6 gene is introduced into the target plant via a recombinant vector containing the LRK6 gene.

[0010] Molecular cloning reagents: Table 2.2 Main experimental instruments used in this study Table 2.2 The main experimental apparatus used in this study

[0011] Synthesis and DNA sequencing using primers from Qingke Biotechnology In this invention: 1. RNA extraction steps: (1) Place the rice tissue to be extracted into a 2 mL centrifuge tube, add steel balls that have been soaked in anhydrous ethanol into the centrifuge tube, cover the centrifuge tube, put the centrifuge tube into liquid nitrogen for quick freezing, and then grind the tissue using a cryo-grinder (the low temperature must be ensured during the grinding process, and the centrifuge tube must be quickly transferred to liquid nitrogen after grinding to prevent RNA degradation). (2) Add 1 mL of lysis buffer CZ to a centrifuge tube, vortex mix for 20 s, and let stand at room temperature for 5 min. (3) Add 200 μL of chloroform, vortex mix for 15 s, and let stand at room temperature for 3 min; (4) Centrifuge for 10 min at 4℃ and 12000 rpm. (5) Take 450 μL of supernatant into a new 1.5 mL RNase-free centrifuge tube, add 200 μL of anhydrous ethanol, mix well, and transfer the mixture to an RNA adsorption column; (6) Centrifuge at 12000rpm for 1 min at 4℃, discard the waste liquid, and add 500 μL of RNA washing buffer (with anhydrous ethanol added) to the adsorption column. (7) Centrifuge at 4℃ and 12000 rpm for 30 s, discard the waste liquid, and repeat once; (8) Place the RNA adsorption column in an empty collection tube and centrifuge at the same speed and temperature as in the previous step for 2 minutes. (9) Transfer the RNA adsorption column to a new 1.5 mL RNase-free centrifuge tube and let it stand at room temperature for 10 min (to dry the ethanol). Add 30 μL of DNase & RNase-free ddH2O to the adsorption column, let it stand for 2 min, centrifuge at 12000 rpm for 2 min at 4℃, discard the adsorption column, and the solution in the centrifuge tube is the RNA. Measure the RNA concentration and store it in a -80℃ refrigerator.

[0012] 2. Steps for RNA reverse transcription to synthesize cDNA: (1) Genomic removal reaction: Prepare the reaction mixture in Table 2.3 on ice, with the RNA sample added last.

[0013] Table 2.3 Reaction system for removing rice genomic DNA Table 2.3 Reaction system for the removal of rice genomic DNA

[0014] Mix the mixture in a small centrifuge, incubate at 42°C for 2 min, and then place it on ice for the next step.

[0015] (2) Reverse transcription reaction: Prepare the reaction mixture in Table 2.4 on ice.

[0016] Table 2.4 RNA reverse transcription system Table 2.4 RNA reverse transcription system

[0017] After mixing the reaction solution, incubate at 37°C for 15 min and at 85°C for 5 s. Store the synthesized cDNA at -20°C.

[0018] 2. Primer design Searching the NCBI database for the genome of rice zhonghua11 yields rice LRK6 Gene sequence, primers designed based on the vector's restriction enzyme sites. Primers are as follows: OsLRK6-F: ggtgttaacttaagcttATGCAGAAGCAGCAG; OsLRK6-R: tgtagtccatggtaccAGTTTTGACTGAATTTTG.

[0019] 3. Carrier Construction The methods include PCR amplification of the target gene, vector linearization, homologous recombination ligation, extraction of E. coli plasmids, enzyme digestion, ligation, transformation of E. coli, PCR identification of positive colonies, plasmid enzyme digestion identification, sequencing, and strain preservation.

[0020] RNA was extracted from wild-type rice and reverse transcribed into cDNA. The coding sequence (CDS) of the target gene was obtained from the National Rice Data Center website. The CDS sequence was imported into SnapGene software to design specific primers for amplifying the target gene. The primers were generally 15-20 bp in size, and adapters (homologous arms) could be added to the primers depending on the vector and ligation method. Primer information is detailed in the appendix. The target gene was amplified by PCR using the high-fidelity enzyme Phanta.

[0021] 4. Carrier linearization The corresponding vectors were digested using relevant restriction endonucleases. The reaction temperature and time were set according to the reaction conditions of different restriction endonucleases. The digestion reaction system is shown below: Table 2.11 Enzyme digestion reaction system Table 2.11 Enzyme digestion reaction system

[0022] Add 2 μL of 10× loading buffer to the product to terminate the reaction. Perform agarose gel electrophoresis on the mixture and use a gel recovery kit (Shanghai Sangon Biotech) to recover the target fragment.

[0023] 5. Homologous recombination linkage (1) The target gene was ligated into the introductory vector using a homologous recombination kit (Nanjing Novizan). The specific reaction system is shown in the table below: Table 2.12 Homologous recombination linkage reaction system Table 2.12 Homologous recombination linkage reaction systems

[0024] Note: The ratio of linearized vector to target gene is 1:2, i.e., X:Y = 1:2. (2) The prepared reaction solution was reacted at 50°C for 5 min, and then immediately placed on ice to cool. (3) Take 2 μL of recombinant product and add it to 100 μL of Escherichia coli competent cells for transformation. The obtained transformation product is plated on LB solid medium containing the corresponding antibiotic and the plate is inverted in an incubator at 37°C for overnight culture. 6. Preparation of competent Escherichia coli cells (1) Take the strain, streak it on LB medium, and incubate it at 37°C overnight.

[0025] (2) Pick a single colony from the LB plate and inoculate it into about 5 mL of LB liquid medium. Incubate overnight at 37°C and 250 rpm.

[0026] (3) Inoculate the bacterial solution into 50 mL of LB liquid medium at a ratio of 1:50, and incubate at 37℃ and 250 rpm for 1-2 h until the OD600 of the bacterial solution is between 0.5 and 0.6.

[0027] (4) Transfer the bacterial culture into a 50mL centrifuge tube and pre-cool for 30 min.

[0028] (5) Centrifuge at 4℃ and 4000 rpm for 10 min. Discard the supernatant.

[0029] (6) Add 5 mL of SSCS solution to suspend the cells.

[0030] (7) Dispense into 1.5mL centrifuge tubes (cooled beforehand), 100µL per tube.

[0031] (8) Quick-freeze with liquid nitrogen, then transfer to -80℃ for storage.

[0032] 7. Escherichia coli transformation (1) Take 100 μL of competent Escherichia coli cells from the -80℃ freezer and place them on ice. After they thaw, add 2 μL of target DNA (plasmid / recombinant product), gently mix, and place the mixture on ice for 25 min. (2) Heat shock in a 42℃ water bath for 1 min, then quickly place on ice and let stand for 2 min; (3) Add 600 μL of antibiotic-free liquid LB medium, mix well, and incubate at 37°C and 220 rpm for 1 h. (4) Centrifuge the cultured bacteria at 10,000 rpm for 1 min, discard the supernatant (keep about 200 μL of bacterial solution to resuspend the bacteria), and spread the resuspended bacteria on LB solid medium containing the corresponding antibiotic. (5) After the culture medium has dried, seal the petri dish, invert it and place it in a 37°C incubator overnight.

[0033] 8. Identification and sequencing of positive clones Single colonies from the plate were picked and incubated in LB broth containing the corresponding antibiotic at 37°C and 220 rpm for 6 hours for bacterial PCR verification. The successfully verified bacterial culture was then expanded and preserved (preservation method: bacterial culture to 50% glycerol in a 1:1 ratio). Plasmids were extracted from the bacterial culture using a plasmid mini-extraction kit (Kangwei Century). The recombinant products were then re-verified using plasmid PCR and enzyme digestion to ensure plasmid accuracy. The colony PCR and plasmid PCR reaction systems are shown in the table below: Table 2.13 PCR reaction system Table 2.13 PCR reaction system

[0034] Note: At least one of the amplification primers must be a universal primer for the vector. The reaction procedures for colony PCR and plasmid PCR are shown in the table below: Table 2.14 PCR Reaction Procedure Table 2.14 PCR reaction procedures

[0035] (2) Enzyme digestion identification Table 2.15 Enzyme digestion reaction system Table 2.15 Enzyme digestion reaction system

[0036] The above reaction solution was digested with restriction endonuclease at the specified temperature and time. After digestion, agarose gel electrophoresis was performed to verify the correctness of the bands. The application described in this invention is to use the aforementioned LRK6 Genes are introduced into target plants to obtain transgenic plants with higher heat resistance than the target plants.

[0037] The present invention LRK6 Genes through the aforementioned containing LRK6 Gene recombination vectors are introduced into target plants.

[0038] In the application described in this invention, the target plant is a monocotyledonous plant or a dicotyledonous plant, and the monocotyledonous plant is specifically rice.

[0039] The method described in this invention is to... LRK6 Genes are introduced into target plants to obtain transgenic plants with higher heat resistance than the target plants.

[0040] In the method described in this invention, the... LRK6 Genes through the aforementioned containing LRK6 Gene recombination vectors are introduced into target plants.

[0041] In the method described in this invention, the one containing LRK6 The gene recombination vector is used to carry out the above-mentioned gene recombination. LRK6 The gene is inserted into the expression vector to obtain the expression vector.

[0042] Experiments of this invention demonstrate that constructing a structure containing LRK6 The gene recombinant vector was used to transfer the expression vector into rice Zhonghua 11 using Agrobacterium-mediated transformation, resulting in transgenic rice lines. Compared with wild-type Zhonghua 11 rice, the transgenic rice lines showed increased heat resistance. Attached Figure Description

[0043] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] Figure 1 Identification of OsLRK6 overexpressing plants A: Construction of OsLRK6 overexpression vector map; B: Identification of T0 generation OsLRK6 transgenic positive plants (M: DNAmarker DL 2000; CK: negative control using water as template; WT: control using wild-type Zhonghua 11 as template); C: Detection of OsLRK6 expression level in T1 generation OsLRK6 transgenic positive plants (WT: wild type; OE: overexpression). Figure 2 Analysis of heat tolerance during germination of LRK6 transgenic plants (AB) Germination rates of wild-type and LRK6 transgenic plants under normal conditions (28℃ / 30℃) and high-temperature environments (42℃ / 40℃); (CE) Phenotypic characteristics of wild-type and LRK6 transgenic plants after 10 days of growth under normal and high-temperature conditions, as well as root and stem length analysis of root development. Transgenic lines improve heat tolerance by promoting lateral root development in rice. Scale bar = 1cm, n = 10 Figure 3 Wild-type and LRK6 transgenic seedling heat tolerance phenotype (A) Wild-type and LRK6 transgenic plants were placed in a high-temperature environment of 42℃ for 3 days after growing for 15 days, and then recovered in a normal environment for 10 days. Phenotypic observation showed that the transgenic lines had obvious heat resistance. Scale bar = 2 cm. (BC) Survival rate of wild-type and LRK6 transgenic plants 10 days after recovery Detailed Implementation

[0045] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0046] Example 1: Constructing a Carrier 1) Primers used to construct the vector: OsLRK6-F: ggtgttaacttaagcttATGCAGAAGCAGCAG; OsLRK6-R: tgtagtccatggtaccAGTTTTGACTGAATTTTG; 2) Recombinant vector A. LRK6 Acquisition of genes: ① Extract rice cDNA, as described in step 1 above.

[0047] ② Using cDNA as a template and OsLRK6-F and OsLRK6-R as primers, the LRK6 gene fragment was amplified by PCR.

[0048] PCR system:

[0049] program:

[0050] B. Construct the graph as follows Figure 1As shown, ①PCR amplification of gene fragments (as shown above); ② PCR product recovery; ③ pCAMBIA1300S vector and gene fragment digestion;

[0051] ④ Gel recovery of enzyme digestion products (same as above, using Shanghai Sangon Gel Recovery Kit); ⑤ Connect; Table 2.12 Homologous recombination linkage reaction system Table 2.12 Homologous recombination linkage reaction systems

[0052] Note: The ratio of linearized vector to target gene is 1:2, i.e., X:Y = 1:2. ⑥ Transformation (as in step 7 above); ⑦ PCR identification of positive colonies; ⑧ Extract plasmids by shaking bacteria (as in step 9 above); ⑨ Enzyme digestion identification; ⑩. Send the sample to Qingke Company for sequencing.

[0053] Example 2: Obtaining and Identifying Transgenic Rice 1. Preparation of Agrobacterium competent cells (1) Preparation: Prepare a 0.15 mol / L NaCl solution and a 20 mmol / L CaCl2 solution containing 15% glycerol, and sterilize them before use.

[0054] (2) Take 20 μL of Agrobacterium strain GV3101 and streak it on LB solid medium (containing 1 / 1000 of rifampicin antibiotic). Invert the culture dish and incubate it in an incubator at 28°C for about 3 days. (3) Pick a single colony of Agrobacterium and inoculate it into 25 mL of LB liquid medium containing 1 / 1000 of rifampicin antibiotic, and shake it overnight at 28°C and 220 rpm. (4) Take 50 μL of the shaken bacterial culture into a new 25 mL tube of LB liquid medium containing 1 / 1000 rifampicin antibiotic, and incubate at 28℃ and 220 rpm for 4-5 h until the OD600 value is about 0.5; (5) Transfer the bacterial culture to a 1.5 mL centrifuge tube and let it stand on ice for 20 min; (6) Centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, add pre-cooled 0.15 mol / L NaCl, suspend the cells, and let stand on ice for 20 min; (8) Centrifuge at 4℃, 5000 rpm for 5 min, discard the supernatant, and pour out as much of the residual waste liquid at the bottom as possible; (9) Add 200 μL of pre-cooled 20 mmol / L CaCl2 solution containing 15% glycerol, suspend the cells, quickly freeze them in liquid nitrogen, and then store them in a -80℃ freezer.

[0055] 3. Genetically modified rice is sent to a company for conversion. 4. Identification of transgenic positive plants Successfully built LRK6 The overexpression vector was transferred into the wild-type Zhonghua 11. Fifteen T0 generation transgenic plants were preliminarily identified as positive. DNA was extracted from leaf tissues of both wild-type and transgenic plants, and PCR was performed using primers specific to the hygromycin phosphotransferase gene on the pCAMBIA1300 vector. Positive plants amplified bands of 400-500 bp, while wild-type and negative controls (using water as a template) did not amplify any bands.

[0056] 3) Agrobacterium infection Select healthy leaves of *Tobacco Bengal* and puncture a relatively flat area with a syringe needle. Generally, two holes can be made in one leaf. Draw the infection solution containing the two types of *Agrobacterium* into the syringe, remove the needle, and gently press the syringe nozzle against the puncture site on the leaf. On the other side of the leaf, gently press the syringe nozzle against the leaf with your finger through the leaf. Inject slowly. You will observe the infection solution entering the leaf and spreading from the puncture site, causing the leaf color to darken (if this phenomenon is not observed, the injection position needs to be adjusted). The diffusion area should be about the size of a one-yuan coin. Continue injecting into the next puncture site.

[0057] 4) Subsequent cultivation and observation After injection, the plant is returned to the incubator and cultured for 3-6 days before the leaves are cut off and observed using a laser scanning confocal microscope.

[0058] Example 3: Reagents and Procedures for Simulating High-Temperature Treatment Experimental methods Plump wild-type Zhonghua 11 rice seeds and overexpressing transgenic rice seeds were selected for an experiment on rice's tolerance to heat and abiotic stress, with Zhonghua 11 seeds serving as the control group.

[0059] 1. Disinfect the surface of the seeds (70% alcohol for 10 min, 10% NaClO for 30 min), rinse several times with tap water, 4. 0 C. Vernalization lasts 3-5 days; 2. Place the seeds at 37°C. 0 Germination in a C-type incubator (water changed daily) will result in sprouting of white hairs in about 3 days; 3. Insert the sprouting seeds into a 96-well plate (with small holes cut at the bottom for water absorption by the rice) using tweezers, label them, place them in a 0.1 mM CaCl2 solution, and then transfer them to a light incubator for hydroponics for 3-4 days. Specific cultivation conditions: light, 28°C. o C, 16h; Darkness, 25 o C, 8 h; 4. When the rice seedlings are growing well (roots about 3-4 cm long), select 15-20 seedlings with relatively uniform growth and insert them into a 96-well plate with 4*5 holes, and place them in rice rooting bottles for cultivation. 5. Treat rice seedlings at 42℃.

[0060] 6. Record the treatment date, measure and photograph the above-ground parts and root length of each rice seedling before treatment, and place them in a light-controlled incubator for hydroponics. Specific cultivation conditions: light, 28°C. o C, 16 h; Darkness, 25 o C, 8 h; observe the phenotypic changes of rice seedlings during the stress treatment period.

[0061] 7. Record the date when the experimental group and the control group show significant phenotypic differences after a period of treatment. Measure and photograph the aboveground parts and root length of each rice seedling after treatment.

[0062] 8. LRK6 Overexpressing plants showed higher tolerance to high-temperature stress than wild-type and mutant plants. LRK6 Genes can positively regulate heat tolerance in rice seedlings.

Claims

1. LRK6 A gene or a recombinant vector containing LRK6 Application of the recombinant vector containing the gene in improving heat tolerance of rice.

2. The method of claim 1, LRK6 a gene or a recombinant vector containing LRK6 application of the gene in improving heat tolerance of rice, characterized in that: The nucleotide sequence of the LRK6 gene is shown as SEQ ID NO.

1. The amino acid sequence of the expression protein of the LRK6 gene is shown as SEQ ID NO.

2. The recombinant vector containing the LRK6 gene is obtained by inserting the LRK6 gene into an expression vector.

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