Mets2 gene for regulating cold tolerance of rice bud stage, protein and application thereof

Abstract

The application relates to the technical field of genetic engineering, and discloses a METS2 gene for regulating cold tolerance of rice sprouts, a protein and application thereof, wherein the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1. The application also discloses application of the METS2 gene in regulating cold tolerance of rice sprouts. The application adopts the above-mentioned METS2 gene for regulating cold tolerance of rice sprouts, protein and application thereof, overexpresses the METS2 gene in rice, can improve the cold tolerance of rice sprouts in the bud stage, and indicates that the related coding gene METS2 or the protein thereof of rice plays an important role in regulating the cold tolerance of rice sprouts in the bud stage. The application not only provides a basis for further elucidating the molecular mechanism of the cold tolerance of rice, but also provides a new gene resource and a breeding resource for rice breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a METS2 gene for regulating cold tolerance of rice at the bud stage, a protein and application thereof. BACKGROUND

[0002] Rice is sensitive to cold stress throughout its entire growth period. Bud stage cold damage (late spring cold) leads to growth point necrosis and a sharp drop in germination rate; cold damage at the booting stage (cold dew wind) reduces pollen fertility and seed setting rate. Cultivation of cold-tolerant varieties is an economically effective solution, but modern cultivated rice has a narrow genetic diversity and has lost cold tolerance genes, making breeding more difficult. Guangxi, as one of the origins of rice, has abundant wild rice resources containing cold tolerance genes, which is the key to breaking through the bottleneck of breeding.

[0003] At present, a number of growth stage-specific cold tolerance genes have been cloned. At the bud stage / seedling stage: COLD1 activates G protein signaling; qLTG3-1 regulates the vacuolization of the aleurone layer; COG2 negatively regulates the cell wall; HAN1 fine-tunes the jasmonic acid pathway. At the booting stage: CTB4a (LRR kinase) enhances ATP synthesis to enhance cold tolerance; CTB5 (HD-Zip transcription factor) maintains gibberellin homeostasis and pollen fertility; OsSRO1c and OsDREB2B dynamically phase transition to regulate cold-responsive genes; bZIP73 Jap enhances reproductive stage cold tolerance. In addition, other cold tolerance molecular regulation mechanisms include OsMKK6 activating trehalose synthesis, ABF1 / 2-OsNAC regulating the network, CTB2 mediating sterol metabolism, and CTB6 regulating ROS homeostasis, etc.

[0004] Although great progress has been made in the discovery of cold tolerance genes and the analysis of molecular mechanisms, cold tolerance germplasm and genes that can be directly used for breeding are still relatively scarce. Therefore, in the future, it is still necessary to deeply mine cold tolerance QTLs / genes from a wide range of wild rice resources, and to analyze the cold tolerance molecular network and regulation mechanism, while creating new germplasm to break through the bottleneck of breeding. SUMMARY

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

[0006] To achieve the above purpose, the present application provides a METS2 gene for regulating cold tolerance of rice at the bud stage, and the nucleotide sequence of the METS2 gene is shown in SEQ ID NO. 1.

[0007] Further, the application further provides application of the METS2 gene in regulating cold tolerance of rice at the bud stage, wherein the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1.

[0008] Further, in the application, the METS2 gene is overexpressed in the rice plant to improve the cold tolerance of the rice at the bud stage.

[0009] Further, the application further provides a METS2 protein for regulating cold tolerance of rice at the bud stage, wherein the amino acid sequence of the METS2 protein is shown as SEQ ID NO. 2, and the coding gene of the METS2 protein is shown as SEQ ID NO. 1.

[0010] Further, the application further provides application of the METS2 protein in regulating cold tolerance of rice at the bud stage, wherein the coding gene of the METS2 protein is the METS2 gene, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1.

[0011] Further, the application further provides a recombinant vector, wherein the recombinant vector comprises the above-mentioned METS2 gene, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1.

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

[0013] A biological material comprises the above-mentioned recombinant vector, wherein the biological material is a recombinant microorganism, a transgenic plant cell line or a transgenic plant tissue, the recombinant microorganism is bacteria, yeast, algae or fungi, the bacteria is one of Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas or Bacillus.

[0014] Further, the application further provides application of the above-mentioned recombinant vector in regulating cold tolerance of rice at the bud stage.

[0015] Further, the application further provides a method for cultivating a transgenic rice with cold tolerance at the bud stage, wherein the METS2 gene is overexpressed in the rice plant, and a transgenic rice with cold tolerance at the bud stage is screened and cultivated, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1.

[0016] The METS2 gene for regulating cold tolerance of rice at the bud stage, the protein and the application thereof have the following advantages and positive effects:

[0017] 1. The application discloses a gene METS2 capable of improving the cold tolerance of rice at the bud stage, and proves through experiments that the rice with improved cold tolerance at the bud stage can be obtained under the condition that the coding gene METS2 related to the cold tolerance of rice at the bud stage is enhanced in function or increased in expression, thereby proving that the coding gene METS2 or the protein thereof of rice plays an important role in controlling the cold tolerance of rice at the bud stage. Not only does the application provide a basis for further elucidating the molecular mechanism of the cold tolerance of rice, but also provides new gene resources and breeding resources for rice breeding.

[0018] 2. The transgenic rice with the enhanced METS2 gene obtained by the application can be used as new rice germplasm material for researching the cold tolerance of rice at the bud stage, and has important application value for effectively regulating the cold tolerance of rice by genetic breeding and genetic engineering methods.

[0019] The technical solutions of the application are further described below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The relative expression amount of the overexpressed gene of the METS2-OE1 and METS2-OE2 plants in the examples of the application and the parent rice Guanghui 998 (GH998) is shown in the table.

[0021] Figure 2 The comparison chart of the survival rate of the transgenic rice with the overexpressed coding gene METS2 related to the cold tolerance of rice at the bud stage under low temperature at the bud stage is shown in the figure, wherein a is the germination of rice, and b is the survival rate statistics of rice. DETAILED DESCRIPTION

[0022] The technical solutions of the application are further described below with reference to the drawings and examples.

[0023] Unless otherwise defined, the technical terms or scientific terms used in the application shall have the usual meanings understood by those skilled in the art to which the application belongs.

[0024] Based on the examples in the application, all other examples obtained by those skilled in the art without making creative efforts fall within the scope of protection of the application. The experimental methods not specified in the following examples are usually determined according to national standards. The experimental instruments, equipment and reagents not specified in the following examples are all commercially available raw materials.

[0025] Unless otherwise defined or explained, all professional and scientific terms used in the application have the same meanings as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the method of the application. It should be noted that the examples in the application and the features in the examples can be combined with each other without conflict.

[0026] Example 1

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

[0028] 1. Construction of overexpression vector:

[0029] 1.1 Obtaining of METS2 gene:

[0030] The DNA of common wild rice Y11 (Oryza rufipogon Griff.) collected by Rice Institute of Guangxi Academy of Agricultural Sciences (Germplasm Bank of Guangxi Academy of Agricultural Sciences) was used as a template, and the following primers primer 1 and primer 2 were used for PCR amplification to obtain the target gene:

[0031] primer 1: 5'-atggcgtctcacattgttgg-3' (SEQ ID NO. 3);

[0032] primer 2: 5'-tcacttcgcgctagcgag-3' (SEQ ID NO. 4).

[0033] After the PCR product was recovered and purified, it was connected into a Zero sequencing vector (purchased from Beijing Zobenjin Company), and DH5a competent cells were transformed. After positive clones were selected, sequencing was performed.

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

[0035]

[0036] The sequence of the METS2 protein is shown as SEQ ID NO. 2:

[0037] MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMADAGIKYIPSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGFSTYFSMARGNATVPAMEMTKWFDTNYHFIVPELGPNTKFSYSSHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKPAKGVEKSFALLSLLSSILPVYKEVIAELKAAGATWIQFDEPTLVLDLDSHQLAAFSAAYTELESALSGLNVLIETYFADIPAESYKTLTSLNSVTAYGFDLIRGFKTLDLVKSAGFPSGKYLFAGVVDGRNIWADDLAASLTTLESLEAIVGKDKLVVSTSCSLMHTAVDLVNETKLDSEIKSWLAFAAQKVVEVNALAKALAGQKDEAYFAANTAAQASRRSSPRVTNEEVQKAAAALRGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTVELRRVRREYKAKKISEDEYVSAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGSRCVKPPIIYGDVSRPNAMTVFWSKMAQSMTSRPMKGMLTGPVTILNWSFVRNDQPRFETCYQIALAIKKEVEDLEAGGIQVIQIDEAALREGLPLRKAEHAFYLDWAVHSFRITNCGVQDTTQIHTHMCYSNFNDIIHSIINMDADVITIENSRSDEKLLSVFREGVKYGAGIGPGVYDIHSPRIPSTEEIADRINKMLAVLDTNILWVNPDCGLKTRKYTEVKPALTNMVLAAKLIRTQLASAK*(SEQ ID NO. 2).

[0038] 1.2 Construction of the overexpression vector of the cold tolerance related gene METS2 of O. officinalis (i.e. the recombinant expression vector PMDC32-OE-METS2):

[0039] 1) The wild rice cDNA was amplified by primer 1 and primer 2 to obtain the sequence of the METS2 gene, and was connected to the vector Zero to obtain a positive clone of the recombinant Zero-METS2, and the recombinant vector Zero-METS2 was digested by restriction endonuclease Kpn I and Pac I to obtain the OE-METS2 fragment;

[0040] 2) The expression vector PMDC32 was digested by restriction endonuclease Kpn I and Pac I to obtain a linear expression vector PMDC32, and the linear fragment was recovered; the fragment OE-METS2 obtained in step 1) was integrated into the linear expression vector PMDC32 by the method of homologous recombination directed cloning (for details, refer to the instruction manual of PMDC32) to obtain a homologous recombination product 1 (i.e. PMDC32-OE-METS2, which is the overexpression vector of the gene METS2 according to the application), and then the homologous recombination product 1 was transformed into DH5α competent cells and cultured at 37°C overnight;

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

[0042] 1.3 Transformation:

[0043] Heat shock method was used to transform E. coli with 5 μL of the recombinant vector PMDC32-OE-METS2, and the specific steps refer to the Transgen kit.

[0044] 1.4 Bacterial liquid PCR verification:

[0045] In the super-clean bench, a single colony was picked as a template for PCR amplification, and Taq DNA polymerase PCR amplification system and program were used.

[0046] 1.5 Plasmid extraction: The plasmid was extracted and the concentration was detected according to the instruction manual of the Transgen plasmid extraction kit, and the recombinant expression vector PMDC32-OE-METS2 was obtained.

[0047] Example 2

[0048] Cultivation of transgenic plants of the overexpression vector of the cold tolerance related coding gene METS2 of rice and identification of the transgenic plants:

[0049] I. Cultivation of the METS2 gene overexpression transgenic plants:

[0050] The recombinant vector PMDC32-OE-METS2 was transformed into GH998 indica rice by Agrobacterium tumefaciens EHA105 mediation, and the method was as follows:

[0051] 1. Plasmid transformation:

[0052] The recombinant vector PMDC32-OE-METS2 obtained in Example 1 was introduced into Agrobacterium tumefaciens EHA105 by heat shock 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 16h, and the bacterial cells were collected; the bacterial cells were diluted with N6 liquid medium (Sigma, product catalog number C1416) containing 100μM acetyl-syringone to obtain a diluted bacterial solution, and the OD600of the diluted bacterial solution was about 0.5;

[0053] 2. Infection:

[0054] The mature embryo somatic callus of GH998 indica rice cultured for one month was mixed with the diluted bacterial solution obtained in step 1 for infection for 30min, and then the bacterial solution was absorbed with filter paper and transferred into N6 solid co-culture medium for co-culture at 24°C for 3d to obtain callus treated by co-culture;

[0055] 3. Selection:

[0056] 3.1 The callus treated by co-culture in step 2 was inoculated on N6 solid selection medium containing 150mg / L hygromycin (N6 solid selection medium was obtained by adding hygromycin to N6 solid medium, and the mass concentration of hygromycin in N6 solid selection medium was 150mg / L) for the first selection;

[0057] 3.2 The healthy callus was picked up on the 16th day after the start of the first selection and transferred into N6 solid selection medium containing 200mg / L hygromycin (N6 solid selection medium was obtained by adding hygromycin to N6 solid medium, and the mass concentration of hygromycin in N6 solid selection medium was 200mg / L) for the second selection, and the callus was subcultured every 15d for a total of 1 time to obtain resistant callus;

[0058] 4. Obtaining overexpression positive plants by differentiation culture:

[0059] The resistant callus obtained in step 3 was transferred to a differentiation medium containing 150 mg / L hygromycin (differentiation medium: 6-BA 2 mg, NAA 0.2 mg, N6 4 g, hydrolyzed casein 1 g, inositol 0.1 g, sucrose 25 g, sorbitol 2.4 g, agar powder 7 g, deionized water 1 L) for differentiation, and cultured at 24°C for 45 d (at this time, the height of the aerial part of the plant was about 15 cm), the bottle opening was opened for 3 d for seedling raising, and then transplanted to a greenhouse for cultivation, which was a PMDC32-OE-METS2 plant (referred to as T0 generation). The obtained positive plants were named as METS2-OE1 and METS2-OE2 plants for different transformation events (transformed into different calli).

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

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

[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 program

[0069] Temperature (°C) Time Cycle number 94 2 min 1 98 10s 1 55 30s 1 68 1 kb / min 25～40

[0070] III. Identification of the expression level of the METS2 gene in the METS2 transgenic plants:

[0071] The RNA of the T1 generation seedlings of the above-mentioned METS2-OE1 and METS2-OE2 plants and the leaf of the receptor parent rice GH998 plant was extracted, the internal reference was set as Actin, the internal reference primers Actin-F and Actin-R and the METS2 gene specific quantitative primers METS2-qRT-F and METS2-qRT-R were used to perform the fluorescent quantitative PCR reaction to detect the change of the expression level of the METS2 gene of different transgenic plants.

[0072] The results show that Figure 1 , the expression level of the METS2 gene in the positive plants into which the recombinant vector PMDC32-OE-METS2 is transferred is significantly higher than that of the METS2 gene of the control strain (GH998), and the 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] Four, phenotype identification of the METS2 transgenic plants:

[0078] The METS2-OE1, METS2-OE2 and GH998 plants were respectively planted in the transgenic test greenhouse of Guangxi Academy of Agricultural Sciences, after the seeds were harvested, germination was induced to 5mm long, the seeds were covered with distilled water, 30 seeds were placed in each sterilized culture dish, 3 repeats were set, and the treatment was performed at 4°C for 7d. After taking out, recovery was performed at 25°C for 7d, and the survival rate was counted. The measurement observation results are shown in Table 3, Figure 2 .

[0079] Table 3: Survival rate of rice cold tolerance related coding gene METS2 overexpression plant under low temperature

[0080] Number Viable rate at low temperature (%) Standard error of viable rate (%) GH998 16.66 7.00 METS2-OE1 36.66 4.70 METS2-OE2 37.77 1.50

[0081] Compared with GH998 plants, the PMDC32-OE-METS2 strains (METS2-OE1, METS2-OE2) all have the phenotype that the survival rate of rice at low temperature at the bud stage is significantly higher than that of the control group (GH998) (P<0.05), thereby proving that the METS2 gene is involved in controlling the cold tolerance of rice, that is, the METS2 gene is a rice bud stage cold tolerance related gene, and overexpression of the METS2 gene can significantly improve the bud stage cold tolerance of rice.

[0082] Therefore, the application of the above-mentioned METS2 gene for regulating the cold tolerance of rice at the bud stage, protein and application can overexpress the METS2 gene in rice, which can improve the cold tolerance of rice at the bud stage, and it is proved that the rice related coding gene METS2 or its protein plays an important role in regulating the cold tolerance of rice at the bud stage; not only provides a basis for further elucidating the molecular mechanism of the cold tolerance of rice, but also provides a new gene resource and breeding resource for rice breeding.

[0083] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Application of the MTS2 gene in regulating cold tolerance at the bud stage of rice, characterized in that: The nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1; the METS2 gene is overexpressed in rice plants to improve the cold tolerance of the rice at the bud stage.

2. Use of a METS2 protein in regulating cold tolerance at the seedling stage of rice, characterized in that: The amino acid sequence of the METS2 protein is shown as SEQ ID NO. 2, and the coding gene of the METS2 protein is shown as SEQ ID NO. 1; the METS2 gene is overexpressed in rice plants to improve the cold tolerance of the rice at the bud stage.

3. Use of the recombinant vector in regulating cold tolerance at the bud stage of rice, characterized in that: The recombinant vector is a plant overexpression vector, and the recombinant vector comprises the METS2 gene shown in claim 1, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO.

1.

4. A method for cultivating cold-resistant transgenic rice during the germination stage, characterized in that: The METS2 gene is overexpressed in rice plants to screen and breed the transgenic rice with cold tolerance at the bud stage, and the nucleotide sequence of the METS2 gene is shown as SEQ ID NO. 1.

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