CTB17 gene for regulating cold resistance of rice in booting stage and application thereof

Abstract

The invention discloses a CTB17 gene for regulating cold resistance of rice at a booting stage and application of the CTB17 gene. The cold resistance of the rice at the booting stage is regulated by regulating the expression or activity of the rice CTB17 gene or the coded protein thereof; and the amino acid sequence coded by the CTB17 gene is as shown in SEQ ID NO. 3. The invention provides the application of the CTB17 gene in regulating and controlling the cold resistance of the rice in the booting stage for the first time; the CTB17 gene plays an important role in regulating and controlling the cold resistance of the rice at the booting stage, provides a key gene resource for cultivating low-temperature-resistant rice varieties, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a CTB17 gene for regulating cold tolerance of rice at booting stage and application thereof. BACKGROUND

[0002] Low temperature environment has obvious adverse effects on the growth and development of crops such as rice, especially at the booting stage. Low temperature can cause pollen development to be blocked, thereby causing the seed setting rate to decrease, the yield to reduce, and even the yield to be zero. Rice is a warm crop and has weak cold tolerance. When the booting stage encounters cold damage, the plant cannot complete the key processes such as pollen development, flowering and pollination, and thus the rice production in the northern rice area of China is seriously affected. Therefore, it is particularly important to improve the adaptability of rice to low temperature, especially the cold tolerance at the key growth stage. In-depth understanding of the molecular mechanism of rice response to low temperature, cloning of key low temperature resistance genes, and then using molecular design to breed low temperature resistant rice varieties are the most effective ways to cope with cold damage and ensure food security.

[0003] Research on the cold tolerance of rice has made some progress, but most of them are concentrated in the germination stage and the seedling stage. Due to the limitation of the accuracy of phenotype identification, the currently identified cold tolerance genes are still very limited. It is urgent to identify more new genes that regulate the cold tolerance of rice at the booting stage, and to analyze their molecular regulation network, so as to provide key gene resources for the molecular design of low temperature resistant rice varieties. SUMMARY

[0004] The present application provides a gene for regulating the cold tolerance of rice at the booting stage and application thereof, and confirms that the CTB17 gene is a key gene for controlling the cold tolerance of rice at the booting stage.

[0005] The present application provides a method for regulating the cold tolerance of rice at the booting stage, which regulates the expression or activity of the CTB17 gene or the encoded protein of rice, so as to regulate the cold tolerance of rice at the booting stage.

[0006] The amino acid sequence encoded by the CTB17 gene is shown in SEQ ID NO. 3.

[0007] In some embodiments, the method is selected from:

[0008] (i) up-regulating the expression or activity of the CTB17 gene or the encoded protein thereof, so as to improve the cold tolerance at the booting stage;

[0009] (ii) down-regulating the expression or activity of the CTB17 gene or the encoded protein thereof, so as to reduce the cold tolerance at the booting stage.

[0010] In some embodiments, the down-regulation of the expression or activity of the CTB17 gene or the protein encoded thereby is specifically any one of the following: knocking out or silencing the CTB17 gene in rice, or inhibiting the activity of the CTB17 protein.

[0011] In some embodiments, the up-regulation of the expression or activity of the CTB17 gene or the protein encoded thereby is specifically any one of the following:

[0012] (a) introducing an expression construct or a vector of the CTB17 gene into rice or rice cells;

[0013] (b) introducing an expression construct or a vector containing the CTB17 gene into rice or rice cells.

[0014] The present application also provides a method for screening a modulator for modulating the cold tolerance of rice at the booting stage, which comprises: adding a candidate substance to a system containing the CTB17 gene or the protein encoded thereby; detecting the system to observe the expression or activity of the CTB17 gene or the protein encoded thereby; the amino acid sequence encoded by the CTB17 gene is shown as SEQ ID NO. 3.

[0015] The present application also provides a method for breeding rice, which comprises the following steps: introducing a substance that increases or inhibits the expression amount of the CTB17 gene, the expression amount of the protein encoded by the CTB17 gene, or the activity of the protein encoded by the CTB17 gene into rice or rice cells to obtain the bred rice; the amino acid sequence encoded by the CTB17 gene is shown as SEQ ID NO. 3.

[0016] The present application also provides the use of the CTB17 gene in modulating the cold tolerance of rice at the booting stage; the amino acid sequence encoded by the CTB17 gene is shown as SEQ ID NO. 3.

[0017] In some embodiments, the modulation is specifically any one of the following:

[0018] (i) reducing the cold tolerance at the booting stage by inhibiting or reducing the activity or expression amount of the protein encoded by the CTB17 gene;

[0019] (ii) reducing the cold tolerance at the booting stage by knocking out, inhibiting, or silencing the expression amount of the CTB17 gene.

[0020] In some embodiments, the modulation is specifically any one of the following:

[0021] (a) increasing the cold tolerance at the booting stage by increasing the activity or expression amount of the protein encoded by the CTB17 gene;

[0022] (b) increasing the cold tolerance of the booting stage by increasing the expression level of the CTB17 gene.

[0023] In some embodiments, the nucleotide sequence of the CTB17 gene is as shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0024] Compared with the prior art, the present application achieves the following technical effects:

[0025] 1. The present application provides the application of the CTB17 gene in regulating the cold tolerance of the booting stage of rice for the first time. The CTB17 gene can play an important regulatory role in the cold tolerance of the booting stage of rice, providing a new gene resource for breeding cold-tolerant rice varieties, and having a broad application prospect.

[0026] 2. The present application uses gene editing technology to knock out or destroy the normal expression level of the CTB17 gene to obtain a mutant with short and whitish anthers, no viable pollen grains, and low seed setting rate, further verifies the application of the CTB17 gene in regulating the cold tolerance of the booting stage of rice, enriches the understanding of the cold tolerance of the booting stage of rice, and provides a theoretical basis for breeding cold-tolerant, stable-yield, and high-yield rice. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 The phenotype identification results of the mutant ctb17 of the present application embodiment 1 ems

[0029] Figure 2 The seed setting rate statistical results of the mutant ctb17 of the present application embodiment 1 ems

[0030] Figure 3 The mutant site diagram of the gene LOC_Os06g05240 of the present application embodiment 2

[0031] Figure 4 The seed setting rate statistical results of the mutant ctb17 and ctb17 of the present application embodiment 2 cr1 cr2

[0032] Figure 5 ​​​​Figure A is a schematic diagram of the conserved motif of the CTB17 protein; Figure B is the subcellular localization result of CTB17;

[0033] Figure 6 Figure A is a sequence alignment of the conserved amino acids of CTB17 and homologous proteins; Figure B is a schematic diagram of the site-directed mutation vector and the conversion sequence of the mutated nucleotide and amino acid; Figure C is the seed setting rate statistics of the mutant obtained by site-directed mutation.

[0034] Figure 7 Figure 4 is a schematic diagram of the chromosome of rice 9311 and KY131 genotypes distribution; wherein, blue (WT) represents homozygous wild type (wild type), green (HT) represents heterozygous (heterozygous), and red (HM) represents homozygous mutant (homozygous mutant).

[0035] Figure 8 Figure 4 is the seed setting rate statistics of the rice 9311 and KY131 isolates; wherein, D10 and D11, D13 and D14 are isolates of the same progeny, D10 and D13 are 9311 fragments, and D11 and D14 are KY131 fragments. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0037] The present application first discovered that CTB17 is an important gene that can regulate cold tolerance at the booting stage in rice. A mutant ctb17 with reduced cold tolerance was obtained by EMS chemical mutagenesis ems, after low temperature treatment, the anther is short and white, no active pollen grain is produced, and the seed setting rate is reduced to almost sterile, indicating that the CTB17 gene is the key gene controlling cold tolerance at the booting stage of rice. Among them, the sequence of CTB17 gene is shown as SEQ ID NO. 1, and the coding sequence of CTB17 gene is shown as SEQ ID NO. 2. Subsequently, through cloning and functional verification of CTB17 gene, it is found that the 121th amino acid H of CTB17 gene is the key amino acid site of CTB17 for cold tolerance at the booting stage of rice. Through the study on the functional differentiation of gene CTB17 in the cold tolerance of rice at the booting stage of different subspecies, it is found that the function of CTB17 gene in indica rice and japonica rice is differentiated, and the function of CTB17 gene in japonica rice is more helpful to improve the cold tolerance at the booting stage of rice, while the function of gene CTB17 in indica rice is weaker in this respect.

[0038] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified.

[0039] Example 1 mutant ctb17 ems phenotype identification

[0040] Select Kongyu131 (KY131) rice seeds as the material, construct an EMS mutagenesis mutant library, and obtain a mutant ctb17 with reduced cold tolerance through cold tolerance detection and screening at the booting stage. ems After treatment, the anther morphology of the mutant and wild type KY131 is observed at the flowering stage, and the anther micrograph is taken using a dissecting microscope to compare the differences between the two under low temperature treatment.

[0041] Low temperature treatment condition: At the booting stage of rice (i.e. 7-10 days before heading), the M2 generation plants in the mutant library are subjected to low temperature treatment, the treatment temperature is set to 15℃, and the treatment duration is 7 days. During the treatment, the natural light condition of the plants is maintained to ensure that the temperature condition is the only variable. After low temperature treatment, all plants are restored to normal growth conditions (CK), the average temperature of rice cultivation season is 28℃, the light condition remains unchanged, and the flowering and seed setting conditions are continuously observed.

[0042] Pollen grain activity detection: Collect rice anthers, stain with iodine-potassium iodide staining solution (I2-KI solution), and observe pollen activity under a microscope after staining. Active pollen appears dark purple after staining, and inactive pollen remains transparent or light colored. The phenotype difference between KY131 and mutant ctb17 ems is recorded.

[0043] The results are shown in Figure 1 , under normal growth conditions (CK), the mutant ctb17ems anther development and morphology, pollen viability (I2-KI staining) showed no significant difference compared with KY131; but after low temperature treatment, the mutant ctb17 ems anther was short and white, and no viable pollen grains were produced.

[0044] Then the seed setting rate of KY131 and ctb17 ems was detected.

[0045] The seed setting rate detection steps are as follows: after low temperature treatment, the normal growth conditions are restored until the seeds mature, the seed setting rate of all rice is counted, the seed setting rate of single plant is calculated, and the difference in seed setting rate between the mutant ctb17 ems and KY131 is recorded.

[0046] The results are shown in Figure 2 , under normal growth conditions (CK), the seed setting rate of the mutant ctb17 ems showed no significant difference compared with KY131, but after low temperature treatment, the seed setting rate of ctb17 ems was reduced to almost sterile.

[0047] The above results show that the mutant ctb17 ems with reduced cold tolerance is successfully screened, and it is proved that the CTB17 gene plays a key regulatory role in the cold tolerance of rice booting stage.

[0048] Example 2 Cloning and functional verification of CTB17 gene

[0049] Backcrossing and construction of BC1F2 population: in order to further fine map the CTB17 gene, the mutant ctb17 ems was backcrossed with the parent KY131 to obtain the BC1F2 population. Leaf blades were taken from individuals in the BC1F2 population, and DNA was extracted by the conventional CTAB method to prepare for subsequent pool sequencing (BSA) and MutMap analysis.

[0050] Pool sequencing and MutMap analysis: BC1F2 individuals with cold tolerance phenotype and non-cold tolerance phenotype were selected to construct two mixed pools of cold tolerance and non-cold tolerance, respectively. Whole genome resequencing was performed on the two mixed pools, and data acquisition was performed using the Illumina sequencing platform.

[0051] As shown in Figure 3 , by comparing the sequences of the two mixed pools, the mutation site was screened, and it was found that the first nucleotide G of the 9th intron of the gene LOC_Os06g05240 located on chromosome 6 was mutated to A.

[0052] Subsequently, the LOC_Os06g05240 gene knockout mutant was created by CRISPR / Cas9 gene editing technology, and a genetic complementation vector of the gene was constructed to verify the mutant ctb17 ems The cold tolerance phenotype of the booting stage was reduced due to the mutation of the gene LOC_Os06g05240, and the specific steps were as follows:

[0053] (1) Design a CRISPR / Cas9 targeting sequence sgRNA for the gene LOC_Os06g05240: SEQ ID NO. 4, and construct a knockout vector for the gene. The vector used is pYLCRISPR / Cas9Pubi-H.

[0054] (2) Use the CRISPR / Cas9 system to create two knockout mutants ctb17 cr1 and ctb17 cr2 of LOC_Os06g05240 in the KY131 background.

[0055] (3) Construct a complementation vector: construct a complementation construct in which the C-terminal Flag tag of the full-length genomic DNA sequence is driven by the promoter of the gene LOC_Os06g05240. The vector is XF675, the upstream primer is CF2918: SEQ ID NO. 5, and the downstream primer is CF2989: SEQ ID NO. 6.

[0056] (4) Transform the CRISPR / Cas9 gene editing vector and the genetic complementation vector into rice by the agrobacterium-mediated genetic transformation method to obtain T0 generation plants, screen positive transgenic lines, and identify the gene editing and exogenous gene insertion.

[0057] Subsequently, the seed setting rate of ctb17 ems , KY131, CRISPR / Cas9 knockout mutants (ctb17 cr1 and ctb17 cr2 ), and the ctb17 ems mutant carrying the complementation construct was counted, and the low temperature treatment (15℃, 7 days) and normal growth conditions were set as the control group.

[0058] The results are shown in Figure 4 After low temperature treatment, the seed setting rate of ctb17 cr1 and ctb17 cr2 was reduced to almost sterile, which was consistent with the phenotype of ctb17 ems , indicating that the mutation of the LOC_Os06g05240 gene affected the cold tolerance of rice at the booting stage. The mutant ctb17 emsThe recovery of the solid rate was not significantly different from KY131, indicating that the exogenous LOC_Os06g05240 gene can restore cold tolerance at the booting stage. The above results show that the gene LOC_Os06g05240 is the CTB17 gene.

[0059] Example 3 Functional and site mutation study of gene CTB17

[0060] The genomic database was searched and analyzed for the gene sequence of CTB17, and the tool was used to predict the functional domain structure of the encoded protein. Through analysis, it was found that CTB17 encodes a carboxypeptidase containing a conserved Zn-peptidase motif (as shown in Figure 5 A), which may be related to the maturation and modification process of proteins.

[0061] Subsequently, subcellular localization analysis was performed on CTB17: rice seedling protoplasts were prepared, and active protoplasts were obtained by enzymolysis according to the conventional method. The CTB17 gene fusion green fluorescent protein (GFP) vector was constructed, and the vector and the specific marker plasmid RFP-SYP61 of trans-Golgi network / early endosome (TGN / EE) were introduced into rice protoplasts by PEG-mediated transformation method. Using a laser confocal scanning microscope to observe the fluorescence signal, it was found that the two were co-localized. It is shown that CTB17 protein is located in the trans-Golgi network / early endosome (TGN / EE) (as shown in Figure 5 B), suggesting that it may be involved in the maturation process of proteins.

[0062] The vector for constructing the CTB17 N-terminal fusion GFP recombinant plasmid was XF2838, the upstream primer was CF4131: SEQ ID NO. 7, and the downstream primer was CF4132: SEQ ID NO. 8.

[0063] Subsequently, the homologous gene protein sequences of rice, Arabidopsis thaliana, humans, and Drosophila were collected, and sequence alignment was performed using tools such as Clustal Omega. As shown in Figure 6 A, the 121st amino acid H (histidine) of CTB17 is highly conserved in the homologous proteins of these species, suggesting that this amino acid may be an important site for enzyme activity.

[0064] A CBE editing system was designed to edit the 2651st and 2652nd bases of the CTB17 gene, so as to mutate the CC encoding the 121st amino acid histidine (H) to TT encoding tyrosine (Y) (as shown in Figure 6 B), a CBE gene editing vector was constructed, and was transformed into KY131 rice by Agrobacterium-mediated method to obtain edited mutant plants.

[0065] The vector of the CBE gene editing construction is XF3631, and the sgRNA is SEQ ID NO. 9.

[0066] The seed setting rate of the statistical editing mutant under normal growth conditions and low temperature treatment (15℃, 7 days) was counted.

[0067] The results are shown in Figure 6 C, the CBE gene editing mutation leads to a significant decrease in the seed setting rate of KY131 rice under low temperature conditions to almost sterile. This phenotype is consistent with the mutant ctb17 ems , further confirming that the 121st amino acid H is a key amino acid site of CTB17 in the cold tolerance of rice booting stage.

[0068] Example 4: Study on the functional differentiation of gene CTB17 in cold tolerance of indica rice and japonica rice at booting stage

[0069] The cold tolerance of indica rice varieties is generally lower than that of japonica rice. This embodiment aims to further explore the functional differences of gene CTB17 in cold tolerance of indica rice and japonica rice at booting stage.

[0070] The cold-tolerant japonica rice variety KY131 and the non-cold-tolerant indica rice variety 9311 were used as parents to construct a Segment Substitution Lines (SSLs) population. Individuals in the SSL population were screened using molecular markers to find two groups of materials in which the CTB17 region was replaced by 9311 and KY131, respectively, in the KY131 genetic background, for direct comparison of the functional differences of CTB17 gene in indica rice and japonica rice. A pair of near-isogenic lines was screened from the SSL population, and the genetic backgrounds of the two materials were both KY131, but the CTB17 region was derived from 9311 and KY131, respectively, as shown in Figure 7 The near-isogenic lines were planted in a greenhouse and subjected to low temperature treatment at booting stage, with the temperature set at 15℃ for 7 days. After treatment, they were moved back to normal growth conditions, and the seed setting rate was counted after the rice matured.

[0071] The results are shown in Figure 8 D11(G) and D14(G) carrying the KY131 fragment had a significantly higher seed setting rate after low temperature treatment than D10(X) and D13(X) carrying the 9311 fragment. This indicates that the functions of CTB17 in indica rice and japonica rice are differentiated, and CTB17 in japonica rice is more helpful to improve the cold tolerance of rice at booting stage, while CTB17 in indica rice has weaker function.

[0072] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

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Claims

1. A method for modulating cold tolerance at the booting stage in rice, characterized by, Adjusting the expression or activity of CTB17 gene of rice or the protein coded by the gene, so as to adjust the cold tolerance of booting stage of rice; The amino acid sequence coded by the CTB17 gene is shown as SEQ ID NO.

3.

2. The method of claim 1, wherein, The method is selected from: (i) up-regulating the expression or activity of CTB17 gene or the protein coded by the gene, so as to improve the cold tolerance of booting stage; (ii) down-regulating the expression or activity of CTB17 gene or the protein coded by the gene, so as to reduce the cold tolerance of booting stage.

3. The method of claim 2, wherein, The down-regulation of the expression or activity of CTB17 gene or the protein coded by the gene is specifically any one of knocking out or silencing CTB17 gene in rice, or inhibiting the activity of CTB17 protein.

4. The method of claim 2, wherein, The up-regulation of the expression or activity of CTB17 gene or the protein coded by the gene is specifically any one of: (a) transferring the expression construct or vector of CTB17 gene into rice or rice cells; (b) transferring the expression construct or vector containing CTB17 gene into rice or rice cells.

5. A method for screening a modulator that modulates cold tolerance at the booting stage of rice, characterized by, The method comprises: adding candidate substance into a system containing CTB17 gene or the protein coded by the gene; detecting the system, and observing the expression or activity of CTB17 gene or the protein coded by the gene; the amino acid sequence coded by the CTB17 gene is shown as SEQ ID NO.

3.

6. A method for breeding rice, characterized by, The method comprises the following steps: introducing a substance that can improve or inhibit the expression amount of CTB17 gene, the expression amount of protein coded by CTB17 gene, or the activity of protein coded by CTB17 gene into rice or rice cells, and obtaining the selected rice; the amino acid sequence coded by the CTB17 gene is shown as SEQ ID NO.

3.

7. Application of CTB17 gene in adjusting the cold tolerance of booting stage of rice; the amino acid sequence coded by the CTB17 gene is shown as SEQ ID NO.

3.

8. Use according to claim 7, characterized in that, The adjustment is specifically any one of: (i) reducing the cold tolerance of booting stage by inhibiting or reducing the activity or expression amount of protein coded by CTB17 gene; (ii) reducing the cold tolerance of booting stage by knocking out, inhibiting or silencing the expression amount of CTB17 gene.

9. Use according to claim 7, characterized in that, The adjustment is specifically any one of: (a) improving the cold tolerance of booting stage by improving the activity or expression amount of protein coded by CTB17 gene; (b) improving the cold tolerance of booting stage by improving the expression amount of CTB17 gene.

10. Use according to claim 7, characterized in that, The nucleotide sequence of the CTB17 gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

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