New gene OsWRKY24 influencing cold resistance of rice and identification method thereof

Abstract

The invention discloses a novel gene OsWRKY24 influencing cold resistance of rice and an identification method thereof. The novel gene OsWRKY24 is prepared by comparative transcriptome research screening carried out by near-isogenic lines Towada and ZL31 in a rice seedling stage under cold stress. A rice seedling stage cold tolerance related gene OsWRKY24 is screened by utilizing a transcriptome re-sequencing method, the function of the gene is verified by constructing a transgenic knockout material, and meanwhile, a gene sequence, an amino acid sequence, a promoter sequence, expression characteristics and a phylogenetic tree are analyzed; theoretical basis and gene resources are provided for cold-resistant improvement and breeding of rice; a novel rice cold sensitive gene OsWRKY24 is identified, and the cold tolerance of rice can be improved by gene mutation.

Description

Technical Field

[0001] The invention relates to the technical field of rice molecular breeding, and in particular to a new gene OsWRKY24 affecting rice cold tolerance and an identification method thereof. Background Art

[0002] Rice is a major food crop that is sensitive to low temperatures. Low temperature damage is one of the important environmental factors affecting rice production. In recent years, the global annual rice planting area is about 140 million hectares, of which more than 15 million hectares of rice-growing area are threatened by low temperatures. my country loses 5 to 10 billion kilograms of rice due to cold damage every year. Low temperatures limit the expansion of rice to high altitudes and high latitudes, thereby limiting the rice planting area; frequent low temperatures in rice-growing areas also seriously affect rice yield and quality; cold stress tolerance is a complex genetic trait controlled by multiple genes / quantitative trait loci; compared with other agronomic traits, genetic research on rice low temperature tolerance has progressed slowly, and only a few low temperature tolerance genes have been identified. Therefore, cloning new rice cold tolerance genes and revealing the molecular mechanisms behind them are the key to improving rice cold tolerance, which is of great significance for the genetic improvement of rice cold tolerance and the breeding of new cold-tolerant varieties. Summary of the invention

[0003] The object of the present invention is to provide a new gene OsWRKY24 affecting rice cold tolerance and an identification method thereof, so as to solve the problems raised in the above background technology.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme: a new gene OsWRKY24 that affects rice cold tolerance is obtained by comparative transcriptome screening of rice seedling near-isogenic lines Towada and ZL31 under cold stress, and its identification method is as follows:

[0005] (1) Construction of knockout families and phenotypic analysis;

[0006] (2) Gene sequence analysis;

[0007] (3) Amino acid sequence analysis;

[0008] (4) Promoter sequence analysis;

[0009] (5) System evolution analysis;

[0010] (6) Expression pattern analysis.

[0011] Preferably, the construction and phenotypic analysis of the knockout family is to mutate the second exon of OsWRKY24 in the parental material Towada background using the CRISPR / Cas9 dual-target knockout system to obtain a transgenic knockout family of OsWRKY24.

[0012] Preferably, in the gene sequence analysis, the OsWRKY24 gene sequences of Nipponbare, ZL31 and Towada materials are compared by sequencing.

[0013] Preferably, in the amino acid sequence analysis, the amino acid sequences of OsWRKY24 proteins from Nipponbare, ZL31 and Towada materials are compared.

[0014] Preferably, in the promoter sequence analysis, 2 kb promoter sequences of Nipponbare, ZL31 and Towada material OsWRKY24 are compared.

[0015] Preferably, the amino acid sequence of OsWRKY24 is used for phylogenetic tree analysis.

[0016] Preferably, the expression pattern of OsWRKY24 in different tissues of Towada seedlings is analyzed by quantitative RT-PCR (qRT-PCR).

[0017] The beneficial effects of the present invention are:

[0018] The present invention screened a rice seedling cold tolerance-related gene OsWRKY24 by transcriptome resequencing, verified the function of the gene by constructing transgenic knockout materials, and analyzed the gene sequence, amino acid sequence, promoter sequence, expression characteristics and phylogenetic tree, providing a theoretical basis and gene resources for rice cold tolerance improvement and breeding.

[0019] The present invention identifies a new rice cold-sensitive gene OsWRKY24, and the gene mutation can improve the cold tolerance of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the overall technical roadmap of the present invention;

[0021] Figure 2 Screening of the cold tolerance-related gene OsWRKY24 in rice seedling stage of the present invention: (A) Phenotypic analysis of Towada and ZL31 after 4°C cold treatment; (B) Comparison of survival rates of Towada and ZL31 after 4°C cold treatment; (C) Comparison of up-regulated and down-regulated genes between Towada and ZL 31 after 3h and 12h of cold treatment; (D) Genes with significant expression differences between Towada and ZL31 after 3 hours or 12 hours of cold treatment; (E) qRT-PCR detection of the expression level of OsWRKY24 in Towada and ZL31 seedlings after different cold treatment times; P value is obtained by two-tailed t test, error bar, sem;

[0022] Figure 3The present invention shows that the CRISPR / Cas9 knockout of OsWRKY24 increases the cold tolerance of rice seedlings: (A) Schematic diagram of CRISPR / Cas9 knockout of OsWRKY24; the upper part shows the gene structure of OsWRKY24, and the lower part shows the sequence variation of the knockout family; (B) The cold tolerance phenotype of the OsWRKY24 knockout family and Towada; (C) The survival rate of the OsWRKY24 knockout family and Towada under 4°C cold treatment; the significance is obtained by two-tailed t test, error bars, sem, number of replicates n=3;

[0023] Figure 4 This is a comparison of the OsWRKY24 gene sequences of Nipponbare, ZL31 and Towada in the present invention, where OsWRKY24-N, Z and T represent the OsWRKY24 gene sequences of Nipponbare, ZL31 and Towada materials, respectively;

[0024] Figure 5 This is a comparison of the amino acid sequences of OsWRKY24 from Nipponbare, ZL31 and Towada of the present invention, where OsWRKY24-N, Z and T represent the amino acid sequences of OsWRKY24 from Nipponbare, ZL31 and Towada materials, respectively;

[0025] Figure 6 The 2 kb promoter sequence analysis of OsWRKY24 in Nipponbare, ZL31 and Towada of the present invention; (A) Comparison of the 2 kb promoter sequences of OsWRKY24 in Nipponbare, ZL31 and Towada, OsWRKY24-N, Z, T represent the 2 kb promoter sequences of OsWRKY24 in Nipponbare, ZL31 and Towada materials, respectively. (B) Analysis of the 2 kb promoter cis-element of OsWRKY24;

[0026] Figure 7 The phylogenetic analysis and expression pattern analysis of OsWRKY24 of the present invention; (A) OsWRKY24 amino acid sequence alignment and structural analysis; NLS, nuclear localization signal peptide, * represents WRKY core sequence; (B) OsWRKY24 amino acid sequence phylogenetic tree evolution analysis; Mega7 was used for analysis, and the evolutionary tree was based on the Neighbor-Joining method;

[0027] Figure 8 Expression pattern analysis of OsWRKY24 of the present invention. Significance was determined by Duncan test. Error bars, sem. Number of replicates n=3. DETAILED DESCRIPTION

[0028] In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

[0029] Screening of candidate gene OsWRKY24:

[0030] There are significant differences in cold tolerance between the near-isogenic lines Towada and ZL31 at the rice seedling stage, and ZL31 shows stronger cold sensitivity than Towada at the rice seedling stage ( Figure 2 A, B). To elucidate the reasons for the differences in cold sensitivity among near-isogenic lines, a comparative transcriptome study under cold stress was conducted, and the cold-related gene OsWRKY24 ( Figure 2 C, D). The expression level of OsWRKY24 in Towada and ZL31 seedlings under different cold treatment times was detected by qRT-PCR. It was found that the gene was up-regulated by cold treatment, and the expression level reached the highest level after 3h of cold treatment. The mRNA abundance in ZL31 was significantly higher than that in Towada ( Figure 2 E), which is consistent with the transcriptome results, so OsWRKY24 is a key candidate gene affecting the difference in cold tolerance between Towada and ZL31.

[0031] CRISPR / Cas9 knockout of OsWRKY24 increases cold tolerance in rice seedlings:

[0032] The second exon of OsWRKY24 was mutated in the Towada background using the CRISPR / Cas9 dual-target knockout system to obtain a transgenic knockout line of OsWRKY24 ( Figure 3 A). Through sequencing and comparison, three different types of transgenic knockout homozygous materials of OsWRKY24 were obtained, oswrky24-1 (+1bp), oswrky24-2 (+2bp), and oswrky24-3 (-2bp), all of which caused frameshift mutations and loss of function of OsWRKY24 ( Figure 3 A). The OsWRKY24 knockout family and wild-type control Towada seedlings were further subjected to 4°C cold treatment for 72 hours, and the survival rate of the seedlings was examined after 14 days of recovery ( Figure 3 B). The results showed that the survival rates of oswrky24-1, oswrky24-2, and oswrky24-3 were 35.42%, 41.67%, and 35.42%, respectively, while the survival rate of the control Towada was only 8.33% ( Figure 3 C), indicating that knocking out OsWRKY24 can significantly increase the cold tolerance of rice at the seedling stage.

[0033] Comparative analysis of OsWRKY24 gene sequence and amino acid sequence:

[0034] The OsWRKY24 gene sequences of Nipponbare, ZL31 and Towada were compared by sequencing, and the results showed that the length of the gene in the three materials was the same, 2175bp. Gene sequence difference analysis showed that the gene sequences of Nipponbare and Towada were the same, while ZL31 had 5 mutations, namely single base mutations of T / C, C / A, C / T, T / C, and A / G, and no sequence frameshift mutation occurred ( Figure 4 );

[0035] The amino acid sequences of OsWRKY24 proteins from Nipponbare, ZL31, and Towada were further compared, and the results showed that the amino acid length of the protein in the three materials was the same, 555. Amino acid sequence difference analysis showed that the amino acid sequences of Nipponbare and Towada were exactly the same, while ZL31 had four mutations, namely Leu / Pro, Ala / Glu, Val / Ala, and Arg / Gly mutations ( Figure 5 ). Comprehensive analysis of the gene sequence and amino acid sequence of OsWRKY24 showed that there were 5 single-base mutations in the gene sequence, all of which occurred in exons, of which 1 was a synonymous mutation and 4 were non-synonymous mutations, causing 4 amino acid mutations, and no frameshift mutations occurred that resulted in large-scale amino acid changes.

[0036] Comparative analysis of OsWRKY24 promoter sequences:

[0037] The 2kb promoter sequences of Nipponbare, ZL31 and Towada material OsWRKY24 were compared. The promoter sequence difference analysis showed that compared with the Nipponbare promoter sequence, the promoter sequence of Towada had one Indel, which was a single base deletion of A / -, while ZL31 had 9 mutations, including 3 Indels (- / A, A / -, G / -) and 6 SNPs (G / A, A / C, T / C, A / G, T / C, C / A) ( Figure 6 A). To understand how OsWRKY24 participates in cold tolerance in rice seedlings, we analyzed the cis-elements of the 2kb promoter sequence of OsWRKY24, which showed that there were some cis-elements related to cold tolerance on the promoter, including ABA response element, MYB binding site and WRKY binding site W-box, etc. ( Figure 6 B), which means that OsWRKY24 may be regulated by related transcription factors and participate in cold tolerance.

[0038] OsWRKY24 phylogenetic analysis:

[0039] Phylogenetic tree analysis (NCBI) was performed using the amino acid sequence of OsWRKY24. The results showed that the gene encodes a WRKY-type transcription factor, which belongs to the first subfamily of the WRKY family. It contains two WRKY domains and a nuclear localization signal peptide, and has the ability to enter the nucleus and bind to DNA. Homologous proteins are found in crops such as rice, Brachypodium distachyon, millet, corn, sorghum, barley and wheat, and the WRKY domain and nuclear localization signal peptide are highly conserved, which means that the protein has certain similarities in terms of function ( Figure 7 ).

[0040] Analysis of OsWRKY24 tissue expression pattern:

[0041] The expression levels of OsWRKY24 in different tissues of Towada seedlings were analyzed by quantitative RT-PCR (qRT-PCR). Figure 8 As shown, OsWRKY24 is expressed in roots, stems, and leaves, and there are differences in expression among tissue locations. The expression level of OsWRKY24 is highest in leaves, followed by roots and then stems.

[0042] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying a new gene OsWRKY24 that affects cold tolerance in rice, characterized by: The novel gene OsWRKY24 was screened by comparative transcriptome studies of near-isogenic rice lines Towada and ZL31 at the seedling stage under cold stress. The identification method is as follows: (1) Construction of knockout families and phenotypic analysis; (2) Gene sequence analysis; (3) Amino acid sequence analysis; (4) Promoter sequence analysis; (5) System evolution analysis; (6) Expression pattern analysis.

2. The method for identifying a new gene OsWRKY24 that affects cold tolerance in rice according to claim 1, characterized in that: The construction and phenotypic analysis of the knockout family are to mutate the second exon of OsWRKY24 in the parental material Towada background using the CRISPR / Cas9 dual-target knockout system to obtain a transgenic knockout family of OsWRKY24.

3. The method for identifying a new gene OsWRKY24 that affects cold tolerance in rice according to claim 1, characterized in that: In the gene sequence analysis, the OsWRKY24 gene sequences of Nipponbare, ZL31 and Towada materials were compared by sequencing.

4. The method for identifying a novel gene OsWRKY24 affecting cold tolerance in rice according to claim 1, characterized in that: In the amino acid sequence analysis, the amino acid sequences of OsWRKY24 proteins from Nipponbare, ZL31 and Towada materials were compared.

5. The method for identifying a novel gene OsWRKY24 affecting cold tolerance in rice according to claim 1, characterized in that: In the promoter sequence analysis, 2 kb promoter sequences of Nipponbare, ZL31 and Towada material OsWRKY24 were compared.

6. The method for identifying a novel gene OsWRKY24 affecting cold tolerance in rice according to claim 1, characterized in that: Phylogenetic tree analysis was performed using the amino acid sequence of OsWRKY24.

7. The method for identifying a novel gene OsWRKY24 affecting cold tolerance in rice according to claim 1, characterized in that: The expression pattern of OsWRKY24 in different tissues of Towada seedlings was analyzed by quantitative RT-PCR (qRT-PCR).

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