Rice heat stress tolerance related rmil gene and application thereof

Abstract

This invention discloses a method for rice to withstand heat stress. RMI1 Genes and their applications. Among them... RMI1 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2; this invention verifies RMI1 The function of the gene, RMI1 protein is located in the cell nucleus, rmi1-1 In mutants RMI1 The deletion of 33 base pairs in the gene results in the loss of 11 amino acids in the RMI1-1 protein, making... rmi1-1 High temperatures during the seedling stage cause a large number of abnormal chromosomes to adhere and fragment during the late mitotic phase of root tip cells, resulting in stunted plant growth and development. rmi1-1 High temperatures during the reproductive growth period of plants lead to a large number of chromosome fragments and adhesions in the late stage of meiosis I, resulting in sterility; high temperatures also affect the interaction between RMI1-1 protein and topoisomerase TOP3α and helicase RECQ4. rmi1-cr3 Frameshift mutations lead to abnormal embryo and endosperm development; overexpression RMI1 This invention can enhance the heat resistance of rice. RMI1 The analysis of gene function provides genetic resources for the breeding of heat-resistant rice varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and particularly to a method for improving rice's tolerance to heat stress. RMI1 Genes and their applications. Background Technology

[0002] Temperature is a crucial environmental factor affecting rice growth and development. With the intensification of the global greenhouse effect and the frequent occurrence of extreme heat events, heat stress has become one of the major threats to rice production. Heat stress triggers various physiological responses in rice, such as short-term avoidance mechanisms, stomatal closure, reduced water loss, leaf senescence, and promotion of nutrient redistribution, thus enabling the plant to survive. The molecular mechanisms by which rice resists high-temperature heat damage mainly include: 1. Scavenging reactive oxygen species: Rice protects itself from reactive oxygen species by synthesizing various antioxidants (superoxide dismutase, catalase, etc.); 2. Increasing the content of saturated fatty acids in membrane lipids to maintain membrane stability and enhance heat resistance; 3. Heat shock protein activation protection: Heat shock factors rapidly activate the expression of a series of enzymes and chaperone proteins to maintain rice growth under high-temperature conditions.

[0003] Heat shock proteins play an important role in maintaining genome stability. In rice, RMI1 The function of the gene has not been reported. This invention shows that... RMI1 Genes involved in rice's tolerance to high temperatures can help us understand the molecular mechanisms of rice's response to high temperatures and the regulatory pathways of rice's heat tolerance, providing genetic resources for the breeding of heat-resistant rice varieties. Summary of the Invention

[0004] This invention addresses the role of rice heat shock proteins in maintaining genome stability in existing technologies, and provides a method for addressing the role of heat shock proteins in rice related to heat stress. RMI1 Genes and their applications to analyze RMI1 To understand the function of genes and gain a deeper understanding of the molecular mechanisms of rice's response to high temperatures and the regulatory pathways of rice heat tolerance, so as to provide genetic resources for the breeding of heat-tolerant rice varieties.

[0005] This invention first provides a method for rice to withstand heat stress. RMI1 Genes, the ones mentioned RMI1 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0006] Furthermore, the aforementioned RMI1 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 2.

[0007] Furthermore, it also includes amino acid sequences or derivatives generated by inserting, substituting, or deleting one or more amino acids or homologous sequences of other species into the said amino acid sequence.

[0008] This invention relates to rice heat stress resistance. RMI1 The application of genes, the application is through 60 A heat stress-sensitive mutant was obtained by irradiating rice cultivar Yandao 8 with Co-γ rays. The method for cloning rice heat stress-related genes was map-based cloning.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0010] This invention reveals for the first time the rice RMI1 Biological function of the gene. Functional verification was performed using gene editing technology: the RMI1 protein is located in the cell nucleus, and the heat stress-sensitive mutant was further demonstrated. rmi1-1 High temperatures during the seedling stage cause a large number of chromosome fragments and abnormal chromosome adhesion in the late mitotic phase of root tip cells, resulting in stunted plant growth and development. rmi1-1 High temperatures during the reproductive growth period of plants can lead to a large number of chromosome fragments and adhesions during the late stage of the first meiotic division, resulting in sterility. rmi1-cr3 Frameshift mutations cause abnormal embryo and endosperm development; overexpression RMII This resulted in rice exhibiting enhanced heat resistance. Yeast two-hybrid studies showed that high temperature affects the interaction between the loss-of-function RMI1-1 protein and the topoisomerase TOP3α and the helicase RECQ4. This invention, through analysis... RMI1 The aforementioned functions of the gene are of great significance for further understanding the molecular mechanism of rice's response to high temperatures and for the breeding of new heat-tolerant rice varieties. Attached Figure Description

[0011] Figure 1 A represents wild-type WT and a temperature-sensitive mutant. rmi1-1 Seedling phenotypes at 28℃ and 38℃; B represents WT and rmi1-1 Plant phenotypes under natural growth conditions in HN (Hainan, low-temperature environment) and YZ (Yangzhou, high-temperature environment); C represents WT and rmi1-1 Iodine staining of anthers under natural growth conditions in HN and YZ.

[0012] Figure 2 Wild-type WT at 28℃ and 38℃ rmi1-1 Chromosomal behavior at different stages of root apical mitosis, shown in the figure: Interphase; Prometaphase; Metaphase; Anaphase.

[0013] Figure 3 WT and YZ under natural growth conditions rmi1-1 Chromosomal behavior at different stages of meiosis in pollen mother cells.

[0014] Figure 4 for RMI1 Map-based cloning of genes and RMI1 Schematic diagram of gene structure.

[0015] Figure 5 This is a schematic diagram showing the location of the RMI1 protein in the cell nucleus.

[0016] Figure 6 for rmi1-cr3 The ovules are observed to be transparent.

[0017] Figure 7 Yeast two-hybrids were performed on RMI1, RMI1-1 and TOP3α, RECQ4 at different temperatures.

[0018] Figure 8 A represents the wild-type (WT) hydroponically grown for 4 days and RMI1 Overexpression ( RMI1-OE Seedlings.

[0019] Figure 8 B represents WT and RMI1-OE Treated at 46°C for 2 days.

[0020] Figure 8 C represents WT and RMI1-OE It was treated at a high temperature of 46℃ for 3 days.

[0021] Figure 8 D represents WT and RMI1-OE After being treated at 46°C for 3 days, it was restored at 28°C for 4 days. Detailed Implementation

[0022] The present invention will be further described below through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0023] Example 1: rmi1-1 Phenotypic identification and genetic analysis

[0024] pass 60 Co-γ ray irradiation of salt-tolerant rice cultivar 8 resulted in a high-temperature-sensitive mutant during the seedling stage. rmi1-1 like Figure 1 As shown. Figure 2 The figures shown are the WT and WT values ​​after one week of growth at 28°C and 38°C. rmi1-1 Chromosomal behavior was observed in the root tips of mutant seedlings, such as... Figure 2 As shown in A-2H, at 28℃ rmi1-1 Root tip chromosome behavior showed no significant difference from WT; Figure 2 L and Figure 2 At 38℃ as shown in P rmi1-1 In the anaphase of mitosis, abnormal chromosome adhesion occurs in the root tip chromosomes, such as... Figure 1 As shown in Figure A, at 38℃ rmi1-1 Plant growth and development have stopped. For example... Figure 3 As shown in A-3G, under the conditions of Hainan (HN). rmi1-1 The chromosome behavior during meiosis in pollen mother cells was not significantly different from that in WT; under Yangzhou (YZ) conditions rmi1-1 During diakinesis, chromosome entanglement forms multivalents, such as... Figure 3 As shown in M ​​and 3R Figure 3 The abnormal adhesion of chromosome I in metaphase N and 3S are shown. Figure 3 O and Figure 3 As shown in Figure T, chromosome fragmentation and adhesion occur in late stage I, resulting in plant sterility. Figure 1 B and Figure 1 As shown in C.

[0025] In addition, genetic analysis was performed using a phenotypic segregating family. rmi1-1 Of the offspring of the heterozygous plants, 71 developed normally under high temperature and 29 developed territories under high temperature (the chi-square test showed a ratio of 3:1). 2 = 0.65, P>0.05), indicating that rmi1-1 The high-temperature sensitivity phenotype is caused by a single recessive mutation in a gene.

[0026] Example 2: RMI1 Cloning of genes

[0027] The target gene was cloned using map-based cloning. rmi1-1 The F2 population, resulting from a cross between the mutant and Huang Huazhan indica rice, was used as the initial mapping population. Preliminary linkage analysis located the target gene within a 603 kb region on the long arm of chromosome 4. Continued cultivation of this population narrowed the region to 55 kb. According to annotations from the Rice Genome Annotation Project website, there were 7 candidate genes within this target region. We sequenced the DNA of these 7 annotated genes and found LOC_Os04g32090 (… RMI1 The ORF region sequence of the rice genome has been mutated. According to published rice genome information, the genome sequence is 5592 bp long, as described in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2. Sequencing results show that... rmi1-1 The mutant has a 33-base deletion in the first exon of the gene, resulting in a 11-amino acid deletion in the encoded protein. We preliminarily identified LOC_Os04g32090 as the target gene.

[0028] A fusion expression vector of RMI1-GFP was constructed and transformed into rice protoplasts. For example... Figure 5 The results showed that the green GFP signal co-localized with the nuclear localization marker fluorescence (red), indicating that RMI1 is a nuclear localization protein.

[0029] Example 3: Knockout using gene editing methods RMI1 Genes cause seedlings to be sensitive to high temperatures

[0030] To verify RMI1 To determine the function of the gene, we used CRISPR-Cas9 gene editing technology to knock it out in the wild type. RMI1 Gene. The resulting mutant. rmi1-cr2 There are 3 base deletions. rmi1-cr3 The presence of a 4-base deletion causes a frameshift in protein translation. For example... Figure 4 As shown, rmi1-cr2 Phenotype and rmi1-1 Phenotypic consistency, characterized by significantly shortened root tips and stunted plant growth due to high temperatures during the seedling stage, demonstrates that the mutant seedlings are sensitive to high temperatures. RMI1 It's caused by a gene mutation. Furthermore... RMI1 Frameshift mutation leads to rmi1-cr3 Embryonic developmental abnormalities, such as Figure 6 As shown, no results were obtained. rmi1-cr3 Homozygous mutant plants.

[0031] After transforming yeast with RMI1, RMI1-1, and TOP3α and RECQ4 yeast two-hybrid vectors, the yeast cells were cultured at 28℃, 33℃, and 36℃, respectively. Figure 7 As shown, RMI1 and RMI1-1 interact with TOP3α and RECQ4 at 28℃ and 33℃. At 36℃, RMI1 also interacts with TOP3α and RECQ4, but RMI1-1 does not interact with TOP3α and RECQ4.

[0032] Example 5: Overexpression RMI1 rice ( RMI1-OE It exhibits stronger heat resistance.

[0033] We expressed [the following] in Salt Rice No. 8 RMII Gene( pACT:RMII And obtained overexpression rice RMI1-OE strains, such as Figure 8 As shown in Figure A, the wild type (WT) and hydroponically grown for 4 days are shown. RMI1 Overexpression rice RMI1-OE Seedlings. Figure 8 B represents the WT hydroponically grown for 4 days and RMI1-OE After being treated at 46℃ for 2 days, approximately 81% of the plants in the WT (Whole Plant) withered, while RMI1-OEOnly 17% of the plants withered, and the withering symptoms were relatively mild. Figure 8 C represents plants for which the 46°C treatment time was extended to 3 days. They were then allowed to recover at 28°C for 4 days, as shown below. Figure 8 As shown in D, WT failed to resume growth. RMI1-OE Approximately 51% of the seedlings recovered and grew, indicating that... RMI1 overexpression strains RMI1-OE It exhibits stronger heat resistance.

[0034] The above examples are merely some specific embodiments of the present invention, but the present invention is not limited to the above embodiments. Modifications and improvements made by those skilled in the art based on the present invention should all be included within the scope of protection of the present invention.

Claims

1. Use of a rice RMI1 gene characterized in that, The RMI1 The nucleotide sequence of the gene is shown in SEQ ID No.

1. Transgenic methods are used to improve rice... RMI1 The expression level of genes is used to improve the heat resistance of rice.

2. The rice plant of claim 1 RMI1 application of the gene, characterized in that, The RMI1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2.

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