Pectin methylesterase coding gene capable of improving cold resistance of rice in seedling stage and application of pectin methylesterase coding gene

Abstract

The invention discloses a pectin methylesterase coding gene OsPME25 for improving cold resistance of rice in a seedling stage and application of the pectin methylesterase coding gene OsPME25, and belongs to the field of rice gene engineering. The nucleotide sequence of the rice pectin methylesterase coding gene Os08g0450100 (OsPME25) is shown as SEQ ID No.1. Overexpression of the gene in rice can significantly reduce the methyl esterification degree of rice pectin and improve the cold stress resistance of rice, a new gene resource is provided for cold-resistant breeding of rice, and the rice pectin methylesterase coding gene Os08g0450100 (OsPME25) has a great application prospect in cultivation of cold-resistant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of rice genetic engineering and relates to a rice pectin methylesterase encoding gene Os08g0450100 (OsPME25) and its application in improving the cold stress resistance of rice seedlings. Background Art

[0002] Rice is a tropical and subtropical crop. When the ambient temperature drops below 16°C, the metabolism and physiological processes of rice plants will be irreversibly damaged, namely, chilling injury (Caton BP, Foin TC, Gibson KD, Hill JE (1998) A temperature-based model of direct-, water-seeded rice (Oryza sativa) stand establishment in California. Agricultural and Forest Meteorology, 90(1):91-102.), resulting in reduced crop yields.

[0003] The cell wall is the first line of defense for plants against adverse stress. It is composed of cellulose microfibrils interconnected with hemicelluloses such as xyloglucan and arabinoxylan and embedded in pectin colloids (Zheng Shaojian, The role of cell wall in plant resistance to nutritional stress and its molecular physiological mechanism. 2014, Chinese Science, 44(4): 334-341.). Previous studies have shown that the pectin component in plant cell walls is closely related to its stress resistance. Studies have shown that plants with high pectin content have enhanced resistance to various abiotic stresses such as salt and drought (Bouton S, Leboeuf E, Mouille G, Leydecker M, Talbotec J, Granier F, Lahaye M, Hōfte H, Truong HN (2002) QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. Plant Cell, 14: 2577-2590.; Liu HH, Ma Y, Chen N, Guo SY, Liu HL, Guo X, Chong K, Xu YY (2014) Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and in), in the model plant Arabidopsis, changes in pectin content directly affect its disease resistance and stress resistance (Vogel JP, Raab TK, Schiff C, Somerville SC (2002) PMR6, a pectate lyase-like gene required for powdery mildew susceptibility in Arabidopsis. Plant Cell, 14(9): 2095-106; Zhu J, Lee BH, Dellinger M, Cui X, Zhang C, Wu S, Nothnagel EA, Zhu JK (2010) A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis. The Plant Journal, 63: 128-140.).At present, there is no report on the correlation between the rice pectin methylesterase encoding gene Os08g0450100 (OsPME25) and rice cold stress. Summary of the invention

[0004] In view of the above problems, the present application provides a rice pectin methylesterase encoding gene OsPME25 and the application of the gene in improving the cold stress tolerance of rice.

[0005] Specifically, the above invention object is achieved through the following technical solutions:

[0006] First, the present application provides a rice pectin methylesterase encoding gene OsPME25, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:1, and the amino acid sequence of the encoded pectin methylesterase is shown in SEQ ID NO:2.

[0007] Secondly, the present application provides the use of overexpressing the OsPME25 gene with the nucleotide sequence as shown in SEQ ID NO: 1 in improving the cold stress tolerance of rice seedlings.

[0008] Third, the present application provides a vector (such as an overexpression vector) or an engineered bacterium (such as Escherichia coli) containing the OsPME25 gene.

[0009] Fourthly, the present application provides a transgenic method for improving the cold stress tolerance of plants using the OsPME25 nucleotide sequence as shown in SEQ ID NO: 1, and the specific steps are as follows:

[0010] 1. Cloning of gene fragments:

[0011] Using the DNA shown in SEQ IN NO.1 as a template, and using the upstream primer shown in SEQ IN NO.3 and the downstream primer shown in SEQ IN NO.4 as primers, PCR amplification was performed:

[0012] PCR amplification system: 2× PhantaFlash Master Mix 25 μL, 10 μM upstream primer 2 μL, 10 μM downstream primer 2 μL, template DNA 1 μg, add ddH2O to a total volume of 50 μL;

[0013] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 30 cycles; and final extension at 72°C for 10 min.

[0014] The amplified product was subjected to agarose gel electrophoresis and then recovered using a gel recovery kit to obtain the amplified product for later use.

[0015] 2. Construction of overexpression vector:

[0016] The original vector pBWA(V)HS (Biorun) and the amplified product obtained in step 1 were digested with BsaI endonuclease.

[0017] The vector digestion system is as follows: 10×Buffer 2μL, BsaI 2μL, pBWA(V)HS2ug, add ddH2O to a total volume of 20μL. After digestion at 37℃ for 1h, purify the digestion product using a PCR purification kit to obtain the digestion vector. This step is performed according to the instructions of the kit.

[0018] The digestion system of the amplified product is as follows: 10× Buffer 2μL, BsaI 2μL, DNA fragment of the amplified product 2ug, add ddH2O to a total volume of 20μL. After digestion at 37℃ for 1h, the above digestion product is purified using a PCR purification kit to obtain the digestion amplification product. This step is performed according to the instructions of the kit.

[0019] T4 DNA ligase was then used to connect the digested vector and the digested fragment. The ligation system was as follows: 10×Buffer 2μL, T4 ligase 2μL, pBWA(V)HS 0.3pmol, OsPME250.03 pmol, and ddH2O was added to a total volume of 20μL. After ligation at 20℃ for 1h, 10μL of the ligation product was transformed into E. coli competent cells and spread on (kanamycin) resistant culture dishes, cultured at 37℃ for 14 hours, and single colonies were picked for colony PCR identification.

[0020] The nucleotide sequence of the upstream primer for colony PCR identification is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; the PCR reaction system is configured as follows: 2×Phanta Flash Master Mix 5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, ddH2O 4 μL.

[0021] The amplified product was identified by agarose gel electrophoresis, and the plasmid was extracted from the single colony with the target band using a plasmid extraction kit and sequenced for identification. The correctly identified plasmid was saved for later use, which was the recombinant plasmid;

[0022] 3. Obtaining transgenic plants:

[0023] The recombinant plasmid was transformed into EHA105 Agrobacterium competent cells and infected with rice callus (conventional methods in the art) to obtain transgenic plants, and stable genetic transgenic strains were obtained after fluorescence quantitative PCR screening and identification. The fluorescence quantitative PCR screening method was as follows: RNA was extracted from rice leaves of the parents and each transgenic strain using an RNA extraction kit, and cDNA was obtained using a reverse cDNA kit. Real-time PCR was performed using a Biorad CFX96 real-time PCR instrument for fluorescence quantitative detection, OsActin was used as an internal reference gene, and three biological replicates were performed. The upstream and downstream primer sequences of OsPME25 fluorescence quantitative detection were shown in SEQ ID No. 5 and SEQ ID No. 6; the transgenic plants with significantly increased expression of OsPME25 in fluorescence quantitative detection were transgenic rice plants with enhanced cold stress tolerance at the seedling stage.

[0024] In the present application, the term "gene knockout" refers to a base deletion, insertion or base substitution in the OsPME25 gene sequence, and a mutation in the corresponding encoded protein. The resulting gene-edited strain is the "gene knockout strain."

[0025] Compared with traditional technologies, the gene-based application provided by this application has the following advantages:

[0026] 1. This application discovered for the first time the correlation between the OsPME25 gene and the cold stress tolerance of rice. Overexpression of this gene can significantly improve the cold stress tolerance of rice plants at the seedling stage, providing a new method for improving the cold stress tolerance of plants.

[0027] 2. The implementation of the method of the present invention is not limited by genetic background, and there is no problem of trait linkage in traditional breeding. By overexpressing genes in target plants through genetic engineering methods, rice plants with improved cold stress tolerance can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the verification results of the interaction between OsPME25 and qPSR10;

[0029] Among them, A is the result of yeast two-hybrid interaction experiment, B is the result of dual luciferase complementation experiment, and C is the result of Co-IP experiment.

[0030] Figure 2 Schematic diagram of cold tolerance phenotype identification and pectin-related phenotype analysis of gene knockout and overexpression strains in OsPME25 under 11 scenes;

[0031] In the figure, A is the phenotype of the gene knockout strain after cold treatment; B is the survival rate (PSR%) of the gene knockout strain after cold treatment; C is the pectin methylesterase activity analysis of the gene knockout strain; D is the methylation degree detection of pectin methyl ester in the gene knockout strain; E is the phenotype of the overexpression strain after cold treatment; F is the survival rate statistics of the gene knockout strain after cold treatment; G is the pectin methylesterase activity analysis of the overexpression strain; H is the methylation degree detection of pectin methyl ester in the overexpression strain. DETAILED DESCRIPTION

[0032] The rice varieties involved in the embodiments are all provided by the laboratory of Jiangsu Lixiahe Agricultural Science Research Institute, and the "Zhonghua 11" used are all conventional rice varieties.

[0033] Example 1: Mining of OsPME25

[0034] In the previous work, the applicant used the map-based cloning to identify a rice cold-tolerance-related gene qPSR10 (“Xiao N, Gao Y, Qian H, Gao Q, WuY Zhang D, Zhang X, Yu L, Li Y, Pan C, Liu G, Zhou C, Jiang M, Huang N, Dai Z, Liang C, Chen Z, Chen J, Li A. (2018) Identification of Genes Related to ColdTolerance and a FunctionalAllele That ConfersColdTolerance. Plant Physiology. 177(3): 1108-1123.”). In the process of studying the function of the qPSR10 gene, the applicant used the yeast two-hybrid screening library method to discover the pectin methylesterase OsPME25 that interacts with qPSR10.

[0035] This example verified the interaction of the gene in vivo and in vitro. Figure 1 The specific verification method is as follows:

[0036] 1.1) Yeast two-hybrid interaction experiment: The coding sequence of qPSR10 was cloned into the pGBKT7 vector, and the coding sequence of OsPME25 was cloned into the pGADT7 vector respectively. The recombinant vectors were co-transformed into the yeast strain AH109 in pairs, and then selected on a medium lacking tryptophan and leucine (SD / Trp / Leu). Positive colonies were picked to SD / Trp / Leu medium and SD / Trp / His / Leu / +20mM 3-AT medium, and the colonies were observed after culturing at 30°C for 4 days. pGADT7-LargeT and pGBKT7-p53 were used as positive controls, and pGADT7-LargeT and pGBKT7-lamic were used as negative controls. The experimental results are shown in the figure. Figure 1 As shown in A;

[0037] 1.2) Dual luciferase complementation experiment: The coding sequence of qPSR10 was cloned into the pCAMBIA1300-cLUC vector, and the coding sequence of OsPME25 was cloned into the pCAMBIA1300-nLUC vector. The recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 and then injected into 4-week-old Nicotiana benthamiana leaves in pairs. After 48 hours of culture, 1mM fluorescent reagent was injected into the leaves and the fluorescent signal was captured using the Tanon-5200 imaging system. AtFLS2-nLUC+ATAG1-cLUC was used as a positive control. The experimental results are shown in Figure 2. Figure 1 As shown in B;

[0038] 1.3) Co-IP experiment: The coding sequence of qPSR10 was cloned into the pCAMBIA-3×Flag vector, and the coding sequence of OsPME25 was cloned into the pBinGFP2 vector, respectively. All recombinant plasmids were transferred into Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens GV3101 strains carrying the recombinant vectors were mixed at OD600=0.5 and infiltrated into leaves of 4-week-old Nicotiana benthamiana leaves. After 48 hours of incubation, proteins were extracted from the infiltrated leaf tissue with extraction buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% TritonX-100 and protease inhibitor cocktail [Sigma-Aldrich]), bound to 30 μL protein A+G agarose (Beyotime, P2012) and 2 μL GFP antibody (Abcam, ab290), and incubated at 4°C for 12 hours or overnight. The agarose beads were then washed 3 to 5 times with 1x PBS buffer and eluted with elution buffer (200 mM glycine [pH = 2.5] and 1 M Tris base [pH = 10.4]). The eluted proteins were used immediately or stored at -80°C. 1x SDS loading buffer was added to the samples, boiled for 10 minutes, and then subjected to SDS-PAGE and IB analysis. The antibodies used for Co-IP and WB experiments in the experiment were anti-GFP (Abcam, ab290) and anti-Flag (Abcam, ab205606). The ChemDoc TouchImaging system (Bio-Rad) was used to capture the stripes. The experimental results are shown in Figure 1 As shown in C.

[0039] The above experiments proved that the pectin methylesterase OsPME25 gene was successfully obtained, the nucleotide sequence of which is shown in SEQ IN NO.1, and the amino acid sequence of the encoded pectin methylesterase is shown in SEQ IN NO.2.

[0040] Example 2 Functional verification of OsPME25

[0041] 2.1 Functional verification of OsPME25 gene knockout

[0042] In this example, the gene knockout vector was constructed using the Biogene CRISPR / Cas9 gene knockout kit, and the gene knockout vector was transferred into the parent Zhonghua 11 (ZH11, carrying the qPSR10 allele) by Agrobacterium transformation to construct a gene knockout plant (commissioned by Biogene Technology (Jiangsu) Co., Ltd. for construction). The gene knockout plants were identified and further generations were performed to obtain a homozygous knockout strain.

[0043] The homozygous knockout strain identification method is as follows: Use a plant DNA extraction kit (NoviZan, DC104) to extract the DNA of the parent Zhonghua 11 and each knockout strain, use the DNA as a template, use the upstream primer (nucleotide sequence as shown in SEQ ID NO: 3) and the downstream primer (nucleotide sequence as shown in SEQ ID NO: 4) of the target gene OsPME25 as primers, and use a high-fidelity DNA polymerase (NoviZan, P510) for PCR amplification:

[0044] The PCR reaction system is: 2×PhantaFlash Master Mix 10μL, upstream primer (10uM) 1μL, downstream primer (10uM) 1μL, template 200ng, add ddH2O to a total volume of 20μL

[0045] The PCR amplification program was pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 30 cycles; and final extension at 72°C for 10 min.

[0046] The amplified products were sequenced, and plants with homozygous gene knockout mutations (OsPME25-KO-1 and OsPME25-KO-2) were selected.

[0047] 2.1.1) Identify the cold-tolerant phenotype of homozygous knockout plants. The specific method is as follows: germinate the seeds of the parental and gene-edited lines and grow them to the 4-leaf stage. Count the total number of seedlings and place them in a 4°C environment for cold treatment. After 48 hours of cold treatment, place the seedlings in normal conditions for 7 days. After 7 days, count the survival rate of the seedlings.

[0048] The results of cold treatment of seedlings are as follows Figure 2 As shown in A, the statistical results of seedling survival rate are as follows Figure 2 As shown in B, the survival rate of the parent Zhonghua 11 was 83%, and the average survival rate of the OsPME25 gene knockout strain was 54.5%. It can be seen that the survival rate of the seedlings of the gene knockout strains decreased significantly.

[0049] 2.1.2) Identify the pectin-related phenotypes of the homozygous plants obtained by screening. The specific method is as follows:

[0050] The pectin esterase activity assay kit (Solarbio, BC2700) was used to detect the activity of pectin methylesterase; the pectin methylation degree test kit (Comin, GJJ-2-W) was used to detect the degree of methylation of pectin.

[0051] Test results such as Figure 2 As shown in C and D, compared with the wild type, the PME activity of the knockout line OsPME25-KO-1 was significantly reduced and the DM of pectin was significantly increased.

[0052] 2.2 Functional verification of overexpression of OsPME25

[0053] In order to explore the application of OsPME25 in enhancing the cold tolerance of rice, this example used japonica rice Zhonghua 11 as the receptor material to create an overexpression strain of the gene. The specific method is as follows:

[0054] 2.2.1) Cloning of gene fragments:

[0055] Using the DNA shown in SEQ IN NO.1 as a template, and using the upstream primer shown in SEQ IN NO.3 and the downstream primer shown in SEQ IN NO.4 as primers, PCR amplification was performed:

[0056] PCR amplification system: 2× PhantaFlash Master Mix 25 μL, 10 μM upstream primer 2 μL, 10 μM downstream primer 2 μL, template DNA 1 μg, add ddH2O to a total volume of 50 μL;

[0057] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 30 cycles; and final extension at 72°C for 10 min.

[0058] The amplified product was recovered by agarose gel electrophoresis using a gel recovery kit (NoviZan, DC301) for later use. The specific method of use was performed according to the instructions of the kit, and the DNA concentration was determined using a nucleic acid concentration detector OD-1000 (One Drop).

[0059] 2.2.2) Construction of overexpression vector:

[0060] The original vector pBWA(V)HS and the amplified product obtained in step 2.2.1) were digested with BsaI endonuclease (Biorun, #RCA02). The digestion system was as follows: 10×Buffer 2μL, BsaI 2μL, DNA 2 ug of the amplified product obtained in step 2.1.1, and ddH2O was added to a total volume of 20μL. After digestion at 37℃ for 1h, the above digestion products were purified using a PCR purification kit to obtain OsPME25 and vector pBWA(V)HS purified products. This step was performed according to the instructions, and the DNA concentration was measured using a nucleic acid concentration detector OD-1000 (One Drop).

[0061] Then, T4 DNA ligase was used to connect the digested vector and the digested fragment. The connection system was as follows: 10×Buffer 2μL, T4 ligase 2μL, pBWA(V)HS 0.3pmol, OsPME25 purified product 0.03pmol, and ddH2O was added to a total volume of 20μL. After 1h of connection at 20℃, 10μL of the connection product was transformed into competent E. coli and spread on a (kanamycin) resistant plate, cultured at 37℃ for 12 hours, and a single colony was picked for plaque PCR identification. The nucleotide sequence of the upstream primer for colony PCR identification is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:4.

[0062] The PCR reaction system was configured as follows: 2×Phanta Flash Master Mix 5 μL, upstream primer (10 uM) 0.5 μL, downstream primer (10 uM) 0.5 μL, and ddH2O 4 μL.

[0063] The amplified product was identified by agarose gel electrophoresis, and the plasmid was extracted from the single colony with the target band using a plasmid extraction kit (NoviZan, DC201) and sequenced. The plasmid with the correct sequence identification was the recombinant plasmid and was stored for future use.

[0064] 2.2.3) Obtaining transgenic plants:

[0065] The recombinant plasmid obtained in step 2.2.2) was transformed into EHA105 Agrobacterium and infect rice callus (for specific methods, refer to the method disclosed in the document "Zhao W, Zheng S, Ling HQ (2011) An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice callus, Livtar Handao 297. Plant Cell Tissue & Organ Culture. 106 (3): 475") to obtain transgenic plants. After fluorescence quantitative PCR screening and identification, an overexpression transgenic strain with significantly increased expression and stable inheritance was obtained.

[0066] The sequences of upstream and downstream primers for fluorescence quantitative analysis of OsPME25 are shown in SEQ ID No.5 and SEQ ID No.6. The fluorescence quantitative PCR screening method is as follows: RNA extraction kit (Tiangen, DP441) is used to extract RNA from rice leaves of each transgenic line, and cDNA is obtained using a reverse cDNA kit (Vazyme, R312). Real-time PCR uses a Biorad CFX96 real-time PCR instrument for fluorescence quantitative detection, OsActin is used as an internal reference gene, and three biological replicates are performed. The specific method of use is operated according to the instructions of the kit. This method can be used to verify the results of transgenic overexpression.

[0067] 2.2.4) Verification of cold tolerance phenotype of overexpression strains and determination of pectin-related phenotypes:

[0068] The cold stress tolerance of the overexpression strains was identified. The specific method was as follows: the seeds of the parents and the overexpression strains (OsPME25-OE1, OsPME25-OE-2) were germinated and grown to the 4-leaf stage, the total number of seedlings was counted and placed in a 4°C environment for cold treatment, and after 72 hours of cold treatment, the seedlings were placed in normal conditions for 7 days, and the survival rate of the seedlings was counted after 7 days.

[0069] Seedling phenotype after cold treatment Figure 2 As shown in E, the survival rate statistics are as follows Figure 2 As shown in F, the survival rate of the parent Zhonghua 11 was 83%, while the survival rate of the OsPME25 overexpression line was significantly increased compared with the parent, reaching 95.67%.

[0070] Refer to the method in step 2.1) above to identify the pectin-related phenotypes. The test results are as follows: Figure 2 As shown in G and H, the pectin methylesterase activity of the OsPME25 overexpression line (OsPME25-OE-1) was significantly increased compared with that of the parent, while the degree of pectin methylesterification was significantly decreased compared with that of the parent. This indicates that overexpression of the OsPME25 gene can significantly increase the pectin methylesterase activity, thereby improving the cold stress tolerance of rice.

Claims

1. A pectin methylesterase encoding gene for improving the cold tolerance of rice seedlings, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The encoded protein of the gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO:

2.

3. A vector or engineered bacteria containing the gene as claimed in claim 1.

4. Use of overexpressing the gene as claimed in claim 1 in improving the cold stress tolerance of rice seedlings, characterized in that: Overexpression in target plants by transgenic methods OsPME25 , so as to achieve the purpose of increasing gene expression.

5. A method for improving the cold tolerance of rice seedlings, characterized in that: The specific steps are as follows: 1) Cloning of gene fragments Using the sequence shown in SEQ IN NO.1 as a DNA template, the sequence shown in SEQ IN NO.3 as an upstream primer, and the sequence shown in SEQ IN NO.4 as a downstream primer, PCR amplification was performed to obtain an amplified product; 2) Construction of overexpression vector The vector and the amplified product obtained in step 1) are digested with BsaI endonuclease, and then the digested products are ligated with DNA ligase. The ligated products are transformed into competent Escherichia coli cells to obtain a recombinant plasmid; 3) Preparation of transgenic plants By transforming the recombinant plasmid into Agrobacterium and infecting the target rice callus tissue, the cold tolerance of the target rice seedling stage can be improved.

6. The method according to claim 5, characterized in that Step 1) The PCR amplification refers to: PCR amplification system: 2 × Phanta Flash Master Mix 25 μL, 10 μM upstream primer 2 uL, 10 μM downstream primer 2 μL, DNA template 1 μg, add ddH2O to make up to 50 μL; The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 30 cycles; and final extension at 72°C for 10 min.

7. The method according to claim 5, characterized in that Step 2) The vector is pBWA(V)HS or other vectors that can be used for rice gene overexpression.

8. The method according to claim 5, characterized in that Step 3) The Agrobacterium is Agrobacterium EHA105 or other Agrobacterium that can be used for rice genetic modification.

Images