Application of cold-tolerant candidate gene NHX2 in rice seedling stage

Abstract

The invention discloses an application of a cold-resistant candidate gene NHX2 in a rice seedling stage, and relates to the technical field of agricultural biology.According to the technical scheme, the cold-resistant candidate gene NHX2 can positively regulate and control the cold resistance of rice in the seedling stage, and the cold-resistant candidate gene NHX2 encodes a Cation / H + antiporter composed of 546 amino acids; the cold-tolerant candidate gene NHX2 belongs to an NHX family, the full length is 4105bp, the full length of a coding region is 1638bp, the cold-tolerant candidate gene NHX2 has 13 exons, and the molecular mass is 59.92 kDa. According to the invention, 1364 strains of a MAGIC multi-parent hybrid population are subjected to seedling-stage cold resistance identification, and then cold resistance related characters, namely the survival rate of a test material and the cold-resistant wilt level of leaves, are subjected to GWAS analysis, so that a seedling-stage cold-resistant candidate gene NHX2 of important QTL qSR11.2 is identified.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and more specifically, it relates to the application of a cold tolerance candidate gene NHX2 at the seedling stage of rice. Background Art

[0002] As one of the important food crops globally, the growth and yield of rice are affected by various environmental factors, especially low-temperature stress. Low temperature is one of the main abiotic stresses restricting the growth and distribution of rice. It can affect the growth and development, physiological metabolism, and ultimately the yield and quality of rice. Improving the cold tolerance of rice is of great significance for ensuring food security and coping with climate change.

[0003] In the study of cold tolerance traits in rice, genetic and molecular biology methods have been widely used to identify and clone key genes controlling cold tolerance traits. Genome-wide association studies (GWAS) and quantitative trait locus (QTL) analysis are two important gene mapping methods, which can help researchers identify molecular markers and genes related to cold tolerance traits without relying on biological background knowledge.

[0004] Traditional methods for improving cold tolerance traits mainly rely on breeding, but this process is often time-consuming and inefficient. With the development of gene editing technologies, such as the CRISPR / Cas9 system, it provides an accurate and efficient means to knockout or modify specific genes, thereby studying gene functions and improving crop traits. In addition, the regulation of gene expression is also an important means to improve crop traits, including the regulatory role of transcription factors and the interaction between proteins.

[0005] Therefore, the present invention aims at the application of a cold tolerance candidate gene NHX2 at the seedling stage of rice to improve the cold tolerance of rice seedlings and provide new strategies and directions for the genetic improvement of cold tolerance traits in rice seedlings. Summary of the Invention

[0006] The object of the present invention is to provide the application of a cold tolerance candidate gene NHX2 at the seedling stage of rice. The present invention identified the cold tolerance candidate gene NHX2 of the important QTL qSR11.2 by identifying the cold tolerance at the seedling stage of 1364 lines of the MAGIC multi-parent hybrid population and then performing GWAS analysis on the traits related to cold tolerance, namely the survival rate of the experimental materials and the cold tolerance wilt degree grade of the leaves.

[0007] The present invention provides the application of a cold tolerance candidate gene NHX2 at the seedling stage of rice in enhancing the cold tolerance of rice seedlings.

[0008] The present invention also provides a cold tolerance candidate gene NHX2 for rice seedlings. The full length of the cold tolerance candidate gene NHX2 is 4105 bp, the full length of the coding region is 1638 bp, it has 13 exons, the molecular mass is 59.92 kDa, and it encodes a Cation / H + antiporter composed of 546 amino acids.

[0009] The present invention is further configured such that: the cold tolerance candidate gene NHX2 is located on the vacuolar membrane.

[0010] In summary, the present invention has the following beneficial effects:

[0011] 1. By overexpressing the NHX2 gene, the present invention significantly improves the cold tolerance of rice. At the same time, using gene editing technology, the expression of the NHX2 gene can be precisely regulated, thereby optimizing the cold tolerance traits of rice under different environmental conditions, solving the problem that traditional breeding methods are time-consuming and inefficient, and being able to accelerate the breeding process of new cold-tolerant varieties;

[0012] 2. Through knockout and overexpression experiments of the NHX2 gene, the present invention shows that this gene plays a key role in regulating the response of rice to low-temperature stress, making the application of the present invention contribute to improving the survival ability and productivity of rice under adverse conditions;

[0013] 3. By improving the cold tolerance traits of rice through overexpression technology, the present invention helps to reduce the impact of low temperature on the growth and development of rice, thereby increasing yield and quality and ensuring food security;

[0014] 4. The rice varieties in the present invention can be planted in more regions, improving the adaptability and stability of crops, thereby bringing higher economic benefits to farmers, and the present invention not only has application value in improving the cold tolerance traits of rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a phenotypic analysis diagram of the cold tolerance of MAGIC population rice in Example 1 of the present invention;

[0016] Figure 2 is an analysis of the cold tolerance of the NHX2 knockout mutant and overexpression lines in Example 1 of the present invention;

[0017] Figure 3 is the subcellular localization and expression pattern of NHX2 in Example 2 of the present invention;

[0018] Figure 4 is a phylogenetic tree analysis diagram of NHX2 in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The following is combined with the attached Figures 1-4The present invention will be further described in detail.

[0020] Example 1: GWAS Analysis of Cold Tolerance Traits in Rice MAGIC Populations

[0021] In this example, 1364 lines (including 8 parents) of the MAGIC multi-parent hybrid population were identified for cold tolerance at the seedling stage. GWAS analysis was performed on traits related to cold tolerance, namely the survival rate (SR, %) of the experimental materials and the leaf score of cold tolerance (SCT). The candidate gene NHX2 for cold tolerance at the seedling stage of the important QTL qSR11.2 was identified.

[0022] The research materials used in this example were MAGIC multi-parent hybrid populations constructed by the International Rice Research Institute in the Philippines using 8 parents. Three rice MAGIC populations were constructed using 8 elite varieties (lines) from different countries as parents. Among them, the 4-parent population DC1 was mainly obtained by pairwise hybridization of parents (A / B / / C / D) followed by paired hybridization and selfing through the single-seed descent method to obtain a population of 392 lines; the 4-parent population DC2 was mainly obtained by pairwise hybridization of parents (E / F / / G / H) followed by paired hybridization and selfing through the single-seed descent method to obtain a population of 386 lines; the 8-parent population 8way was selected from 25 F1 single plants each from the 4-parent hybrids A / B / / C / D and E / F / / G / H, and 8-parent hybrids (A / B / / C / D / / / E / F / / G / H) were produced through multiple reciprocal crosses and then advanced by the single-seed descent method to construct a population of 578 lines; the DC1 and DC2 populations were combined and called the DC12 combined population, the DC1 and 8way populations were combined and called the DC18 combined population, the DC2 and 8way populations were combined and called the DC28 combined population, and the DC1, DC2, and 8way populations were combined and called the DC128 population, as shown in Table 1:

[0023] Table 1 Line Sources of 8 Parents of MAGIC Populations

[0024]

[0025] In the phenotypic analysis of cold tolerance at the seedling stage of the MAGIC population, by evaluating the cold tolerance of 8 parents of the MAGIC multi-parent hybrid population, parent F (CT16658-5-2-2SR-2-3-6MP from Colombia, CT) had the best cold tolerance, while parent G (IR68 from the International Rice Research Institute) was cold-sensitive. Among the 3 single populations, the survival rate (SR) of the DC2 population was significantly higher than that of other populations, and the leaf blight degree grade (SCT) was also significantly lower than that of other populations. The SR of the 8way population was significantly lower than that of other populations, and the SCT was also significantly higher than that of other populations, asFigure 1 (Shown in (a) is the SR box plot of the DC128, DC1, DC2, and 8way populations; (b) is the SCT box plot of the DC128, DC1, DC2, and 8way populations; (c) is the average of SR and SCT of 8 parents in the MAGIC population. In a - b, different letters indicate significant differences). The average SR of the DC1 population is 64%, and the average SCT is 4.60. The average SR of the DC2 population is 74%, and the average SCT is 3.80. The average SR of the 8way population is 45%, and the average SCT is 5.42. The results show that among the three single populations of the MAGIC population, the cold tolerance of the DC2 population is significantly higher than that of other populations; the 8way population contains more cold - sensitive materials, and its cold tolerance is much lower than that of other populations.

[0026] In the GWAS analysis of the cold - tolerance traits of the MAGIC population, in this example, the MLM of TASSEL 5.2.3 was used to perform GWAS analysis on the traits SR and SCT related to cold tolerance in the DC1, DC2, 8way, DC12, DC18, DC28, and DC128 populations. In 3 single populations (DC1, DC2, and 8way) and 4 combined populations (DC12, DC18, DC28, and DC128), a total of 9 QTLs were identified, including 3 QTLs mapped from the SR trait and 6 QTLs mapped from the SCT trait, as shown in Table 2:

[0027] Table 2 QTL table obtained from GWAS analysis of SR and SCT

[0028]

[0029]

[0030] Since qSR11.2 affects both SR and SCT traits and was detected in 6 populations including DC128, DC2, 8way, DC12, DC18, and DC28, it was studied as an important QTL.

[0031] The extremely significant SNP locus rs11 - 25779115, which is significantly associated with SR and SCT for the QTL qSR11.2 detected on chromosome 11. Through qRT - PCR experiments on the genes in this interval and transgenic functional verification, a candidate gene NHX2 regulating cold tolerance at the seedling stage of rice was mined. This gene encodes a cation transporter ( Figure 4 ). Through qRT - PCR experiments, it was found that after cold treatment, the expression level of NHX2 in the cold - tolerant CT increased significantly compared with the cold - sensitive parent IR68. Figure 2b). The gene NHX2 was knocked out using the cold-tolerant parent CT as the background, and two types of homozygous mutants, KO1 and KO2, were obtained. They had a 1-base insertion and a 34-base deletion respectively, both of which led to premature termination of amino acids ( Figure 2 d). NHX2 overexpression lines were constructed using the cold-sensitive parent IR68 as the background. The overexpression levels of OE1 and OE2 increased by 376-fold and 500-fold respectively ( Figure 2 f). The NHX2 gene knockout and overexpression lines were subjected to cold treatment at the seedling stage, and the plant survival rate was counted after recovery ( Figure 2 c). The results showed that the survival rate of the NHX2 knockout mutants decreased significantly compared with the cold-tolerant parent CT ( Figure 2 e); the survival rate of the NHX2 overexpression materials increased significantly compared with the cold-sensitive parent IR68 ( Figure 2 g).

[0032] In summary, NHX2 is a positively regulated cold-responsive gene. After knocking out this gene, cold sensitivity of rice seedlings will occur; while overexpressing the NHX2 gene will enhance the cold tolerance of rice seedlings.

[0033] Figure 2 Figure legend ((a) Manhattan plot and Q-Q plot of SR in the DC128 population. (b) Expression level difference of NHX2 in parents CT and IR68 under cold stress. (c) Phenotypes of NHX2 knockout mutants and overexpression lines during cold treatment. (d) Sequence analysis of NHX2 knockout mutants. (e) Statistical analysis of the survival rate (SR) of NHX2 knockout mutant plants. (f) Expression level detection of NHX2 overexpression lines. (g) Statistical analysis of the survival rate (SR) of NHX2 overexpression line plants. Error bars represent the standard deviation of 3 biological replicates. The significant difference between two groups was calculated using a two-tailed Student's t-test.)

[0034] Example 2: Subcellular localization and expression pattern of NHX2

[0035] The full length of NHX2 is 4105 bp, and the full length of the coding region is 1638 bp. It has 13 exons and encodes a Cation / H + antiporter composed of 546 amino acids, with a molecular mass of approximately 59.92 kDa. It belongs to the NHX transporter family. As Figure 4 shown (phylogenetic tree analysis of the NHX family in different species), NHX2 contains a Cation / H +exchanger domain. To further study the molecular functional characteristics of NHX2, an NHX2-EGFP vector was constructed. The fusion vector was driven by CaMV 35S. The NHX2-EGFP vector was transformed into rice protoplasts, and the GFP fluorescence signal was observed at a wavelength of 488nm using a laser confocal microscope. The transient expression analysis of the NHX2-EGFP fusion protein in rice protoplasts showed that the NHX2 protein was localized on the vacuole membrane of rice protoplasts ( Figure 3 a). We used Nipponbare as material and found that NHX2 was expressed in roots, stems, leaves at the seedling stage and roots, stems, leaves on the ear, ears, and leaf sheaths at the booting stage using qRT-PCR. As shown in the figure, NHX2 is a constitutively expressed gene ( Figure 3 c). The results of GUS staining showed that in NHX2::GUS transgenic plants, NHX2 was expressed in the roots, stems, and leaves of rice seedlings, and the expression level was higher in the root tip ( Figure 3 b).

[0036] Figure 3 Figure legend ((a) Subcellular localization of NHX2, Bar = 5 μm. (b) Staining of GUS materials driven by NHX2 promoter, in order: leaves, stems, root maturation zone, root elongation zone, root meristem and root tip, Bars = 500 μm. (c) Expression analysis of NHX2 in different tissues at seedling and booting stages.)

[0037] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Application of the cold tolerance candidate gene NHX2 in rice seedling stage in enhancing cold tolerance of rice seedlings.

2. A cold tolerance candidate gene NHX2 at the rice seedling stage, characterized in that: The cold-tolerant candidate gene NHX2 is 4105 bp in full length, with a coding region of 1638 bp in full length, 13 exons, a molecular mass of 59.92 kDa, and encodes a Cation / H + antiporter consisting of 546 amino acids.

3. A rice seedling cold tolerance candidate gene NHX2 according to claim 2, characterized in that: The cold tolerance candidate gene NHX2 is located on the vacuolar membrane.

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