CTB5 protein related to low-temperature tolerance of rice as well as related biological materials and application of CTB5 protein

Abstract

The invention discloses a CTB5 protein related to low-temperature tolerance of rice as well as a related biological material and application of the CTB5 protein. The amino acid sequence of the CTB5 protein is as shown in SEQ ID NO. 1. Experiments prove that when the CTB5 gene is over-expressed in the rice variety Towada, the cold resistance in the seedling stage and the cold resistance in the booting stage are remarkably improved; the CTB5 gene is knocked out from a rice variety Nipponbare, and the cold resistance in the seedling stage and the cold resistance in the booting stage are remarkably reduced. Therefore, the CTB5 protein positively regulates and controls the low-temperature tolerance of the rice. In addition, the invention provides a primer pair CTB5-FM01 which can be applied to auxiliary breeding of low-temperature-resistant japonica rice varieties. The invention has important application value, can be used for genetic improvement of low-temperature tolerance of rice, and has important significance for cultivation of new varieties of low-temperature-resistant rice.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to CTB5 protein related to low temperature tolerance of rice and related biological materials and applications thereof. Background Art

[0002] Rice (Oryza sativa L.) is the main food crop in my country, and 60% of the country's population relies on rice as their basic food. Rice originated in tropical and subtropical regions. It is a thermophilic crop and is extremely sensitive to low temperature and chilling damage. Low temperature not only affects the planting season and geographical distribution of rice, but also seriously affects the normal growth and development of rice, resulting in reduced rice production and quality. my country has a wide range of rice planting, and low temperature and chilling damage frequently occurs in rice-growing areas. In high-altitude rice-growing areas such as the high-latitude areas in Northeast my country, the plateau mountains in the southwest, and the early rice sowing and seedling raising and late rice heading and flowering periods in South China and the middle and lower reaches of the Yangtze River, low temperature and chilling damage are often encountered, resulting in a large reduction in rice production, which seriously affects local rice production. According to statistics, the annual rice production reduction caused by chilling damage in my country is about 3-5 million tons, and low temperature and chilling damage has become one of the main abiotic stresses affecting rice production in my country.

[0003] Rice is extremely sensitive to low temperature and chilling damage throughout its entire growth period. According to the period of occurrence of chilling damage, rice chilling damage can be divided into chilling damage during the bud stage, chilling damage during the seedling stage, chilling damage during the booting stage, and chilling damage during the grain filling stage. Chilling damage during the bud stage refers to the phenomenon that rice is attacked by low temperature from the germination to the first complete leaf, resulting in prolonged germination time or mildew of young shoots. The survival rate of young shoots is often used as an evaluation index for cold tolerance. Chilling damage during the seedling stage refers to the phenomenon that rice is attacked by low temperature during the entire vegetative growth period starting from the first complete leaf, resulting in seedlings losing green, becoming stiff, reducing tillering, withering and even death of seedlings. The survival rate of seedlings is often used as an evaluation index for cold tolerance. Chilling damage during the booting stage refers to the phenomenon that rice is affected by low temperature from the beginning of reproductive growth to the beginning of heading and flowering, resulting in the obstruction of pollen development, which in turn affects normal flowering and pollination, and forms empty loquats. The fruit set rate is often used as an evaluation index for cold tolerance. Chilling damage during the grain filling period refers to the phenomenon that when rice encounters low temperatures after fertilization, leaf photosynthesis and photosynthate transport are inhibited, which in turn leads to deterioration of rice quality. Among the above types of chilling damage, chilling damage during the booting period occurs during the critical period of rice pollen mother cell meiosis and microspore formation, and is most sensitive to low temperatures. Chilling damage during the booting period causes pollen abortion and a decrease in the fruit setting rate, which will cause an irreversible reduction in rice yield. It is the most harmful barrier-type chilling damage among all chilling damage periods.

[0004] Breeding cold-resistant and high-quality rice varieties is the fundamental way to reduce the harm of low temperatures. Traditional breeding methods have problems such as long breeding cycles and high chance, while modern biological breeding methods based on genetic resources can greatly improve breeding efficiency and shorten breeding cycles. Discovering and cloning rice cold-resistant related regulatory genes and developing their functional markers will provide important genetic resources for rice cold-resistant breeding, which has important theoretical significance and application value. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a CTB5 protein related to low temperature tolerance of rice and related biological materials and applications thereof. The present invention provides a method for regulating the low temperature tolerance of plants.

[0006] The present invention provides a protein named CTB5, which is the following protein A1), A2) or A3):

[0007] A1) The amino acid sequence is the protein shown in SEQ ID No. 1 in the sequence list;

[0008] A2) a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1);

[0009] A3) A protein related to plant low temperature tolerance having 80% or more homology to the amino acid sequence defined in SEQ ID No. 1 obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in A1) or A2).

[0010] Among them, SEQ ID No. 1 consists of 244 amino acid residues, and the protein is derived from rice.

[0011] The above proteins can be synthesized artificially; or the coding genes can be synthesized first and then expressed biologically.

[0012] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein; the protein tag can be a Flag tag, a His tag, an MBP tag, a HA tag, a Myc tag, a GST tag and / or a SUMO tag, etc.

[0013] In the above proteins, homology refers to the homology of amino acid sequences; the homology of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage; in the above proteins, the above 80% homology can be at least 81%, 85%, 91%, 95% or 100% homology.

[0014] Biological materials related to protein CTB5 also fall within the protection scope of the present invention.

[0015] The biomaterial related to protein CTB5 provided by the present invention is any one of the following B1) to B7):

[0016] B1) a DNA molecule encoding the protein according to claim 1;

[0017] B2) an expression cassette containing the DNA molecule described in B1);

[0018] B3) a recombinant vector containing the DNA molecule described in B1) or the expression cassette described in B2);

[0019] B4) a recombinant microorganism containing the DNA molecule described in B1), or containing the expression cassette described in B2), or containing the recombinant vector described in B3);

[0020] B5) a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule described in B1) or the expression cassette described in B2);

[0021] B6) a nucleic acid molecule that reduces the expression of the DNA molecule described in B1);

[0022] B7) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B6).

[0023] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0024] In the above biological material, the DNA molecule described in B1) is the gene shown in b1) or b2) below:

[0025] b1) the coding sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

[0026] b2) The nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No.2.

[0027] Among them, SEQ ID No. 2 in the sequence list consists of 735 nucleotides and encodes the protein shown in SEQ ID No. 1 in the sequence list.

[0028] In the above biological materials, the expression cassette containing the DNA molecule described in B2) refers to a DNA molecule capable of expressing CTB5 in a host cell, and the DNA molecule may include not only a promoter for initiating transcription of the CTB5 gene, but also a terminator for terminating transcription of CTB5, and may also include an enhancer sequence.

[0029] A plant expression vector can be used to construct a recombinant vector containing the protein CTB5 encoding gene or the protein CTB5 encoding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, etc. When the CTB5 gene is used to construct a recombinant vector, any enhancing, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide.

[0030] In the above biological materials, the recombinant microorganism can be bacteria, fungi, yeast or algae; for example, the bacteria can be Agrobacterium EHA105 strain, and the yeast can be yeast Y2HGold strain.

[0031] The application of the above-mentioned protein or the above-mentioned biological material in any of the following C1-C2 also falls within the protection scope of the present invention:

[0032] C1) Regulate plant low temperature tolerance;

[0033] C2) Preparation of products for regulating low temperature tolerance of plants.

[0034] The above-mentioned regulation of low temperature tolerance is to improve low temperature tolerance or reduce low temperature tolerance; the improvement of low temperature tolerance is to improve the fruit setting rate under low temperature at the booting stage and / or the survival rate under low temperature at the seedling stage, and the reduction of low temperature tolerance is to reduce the fruit setting rate under low temperature at the booting stage and / or the survival rate under low temperature at the seedling stage; the improvement of low temperature tolerance is achieved by promoting or increasing the expression amount of the protein CTB5 encoding gene, or / and the activity or / and content of the protein; the reduction of low temperature tolerance is achieved by inhibiting or reducing the expression amount of the protein CTB5 encoding gene, or / and the activity or / and content of the protein.

[0035] The present invention also provides a method for improving plant low temperature tolerance, comprising the following steps: promoting or improving the expression of the CTB5 gene in a recipient plant, or / and the activity and / or content of the protein CTB5, to obtain a target plant with higher low temperature tolerance than the recipient plant; the CTB5 gene is a gene encoding the above-mentioned CTB5 protein.

[0036] In the above method, the promotion or increase of the expression level of the CTB5 gene in the recipient plant, and / or the activity and / or content of the protein CTB5 is achieved by introducing a nucleic acid molecule encoding the CTB5 protein into the recipient plant.

[0037] In the above method, the method of introducing a nucleic acid molecule encoding a CTB5 protein into a recipient plant comprises hybridizing a plant containing a CTB5 protein encoding gene with a recipient plant not containing the gene, or / and inserting the nucleic acid molecule encoding the CTB5 protein into a plant expression vector, and then transferring it into the recipient plant through genetic engineering.

[0038] Experiments on knocking out the CTB5 gene in rice have shown that the fruit set rate and pollen fertility of transgenic rice with CTB5 gene knocked out at low temperature during the booting stage were reduced, and the cold tolerance during the booting stage was reduced; the survival rate at low temperature during the seedling stage was reduced, and the cold tolerance during the seedling stage was reduced; genetic complementation and overexpression experiments of introducing the CTB5 gene coding sequence into rice have shown that compared with the recipient rice, the transgenic rice that genetically transformed and introduced the CTB5 protein coding gene into the recipient plant, or / and overexpressed the CTB5 protein had increased fruit set rate and pollen fertility at low temperature during the booting stage, and enhanced cold tolerance during the booting stage; increased survival rate at low temperature during the seedling stage, and enhanced cold tolerance during the seedling stage; knockout experiments, genetic complementation experiments and overexpression experiments all indicate that CTB5 protein is a gene related to plant low temperature tolerance, and improving CTB5 protein enhances plant low temperature tolerance.

[0039] The invention provides a primer pair for screening cold-resistant rice strains, which is named CTB5-FM01.

[0040] The forward primer CTB5-FM01-F in the primer pair consists of 21 nucleotides, and the reverse primer CTB5-FM01-R consists of 22 nucleotides; the sequences are as follows:

[0041] CTB5-FM01-F:5'-GCGGAGACATCATGGGCTTAG-3'

[0042] CTB5-FM01-R:5'-CCGGGACATACACCAGGTCATC-3'

[0043] The present invention provides a kit or PCR reagent for detecting cold-resistance related genes, and the use of the special primer pair, the kit or PCR reagent related to the special primer pair in detecting cold-resistance related genes or assisting in screening cold-resistance plant strains; the plant is rice.

[0044] The present invention provides a method for assisting in identifying cold-resistant rice strains, comprising the following steps: using the genomic DNA of the rice strain to be detected as a template, amplifying with the primer pair, and if the size of the obtained PCR amplification product is 150 bp, determining that the rice strain to be detected is a cold-resistant strain.

[0045] The cold resistance mentioned in the present invention specifically refers to that a certain rice strain has a higher fruit setting rate at an air temperature below 20°C during the booting stage than the cold-sensitive variety Towoda; the fruit setting rate of Towoda at an air temperature below 20°C during the booting stage is 40%; the higher the fruit setting rate of the identified rice strain after treatment, the stronger the cold resistance.

[0046] Any of the above plants may be any one of the following D1) to D4): D1) a plant of the Poaceae family; D2) a plant of the genus Oryza; D3) rice; D4) japonica rice.

[0047] The method of the invention is simple to operate, low in cost, can shorten the breeding cycle, is easy to promote and operate, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The results of positional cloning of the CTB5 gene and the statistics of cold tolerance phenotypes of near-isogenic lines at the booting stage under normal conditions (NC) and natural low temperature conditions (CS-HAA). ORF4 in the figure is the CTB5 gene, and the scale in the ear morphology photo in the figure is 5 cm. The P value is calculated by a two-tailed t test;

[0049] Figure 2 Schematic diagram of expression level detection of CTB5 gene overexpression transgenic strains and sequencing identification of knockout strains. Different letters a, b and c in the figure represent significant differences, which are calculated by one-way analysis of variance (Duncan test);

[0050] Figure 3 This is the cold tolerance phenotype identification of CTB5 gene overexpressing transgenic lines at the booting stage under three cold treatment conditions: natural low temperature (CS-HAA), circulating deep cold water irrigation (CS-DW) and artificial climate chamber (CS-PT). The scale in the ear morphology photos in the figure is 2 cm, and the different letters a, b and c represent significant differences, which were calculated by one-way analysis of variance (Duncan test);

[0051] Figure 4 This is the cold tolerance phenotype identification of CTB5 knockout strains at the booting stage under three cold treatment conditions: natural low temperature (CS-HAA), circulating deep cold water irrigation (CS-DW) and artificial climate chamber (CS-PT). The scale in the ear morphology photos in the figure is 2 cm, and the different letters a, b and c represent significant differences, which were calculated by one-way analysis of variance (Duncan test);

[0052] Figure 5 This is the identification of the cold tolerance phenotype of the CTB5 gene genetic complementation transgenic lines at the booting stage under three cold treatment conditions: natural low temperature (CS-HAA), circulating deep cold water irrigation (CS-DW) and artificial climate chamber (CS-PT). The scale in the ear morphology photos in the figure is 2 cm, and the different letters a, b and c represent significant differences, which were calculated by one-way analysis of variance (Duncan test);

[0053] Figure 6Pollen fertility statistics of CTB5 gene overexpression strains and knockout strains under normal conditions (NC) and natural low temperature (CS-HAA). The scale in the pollen iodine-stained photos in the figure is 100 μm. Different letters a, b and c represent significant differences, which were calculated by one-way analysis of variance (Duncan test). P values ​​were calculated by two-tailed t test.

[0054] Figure 7 The cold tolerance phenotypes and statistical diagrams of the CTB5 gene overexpression and knockout strains at the seedling stage, with the P value calculated by a two-tailed t test;

[0055] Figure 8 is the tissue and cold-induced expression pattern of CTB5 gene, P-3cm, P-6cm, P-9cm, P-12cm and P-15cm in the figure represent ears with lengths of 3cm, 6cm, 9cm, 12cm and 15cm, respectively, 0h, 4h, 8h, 12h, 16h, 1d, 2d, 3d, 4d, 5d, 6d and 7d in the figure represent the time points before and after cold treatment at the booting stage, and the P value in the figure is calculated by two-tailed t test;

[0056] Fig. 9 Identification of functional markers for the CTB5 gene. DETAILED DESCRIPTION

[0057] The present invention provides CTB5 protein related to low temperature tolerance of rice and its related biomaterials and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0058] The vector SK-gRNA and the vector pC1300-Cas9 in the following embodiments are both recorded in the non-patent document "Chun Wang et al., A simple CRISPR / Cas9 system for multiplex genome editing in rice. Journal of Genetics and Genomics", and the vectors pCM1307 and pMDC163 are recorded in the non-patent document "Gangling Li et al., RGN1 controls grain number and shapes panicle architecture in rice. Plant Biotechnology Journal". The public can obtain them from China Agricultural University (i.e., the applicant) to repeat the experiments of this application and cannot be used for other purposes.

[0059] In the low temperature tolerance identification experiment at the booting stage, the normal conditions (NC) refer to sowing the test materials at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing, transplanting them to the field 30 days later, without low temperature treatment during the entire growth period, and examining the fruit set rate (number of filled grains / total number of grains) after harvest. At least 10 plants were examined for each material, with 3-4 main ears per plant.

[0060] The circulating deep cold water irrigation treatment (Cold Stress in ColdWater, CS-DW) refers to sowing the test materials at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing, transplanting them to the field after 30 days, selecting the tillers with the upper and lower 2 cm distance from the flag leaf pulvinus to the second leaf pulvinus at the booting stage for hanging, hanging 3 tillers per plant, and treating at least 10 plants for each material; moving the hung single plants to a deep water pool and treating them with circulating cold water (water temperature 18°C) for one week, and then transplanting them to the field to grow under normal conditions until maturity; harvesting the ears corresponding to the hung tillers, and examining the fruit set rate (number of filled grains / total number of grains).

[0061] The artificial climate chamber treatment (Cold Stress in a Phytotron, CS-PT) refers to sowing the test materials at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing, transplanting them to the field after 30 days, selecting the tillers with the upper and lower 2 cm distance from the flag leaf pulvinus to the second leaf pulvinus at the booting stage for hanging, hanging 3 tillers per plant, and treating at least 10 plants for each material; moving the hung single plants to the artificial climate chamber (temperature 17°C) for one week, and then transplanting them to the field to grow to maturity under normal conditions; harvesting the ears corresponding to the hung tillers, and examining the fruit setting rate (number of filled grains / total number of grains).

[0062] The natural low temperature (Cold Stress in a High-altitude Area, CS-HAA) refers to sowing the test materials in Baiyi District, Kunming City, Yunnan Province (1974 meters above sea level, average daily temperature <20°C), and transplanting them to the field after 30 days; after maturity and harvest, at least 10 plants of each material were selected to examine the average fruit setting rate of the main ear.

[0063] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0064] Example 1. Acquisition of CTB5 gene

[0065] A QTL qCTB7 for cold tolerance at the booting stage on rice chromosome 7 was detected through linkage analysis in a BC7F2 backcross population constructed from the cold-tolerant highland japonica rice Kunming Xiaobaigu (donor parent) and the cold-sensitive japonica rice Towada (recurrent parent). This QTL explained 9% to 21% of the phenotypic variation and was located in the 330.2 kb interval of molecular markers RM7237-RM5508 (Zhou et al., Fine mapping a QTL qCTB7 for cold tolerance at the booting stage on rice chromosome 7 using a near-isogenic line, Theoretical and Applied Genetics).

[0066] Using a BC8F2 backcross population containing 8075 plants, 44 recombinant plants were screened between RM7237 and RM5508. Through progeny testing, qCTB7 was further finely located to the 35 kb interval of molecular markers RM21868-RM5508, which contains 4 predicted open reading frames (ORFs) (such as Figure 1 By comparing the sequences between the parents, it was found that only the CTB5 gene (ORF4) had 18 SNPs in the promoter region of the parents and 1 non-synonymous SNP and 1 InDel in the coding region.

[0067] The near-isogenic line NIL of CTB5 gene was screened from the BC8F5 backcross population by backcrossing and molecular marker-assisted selection. CTB5 ; The results of cold tolerance phenotype evaluation at the booting stage are as follows Figure 1 As shown, under normal conditions NIL CTB5 There was no significant difference in fruit setting rate between NIL and Towada, but under natural low temperature conditions, CTB5 The fruiting rate of NIL was significantly higher than that of Towada. CTB5It has stronger cold tolerance during the booting stage than Towada.

[0068] The CTB5 gene was obtained by PCR amplification using the cDNA of the panicle of cold-tolerant japonica rice Kunming Xiaobaigu as a template. The primers used are as follows:

[0069] CTB5-F:5'-ATGGACCGATACGGCGAGAAGC-3';

[0070] CTB5-R:5'-TTAGATCAATCCATACAGGCTG-3'.

[0071] After sequencing, the coding nucleotide sequence of the CTB5 gene is shown as SEQ ID No.2, encoding protein CTB5, and the amino acid sequence of the protein is SEQ ID No.1.

[0072] SEQ ID No.1: CTB5 protein sequence

[0073] MDRYGEKQQQQQMFASYVDASLLAASGEVQGERPRARRRRRRGARCVGGGGGGGEVDGGDPKKRRLSDEQVEMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSKLLEEE FSKLKHAHDAAILHKCHLENEVLRLKERLVVAEEEVRRLRSAAGSHTASGEGGDIMGLGGSGACVAGPSSSFSTGTCQPPSFGGGGGGGGDHLGDDDLVYVPEYGGYADNSVVEWFSLYGLI.

[0074] SEQ ID No.2: CTB5 protein coding sequence

[0075] ATGGACCGATACGGCGAGAAGCAGCAGCAGCAGCAGATGTTTGCCTCCTACGTGGACGCCTCCCTCCTAGCAGCCAGCGGTGAGGTGCAGGGCGAGAGGCCGAGAGCGCGGAGGAGGAGGAGGCGTGGGGCGAGGTGCGTTGGCGGCGGCGGCGGTGGTGGCGAGGTGGACGGAGGGGACCCGAAGAAGCGGCGGCTGAGCGACGAGCAGGTGGAGATGCTGGAGCTGAGCTTCCGG GAGGAGCGGAAGCTGGAGACCGGGCGGAAGGTGCACCTTGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTGGCCGTCTGGTTCCAGAACCGCCGCGCCCGCCACAAGCAAGCTGCTCGAGGAGGAGTTCTCCAAGCTCAAGCACGCCCACGACGCCGCCATCCTCCACAAATGCCACCTCGAGAACGAGGTGCTGAGGCTGAAGGAGAGGCTGGTGGTCGCCGAGGAGGAAGTG AGGCGGCTCAGATCAGCGGCGGGGAGCCACACGGCCTCCGGAGAAGGCGGAGACATCATGGGCTTAGGCGGCAGCGGCGCCTGCGTCGCCGGGAGCCCGAGCTCGTCGTTCTCGACGGGTACCTGCCAGCC GCCGAGCTTCGGCGGCGGGGGCGGCGGCGGAGATCACCTCGGAGACGATGACCTGGTGTATGTCCCGGAGTACGGCGGCTACGCTGATAACAGTGTGGTTGAGTGGTTCAGCCTGTATGGATTGATCTAA.

[0076] Example 2: Functional verification of CTB5 protein

[0077] 1. Construction of overexpression vector

[0078] The overexpression vector used in this experiment was named CTB5-OE. The CDS (SEQ ID No. 2) of the CTB5 gene in Kunming Xiaobaigu was connected to the plant expression vector PCM1307 with a strong 35S promoter by homologous recombination. The restriction sites were SpeI and KpnI. The amplification primers used were as follows:

[0079] CTB5-OE-F:5'-TCGACGAGCTCTCTAGAACTATGGACCGATACGGCGA GAAGC-3';

[0080] CTB5-OE-R:5'-TTTTTGCGGAGTACCCCGGGTACCTTAGATCAATCCAT ACAGGCTG-3'.

[0081] The structure of the CTB5-OE vector is described as follows: a small fragment between the restriction sites SpeI and KpnI of the PCM1307 vector is replaced with the CTB5 gene coding region sequence (as shown in SEQ ID No. 2), and the other sequences of the PCM1307 vector are kept unchanged to obtain a positive plasmid, which is a recombinant overexpression vector of the CTB5 protein.

[0082] 2. Construction of knockout vector

[0083] (1) The genomic nucleotide sequence of CTB5 is SEQ ID No. 3, which has 3 exons and 2 introns (wherein, positions 1-166 of SEQ ID No. 3 are 5'UTR, positions 167-245 are the first exon, positions 246-362 are the first intron, positions 363-712 are the second exon, positions 713-800 are the second intron, positions 801-1106 are the third exon, and positions 1107-1314 are 3'UTR). Log in to the website CRISPR-plant (https: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) to screen target sites, and evaluate off-target conditions at the website http: / / www.rgenome.net / cas-offinder / ; select sequences with low off-target rates as target sequences for this study, and the target sequences are as follows: 5'-GGCGTGGGGCGAGGTGCGT-3' (same as positions 122-140 of SEQ ID No.2, positions 405-423 of SEQ ID No.3); the location of the CTB5 gene knockout target sequence on its genome is as follows Figure 2 As shown, it is located in the second exon.

[0084] (2) Design two complementary DNA sequences, add GGCA before the forward target sequence and add AAAC before the reverse complementary target sequence, as follows:

[0085] CTB5-sgRNA-F: 5'-GGCA GGCGTGGGGCGAGGTGCGT -3';

[0086] CTB5-sgRNA-R: 5'-AAAC ACGCACCTCGCCCCACGCC-3' (the underlined sequence is the reverse complement of the underlined sequence in F).

[0087] (3) Construction of intermediate vector:

[0088] a. The vector SK-gRNA was digested with AarI (Ferment) to form a linear vector with sticky ends;

[0089] bF chain and R chain are mixed and denatured and annealed to form fragments with sticky ends;

[0090] c. The linear vector obtained in step a and the fragment obtained in step b were connected (molar concentration 1:3-10), transformed into E. coli DH5α, and a recombinant plasmid was obtained; the colony PCR positive detection was performed using primers T3 and R chain, and the primer sequences are as follows:

[0091] T3: 5'-ATTAACCCTCACTAAAGGGA-3';

[0092] CTB5-sgRNA-R: 5'-AAACACGCACCTCGCCCCACGCC-3'.

[0093] d. Verified by T3 sequencing, the correct vector was named SK-gRNA-CTB5.

[0094] (4) Construction to the final vector:

[0095] The vector pC1300-Cas9 was digested with KpnI and BamHI to obtain a linearized pC1300-Cas9 vector; the SK-gRNA-CTB5 constructed in step (3) was digested with KpnI and BglII, the target fragment was recovered, and it was ligated to the linearized pC1300-Cas9 vector to obtain a knockout vector. After correct sequencing, the resulting knockout vector was named CTB5-CR.

[0096] The structure of the CTB5-CR vector is described as follows: the fragment between the restriction endonuclease KpnI and BamHI recognition sites of the vector pC1300-Cas9 is replaced by the target fragment after SK-gRNA-CTB5 digestion, and the other sequences of the pC1300-Cas9 vector are kept unchanged to obtain a recombinant vector, which is a knockout vector of the CTB5 gene; the knockout vector CTB5-CR expresses gRNA, and the target sequence of the gRNA is located on the second exon of the rice CTB5 gene. The specific target sequence is shown in positions 122-140 of SEQ ID No.2.

[0097] 3. Construction of genetic complementation vector

[0098] The genetic complementation vector used in this experiment was named CTB5-CP. A 5079 bp genomic fragment containing a 2573 bp fragment upstream of the start codon ATG, a coding region fragment containing 3 exons and 2 introns, and a 1592 bp fragment downstream of the stop codon was amplified from the genomic DNA of Kunming Xiaobaigu spikelets. It was connected to the plant expression vector pMDC163 by homologous recombination, and the restriction sites were PmeI and SacI. The amplification primers used are as follows:

[0099] CTB5-CP-F:5'-TGTCAAACACTGATAGTTTAAAACCGATACGATTCTCG GTTGGTCG-3';

[0100] CTB5-CP-R:5'-AACGATCGGGGAAATTCGAGCTCCCACCTTTCTCTTGC TGTCGACA-3'.

[0101] The structure of the CTB5-CP vector is described as follows: a small fragment between the restriction sites PmeI and SacI of the pMDC163 vector is replaced with a full-length 5079bp genomic fragment of the CTB5 gene, including a 2573bp fragment upstream of ATG, a coding region, and a 1592bp fragment downstream of the stop codon, while keeping other sequences of the pMDC163 vector unchanged to obtain a positive plasmid, which is a recombinant genetic complementation vector of the CTB5 protein; the coding sequence of the genomic fragment (5079bp) containing CTB5 is shown in SEQ ID No.4.

[0102] SEQ ID No.3: CTB5 protein coding gene genomic sequence

[0103] GTGGTGGTGGTGGTGCGTGGTCCTCTTCTCCATCCAGAGACAATCCAGTTCAGTTCAGTTTCCTCTCTCCCTCCTGTCCTCACCTCACACTTGACAAGAGAGAGAGAGAGAGAGAGAGAGAAAGGCTGAACTGAGCTGAGCTGATCGAGCTAGTGTGCGAGACGGCATGGACCGATACGGCGAGAAGCAGCAGCAGCAGCAGATGTTTGCCTCCTACGTGGACGCCTCCCTCCTAGCAGCCAGCGGTTCGTGCTACTTAGCTTTTGCTTGCTAGCTCCCGCCATTTTCTTGCCTCTCCATCTCATGGCAAGTGTCAACTGTGTACTGATCATGCTGCCACGATCGATCGATCTTTTTTTCAGGTGAGGTGCAGGGCGAGAGGCCGAGAGCGCGGAGGAGGAGGAGGCGTGGGGCGAGGTGCGTTGGCGGCGGCGGCGGTGGTGGCGAGGTGGACGGAGGGGACCCGAAGAAGCGGCGGCTGAGCGACGAGCAGGTGGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACCGGGCGGAAGGTGCACCTTGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTGGCCGTCTGGTTCCAGAACCGCCGCGCCCGCCACAAGAGCAAGCTGCTCGAGGAGGAGTTCTCCAAGCTCAAGCACGCCCACGACGCCGCCATCCTCCACAAATGCCACCTCGAGAACGAGGTAACAACACATCGTCCGTCCTTGCATGTACACACATGTATACTTATTGCTGCCTCAATTCCTCTGATCCATCCATGGCGGCATGCAGGTGCTGAGGCTGAAGGAGAGGCTGGTGGTCGCCGAGGAGGAAGTGAGGCGGCTCAGATCAGCGGCGGGGAGCCACACGGCCTCCGGAGAAGGCGGAGACATCATGGGCTTAGGCGGCAGCGGCGCCTGCGTCGCCGGGAGCCCGAGCTCGTCGTTCTCGACGGGTACCTGCCAGCCGCCGAGCTTCGGCGGCGGGGGCGGCGGCGGAGATCACCTCGGAGACGATGACCTGGTGTATGTCCCGGAGTACGGCGGCTACGCTGATAACAGTGTGGTTGAGTGGTTCAGCCTGTATGGATTGATCTAACGAAGATCAAGTTGATGATGGCATGTGCGGGGAATTTAGTTAGTCATAGTGTTAATTAAGCTGTATGGTGATCGATCGAACTGATATGGTCAGCTCTAAGTGTGTGTGTTTAATTATGTACATAAAGTGATTAATGGCAAAACCTGGCCAGCTAGCTAGTAATGTATTAATATTAGTAGTTCGCTTAAAAAAGATCAGATGTGTGTTC。

[0104] SEQ ID No.4: Genomic sequence for genetic complementation of the CTB5 protein-encoding gene

[0105]

[0106] IV. Obtaining genetically modified rice

[0107] (1) Recombinant bacteria

[0108] The overexpression vector CTB5-OE prepared in step 1 above was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the recombinant bacterium EHA105-CTB5-OE;

[0109] The knockout vector CTB5-CR prepared in step 2 above was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the recombinant bacterium EHA105-CTB5-CR;

[0110] The genetic complementation vector CTB5-CP prepared in the above step 3 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the recombinant bacterium EHA105-CTB5-CP.

[0111] (2) Transgenic rice

[0112] Using the Agrobacterium-mediated callus infection method, the recombinant bacteria EHA105-CTB5-OE and EHA105-CTB5-CP were used to infect the callus tissue of the recipient rice variety Towada, and the recombinant bacteria EHA105-CTB5-CR was used to infect the callus tissue of the recipient rice variety Nipponbare. The specific steps are as follows:

[0113] a. Obtaining embryonic callus: After shelling the mature seeds, disinfect them with 75% alcohol, then with 20% sodium hypochlorite solution, rinse with sterile water for 4-5 times, and air-dry for 6 hours; inoculate them on NB solid culture medium, culture them in the dark at 28℃ for 2 weeks, peel off the embryonic callus, subculture them to new NB culture medium, and subculture them for 2 weeks.

[0114] b. Prepare infection solution: aspirate the preserved Agrobacterium solution, spread it on YEP solid culture medium containing rifampicin and kanamycin, invert and culture in the dark at 28°C for 2 days, scrape a small amount of Agrobacterium into AAM liquid culture medium, pipette and mix, and measure the bacterial solution concentration OD600 to be about 0.3.

[0115] c. Co-cultivation: Select naturally dispersed, bright yellow granular callus tissue with a diameter of about 3-5 mm and put it into a conical flask, add the prepared infection solution, infect for 15 minutes, absorb the excess infection solution with sterile filter paper, place it on the co-cultivation medium covered with a layer of filter paper, and co-cultivate at 20℃ for 2-3 days.

[0116] d. Screening of resistant callus: Take out the callus after co-cultivation, wash it with sterile water for 5-6 times by rapid shaking, then wash it with sterile water containing cephalosporin and carbenicillin for 20 minutes, and finally place it on sterile filter paper to drain for 3 hours. Then move it to the delayed screening medium. After one week, move it to the first round screening medium, and after two weeks, move it to the second round screening medium and continue to culture for two weeks.

[0117] e. Differentiation culture: The resistant callus obtained by screening was inoculated into a pre-differentiation medium and cultured in the dark at 28°C for 2 weeks, then transferred to a differentiation medium and cultured in the light for 2-3 weeks to obtain regenerated transgenic seedlings.

[0118] f. Move the seedlings to the seedling culture medium. After the seedlings take root and grow, remove them from the culture bottle, wash the culture medium on the roots, harden the seedlings for 1-2 weeks, and move them to the field for planting until they mature.

[0119] The formula of the culture medium used in the plant tissue culture process is as follows:

[0120] NB solid culture medium: Dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30 g of sucrose and 3 g of plant gel in 1 L of distilled water, adjust the pH to 5.8, and sterilize under high temperature and high pressure for 20 min.

[0121] YEP solid culture medium: Dissolve 10 g yeast extract, 5 g NaCl and 10 g tryptone in distilled water, make up to 1 L with distilled water, add 15 g agar powder, and sterilize by high pressure at 121°C for 15 min.

[0122] AAM liquid culture medium: dissolve 50 mL of AAM macro (20×), 5 mL of MS micro (200×), 10 mL of AAM organic (100×), 10 mL of iron salt (100×), 500 mg of hydrolyzed casein, 68.5 g of sucrose, 36 g of glucose, 75 mg of glycine, 877 mg of glutamine, 266 mg of aspartic acid and 228 mg of arginine in appropriate amount of deionized water, make up to 1 L with deionized water, adjust the pH to 5.5, and sterilize at 115°C for 15 min; finally, add 1 ml of 20 mg / ml acetosyringone solution and mix well.

[0123] Co-culture medium: Dissolve 20 mL of N6 macro-mother solution (50×), 10 mL of B5 micro-mother solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2 mg of 2,4-D in 1 L of distilled water, adjust the pH to 5.4, sterilize at 121°C for 15 min, and add 10 g of 0.22 μM filtered sterilized glucose and 20 mg of acetosyringone when cooled to 50-60°C.

[0124] Delayed screening medium: Dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 500 mg of cephalosporin sterilized by 0.22 μM filtration when cooled to 50-60°C.

[0125] First round screening culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 500 mg of cephalosporin and 50 mg of hygromycin sterilized by 0.22 μM filtration when cooled to 50-60°C.

[0126] Second round screening culture medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gelatin and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of 0.22 μM filtered sterilized hygromycin when cooled to 50-60°C.

[0127] Predifferentiation medium: dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, 1 mg of 6-BA, 2 mg of NAA and 5 mg of ABA in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of hygromycin sterilized by 0.22 μM filtration when cooled to 50-60°C.

[0128] Differentiation medium: Dissolve 20 mL of N6 macromolecular solution (50×), 10 mL of B5 micromolecular solution (100×), 1 mL of B5 organic mother solution (1000×), 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of phytogenin, 2 mg of 6-BA, 1 mg of NAA and 1 mg of KT in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121°C for 15 min, and add 50 mg of 0.22 μM filtered sterilized hygromycin when cooled to 50-60°C.

[0129] Seedling culture medium: Dissolve 25 mL of MS large mother solution (20×), 2.5 mL of MS trace mother solution (200×), 2.5 mL of MS iron salt mother solution (200×), 2.5 mL of MS organic mother solution (200×), 3 g of plant gel, 0.5 mg of NAA and 0.25 mg of paclobutrazol in 1 L of distilled water, adjust the pH to 5.8, and sterilize at 121°C for 15 min.

[0130] (3) PCR identification to obtain positive transgenic materials

[0131] The T0 generation CTB5-OE transgenic rice plants obtained in step (2) were subjected to PCR identification at the DNA level and expression level identification at the RNA level, respectively. The PCR identification primers for the CTB5-OE transgenic plants were CTB5-OE-check-F (5'-ACCATTTACGAACGATAGCC-3') and CTB5-OE-check-R (5'-GTGGCATTTGTGGAGGAT-3'); the target fragment size was 660 bp, and the plants containing the target fragment in the PCR amplification product were positive, and the plants without the target fragment were negative. The positive plants were T0 generation CTB5-OE transgenic materials (i.e., CTB5 gene overexpression materials).

[0132] The T0 generation CTB5-CR transgenic rice plants obtained in step (2) were sequenced and identified at the DNA level. The sequencing identification primers for the CTB5-CR transgenic plants were CTB5-CR-check-F (5'-CCA GTTCAGTTCAGTTTCCTC-3') and CTB5-CR-check-R (5'-CGGACGATGTGTT GTTACC-3'); in the sequencing results, the plants with sequence variation in the target region were positive, and the plants with no sequence variation were off-target. The positive plants were T0 generation CTB5-CR transgenic materials (i.e., CTB5 gene knockout materials).

[0133] The T0 generation CTB5-CP transgenic rice plants obtained in step (2) were subjected to PCR identification at the DNA level. The PCR identification primers for the CTB5-CP transgenic plants were CTB5-CP-check-F (5'-CG GCTCCACCACTAATAG-3') and CTB5-CP-check-R (5'-TGCGGGACTCTAATC ATA-3'); the target fragment size was 251 bp, and the plants containing the target fragment in the PCR amplification product were positive, and the plants without the target fragment were negative. The positive plants were T0 generation CTB5-CP transgenic materials (i.e., CTB5 gene genetic complementation materials).

[0134] 5. Identification of cold tolerance of transgenic materials at the booting stage

[0135] After the T0 positive strains of CTB5-OE were harvested, they were soaked in 20 mg / L hygromycin solution for germination before sowing the T1 generation. The seedlings that could take root normally were selected and transplanted to the field. After 30 days, the leaves were taken to extract DNA, and the DNA level was identified using the above CTB5-OE-check-F and CTB5-OE-check-R to obtain positive individual plants. Before sowing the T2 generation, all positive individual seeds of the T1 generation were treated with 20 mg / L hygromycin solution. All T1 individual plants that could take root normally after soaking seeds were homozygous strains, recorded as CTB5-OE1 and CTB5-OE2. The homozygous positive T2 generation CTB5 gene overexpression strains (CTB5-OE1 and CTB5-OE2) were sown at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing and the Experimental Station of Yunnan Academy of Agricultural Sciences in Kunming, Yunnan Province, and transplanted to the field 30 days later.

[0136] When the seeds grew to the booting stage, the panicles of CTB5-OE 1 and CTB5-OE2 were collected at the Shangzhuang Experimental Station of China Agricultural University in Haidian District, Beijing to extract total RNA, and the first-chain cDNA was synthesized by reverse transcription using reverse transcriptase M-MLV. The first-chain cDNA was used as a template, and the primers CTB5-qRT-F (5'-GGAGACG ATGACCTGGTGTATG-3') and CTB5-qRT-R (5'-CAGGCTGAACCACTCA ACCA-3') were used to amplify the specific fragment of the CTB5 gene. The primers Actin-F (5'-CACAGGTATTGTGTTGGACTCTG-3') and Actin-R (5'-AGTAACCACGCTCCGTCAGG-3') were used to amplify the specific fragment of the rice Actin gene (Os03g0718100) as an internal reference for real-time quantitative analysis. Real-time fluorescence quantitative PCR was performed on a real-time fluorescence quantitative PCR instrument Applied Biosystems. The experiments were performed on 7500 Real Time PCR system (ABI, USA). Three biological replicates were set for each experiment and three mechanical replicates were performed for each biological replicate. The relative expression level was calculated using the method reported by Livak KJ and Schmittgen TD (2001), i.e., 2-ΔΔCT.

[0137] The results are as follows Figure 2 As shown, the expression levels of two strains of CTB5-OE transgenic plants, CTB5-OE1 and CTB5-OE2, were significantly higher than those of the wild type Towada.

[0138] The cold tolerance of Towada and CTB5 gene overexpression lines at the booting stage was identified using circulating deep cold water irrigation and artificial climate chamber treatment at the Shangzhuang Experimental Station of China Agricultural University.

[0139] The natural low temperature identification method was used at the experimental station of Yunnan Academy of Agricultural Sciences to identify the cold tolerance of Towada and CTB5 gene overexpression lines at the booting stage.

[0140] The fruit setting rate was used as an evaluation index for cold tolerance at the booting stage. Figure 3 As shown, compared with the wild-type Towada, the cold tolerance of the CTB5 overexpression lines CTB5-OE1 and CTB5-OE2 at the booting stage was significantly enhanced.

[0141] The T0 generation positive strains of CTB5-CR were harvested to obtain the T1 generation, and two CTB5-CR homozygous positive strains Nip-CTB5-cr1 and Nip-CTB5-cr2 were obtained by sequencing.

[0142] Compared with the wild-type rice Nipponbare, the homozygous positive strain Nip-CTB5-cr1 of CTB5-CR showed a mutation in the CTB5 gene in the rice genome, such as Figure 2 As shown: in the two homologous chromosomes, the genomic gene of CTB5 whose nucleotide sequence is sequence 3 in the sequence list has undergone the following changes: 29 nucleotides from positions 404 to 433 of sequence 3 in the sequence list are deleted, that is, 29 nucleotides from positions 121 to 150 of sequence 2 in the sequence list are deleted, resulting in a frameshift mutation in the amino acid sequence after the 42nd amino acid in sequence 1 in the sequence list, thereby causing the CTB5 gene to be knocked out.

[0143] Compared with the wild-type rice Nipponbare, the homozygous positive strain Nip-CTB5-cr2 of CTB5-CR has a mutation in the CTB5 gene in the rice genome, such as Figure 2 As shown: in the two homologous chromosomes, the genomic gene of CTB5 whose nucleotide sequence is sequence 3 in the sequence list has undergone the following changes: 2 nucleotides are inserted after the 420th position of sequence 3 in the sequence list, that is, 2 nucleotides are inserted after the 137th position of sequence 2 in the sequence list, resulting in premature termination of the amino acid sequence after the 45th amino acid of sequence 1 in the sequence list, thereby causing the CTB5 gene to be knocked out.

[0144] The homozygous positive T2 generation CTB5 gene knockout lines Nip-CTB5-cr1 and Nip-CTB5-cr2 were treated with circulating deep cold water irrigation, artificial climate chamber and natural low temperature to identify the cold tolerance of Nipponbare and CTB5 gene knockout lines at the booting stage.

[0145] The fruit setting rate was used as an evaluation index for cold tolerance at the booting stage. Figure 4 As shown, compared with the wild-type Nipponbare, the cold tolerance of the CTB5 knockout lines Nip-CTB5-cr1 and Nip-CTB5-cr2 at the booting stage was significantly reduced.

[0146] After the T0 positive strains of CTB5-CP were harvested, they were soaked in 20 mg / L hygromycin solution for germination before sowing the T1 generation. The seedlings that could take root normally were selected and transplanted to the field. After 30 days, the leaves were taken to extract DNA, and the DNA level was identified using the above CTB5-CP-check-F and CTB5-CP-check-R to obtain positive individual plants. Before sowing the T2 generation, all positive individual seeds of the T1 generation were treated with 20 mg / L hygromycin solution. All T1 individual plants that could take root normally after soaking were homozygous strains, recorded as CTB5-CP1 and CTB5-CP2.

[0147] The homozygous positive T2 generation CTB5 gene genetic complementation lines CTB5-CP1 and CTB5-CP2 were treated with circulating deep cold water irrigation, artificial climate chamber and natural low temperature to identify the cold tolerance of Towada and CTB5 gene genetic complementation lines at the booting stage.

[0148] The fruit setting rate was used as an evaluation index for cold tolerance at the booting stage. Figure 5 As shown, compared with the wild-type Towada, the cold tolerance of the CTB5 complementation lines CTB5-CP1 and CTB5-CP2 at the booting stage was significantly enhanced.

[0149] To investigate the effect of CTB5 gene on rice pollen fertility, spikelets of T2 generation CTB5 gene overexpression lines CTB5-OE1, CTB5-OE2 and wild-type Towada, as well as CTB5 gene knockout lines Nip-CTB5-cr1, Nip-CTB5-cr2 and wild-type Nipponbare were taken from Shangzhuang Experimental Station of China Agricultural University and Experimental Station of Yunnan Academy of Agricultural Sciences. Spikelets of 3-5 well-grown main stem spikes of each test material were selected for microscopic examination. A drop of 1% I2-KI stain was added to the slide. Three anthers were taken from each spikelet and placed in the staining solution. Pollen grains were squeezed out of the anthers with clean tweezers, covered with a coverslip, and observed in three different fields of view under a 10x microscope of an ordinary optical microscope. Random photos were taken for data statistics. Fertile pollen grains were judged based on the different morphology and staining of the anthers. The average pollen fertility rate of 5 spikelets of each material represented the pollen fertility of the material.

[0150] The results are as follows Figure 6 As shown, compared with the wild-type Towada, the fertile pollen rate of the CTB5 gene overexpression lines CTB5-OE1 and CTB5-OE2 was significantly increased; while compared with the wild-type Nipponbare, the fertile pollen rate of the CTB5 gene knockout lines Nip-CTB5-cr1 and Nip-CTB5-cr2 was significantly reduced.

[0151] The above results show that low temperature chilling damage at the booting stage affects the fruit set rate and pollen fertility of rice. Under the three cold treatments of natural low temperature, circulating deep cold water irrigation and artificial climate chamber, the cold tolerance of the CTB5 gene overexpression strain and the genetic complementation strain at the booting stage was significantly stronger than that of Towada; while the cold tolerance of the CTB5 gene knockout strain at the booting stage was significantly lower than that of Nipponbare, indicating that the CTB5 gene can positively regulate the cold tolerance of rice at the booting stage and reduce the effect of low temperature on the fruit set rate and pollen fertility under cold stress.

[0152] VI. Identification of cold tolerance of transgenic materials at the seedling stage

[0153] Seeds of homozygous positive T2 generation CTB5 gene overexpression strains CTB5-OE1, CTB5-OE2 and wild-type Towada, as well as CTB5 gene knockout strains Nip-CTB5-cr1, Nip-CTB5-cr2 and wild-type Nipponbare were taken, and water was added to place them in a 28°C artificial climate box for germination. After the seeds were fully germinated, they were transferred to a 96-well PCR plate and placed in a 28°C artificial climate box (16h light / 8h dark, 85% relative humidity) for two weeks. During the culture period, the nutrient solution was replaced every two days.

[0154] The solutes and their concentrations of the rice hydroponic nutrient solution are 116.0g / L NH4NO3, 49.9g / L NaH2PO4·2H2O, 87.0g / LK2SO4, 111.0g / L CaCl2, 418.0g / LMgSO4·7H2O, 1801.8mg / L MnCl2·4H2O, 91.8mg / L(NH4)6MoO24·4H2O, 1098.0mg / L H3BO3, 44.55mg / L ZnSO4·7H2O, 41.6mg / LCuSO4·5H2O, and 9738.0mg / L FeCl3·6H2O. The solvent is water and the pH is 5.5.

[0155] When the plants grew to the three-leaf stage, they were placed in an artificial climate box at 4°C (16 h light / 8 h dark, 85% relative humidity) for 3 days, after which the test materials were transferred to a 28°C artificial climate box (16 h light / 8 h dark, 85% relative humidity) for one week of recovery, and the survival rate of each material was counted. Three biological replicates were set for each material per test, with 40 seedlings per replicate.

[0156] The results are as follows Figure 7 As shown, compared with the wild-type Towada, the survival rates of the CTB5 gene overexpression strains CTB5-OE1 and CTB5-OE2 were significantly improved; while compared with the wild-type Nipponbare, the survival rates of the CTB5 gene knockout strains Nip-CTB5-cr1 and Nip-CTB5-cr2 were significantly reduced.

[0157] The above results show that low temperature and chilling damage at the seedling stage affect the survival rate of rice seedlings. Under low temperature stress, the seedling cold tolerance of the CTB5 gene overexpression strain was significantly stronger than that of Towada, while the seedling cold tolerance of the CTB5 gene knockout strain was significantly lower than that of Nipponbare, indicating that the CTB5 gene can positively regulate the cold tolerance of rice seedlings and improve the survival rate of rice plants under cold stress.

[0158] Example 3: Tissue and cold-induced expression pattern of CTB5 gene

[0159] In order to analyze the tissue expression of CTB5 gene, the NILCTB5 Tissue samples including roots, leaf sheaths, leaves, stems at the heading period and ears at different developmental stages (lengths are 3cm, 6cm, 9cm, 12cm, and 15cm respectively).

[0160] In order to analyze the cold-induced expression of CTB5 gene, the near-isogenic line NIL was selected at the booting stage. CTB5 The seeds were labeled with Towada and moved to an artificial climate room (air temperature 16°C), and young ear samples were taken before treatment and at 4h, 8h, 12h, 16h, 1d, 2d, 3d, 4d, 5d, 6d, and 7d of cold treatment.

[0161] The total RNA of the samples was extracted by TRIZOL method, and the first chain of cDNA was synthesized by reverse transcription using reverse transcriptase M-MLV. The first chain of cDNA was used as a template, and primers CTB5-qRT-F and CTB5-qRT-R were used to amplify the specific fragment of the CTB5 gene. Primers Actin-F and Actin-R were used to amplify the specific fragment of the rice Actin gene (Os03g0718100) as an internal reference for real-time quantitative analysis.

[0162] The primer sequences used are as follows:

[0163] CTB5-qRT-F: 5'-GGAGACGATGACCTGGTGTATG-3';

[0164] CTB5-qRT-R: 5'-CAGGCTGAACCACTCAACCA-3';

[0165] Actin-F: 5'-CACAGGTATTGTGTTGGACTCTG-3';

[0166] Actin-R: 5'-AGTAACCACGCTCCGTCAGG-3'.

[0167] Real-time fluorescence quantitative PCR was performed on the real-time fluorescence quantitative PCR instrument Applied Biosystems 7500 Real Time PCR system (ABI, USA), with three biological replicates for each experiment, and three mechanical replicates for each biological replicate. The relative expression level was calculated using the method reported by Livak KJ and Schmittgen TD (2001), namely 2-ΔΔCT.

[0168] The results are as follows Figure 8 As shown in Figure 2, the CTB5 gene is expressed in rice roots, leaf sheaths, leaves, and panicles at different developmental stages, and is significantly induced by low temperature. CTB5 The induction level in was significantly higher than that in Towada.

[0169] Example 4. Development of CTB5 gene functional markers

[0170] 1. Preparation of primer pair CTB5-FM01

[0171] The following primer sequences were artificially synthesized:

[0172] CTB5-FM01-F: 5'-GCGGAGACATCATGGGCTTAG-3';

[0173] CTB5-FM01-R: 5'-CCGGGACATACACCAGGTCATC-3'.

[0174] The primer pair was named CTB5-FM01, the forward primer CTB5-FM01-F consisted of 21 nucleotides, and the reverse primer CTB5-FM01-R consisted of 22 nucleotides.

[0175] 2. Method for screening cold-tolerant rice lines using primer pair CTB5-FM01

[0176] The genomic DNA of the rice strain to be tested is used as a template and amplified using CTB5-FM01. If the size of the obtained PCR amplification product is 150 bp, the rice strain to be tested is determined to be a cold-tolerant strain.

[0177] PCR amplification reaction system (10 μL): 6.5 μL ddH2O, 1 μL 10×Taq buffer, 0.4 μL dNTPs (10 mM), 0.5 μL primer F chain, 0.5 μL primer R chain, 0.1 μL Taq enzyme and 1 μL template DNA.

[0178] PCR amplification program: pre-denaturation (95°C, 5 min), denaturation (95°C, 30 sec), annealing (55°C, 30 sec), extension (72°C, 40 sec), repeated for 35 cycles.

[0179] After amplification, 1 / 2 volume of denaturant (50 mL formamide, 50 mg bromophenol blue, 50 mg xylene nitrile) was added to the PCR product and mixed evenly, and the amplified product bands were detected by 5% (w / v) agarose gel electrophoresis.

[0180] 3. Results of screening cold-tolerant rice lines using primer pair CTB5-FM01

[0181] The CTB5 genotype of rice germplasm resources at home and abroad was analyzed and haplotypes were divided. The cold-tolerant parent Kunming Xiaobaigu type haplotype was the dominant haplotype, and the cold-sensitive parent Towada type haplotype was the inferior haplotype. Eight japonica rice germplasms with dominant and inferior haplotypes of the CTB5 gene were selected, respectively, for a total of 16 japonica rice germplasms (all rice varieties can be obtained from the core germplasm bank of the China National Rice Research Institute) to identify and screen cold tolerance at the booting stage according to the above method.

[0182] Filter results such as Fig. 9 As shown, the PCR product amplified from the superior haploid germplasm using CTB5-FM01 is 150bp, and the PCR product amplified from the inferior haploid germplasm is 135bp. There are obvious differences in the bands, indicating that the CTB5 gene functional marker primer pair CTB5-FM01 can be used to effectively screen cold-tolerant germplasm from japonica rice germplasm screening germplasm. The PCR product amplified from the superior haploid germplasm of the CTB5 gene is larger than the PCR product amplified from the inferior haploid germplasm of the CTB5 gene, indicating that the germplasm is cold-tolerant.

[0183] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experiments. Although the present invention has given specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that are made by conventional techniques known in the art that are out of the scope of the present application. The application of some basic features can be carried out within the scope of the following claims.

[0184] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A protein, characterized in that The protein is the following protein A1), A2) or A3): A1) The amino acid sequence is the protein shown in SEQ ID No. 1 in the sequence list; A2) a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1); A3) A protein related to plant low temperature tolerance having 80% or more homology to the amino acid sequence defined in SEQ ID No. 1 obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in A1) or A2).

2. The protein according to claim 1, characterized in that: The protein is derived from rice.

3. The biological material related to the protein according to claim 1 or 2, which is any one of the following B1) to B7): B1) a DNA molecule encoding the protein according to claim 1; B2) an expression cassette containing the DNA molecule described in B1); B3) a recombinant vector containing the DNA molecule described in B1) or the expression cassette described in B2); B4) a recombinant microorganism containing the DNA molecule described in B1), or containing the expression cassette described in B2), or containing the recombinant vector described in B3); B5) a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule described in B1) or the expression cassette described in B2); B6) a nucleic acid molecule that reduces the expression of the DNA molecule described in B1); B7) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B6).

4. The biomaterial according to claim 3, characterized in that: B1) The DNA molecule is the gene shown in b1) or b2) below: b1) the coding sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No.

2.

5. The use of the protein according to claim 1 or 2, or the biomaterial according to claim 3 or 4 in any of the following C1-C2 also falls within the scope of protection of the present invention: C1) Regulate plant low temperature tolerance; C2) Preparation of products for regulating low temperature tolerance of plants.

6. The use according to claim 5, characterized in that: The regulation of low temperature tolerance is to increase or / and decrease low temperature tolerance; the increase or / and decrease of low temperature tolerance is to increase or / and decrease the fruit setting rate under low temperature at the booting stage or / and the survival rate under low temperature at the seedling stage; the increase or / and decrease of low temperature tolerance is achieved by promoting or / and decreasing the expression level of the protein encoding gene according to claim 1, or / and the activity or / and content of the protein.

7. A method for improving low temperature tolerance of plants, characterized in that: Genetically hybridizing a plant containing a gene encoding the protein according to claim 1 with a recipient plant that does not contain the gene, or / and inserting the gene encoding the protein according to claim 1 into a plant expression vector, and then transferring the gene encoding the protein according to claim 1 into a recipient plant through genetic engineering, so as to introduce a nucleic acid molecule encoding the protein according to claim 1 into the recipient plant, thereby promoting or increasing the expression of the gene encoding the protein according to claim 1 in the recipient plant, or / and the activity or / and content of the protein, and obtaining a target plant with higher low temperature tolerance than the recipient plant.

8. A special primer pair for screening cold-tolerant rice strains, and a kit or PCR reagent related to the primer pair for detecting cold-tolerant related genes.

9. Use of the special primer pair, the special primer pair-related kit or the PCR reagent according to claim 8 in detecting cold-resistance-related genes or assisting in screening cold-resistance plant strains; the plant is rice.

10. A method for assisting identification of whether a rice strain to be tested is a cold-tolerant rice strain, comprising the following steps: using the genomic DNA of the rice strain to be tested as a template, performing PCR amplification using the primer pair of claim 8, and if the size of the obtained PCR amplification product is 150 bp, determining that the rice strain to be tested is a cold-tolerant rice strain.

11. The use or method according to any one of claims 5 to 10, characterized in that: The plant is any one of the following D1) to D4): D1) a plant of the Poaceae family; D2) a plant of the genus Oryza; D3) rice; D4) japonica rice.

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