Rice bud-stage cold-tolerant gene and application thereof

Abstract

The invention relates to a rice bud-stage cold tolerance related coding gene Leng8. The gene comprises (a) a nucleotide sequence as shown in SEQ ID No: 1, (b) a nucleotide sequence as shown in SEQ ID No: 2, and (c) a nucleotide sequence as shown in SEQ ID No: 3; and (b) a nucleotide sequence which is generated by adding, substituting or deleting one or more basic groups from the nucleotide sequence as shown in SEQ ID No: 1 and is used for coding the cold-tolerant functional protein in the rice bud stage. The invention belongs to the technical field of gene engineering. The cold resistance of the rice in the bud stage is improved by overexpressing the gene.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a cold-resistant gene in the booting stage of rice and an application thereof. Background Art

[0002] Rice originated in tropical regions and is a low-temperature sensitive crop. Low temperatures severely restrict the cultivation of rice in high-latitude and high-altitude areas. The rice bud stage refers to the stage from seed germination to the unfolding of the first true leaf. This stage lasts about 7 to 10 days. The seedlings mainly rely on the nutrients stored inside the seeds for growth, and the root system begins to develop to absorb water and nutrients from the soil. During the bud stage, the germination process of rice seeds requires suitable temperature and humidity conditions. After the seeds absorb water, the embryo begins to grow and breaks through the seed coat to form a seedling. The length of the germination period is affected by environmental conditions. If the temperature is too low, the germination rate will be low, affecting the growth of rice.

[0003] In addition, the rich rice resources contain many cold-resistant genes. Discovering and identifying excellent genes can broaden the gene sources for rice breeding and enrich the gene background. Discovering and utilizing the cold-resistant genes of rice is a practical problem that needs to be solved urgently. However, cold-resistant genes need to be verified by transgenic verification or by constructing populations and near-isogenic lines. It is difficult to discover cold-resistant genes obtained through natural variation of rice. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a rice cold-tolerant gene at the booting stage and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a rice bud stage cold tolerance-related coding gene Leng8, wherein the gene has the following nucleotide sequence (a) or (b):

[0007] (a) having a nucleotide sequence as shown in SEQ ID No: 1;

[0008] (b) The nucleotide sequence shown in SEQ ID No: 1 is generated by adding, replacing or deleting one or more bases, and encodes a nucleotide sequence of a rice sprout stage cold-tolerant functional protein.

[0009] In a second aspect, the present invention provides the use of the Leng8 gene described in the first aspect in regulating the cold tolerance of rice at the bud stage.

[0010] As a preferred embodiment of the second aspect, the Leng8 gene improves the cold tolerance of rice at the bud stage by overexpression.

[0011] In a third aspect, the present invention provides a rice bud stage cold-tolerant protein, wherein the protein is encoded by the gene described in the first aspect.

[0012] In a fourth aspect, the present invention provides use of the protein described in the third aspect in regulating cold tolerance of rice during the bud stage.

[0013] In a fifth aspect, the present invention provides a recombinant vector comprising the gene sequence described in the first aspect.

[0014] In a sixth aspect, the present invention provides a biomaterial comprising the recombinant vector described in the fifth aspect.

[0015] As a preferred embodiment of the sixth aspect, the biological material is a recombinant microorganism, a transgenic plant cell line or a transgenic plant tissue; the recombinant microorganism is a bacterium, a yeast, an algae or a fungus; the bacterium is one of Escherichia, Erwinia, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas or Bacillus.

[0016] The recombinant microorganism is a bacterium, yeast, algae or fungus; the bacterium is one of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas or Bacillus.

[0017] In a seventh aspect, the present invention provides use of the recombinant vector described in the fifth aspect or the recombinant microorganism described in the sixth aspect in regulating cold tolerance of rice at the bud stage.

[0018] In an eighth aspect, the present invention provides a method for cultivating transgenic rice that is cold-resistant at the bud stage, the method utilizing transgenic technology to transform a nucleotide sequence encoding the Leng8 gene into a recipient rice, thereby increasing the expression level of the Leng8 gene in the recipient rice and obtaining transgenic rice that is cold-resistant at the bud stage.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention has discovered a gene Leng8 encoding rice cold tolerance during the bud stage. When the function of the gene Leng8 encoding rice cold tolerance during the bud stage is enhanced or the expression level is increased, cold-tolerant rice can be obtained, proving that the protein of the gene encoding rice cold tolerance during the bud stage or its protein plays an important role in controlling the cold tolerance of rice during the bud stage. The present invention 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. The Leng8 gene-enhanced transgenic rice obtained by the present invention, as a new rice germplasm material, can be used to study the mechanism of rice cold tolerance during the bud stage and discover more genes regulating rice cold tolerance during the bud stage, and has important application value for effectively regulating rice grain shape by using the gene resources through genetic breeding and genetic engineering methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of the transcription level of Leng8 in the overexpressing transgenic rice with increased expression level of the cold-tolerant gene Leng8 at the rice bud stage;

[0022] Figure 2 This is a schematic diagram of the expression level of Leng8 gene in transgenic rice overexpressing the cold-tolerant gene Leng8 at the rice bud stage;

[0023] Figure 3 This is a schematic diagram of the survival rate of transgenic rice under low temperature that overexpresses the cold-tolerant gene Leng8 at the rice bud stage. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] Example 1

[0026] Construction of overexpression vector of genes encoding cold tolerance at bud stage in rice:

[0027] 1. Construction of overexpression vector

[0028] 1.1 Acquisition of Leng8 gene

[0029] Using DNA from common wild rice Y12 (Oryzarufipogon Griff.) as a template, PCR amplification was performed using the following primers primer1 and primer2 to obtain the target gene:

[0030] primer1: 5'-ATGGCCGGGCGTGGGCGTGGTAGTAG-3' (SEQ ID No. 2);

[0031] Primer2: 5'-TTACAAATCCGTTTGAGATGAGACG-3' (SEQ ID No. 3).

[0032] 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 the positive clones were selected and sequenced.

[0033] The sequencing results showed that the PCR product sequence was as shown in SEQ ID No. 1, with a length of 1347 bp, and it was named Leng8 gene.

[0034]

[0035] 1.2 Construction of the overexpression vector of the common wild rice grain type-related gene Leng8 (i.e., the recombinant expression vector PMDC32-OE-Leng8):

[0036] 1) Using primer 1 and primer 2 to amplify wild rice cDNA, the sequence of Leng8 gene was obtained, and the sequence was connected to the vector Zero to obtain the positive clone of recombinant Zero-Leng8. The recombinant vector Zero-Leng8 was digested with restriction endonucleases KpnI and PacI to obtain the OE-Leng8 fragment;

[0037] 2) digesting the expression vector PMDC32 with restriction endonucleases Kpn I and Pac I to obtain a linear expression vector PMDC32, and recovering the linear fragment; integrating the fragment OE-Leng8 obtained in step 1) into the linear expression vector PMDC32 by homologous recombination directional cloning (for specific methods, refer to the instruction manual of PMDC32) to obtain a homologous recombination product 1 (i.e. PMDC32-OE-Leng8, which is the gene Leng8 overexpression vector of the present invention), and then transferring the homologous recombination product 1 into DH5α competent cells and culturing at 37° C. overnight;

[0038] 3) The recombinant vector PMDC32-OE-Leng8 obtained in step 2) was sequenced, and the results showed that the recombinant vector was inserted with the nucleotide sequence shown in SEQ ID No.1 in the forward direction at the Kpn I restriction site of the expression vector PMDC32, that is, the DNA sequence between the Kpn I and PacI recognition sites (recognition sequences) of PMDC32 was successfully replaced with the DNA sequence shown in SEQ ID No.1.

[0039] 1.3 Conversion

[0040] Heat shock method to transform E. coli: Take 5 μL of the recombinant vector PMDC32-OE-Leng8 and transform E. coli by heat shock method. The specific steps refer to the Tiangen transformation kit.

[0041] 1.6 Bacterial liquid PCR verification

[0042] In the clean bench, a single colony was picked as a template for PCR amplification, using the Taq DNA polymerase PCR amplification system and procedure.

[0043] 1.7 Plasmid extraction In this experiment, the plasmid was extracted and the concentration was detected according to the instructions of the Tiangen Plasmid Extraction Kit.

[0044] Example 2

[0045] Cultivation of transgenic plants carrying the overexpression vector of Leng8, a gene encoding rice grain shape, growth and development, and identification of transgenic plants

[0046] 1. Cultivation of transgenic plants with Leng8 gene overexpression vector

[0047] The recombinant vector PMDC32-OE-Leng8 was transformed into Nipponbare japonica rice mediated by Agrobacterium tumefaciens EHA105. The specific method is as follows:

[0048] 1. Plasmid transformation:

[0049] The recombinant vector PMDC32-OE-Leng8 obtained in Example 1 was introduced into Agrobacterium tumefaciens EHA105 by heat shock method to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE-Leng8; the recombinant Agrobacterium tumefaciens EHA105 containing the recombinant vector PMDC32-OE-Leng8 was cultured at 28° C. for 16 h, and the cells were collected; the cells were diluted with N6 liquid culture medium (Sigma, product catalog number C1416) containing 100 μM acetosyringone to obtain a diluted bacterial solution, and the OD600 of the diluted bacterial solution was ≈0.5;

[0050] 2. Infection:

[0051] Mix the rice mature embryonic callus cultured for one month with the diluted bacterial solution obtained in step 1 and infect for 30 minutes, use filter paper to dry the bacterial solution and then transfer it to N6 solid co-cultivation medium, co-cultivate at 24° C. for 3 days, and obtain co-cultivated callus;

[0052] 3. Screening:

[0053] 3.1 The callus after the co-cultivation treatment in step 2 was inoculated on N6 solid screening medium containing hygromycin at a mass concentration of 150 mg / L (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;

[0054] 3.2 On the 16th day after the first screening, healthy callus tissue was selected and transferred to N6 solid screening medium containing hygromycin at a mass concentration of 200 mg / L (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 200 mg / L) for the second screening, subcultured once every 15 days, and subcultured once in total to obtain resistant callus tissue;

[0055] 4. Differentiation culture to obtain overexpression positive plants:

[0056] The resistant callus obtained in step 3 was selected and transferred to a differentiation medium containing 150 mg / L hygromycin (differentiation medium: 2 mg 6-BA, 0.2 mg NAA, 4 g N6, 1 g hydrolyzed casein, 0.1 g inositol, 25 g sucrose, 2.4 g sorbitol, 7 g agar powder, 1 L deionized water) for differentiation, cultured at 24°C for 45 days (at this time, the height of the aboveground part of the plant was about 15 cm), opened the bottle mouth for 3 days of hardening, and then transplanted to a greenhouse for cultivation, which is the PMDC32-OE-Leng8-transformed plant (recorded as T0 generation). Different transformation events were named N-OE1 and N-OE2, representing the overexpression positive plants transferred into the recombinant vector PMDC32-OE-Leng8.

[0057] 2. PCR identification of Leng8 transgenic plants:

[0058] The genomic DNA of the T0 generation seedlings of the above-mentioned N-OE1 and N-OE2 plants and the recipient parent rice Guanghui 998 plant (abbreviated as GH998 in the attached figure) was extracted, and the primers primer3 and primer4 were used to perform PCR molecular detection to identify the positive seedlings. After sequencing verification, the homozygous plants with sequence mutations, namely the above-mentioned N-OE1 and N-OE2 ( Figure 1 ).

[0059] primer3: 5'-ATGGCCGGGCGTGGGCGTGGTAGTAG-3'(SEQ ID No.4)

[0060] primer4: 5'-TTACAAATCCGTTTGAGATGAGACG-3' (SEQ ID No.5)

[0061] PCR system:

[0062] 2× PCR SuperMix (+dye) 5μl, 100ng / ml primer3 1μl, primer4 1μl, DNA 1μl, ddH 2 O 2μl amplification procedure:

[0063] 94℃2min;

[0064] ↓

[0065] 98℃10sec;

[0066] 55℃30sec;

[0067] 68℃1min / kb 25~40cycles.

[0068] 3. Identification of Leng8 gene expression level in Leng8 transgenic plants:

[0069] RNA was extracted from leaves of the T0 generation seedlings of the above-mentioned N-OE1 and N-OE2 plants (abbreviated as OE1 and OE2 in the attached figure) and the recipient parent rice Riguanghui 998 plant (abbreviated as GH998 in the attached figure), and Actin was set as the internal reference. The internal reference primers Actin-F and Actin-R, and the Leng8 gene-specific quantitative primers Leng8-qRT-F and Leng8-qRT-R were used to perform fluorescence quantitative PCR reaction to detect the changes in the expression level of the Leng8 gene in different transgenic plants; the results showed that ( Figure 2 ), the expression level of Leng8 gene in the positive plants (OE1, OE2) transformed with the recombinant vector PMDC32-OE-Leng8 was significantly increased compared with the expression level of Leng8 gene in the control strain (GH998). The above primers are as follows:

[0070] Actin-F: 5'-ATTTGGCACCACACATTCTAC-3' (SEQ ID No. 6)

[0071] Actin-R: 5'-ATAACCTTCGTAGATTGGGACT-3' (SEQ ID No. 7)

[0072] Leng8-qRT-F:5'-GCGTGGTAGTAGATCCGGAG-3'(SEQ ID No.8)

[0073] Leng8-qRT-R: 5'-CGGTGATGGCGTTGTAGATG-3' (SEQ ID No. 9)

[0074] 4. Phenotypic identification of Leng8 transgenic plants

[0075] N-OE1 and N-OE2 plants (abbreviated as OE1 and OE2 in the attached figure) and the recipient parent rice Guanghui 998 plants (abbreviated as GH998 in the attached figure) were planted in the Hainan experimental base to observe the phenotypic differences between N-OE1 and N-OE2 plants and the recipient parent rice Guanghui 998 plants under low temperature (rice with 3-day germination and 5mm buds were selected and treated at 4℃ for 7 days and at room temperature for 5 days to calculate the survival rate under low temperature). The measurement and observation results are as follows Figure 3As shown in Table 1, compared with the recipient parent rice Guanghui 998 plants, PMDC32-OE-Leng8 plants all showed a phenotype of higher rice germination rate under low temperature than the control group, which proved that the Leng8 gene is involved in controlling the cold tolerance of rice during the bud stage, that is, the Leng8 gene is a gene related to the cold tolerance of rice during the bud stage, and overexpression of this gene can increase the survival rate of rice seedlings under low temperature environment.

[0076] Table 1: Grain length performance of rice plants with knockout gene Leng8 encoding grain shape growth and development

[0077] Plant name Survival rate of seedlings under low temperature (%) Standard error of survival rate (%) Guanghui 998 4.44 3.14％ OE1 56.67 7.20％ OE2 37.77 3.14％

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A rice bud stage cold tolerance-related coding gene Leng8, characterized in that: The gene has the following nucleotide sequence (a) or (b): (a) having a nucleotide sequence as shown in SEQ ID No: 1; (b) The nucleotide sequence shown in SEQ ID No: 1 is generated by adding, replacing or deleting one or more bases, and encodes a nucleotide sequence of a rice sprout stage cold-tolerant functional protein.

2. Use of the Leng8 gene according to claim 1 in regulating cold tolerance of rice during the bud stage.

3. The use according to claim 2, characterized in that: The Leng8 gene improves the cold tolerance of rice during the bud stage through overexpression.

4. A rice sprout cold-resistant protein, characterized in that: The protein is encoded by the gene according to claim 1.

5. Use of the protein according to claim 4 in regulating the cold tolerance of rice during the budding period.

6. A recombinant vector, characterized in that: Containing the gene according to claim 1.

7. A biomaterial, characterized in that: Containing the recombinant vector according to claim 6.

8. The biomaterial according to claim 7, characterized in that The biological material is a recombinant microorganism, a transgenic plant cell line or a transgenic plant tissue; the recombinant microorganism is a bacterium, a yeast, an algae or a fungus; the bacterium is one of Escherichia, Erwinia, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas or Bacillus.

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

10. A method for cultivating cold-tolerant transgenic rice at the bud stage, characterized in that: The method utilizes transgenic technology to transform a nucleotide sequence encoding the Leng8 gene into a recipient rice, thereby increasing the expression level of the Leng8 gene in the recipient rice and obtaining a transgenic rice that is cold-resistant during the bud stage.

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