Use of qcts11.1 gene in regulating cold tolerance of rice

Abstract

The application relates to application of a qCTS11.1 gene in regulating cold tolerance of rice, and a method for improving cold tolerance of rice, comprising the steps of replacing the original qCTS11.1 gene in the genome of a starting rice variety with a low-function qCTS11.1 gene, or knocking out the qCTS11.1 gene from the genome of the starting rice variety. The application finds a new cold tolerance related gene qCTS11.1, provides a theoretical basis and gene resource for rice cold tolerance improvement and breeding, and can be used for hybrid breeding and transgenic breeding.

Description

Technical Field

[0001] This invention relates to the field of rice molecular breeding, and more particularly to the application of the qCTS11.1 gene in regulating cold tolerance in rice. Background Technology

[0002] Rice (Oryza sativa) is my country's most important staple food crop, with more than half of the population relying on it as their primary food source. Because rice originated in tropical and subtropical regions, it is more sensitive to low-temperature stress than other crops. Low-temperature stress can lead to stunted growth and disrupted physiological metabolism in rice seedlings, and in severe cases, can cause yellowing, wilting, and even death of rice leaves, resulting in a significant decline in yield and quality. Therefore, conducting research on rice cold tolerance and identifying superior cold-resistant genes is of great theoretical and practical significance for promoting increased and stable rice production in my country and ensuring national food security.

[0003] In this study, we constructed a genetic population of recombinant inbred lines from Nipponbare and WD16343, used BSA-seq to perform QTL mapping for cold tolerance, and predicted qCTS11.1 as a candidate gene for cold tolerance in rice based on candidate gene functional annotation analysis. We then constructed transgenic materials to verify this prediction. In addition, we analyzed the gene's phylogenetic evolution and expression characteristics, providing a theoretical basis and genetic resources for breeding superior rice varieties that can withstand low-temperature damage. Summary of the Invention

[0004] In our study, we discovered two haplotypes of the qCTS11.1 gene in rice, one of which confers cold tolerance to rice. Furthermore, we found that when the qCTS11.1 gene was knocked out, the cold tolerance of the transgenic rice was also improved.

[0005] Based on the above research, this invention provides the application of the qCTS11.1 gene in regulating cold tolerance in rice.

[0006] The present invention also provides a method for improving the cold tolerance of rice, including replacing the original qCTS11.1 gene in the genome of the starting rice variety with a low-functioning qCTS11.1 gene, or knocking out the qCTS11.1 gene from the genome of the starting rice line.

[0007] In one specific implementation, the method includes the following steps:

[0008] Rice lines containing the low-function qCTS11.1 gene are crossed with rice lines containing the normal-function qCTS11.1 gene. Then, the low-function qCTS11.1 gene is used as a selection marker to ensure that the target rice line contains only the low-function qCTS11.1 gene.

[0009] In one specific implementation, the rice line containing the low-functioning qCTS11.1 gene is NIP, or contains the same qCTS11.1 gene as NIP.

[0010] In one specific implementation, the low-function qCTS11.1 gene sequence is shown in SEQ ID NO:1.

[0011] In one specific implementation, the qCTS11.1 gene is knocked out from the genome of the starting rice line through gene editing.

[0012] This invention discovers a new cold tolerance-related gene, qCTS11.1, which provides a theoretical basis and gene resource for cold tolerance improvement and breeding of rice. It can be used for both hybridization breeding and transgenic breeding. Attached Figure Description

[0013] Figure 1 This study aimed to screen for the cold tolerance-related gene qCTS11.1 in rice seedlings. A shows the cold tolerance phenotypes of NIP and WD16343; B is a schematic diagram of the RI L population construction; C compares the survival rates of the RI L population, Nipponbare, and WD16343 after 4℃ cold treatment; D shows the distribution of Δ(SNP-index) values ​​on each chromosome; E is a detailed genetic map of the cold tolerance gene; and F shows the expression level of qCTS11.1 in Nipponbare and WD16343 seedlings after different cold treatment times, detected by qRT-PCR. P-values ​​were obtained from a two-tailed t-test, and error bars and SEM values ​​are included.

[0014] Figure 2 The study aimed to increase cold tolerance in rice seedlings by knocking out qCTS11.1 using CRISPR / Cas9. Figure A shows a schematic diagram of qCTS11.1 knockout, with the gene structure of qCTS11.1 at the top and sequence variations in knockout families shown below. Figure B shows the cold tolerance phenotypes of qCTS11.1 knockout families and NIPs. Figure C shows the survival rate of qCTS11.1 knockout families and NIPs under 4℃ cold treatment. Significance was determined using a two-tailed t-test, with error bars, SEM, and a replication factor of n=3. Detailed Implementation

[0015] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

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

[0017] We selected two parental rice varieties, Nipponbare (NIP) and WD16343, which showed significant differences in cold tolerance phenotypes at the seedling stage. NIP exhibited stronger cold tolerance at the seedling stage compared to WD16343. Figure 1 A); More than 200 recombinant inbred line populations (RILs) were constructed by self-pollinating for more than 8 generations after crossing Nipponbare with WD16343 as experimental materials. Figure 1 B); Analysis of the cold tolerance of the recombinant inbred line population and statistical results showed that 41 seedlings had a survival rate as high as 80%, belonging to highly cold-resistant families, while 38 had a lower survival rate, belonging to cold-sensitive families. Significant phenotypic segregation was observed, and the survival rate distribution within the population followed a normal distribution. Figure 1 C) indicates that cold tolerance is determined by multiple genes, and quantitative trait (QTL) mapping analysis can be performed on it.

[0018] Using seedling survival rate at low temperatures as an indicator of cold tolerance, two DNA pools were constructed: a pool for high cold tolerance genes and a pool for low cold tolerance genes. Genome sequencing was then performed on the parents and the pools. Blued Segregant Analysis (BSA) was used to identify QTLs for cold tolerance in rice seedlings. Genome-wide SNP-index analysis revealed a highly significant locus in a 3.6 Mb region on chromosome 11. Figure 1 D). Using the remaining heterozygous lines and molecular markers at this site, six recombinant single plants were screened. Co-segregation detection in their progeny allowed for fine localization of this site, narrowing the range to within 1.4 Mb, and it was named qCTS11. Figure 1 E).

[0019] To identify candidate genes influencing differences in cold sensitivity, we screened a gene, LOC_Os11 g38800, whose expression was upregulated by cold and exhibited sequence differences through transcriptomic and genomic analyses under cold stress. This gene was named qCTS11.1. We validated the expression level of qCTS11.1 in Nipponbare and WD16343 seedlings under different cold treatment times using qRT-PCR, finding that the gene was indeed upregulated by cold, reaching its highest expression level after 12 hours of cold treatment. Figure 1 F), it is speculated that qCTS11.1 is a key candidate gene affecting the difference in cold tolerance between Nipponbare and WD16343.

[0020] 2. CRI SPR / Cas9 knockout qCTS11.1 increases cold tolerance in rice seedlings.

[0021] To investigate the biological function of qCTS11.1, we used the CRI SPR / Cas9 dual-target knockout system to mutate qCTS11.1 in the parental Nipponbare background to obtain transgenic knockout families of qCTS11.1. Figure 2 A). Through sequencing and alignment, we obtained two transgene knockout homozygous materials with different mutation types in qCTS11.1, qCTS11.1-1 (+1bp) and qCTS11.1-2 (-2bp), both of which led to frameshift mutations and premature termination in qCTS11.1.

[0022] Furthermore, we subjected qCTS11.1 knockout families and wild-type control Nipponbare seedlings to a 72-hour cryogenic treatment at 4°C. After a 14-day recovery period, the survival rate of the seedlings was assessed. After the recovery culture was completed, the survival rate was statistically analyzed and cold tolerance was evaluated. The results showed that the survival rate of the qCTS11.1-1 and qCTS11.1-2 mutant materials was significantly higher than that of the wild-type Nipponbare. Figure 2 B and Figure 2 C) indicates that knocking out qCTS11.1 can significantly increase the cold tolerance of rice seedlings.

[0023] 3. Comparative analysis of qCTS11.1 gene sequence and amino acid sequence

[0024] We sequenced and compared the qCTS11.1 gene sequences of Nipponbare (N ip) (SEQ ID NO:1) and WD16343 (SEQ ID NO:2). Gene sequence difference analysis showed that WD16343 had 36 variations compared with the Nipponbare gene sequence. These variations occurred in the 5' untranslated region (UTR), introns, exons and 3' UTR.

[0025] We further compared the amino acid sequences of the qCTS11.1 protein from Nipponbare and WD16343 materials, and the results showed that the protein in both materials had the same amino acid length of 278. Amino acid sequence difference analysis showed that the amino acid sequences of Nipponbare and WD16343 were completely identical, and the three variations in the exons of the qCTS11.1 gene were all synonymous mutations and did not lead to changes in amino acids.

[0026] 4. Comparative Analysis of qCTS11.1 Promoter Sequences

[0027] We compared the 2kb promoter sequences of Nipponbare (SEQ ID NO:3) and WD16343 (SEQ ID NO:4) materials qCTS11.1. Differential analysis of the promoter sequences revealed 13 variations in the WD16343 promoter sequence compared to the Nipponbare promoter sequence, including 3 Indels (-- / AT, GGA / ---, - / A) and 10 SNPs (A / G, G / A, A / G, C / T, C / T, C / T, A / G, G / A, G / A, A / T).

[0028] The above experiments demonstrate that we discovered two haplotypes of the qCTS11.1 gene in different rice varieties. The qCTS11.1 haplotype from Nipponbare has low function, while the qCTS11.1 haplotype from WD16343 has high function. When the high-functioning qCTS11.1 haplotype in a rice variety is replaced with the low-functioning qCTS11.1 haplotype through hybridization, the resulting hybrid rice variety exhibits higher cold tolerance. Furthermore, when the qCTS11.1 gene is knocked out in a rice variety containing the high-functioning qCTS11.1 haplotype, the resulting transgenic rice variety also exhibits 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the cold tolerance of rice, characterized in that, This includes knocking out [the virus] from the genome of the starting rice line. qCTS11.1 The genetic steps, the starting rice variety is Nipponbare, the... qCTS11.1 The gene sequence is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that, Knockout from the genome of the starting rice line through gene editing qCTS11.1 Gene.

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