METS2 gene for regulating and controlling cold resistance of rice in bud stage, protein and application of METS2 gene and protein

Abstract

The invention relates to the technical field of gene engineering, and discloses an METS2 gene for regulating and controlling cold resistance of rice in a bud stage, a protein and application of the METS2 gene, and a nucleotide sequence of the METS2 gene is shown as SEQ ID NO.1. The invention also discloses an application of the METS2 gene in regulating and controlling the cold resistance of rice in the bud stage. According to the METS2 gene for regulating and controlling the cold resistance of the rice in the bud stage, the protein and the application of the METS2 gene, the METS2 gene is overexpressed in the rice, and the cold resistance of the rice in the bud stage can be improved, so that the rice related coding gene METS2 or the protein thereof plays an important role in regulating and controlling the cold resistance of the rice in the bud stage; a foundation is provided for further clarification of the molecular mechanism of the cold tolerance of the rice, and new gene resources and breeding resources are provided for rice breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a METS2 gene and protein for regulating cold tolerance of rice during bud stage, and applications thereof. Background Art

[0002] Rice is sensitive to cold stress throughout its growth period. Global climate change has brought severe low-temperature challenges to rice production in Guangxi and across the country. Chilling damage during the budding stage (late spring cold) causes necrosis of the growing point and a sharp drop in the germination rate; chilling damage during the heading stage (Cold Dew Wind) reduces pollen fertility and fruit set rate. Breeding cold-tolerant varieties is an economical and effective solution, but the difficulty of breeding modern cultivated rice has increased dramatically due to its narrow genetic diversity and loss of cold-tolerant genes. As one of the origins of rice, Guangxi's wild rice resources are rich in cold-tolerant genes, which are the key to breaking through the bottleneck of breeding.

[0003] Currently, several growth-stage-specific cold-tolerance genes have been cloned. Bud / seedling stage: COLD1 activates G protein signaling; qLTG3-1 regulates aleurone vacuolization; COG2 negatively regulates the cell wall; and HAN1 fine-tunes the jasmonic acid pathway. Booting stage: CTB4a (LRR kinase) enhances cold tolerance by enhancing ATP synthesis; CTB5 (HD-Zip transcription factor) maintains gibberellin homeostasis and pollen fertility; OsSRO1c and OsDREB2B dynamically change phases to regulate cold-responsive genes; bZIP73 Jap Improve cold tolerance during the reproductive period. In addition, other molecular regulatory mechanisms of cold tolerance include OsMKK6 activation of trehalose synthesis, ABF1 / 2-OsNAC regulatory network, CTB2-mediated sterol metabolism, and CTB6 regulation of ROS homeostasis.

[0004] Despite significant progress in identifying cold-tolerance genes and elucidating their molecular mechanisms, cold-tolerant germplasm and genes that can be directly used in breeding remain relatively scarce. Therefore, further research is needed to identify cold-tolerance QTLs / genes within a broad range of wild rice resources, further elucidate the molecular networks and regulatory mechanisms of cold tolerance, and simultaneously develop new germplasm to overcome breeding bottlenecks. Summary of the Invention

[0005] The purpose of the present invention is to provide the METS2 gene, protein and application thereof for regulating the cold tolerance of rice at the bud stage. Overexpression of the METS2 gene in rice can improve the cold tolerance of rice at the bud stage, indicating that the rice-related encoding gene METS2 or its protein plays an important role in regulating the cold tolerance of rice at the bud stage. This not only provides a basis for further clarifying the molecular mechanism of rice cold tolerance, but also provides new gene resources and breeding resources for rice breeding.

[0006] To achieve the above object, the present invention provides a METS2 gene for regulating cold tolerance of rice during the budding period. The nucleotide sequence of the METS2 gene is shown in SEQ ID NO.1.

[0007] Furthermore, the present invention also provides the use of the METS2 gene in regulating the cold tolerance of rice during the budding period. The nucleotide sequence of the METS2 gene is shown in SEQ ID NO.1.

[0008] Furthermore, when used, the METS2 gene is overexpressed in rice plants to improve the cold tolerance of rice during the bud stage.

[0009] Furthermore, the present invention also provides a METS2 protein for regulating the cold tolerance of rice during the budding period. The amino acid sequence of the METS2 protein is shown in SEQ ID NO.2, and the gene encoding the METS2 protein is shown in SEQ ID NO.1.

[0010] Furthermore, the present invention also provides the use of METS2 protein in regulating the cold tolerance of rice during the budding period. The gene encoding the METS2 protein is the METS2 gene, and the nucleotide sequence of the METS2 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the present invention also provides a recombinant vector comprising the above-mentioned METS2 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] Furthermore, the recombinant vector is a plant overexpression vector.

[0013] A biological material containing the aforementioned recombinant vector. The biological material is a recombinant microorganism, a transgenic plant cell line, or a transgenic plant tissue. The recombinant microorganism is a bacterium, yeast, algae, or fungus; and the bacterium is one of the genera Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, or Bacillus.

[0014] Furthermore, the present invention also provides the use of the above-mentioned recombinant vector in regulating the cold tolerance of rice during the budding period.

[0015] Furthermore, the present invention also provides a method for cultivating transgenic rice with cold tolerance at the budding stage, wherein the METS2 gene is overexpressed in rice plants, and transgenic rice with cold tolerance at the budding stage is obtained by screening and cultivation; the nucleotide sequence of the METS2 gene is shown in SEQ ID NO.1.

[0016] The advantages and positive effects of the METS2 gene and protein for regulating cold tolerance in rice bud stage and their application according to the present invention are:

[0017] 1. The present invention discloses the METS2 gene, which can improve rice cold tolerance during the budding stage. Experiments demonstrate that enhancing the function or increasing the expression of the METS2 gene, which codes for rice cold tolerance during the budding stage, can produce rice with improved cold tolerance during the budding stage. This demonstrates that the METS2 gene or its protein plays an important role in controlling rice cold tolerance during the budding stage. This research not only provides a basis for further elucidating the molecular mechanism of rice cold tolerance, but also provides new genetic and breeding resources for rice breeding.

[0018] 2. The METS2 gene-enhanced transgenic rice obtained in the present invention can be used as a new rice germplasm material to study the cold tolerance of rice during the bud stage, and has important application value for effectively regulating the cold tolerance of rice by using this gene resource through genetic breeding and genetic engineering methods.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The relative expression results of the overexpressed genes in METS2-OE1, METS2-OE2 plants and the parent rice Guanghui 998 (abbreviated as GH998) in the examples of the present invention are shown;

[0021] Figure 2 This is a comparison chart of the survival rate of transgenic rice overexpressing the METS2 gene encoding rice related to rice cold tolerance under low temperature during the germination period in an embodiment of the present invention, wherein a represents the germination situation of rice and b represents the survival rate statistics of rice. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0024] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0025] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0026] Example 1

[0027] Construction of rice cold tolerance-related gene overexpression vector:

[0028] 1. Construction of overexpression vector:

[0029] 1.1 Acquisition of METS2 gene:

[0030] The target gene was obtained by PCR amplification using DNA from common wild rice Y11 (Oryza rufipogon Griff.) collected from the Rice Research Institute of Guangxi Academy of Agricultural Sciences (Guangxi Academy of Agricultural Sciences Germplasm Bank) as a template using the following primers: primer1 and primer2:

[0031] primer1:5'-atggcgtctcacattgttgg-3'(SEQ ID NO.3);

[0032] primer2:5'-tcacttcgcgctagcgag-3' (SEQ ID NO. 4).

[0033] The PCR product was recovered and purified, and then connected to the Zero sequencing vector (purchased from Beijing Quanshijin Company), transformed into DH5α competent cells, and positive clones were selected and sequenced.

[0034] The sequencing results showed that the sequence of the PCR product was as shown in SEQ ID NO. 1, with a length of 2301 bp, and it was named METS2 gene.

[0035]

[0036] The amino acid sequence of METS2 is shown in SEQ ID NO.2:

[0037] MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMADAGIKYIPSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGFSTYFSMA RGNATVPAMEMTKWFDTNYHFIVPELGPNTKFSYSSHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKPAKGVEKSFALLSLLSSILPVYKEVIAELKAAGATWIQFDEPTLVLDLDSHQLAAFSAAYTELESALSGLNVLIETYFADIPAESYKTLTSLNSVTAYGFDLIRGFKTLDLVKSAGFPSGKYLFAGVVDGRNIWADDLAASLTTLESLEAIVGKDKLVVSTSCSLMHTAVDLVNETKLDSEIKSWLAFAAQKVVEVNALAKALAGQKDEAYFAANTAAQASRRSSPRVTNEEVQKAAAALRGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTVELRRVRREYKAKKISEDEYVSAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGSRCVKPPIIYGDVSRPNAMTVFWSKMAQSMTSRPMKGMLTGPVTILNWSFVRNDQPRFETCYQIALAIKKEVEDLEAGGIQVIQIDEAALREGLPLRKAEHAFYLDWAVHSFRITNCGVQDTTQIHTHMCYSNFNDIIHSIINMDADVITIENSRSDEKLLSVFREGVKYGAGIGPGVYDIHSPRIPSTEEIADRINKMLAVLDTNILWVNPDCGLKTRKYTEVKPALTNMVLAAKLIRTQLASAK*(SEQ ID NO.2).

[0038] [[ID=*]]1.2 Construction of the overexpression vector of the cold tolerance-related gene METS2 in common wild rice (i.e., the recombinant expression vector PMDC32-OE-METS2):

[0039] 1) Using primers 1 and 2 to amplify wild rice cDNA, the METS2 gene sequence was obtained and ligated into the vector Zero to obtain a positive clone of recombinant Zero-METS2. The recombinant vector Zero-METS2 was digested with restriction endonucleases KpnI and PacI to obtain the OE-METS2 fragment;

[0040] 2) digesting the expression vector PMDC32 with the restriction endonucleases Kpn I and Pac I to obtain a linear expression vector PMDC32, and recovering the linear fragment; integrating the OE-METS2 fragment obtained in step 1) into the linear expression vector PMDC32 using homologous recombination directional cloning (for specific methods, refer to the PMDC32 instruction manual) to obtain a homologous recombination product 1 (i.e., PMDC32-OE-METS2, which is the METS2 gene overexpression vector of the present invention); and then transforming the homologous recombination product 1 into DH5α competent cells and culturing at 37° C. overnight;

[0041] 3) The recombinant vector PMDC32-OE-METS2 obtained in step 2) was sequenced. The results showed that the recombinant vector had the nucleotide sequence shown in SEQ ID NO. 1 inserted in the forward direction of the Kpn I restriction site of the expression vector PMDC32, that is, the DNA sequence between the Kpn I and PacI recognition sites (recognition sequence) of PMDC32 was successfully replaced with the DNA sequence shown in SEQ ID No. 1.

[0042] 1.3 Conversion:

[0043] Heat shock method to transform E. coli: Take 5 μL of recombinant vector pMDC32-OE-METS2 and transform E. coli by heat shock method. The specific steps refer to Tiangen transformation kit.

[0044] 1.4 Bacterial liquid PCR verification:

[0045] In a clean bench, a single colony was picked as a template for PCR amplification using the Taq DNA polymerase PCR amplification system and procedure.

[0046] 1.5 Plasmid extraction: Extract the plasmid and detect the concentration according to the instructions of the Tiangen Plasmid Extraction Kit to obtain the recombinant expression vector PMDC32-OE-METS2.

[0047] Example 2

[0048] Cultivation of transgenic plants overexpressing the rice gene METS2 encoding cold tolerance and identification of transgenic plants:

[0049] 1. Cultivation of transgenic plants overexpressing METS2 gene:

[0050] The recombinant vector PMDC32-OE-METS2 was transformed into GH998 indica rice via Agrobacterium tumefaciens EHA105. The specific method is as follows:

[0051] 1. Plasmid transformation:

[0052] The recombinant vector PMDC32-OE-METS2 obtained in Example 1 was introduced into Agrobacterium tumefaciens EHA105 using a heat shock method to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE-METS2; the recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE-METS2 was cultured at 28° C. for 16 h, and the cells were collected; the cells were diluted with N6 liquid medium (Sigma, catalog number C1416) containing 100 μM acetosyringone to obtain a diluted bacterial solution, wherein the OD600 of the diluted bacterial solution was ≈0.5;

[0053] 2. Infection:

[0054] The mature embryonic callus of GH998 indica rice cultured for one month was mixed with the diluted bacterial solution obtained in step 1 and infected for 30 minutes. The bacterial solution was dried with filter paper and then transferred to N6 solid co-cultivation medium. The callus was co-cultivated at 24°C for 3 days to obtain the co-cultivated callus.

[0055] 3. Screening:

[0056] 3.1 The callus tissue after the co-cultivation treatment in step 2 was inoculated on N6 solid screening medium containing 150 mg / L hygromycin (hygromycin was added to N6 solid screening medium to obtain N6 solid screening medium, and the mass concentration of hygromycin in N6 solid screening medium was 150 mg / L) for the first screening;

[0057] 3.2 On the 16th day after the first screening, healthy calli were selected and transferred to N6 solid screening medium containing 200 mg / L hygromycin (hygromycin was added to N6 solid screening medium to obtain N6 solid screening medium, and the hygromycin concentration in N6 solid screening medium was 200 mg / L) for a second screening. Subculture was performed every 15 days for a total of one subculture to obtain resistant calli.

[0058] 4. Differentiation culture to obtain overexpression positive plants:

[0059] Resistant calli obtained in step 3 were selected and transferred to differentiation medium containing 150 mg / L hygromycin (differentiation medium: 2 mg 6-BA, 0.2 mg NAA, 4 g N6, 1 g casein hydrolyzate, 0.1 g inositol, 25 g sucrose, 2.4 g sorbitol, 7 g agar powder, 1 L deionized water) for differentiation. The culture medium was incubated at 24°C for 45 days (at which point the plant's aerial height was approximately 15 cm). The seedlings were opened and hardened for 3 days before being transplanted to a greenhouse for cultivation. These were PMDC32-OE-METS2 plants (denoted as the T0 generation). Positive plants obtained for different transformation events (transformed into different calli) were designated METS2-OE1 and METS2-OE2.

[0060] 2. PCR identification of METS2 transgenic plants:

[0061] Genomic DNA was extracted from the leaves of T1 seedlings of the above-mentioned METS2-OE1 and METS2-OE2 plants and the seedlings of the recipient parent rice GH998 plants, and PCR molecular detection was performed using primers hyg-F and hyg-R to identify positive seedlings. After PCR verification, the individual plants with hyg-positive bands, namely the above-mentioned METS2-OE1 and METS2-OE2, were identified as transgenic plants.

[0062] hyg-F:5'-AAAAGTTCGACAGCGTCTCCGACC-3'(SEQ ID NO.5)

[0063] hyg-R:5'-TCTACACAGCCATCGGTCCAGACG-3'(SEQ ID NO.6)

[0064] The PCR system is shown in Table 1:

[0065] Table 1 PCR system

[0066]

[0067] The amplification program is shown in Table 2:

[0068] Table 2 Amplification procedures

[0069] Temperature (℃) time Number of cycles 94 2min 1 98 10s 1 55 30s 1 68 1kb / min 25～40

[0070] 3. Identification of METS2 gene expression levels in METS2 transgenic plants:

[0071] RNA was extracted from the leaves of the T1 seedlings of the above-mentioned METS2-OE1 and METS2-OE2 plants and the recipient parent rice GH998 plants. Actin was set as the internal reference. Fluorescence quantitative PCR reactions were performed using the internal reference primers Actin-F and Actin-R and the METS2 gene-specific quantitative primers METS2-qRT-F and METS2-qRT-R to detect changes in the expression levels of the METS2 gene in different transgenic plants.

[0072] The results showed that ( Figure 1 ), the expression level of METS2 gene in the positive plants transformed with the recombinant vector PMDC32-OE-METS2 was significantly increased compared with the expression level of METS2 gene in the control strain (GH998). The above primers are as follows:

[0073] METS2-qRT-F:5'-GCGCTGAGGATTTGGAGAAG-3'(SEQ ID NO.7)

[0074] METS2-qRT-R:5'-ATAGCAGGGACAGTGGCATT-3'(SEQ ID NO.8)

[0075] Actin-F:5'-ATTTGGCACCACACATTCTAC-3'(SEQ ID NO.9)

[0076] Actin-R:5'-ATAACCTTCGTAGATTGGGACT-3'(SEQ ID NO.10)

[0077] IV. Phenotypic identification of METS2 transgenic plants:

[0078] METS2-OE1, METS2-OE2, and GH998 plants were planted in the transgenic experimental greenhouse of the Guangxi Academy of Agricultural Sciences. After harvesting, the seeds were germinated to 5 mm in length and lightly covered with distilled water. Thirty seeds were placed in each sterilized culture dish, with three replicates set up. The treatment was carried out at 4°C for 7 days. After removal, the seeds were allowed to recover at room temperature at 25°C for 7 days, and the survival rate of the seedlings was counted. The measurement and observation results are shown in Table 3. Figure 2 shown.

[0079] Table 3 Survival rate of rice plants overexpressing the cold tolerance-related gene METS2 under low temperature

[0080] serial number Survival rate of seedlings at low temperature (%) Standard error of survival rate (%) GH998 16.66 7.00 METS2-OE1 36.66 4.70 METS2-OE2 37.77 1.50

[0081] Compared with GH998 plants, PMDC32-OE-METS2 lines (METS2-OE1, METS2-OE2) showed a phenotype in which the survival rate of rice seedlings under low temperature at the bud stage was significantly higher than that of the control group (GH998) (P < 0.05), which proved that the METS2 gene is involved in controlling the cold tolerance of rice, that is, the METS2 gene is a gene related to the cold tolerance of rice at the bud stage, and overexpression of the METS2 gene can significantly improve the cold tolerance of rice at the bud stage.

[0082] Therefore, the present invention adopts the above-mentioned METS2 gene, protein and application thereof for regulating the cold tolerance of rice at the bud stage, and overexpresses the METS2 gene in rice, which can improve the cold tolerance of rice at the bud stage, indicating that the rice-related encoding gene METS2 or its protein plays an important role in regulating the cold tolerance of rice at the bud stage; it not only provides a basis for further clarifying the molecular mechanism of rice cold tolerance, but also provides new gene resources and breeding resources for rice breeding.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The METS2 gene for regulating cold tolerance in rice during the budding period is characterized by: The nucleotide sequence of the METS2 gene is shown in SEQ ID NO.

1.

2. Application of the METS2 gene in regulating cold tolerance at the budding stage of rice, characterized by: The nucleotide sequence of the METS2 gene is shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that: Overexpression of the METS2 gene in rice plants improves cold tolerance during the bud stage.

4. A METS2 protein for regulating cold tolerance in rice during the budding period, characterized in that: The amino acid sequence of the METS2 protein is shown in SEQ ID NO.2, and the gene encoding the METS2 protein is shown in SEQ ID NO.

1.

5. Application of METS2 protein in regulating cold tolerance at the budding stage of rice, characterized by: The amino acid sequence of the METS2 protein is shown in SEQ ID NO.2, and the gene encoding the METS2 protein is shown in SEQ ID NO.

1.

6. A recombinant vector, characterized in that: The recombinant vector comprises the METS2 gene described in claim 1, and the nucleotide sequence of the METS2 gene is shown in SEQ ID NO.

1.

7. The recombinant vector according to claim 6, characterized in that: The recombinant vector is a plant overexpression vector.

8. Use of the recombinant vector according to claim 6 or 7 in regulating cold tolerance of rice at the bud stage.

9. A method for cultivating cold-tolerant transgenic rice at the bud stage, characterized by: The METS2 gene was overexpressed in rice plants, and transgenic rice with cold tolerance at the bud stage was obtained by screening and cultivation; the nucleotide sequence of the METS2 gene is shown in SEQ ID NO.1.

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