Application of qCTS11.3 gene in regulation of cold tolerance of rice

Abstract

The invention relates to application of a qCTS11.3 gene in regulation of cold tolerance of rice and a method for improving cold tolerance of rice, and the method comprises the step of replacing the original qCTS11.3 gene in a genome of a starting rice variety with a low-function qCTS11.3 gene or knocking out the qCTS11.3 gene from the genome of the starting rice variety. The invention finds a novel cold tolerance related gene qCTS11.3, provides a theoretical basis and gene resources for cold tolerance improvement and breeding of rice, and can be used for crossbreeding and transgenic breeding.

Description

Technical Field

[0001] The present invention relates to the field of rice molecular breeding, and more particularly to the application of qCTS11.3 gene in regulating the cold tolerance of rice. Background Art

[0002] Rice (Oryza sativa L.) is the most important staple food crop in my country, and more than half of the population relies on rice as their staple food. Since rice originates from tropical and subtropical regions, it is more sensitive to low temperature stress than other crops. Low temperature stress can cause growth stagnation and physiological metabolic disorders in rice seedlings. In severe cases, it may cause yellowing, withering, or even death of rice leaves, resulting in a significant decrease in yield and quality. Therefore, conducting research on rice cold tolerance and exploring excellent cold tolerance genes are of great theoretical and practical significance for promoting the increase and stability of rice production in my country and ensuring national food security.

[0003] In this study, we constructed a recombinant inbred line genetic population of Nipponbare and WD16343, used BSA-seq to locate QTLs for cold tolerance traits, and predicted qCTS11.3 as a candidate gene for rice cold tolerance based on the functional annotation analysis of candidate genes. We also constructed transgenic materials for verification and analyzed the systematic evolution and expression characteristics of the gene, providing a theoretical basis and genetic resources for breeding excellent rice varieties that can resist low temperature damage. Summary of the invention

[0004] In our study, we found that there are two haplotypes of the qCTS11.3 gene in rice, one of which can confer cold tolerance to rice; further, our study found that when the qCTS11.3 gene was knocked out, the cold tolerance of the transgenic rice could also be improved.

[0005] The invention provides application of qCTS11.3 gene in regulating cold tolerance of rice.

[0006] The present invention also provides a method for improving the cold tolerance of rice, comprising the steps of replacing the original qCTS11.3 gene in the genome of a starting rice variety with a low-function qCTS11.3 gene, or knocking out the qCTS11.3 gene from the genome of a starting rice line.

[0007] In a specific embodiment, the method comprises the following steps:

[0008] A rice line containing a low-function qCTS11.3 gene is hybridized with a rice line containing a normal-function qCTS11.3 gene, and then the low-function qCTS11.3 gene is used as a screening marker to make the target rice line contain only the low-function qCTS11.3 gene.

[0009] In a specific embodiment, the rice line containing the low-function qCTS11.3 gene is NIP, or contains the qCTS11.3 gene identical to NIP.

[0010] In a specific embodiment, the low-function qCTS11.3 gene sequence is shown as SEQ ID NO:1.

[0011] In one specific embodiment, the qCTS11.3 gene is knocked out from the genome of a starting rice line by gene editing.

[0012] The present invention discovered a new cold tolerance-related gene qCTS11.3, which provided a theoretical basis and gene resources for rice cold tolerance improvement and breeding, and can be used for hybrid breeding as well as transgenic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Screening of the cold-tolerant gene qCTS11.3 in rice seedlings. A is the cold-tolerant phenotype of NIP and WD16343; B is the schematic diagram of the construction of the RIL population; C is the comparison of the survival rates of the RIL population and Nipponbare and WD16343 after 4°C cold treatment; D is the distribution of Δ(SNP-i ndex) values ​​on each chromosome; E is the fine genetic map of cold-tolerant genes; F is the expression level of qCTS11.3 detected by qRT-PCR after different cold treatment times in Nipponbare and WD16343 seedlings. P values ​​are obtained by two-tailed t test, error bars, sem.

[0014] Figure 2 CRISPR / Cas9 knockout of qCTS11.3 increases cold tolerance in rice seedlings. A is a schematic diagram of CRISPR / Cas9 knockout of qCTS11.3, with the gene structure of qCTS11.3 above and the sequence variation of the knockout family below. B is the cold tolerance phenotype of the qCTS11.3 knockout family and NIP. C is the survival rate of the qCTS11.3 knockout family and NIP under 4℃ cold treatment. Significance was obtained by two-tailed t test, error bars, sem, number of replicates n=3. DETAILED DESCRIPTION

[0015] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0016] 1. Screening of candidate gene qCTS11.3

[0017] We selected two parental materials, Nipponbare (NIP) and WD16343, which have significant differences in the cold tolerance phenotype at the seedling stage. NIP showed stronger cold tolerance at the seedling stage than WD16343 ( Figure 1 A and Figure 1 B); more than 200 recombinant inbred line populations (RIL) were constructed by hybridizing Nipponbare and WD16343 and selfing for more than 8 generations as experimental materials ( Figure 1 C); The cold tolerance identification and statistical analysis of the recombinant inbred line population showed that 41 seedling survival rates were as high as 80%, belonging to the high cold tolerance family, and 38 survival rates were low, belonging to the cold sensitive family. There was a significant phenotypic separation phenomenon, and the distribution of survival rates in the population showed a normal distribution ( Figure 1 D), indicating that the cold tolerance trait is determined by the participation of multiple genes, and quantitative trait QTL positioning analysis can be performed on it.

[0018] Using the survival rate of seedlings at low temperatures as the cold tolerance identification index, two DNA pools were constructed, namely a high cold tolerance gene pool and a low cold tolerance gene pool. Then the genomes of the parents and the pools were sequenced, and the QTLs for cold tolerance in rice seedlings were identified by the Bulked Segregant Analysis (BSA). The whole genome SNP-index analysis showed that a highly significant locus ( Figure 1 E). Using the remaining heterozygous lines and molecular markers of the locus, six recombinant strains were screened, and the locus was precisely located through co-segregation detection of their progeny, narrowing the range to a 1.4Mb interval, which was named qCTS11 ( Figure 1 F).

[0019] In order to explore candidate genes that affect cold sensitivity, we screened a gene LOC_Os11 g36160 whose expression was induced by cold and had gene sequence differences through transcriptome and genome analysis under cold stress, and named it qCTS11.3. We verified the expression level of qCTS11.3 in Nipponbare and WD16343 seedlings under different cold treatment times by qRT-PCR and found that the gene was indeed up-regulated by cold treatment, and the expression level reached the highest level after 12 hours of cold treatment. It is speculated that qCTS11.3 is a key candidate gene that affects the cold tolerance difference between Nipponbare and WD16343.

[0020] 2. CRISPR / Cas9 knockout of qCTS11.3 increases cold tolerance in rice seedlings

[0021] In order to study the biological function of qCTS11.3, we used the CRISPR / Cas9 dual-target knockout system to mutate qCTS11.3 in the parental material Nipponbare background to obtain a transgenic knockout family of qCTS11.3 ( Figure 2 A). Through sequencing and comparison, we obtained two different types of transgenic knockout homozygous materials of qCTS11.3, qCTS11.3-1 (-4bp) and qCTS11.3-2 (-5bp), both of which resulted in frameshift mutation and loss of function of qCTS11.3.

[0022] We further subjected the qCTS11.3 knockout family and the wild-type control Nipponbare seedlings to a 4°C cold treatment for 72 hours, and examined the survival rate of the seedlings after 14 days of recovery. After the recovery culture was completed, the survival rate was statistically analyzed and the cold tolerance was evaluated. The results showed that the survival rate of the qCTS11.3-1 and qCTS11.3-2 mutant materials was higher than that of the wild-type Nipponbare ( Figure 2 B and Figure 2 C), indicating that knocking out qCTS11.3 can significantly increase the cold tolerance of rice at the seedling stage.

[0023] 3. Comparative analysis of qCTS11.3 gene sequences

[0024] We compared the qCTS11.3 gene sequences of Nipponbare (Nip) (SEQ ID NO:1) and WD16343 (SEQ ID NO:2) materials by sequencing. Gene sequence difference analysis showed that compared with the gene sequence of Nipponbare, the gene sequence of WD16343 had 48 mutations, which occurred in the 3' untranslated region (UTR), introns and exons.

[0025] We further compared the amino acid sequences of qCTS11.3 proteins from japonica rice Nipponbare (Nip) and indica rice WD16343, and the results showed that the amino acid length of the protein in the two materials was the same, 1102. Amino acid sequence difference analysis showed that compared with the amino acid sequence of Nipponbare, the amino acid sequence of WD16343 had five mutations, namely His / Gln, Al a / Thr, Thr / Asn, Phe / Leu, and Lys / Glu. Comprehensive analysis of the gene sequence and amino acid sequence of qCTS11.3 showed that there were 48 mutations in the gene sequence, which occurred in the 3' untranslated region (UTR), introns and exons, and there were 10 mutations in exons, 5 of which were synonymous mutations and 5 were non-synonymous mutations, causing 5 amino acid mutations, and no frameshift mutations occurred, resulting in large-scale amino acid changes.

[0026] 4. Comparative analysis of qCTS11.3 promoter sequences

[0027] We compared the 2kb promoter sequences of qCTS11.3 from Nipponbare (SEQ ID NO:3) and WD16343 (SEQ ID NO:4). Promoter sequence difference analysis showed that compared with the promoter sequence of Nipponbare, the promoter sequence of WD16343 had 24 variations, including 7 Indels (- / C, - / T, - / T, A / -, C / -, - / A, - / A) and 17 SNPs (C / T, T / A, G / T, A / C, C / G, G / A, T / C, T / G, T / A, T / C, T / C, C / T, G / T, C / T, G / A, T / C, T / C).

[0028] The above experiments show that we found two qCTS11.3 gene haplotypes in different rice lines. Among them, the qCTS11.3 gene haplotype from Nipponbare has low function, and the qCTS11.3 gene haplotype from WD16343 has high function. When the original high-function qCTS11.3 gene haplotype in the rice line is replaced with a low-function qCTS11.3 gene haplotype through hybridization, the resulting hybrid rice line has higher cold tolerance; when the qCTS11.3 gene is knocked out in the rice line containing the high-function qCTS11.3 gene haplotype, the resulting transgenic rice line also has higher cold tolerance.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of qCTS11.3 gene in regulating cold tolerance of rice.

2. A method for improving cold tolerance of rice, characterized in that: The method includes the steps of replacing the original qCTS11.3 gene in the genome of the starting rice variety with the low-function qCTS11.3 gene, or knocking out the qCTS11.3 gene from the genome of the starting rice line.

3. The method according to claim 2, characterized in that The following steps are involved: A rice line containing a low-function qCTS11.3 gene is hybridized with a rice line containing a normal-function qCTS11.3 gene, and then the low-function qCTS11.3 gene is used as a screening marker to make the target rice line contain only the low-function qCTS11.3 gene.

4. The method according to claim 3, characterized in that The rice line containing the low-function qCTS11.3 gene is NIP, or contains the same qCTS11.3 gene as NIP.

5. The method according to claim 4, characterized in that The low-function qCTS11.3 gene sequence is shown in SEQ ID NO:

1.

6. The method according to claim 2, characterized in that The qCTS11.3 gene was knocked out from the genome of the starting rice line by gene editing.

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