CN119614611A - Application of Rice High Temperature Response Gene OsTOGR4 - Google Patents

Abstract

The invention discloses application of a rice high-environmental-temperature response gene OsTOGR. The invention firstly identifies a key factor for regulating the growth and development of rice plants at high environmental temperature, and discloses a method for cultivating high-environmental-temperature rice by mutating OsTOGR gene, thereby providing gene resources and research basis for cultivating high-environmental-temperature rice varieties.

Description

Application of rice high-environmental-temperature response gene OsTOGR4

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of a high-environment temperature response gene OsTOGR of rice.

Background

The global air temperature is continuously increased and the climate is continuously changed under the influence of the greenhouse effect. The artificially domesticated crops lose a plurality of excellent genes derived from wild ancestor species, the genetic diversity is reduced, and the crops are difficult to adapt to changing natural environments, so that the agricultural production is restricted. From 2000 to 2009, sorghum and soybeans in western africa lost 10-20% and 5-15% of their yield, respectively, due to climate change (Sultan et al, 2019). The global wheat, rice, corn and soybean also experience 6%, 3.2%, 7.4% and 3.1% yield reductions, respectively, at a global average temperature rise of 1 ℃ (Zhao et al, 2017), affected by temperature. The temperature change affects the grain yield, and the research on the relationship between the plant development and the temperature has important practical significance for cultivating the crop varieties with stable and high yield.

In the case of changes in ambient temperature, plant morphology changes (Casal and Balasubramanian, 2019), and a series of changes in plant morphology from temperatures below heat stress with increasing temperature are collectively referred to as thermo-morphogenesis. The hypocotyl of Arabidopsis is accelerated to grow at high ambient temperature to protect cotyledons from soil with higher temperature, the petioles are also elongated, the distance between the leaves is increased, a looser plant type is formed, and heat dissipation of the plant is facilitated so as to adapt to the environment (Quint et al 2016). The plant height of rice stretches with increasing ambient temperature (KRISHNAN ET al., 2011), a core element affecting the plant type and yield of rice (Liao et al., 2019). However, little research has been done so far on the molecular mechanism by which rice adjusts plant height in response to ambient temperature.

Disclosure of Invention

The invention aims to provide application of a rice high-environmental-temperature response gene OsTOGR 4.

To achieve the object of the present invention, in a first aspect, the present invention provides the use of the rice high environmental temperature response gene OsTOGR4 for regulating plant sensitivity to high environmental temperature (i.e., for regulating plant growth sensitivity at high environmental temperature).

In the present invention, gene OsTOGR4 is a gene encoding the following protein (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2, or

(B) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

Further, the plants include plants of the Gramineae family, preferably rice.

Further, by modifying the high-ambient-temperature responsive gene OsTOGR of rice, the gene function is deleted, so that the sensitivity of the rice to the high ambient temperature (namely, the sensitivity of the rice growth to the high ambient temperature) is reduced.

Gene OsTOGR may be engineered using genome editing techniques such as CRISPR, TALEN, ZFN.

In a second aspect, the present invention provides a method for reducing the sensitivity of rice to high ambient temperature comprising weakening the high ambient temperature responsive gene OsTOGR to rice by genetic engineering means.

Further, a gene OsTOGR4 is used as a target, a sgRNA sequence based on CRISPR/Cas9 is designed, a DNA fragment containing the coding the sgRNA sequence is connected into a carrier carrying the CRISPR/Cas9, and rice is transformed or transfected, so that transgenic rice with the gene function deletion is obtained.

Preferably, the original vector for CRISPR/Cas9 is constructed and provided by the hundred gene company under the number SG11588.

Preferably, the nucleotide sequence of the sgRNA site of action is 5'-TGGCACACCGCTGTTAG-3'.

Further, the transformation or transfection may be performed using Agrobacterium-mediated methods or gene gun methods.

In a third aspect, the present invention provides the use of transgenic rice obtained according to the method in plant breeding.

Further, breeding methods include, but are not limited to, transgenesis, crosses, backcrosses, selfing, or asexual propagation.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

The invention firstly identifies a key factor for regulating the growth and development of the rice plants at high environmental temperature, and discloses a method for reducing the sensitive growth of the rice at high environmental temperature by knocking-out OsTOGR gene, thereby providing gene resources and research basis for cultivating good rice varieties adapting to high environmental temperature.

Drawings

FIG. 1 is a phenotype comparison of togr4 in the preferred embodiment of the invention with wild-type flower 11. (a) Mutant togr4 and wild type ZH11 were used for field phenotype statistics in tomb water, beijing and Yangzhou. (b) And (5) counting the plant heights of seedlings grown for 2 weeks under different temperature conditions. (a) And (b) bar black bars represent ZH11, gray bars represent mutant togr4,. The values are expressed as mean.+ -. Standard error (n.gtoreq.10) and the scales are 10 cm.

FIG. 2 shows the cell phenotype of togr and wild-type medium flower 11 at high ambient temperature in a preferred embodiment of the invention. (a) Tissue longitudinal cell maps of the inverted internodes of WT and togr4 at high ambient temperature were scaled to 100 μm. (b) The transection of the tissues between the inverted nodes of WT and togr4 at high ambient temperature was 100 μm on a scale. (c) Leaf epidermal cells of WT and togr4 at high ambient temperature. The scale length was 100. Mu.m. The corresponding graph includes the width of the inverted internode, the stem thickness, the length, width, area size and total number of cells of the inverted internode, the length of the leaf blade and the length of the leaf epidermis stomata cells. The values are expressed as mean ± standard error, the difference is expressed as。

FIG. 3 shows subcellular localization of proteins OsFIE and togr4 according to the preferred embodiment of the invention. (a) Subcellular localization of protein OsFIE2 in rice root cells. The roots of the transgenic lines (up) and pUb:: osFIE2-GFP complementation togr mutant, respectively, are shown as pUb:: GFP over-expression wild type material. The scale is 50 μm long. (b) subcellular localization in rice protoplasts. The fusion protein OsbZIP52-mCherry is used as a nuclear localization marker. The scale is 10 μm long. (c) tobacco leaf subcellular localization. The scale is 50 μm long.

FIG. 4 shows the gene targeting of OsTOGR4 and candidate gene determination in a preferred embodiment of the invention. (a) togr phenotype of togr, wild ZH11 and F1 generation plants crossed with the wild ZH11 in Yangzhou, scale is 10 cm. (b) OsTOGR4 linkage map. M1 to M7 are molecular markers, osTOGR4 candidate genes are shown in the figure, black rectangular boxes represent exons, and middle connecting line segments represent introns. (c) the recombination frequency of each molecular marker in the linkage map.

FIG. 5 shows the OsTOGR gene over-expression vector in the preferred embodiment of the invention.

FIG. 6 shows a part of phenotype of OsTOGR gene affecting plant thermo-morphogenesis in a preferred embodiment of the invention. (a) WT, togr4 and complementary material were planted in Yangzhou field phenotypes, scale 10cm, respectively. (b) The seedling stage phenotype of WT, togr4 and togr4/OsFIE2 complementary materials at different culture temperatures was rated at 10cm.

FIG. 7 shows the transcriptional expression analysis of the OsFIE2 gene in togr and wild-type flowers 11 according to the preferred embodiment of the invention. (a) OsFIE analysis of transcript levels in different tissues of rice. (b) Transcript levels analysis of OsFIE in leaf, leaf sheath and aerial tissue of two week seedlings at different temperatures. (a) And (b) data are expressed as mean ± standard error, n=3. With the gene action as an internal reference, data analysis used Tukey HSD test (p < 0.05), the different letters indicated significant differences. (c) Analysis of the expression of the above-ground protein OsFIE from two weeks of seedlings treated at different temperatures. (d) analysis of protein expression of OsFIE2 after heat treatment. OsFIE2-GFP seedlings grown for two weeks at 25℃were transferred to 35℃for heat treatment. (c) And (d) protein immunoblots, the following table shows the gray scale analysis of protein expression, taking protein Actin as an internal reference. 3 experiments were repeated, data analysis was performed using Student's test,。

Fig. 8 is an example of altering the sensitivity of rice to high ambient temperatures using CRISPR/Cas9 technology. Wherein osfie-CRISPR is a OsFIE allelic mutant generated using CRISPR/Cas9 technology. Schematic of allelic mutation of (a) gene OsFIE. The black boxes represent exons and the triangular connected segments indicate the mutation sites of the allelic mutants on the gene. The red line box shows the sequencing peak pattern of the mutation site. (b) field plant height and tillering of both allelic mutant forms. The data n is more than or equal to 15. (c) both allelic mutant phenotypes under temperature treatment. The length of the scale is 10 cm, and n is more than or equal to 16. (b) And (c) the data are represented as mean ± standard error, the data analysis employs a one-way analysis of variance, and the significance of the differences is represented by asterisks, respectively。

Detailed Description

The invention provides a rice response environmental temperature change gene OsTOGR (Thermotolerant Growth Required) 4, a coding protein and a functional analogue of the gene, a vector containing a nucleotide sequence of the gene and a host cell containing the nucleotide sequence of the gene or the vector. The invention also provides a method for cultivating the rice high-environmental-temperature adaptive variety capable of growing phenotypes at high environmental temperature close to normal temperature by using the gene OsTOGR for reducing the sensitivity of the rice to high environmental temperature, which is expected to reduce the problems of lodging, brittle bars and the like in the later stage caused by rapid growth of the rice at high environmental temperature.

A rice plant height mutant OsTOGR4 (Thermotolerant Growth Required) was isolated and identified that changed morphology in response to high ambient temperatures using Ethyl Methanesulfonate (EMS) mutagenesis and genetic screening. The morphological features are very similar to those of wild type when togr4 is grown under low ambient temperature conditions (e.g., 12 months to 4 months of the next year in Hainan) and the growth is significantly inhibited when grown under higher ambient temperature conditions (e.g., 5-9 months in Yangzhou) and a dwarf phenotype is exhibited. To verify the phenotype, the phenotype of togr4 in response to ambient temperature was further confirmed by counting the growth phenotype of rice material at different temperatures using an artificial incubator environment to simulate the growth environment. On the basis of preliminary establishment of phenotype, a key gene OsTOGR4 for regulating and controlling the change of the rice response to the environmental temperature is positioned and cloned by a method of phenotype screening and molecular marker identification. The main functions and associated phenotypes of the gene are proved by phenotypic analysis of the mutant and genetic complementation experiments. The biochemical experiment shows that OsTOGR4 gene encoded protein content tends to decrease with increasing ambient temperature. Based on the experiments, the invention firstly identifies a seedling hypersensitive gene OsTOGR4 for regulating and controlling the high environmental temperature response of rice, and the gene codes OsFIE subunit of the PRC2 protein complex of the rice.

PRC2 is a Polycomb group (PcG) consisting of four core subunits and some accessory subunits, and is prepared by methylation modification of histone H3 in the form of complex to regulate the open state of chromatin, regulate the silencing and activation state of genes at apparent level, and play an important role in early embryo development, cancer regulation, plant growth and development, environmental response, etc. of animals (Goodrich et al, 1997; mozgova AND HENNIG, 2015; baile et al, 2022). In rice, PRC2 contains subunit OsEZ1, which is a methyltransferase function, and can carry out trimethylation modification on the 27 th Lys residue of histone H3, thereby carrying out apparent regulation. OsFIE2 is one of the core subunits of rice PRC2, interacts with EZ1 in a binding manner, affects the histone H3 methyltransferase activity of PRC2, regulates rice plant height, seed development and grain filling (NALLAMILLI ET al., 2013), and has additive effects on the synthesis of some storage proteins and the expression regulation of photosynthesis genes (Liu et al., 2016; cheng et al., 2020). OsFIE2 (Cheng et al, 2020), the reduced expression levels will result in smaller rice seeds, inadequate filling, and some seeds will not sleep normally (NALLAMILLI ET al, 2013). The mutant osfie-1 has a reduced internode longitudinal cell number, a significantly thinner internode lateral cell layer, and a reduced number of large and small vascular bundles and cell numbers and sizes in glumes (Liu et al, 2016), exhibiting dwarf, reduced seed setting, flower organ defects, and reduced grain size phenotypes. However, so far, no study has been conducted to show that OsFIE2 is involved in the response of rice temperature.

The core subunit OsTOGR/OsFIE 2 of the rice PRC2 protein complex identified by the invention participates in the growth and development regulation of rice in response to high environmental temperature, and the normal expression and protein content of the gene ensure the normal growth of rice at high environmental temperature.

The invention adopts the following technical scheme:

In a first aspect, the invention identifies a rice plant height gene OsTOGR4 which codes for a protein as shown in SEQ ID NO. 2 or a protein derived from SEQ ID NO. 2 which has the same function as the protein as shown in SEQ ID NO. 2 by substitution, deletion or addition of one or more amino acids to the amino acid sequence as shown in SEQ ID NO. 2. Wherein the protein shown in SEQ ID NO. 2 consists of 376 amino acids.

The plant height gene OsTOGR4 of the rice responding to high environmental temperature is an isolated nucleotide sequence. In a preferred embodiment, the nucleotide sequence of gene OsTOGR4 for controlling the response of rice to high ambient temperatures is shown by SEQ ID NO. 1 (the sequence shown in SEQ ID NO. 1 also includes promoter and 3' UTR sequences). Moreover, it will be appreciated by those skilled in the art that in a broad sense, the rice plant height gene OsTOGR4 which responds to high ambient temperatures is a nucleotide sequence which has 90% or more, preferably 99% or more homology with the nucleotide sequence shown in SEQ ID NO. 1 and encodes a protein having the same function.

In a second aspect, the present invention provides a protein encoded by the gene OsTOGR for controlling a high-environmental temperature response of rice, which is (a) or (b) below:

(a) A protein consisting of an amino acid sequence shown in SEQ ID NO. 2;

(b) And (3) the protein which is derived from the SEQ ID NO. 2, has the same function as the protein shown in the SEQ ID NO. 2 through substitution, deletion or addition of one or more amino acids by the amino acid sequence shown in the SEQ ID NO. 2.

In a preferred embodiment, the protein encoded by the gene OsTOGR for controlling the response of rice to high ambient temperatures is an isolated protein having the amino acid sequence shown in SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant vector comprising the gene OsTOGR for controlling the response of rice to high ambient temperature.

Preferably, the recombinant vector comprises an exogenous nucleotide fragment encoding the amino acid sequence shown in SEQ ID NO. 2. More preferably, the recombinant vector comprises an exogenous nucleotide fragment as shown in SEQ ID NO. 1.

In one embodiment, the plasmid used to construct the recombinant vector may be selected from, but is not limited to, pCAMBIA1300.

Host cells comprising the recombinant vector are also within the scope of the invention. Host cells comprising the recombinant vector may be obtained by transformation or transfection of the recombinant vector into cells for further use in amplifying expression vectors, expressing the protein or obtaining transgenic plants, etc. The cells used for transformation or transfection may be selected from, but are not limited to, bacterial cells, such as E.coli cells or Agrobacterium cells, fungal cells, such as yeast cells, or plant cells, such as rice cells, and the like.

In a fourth aspect, the present invention provides the use of gene OsTOGR4, which modulates the high environmental temperature sensitivity of rice, in breeding rice varieties capable of normal response and growth at high environmental temperatures.

In a fifth aspect, the present invention provides a method for breeding rice varieties capable of normal response and growth at high ambient temperatures, said method comprising reducing the amount of protein encoded by the OsTOGR gene. The method of reducing OsTOGR.sup.4 gene-encoded protein content can be achieved by knocking out OsTOGR gene using CRISPR/Cas9 gene editing technology.

Transformation or transfection of OsTOGR gene and OsTOGR gene-editing vector may be performed by agrobacterium-mediated or biolistic methods.

The invention provides theory and material foundation for cultivating crop varieties with reduced high-environment temperature sensitivity and improved high-environment temperature adaptability. When the gene of the present invention is used for improving the response of rice to high environmental temperature, the following methods may be employed (1) constructing a vector for gene editing of OsTOGR gene, (2) transforming the constructed vector into a regenerable rice tissue or organ, (3) cultivating the transformed tissue or organ into a plant and selecting the plant causing the mutation of the gene.

Further, the invention provides an isolated gene for regulating and controlling the sensitivity of rice to high environmental temperature, wherein the gene codes for a protein shown as SEQ ID NO.2 or codes for a protein which is derived from the SEQ ID NO.2, has the same function as the protein shown as the SEQ ID NO.2 through insertion, deletion or substitution of one or more amino acids of the amino acid sequence shown as the SEQ ID NO. 2.

Further, it is a nucleotide sequence having 90% or more, preferably 99% or more homology with the nucleotide sequence shown in SEQ ID NO.1 and encoding a protein having the same function.

Further, it is the nucleotide sequence shown in SEQ ID NO. 1.

The invention also provides an isolated protein which is encoded by the gene OsTOGR and used for controlling the high environmental temperature sensitivity of rice, and is shown as SEQ ID NO.2, or a protein which is derived from the SEQ ID NO.2, has the same function as the protein shown as the SEQ ID NO.2 through insertion, deletion or substitution of one or more amino acids of the amino acid sequence shown as the SEQ ID NO. 2.

Further, the protein is shown as SEQ ID NO. 2.

The invention also provides a recombinant vector containing the gene OsTOGR or a fragment thereof.

Further, the recombinant vector is a plant expression vector, preferably a vector suitable for expression in rice.

Preferably, the recombinant vector is constructed from plasmid pCAMBIA 1300.

Further, the plant expression vector also comprises OsTOGR promoter or a Ubiquitin promoter, and a gene sequence encoding a tag protein, such as GFP.

Further, the host cell is selected from bacterial, fungal or plant cells, preferably E.coli (ESCHERICHIA COLI), agrobacterium (Agrobacterium tumefaciens) or plant cells.

The present invention also provides a method for breeding a high ambient temperature-adapted plant having excellent high ambient temperature insensitivity, which comprises designing a gene editing target using the gene OsTOGR or a fragment thereof as a template and constructing a vector by using a gene editing technique, then transforming cells or tissues of the plant, and growing the transformed plant cells or tissues into a plant with OsTOGR function deletion and reduced sensitivity of rice to high ambient temperature.

Further, the transformation is performed by agrobacterium-mediated or biolistic methods.

Further, the plant is a gramineous plant, preferably rice.

The invention also provides application of the gene OsTOGR or the protein for regulating the response of the plant to high environmental temperature in cultivating high environmental temperature adaptive plants with excellent high environmental temperature and low sensitivity.

The present invention also provides a method for breeding rice having excellent high environmental temperature and low sensitivity and high environmental temperature adaptability, which comprises transforming cells or tissues of the target plant with host cells comprising the plant gene editing vector, and breeding the transformed cells or tissues to obtain transgenic plants in which the polynucleotide sequences are edited.

Further, the method comprises transforming rice cells or tissues with the recombinant vector and cultivating the transformed cells or tissues into plants that overexpress genes controlling high environmental temperature sensitivity of rice.

Further, the transformation is performed by agrobacterium-mediated or gene gun methods, the plants including gramineous plants, preferably rice.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

EXAMPLE 1 isolation of high environmental temperature sensitivity control Gene of Rice

A mutant togr with dwarf plant height at high ambient temperature was first selected from the EMS (Ethylmethylsulfone) -mutated non-glutinous rice ZH11 mutant pool (FIG. 1, a). To further determine whether the mutant was sensitive to environmental temperature change response, we grown it in dark cultures at three temperature conditions of 25℃/20℃for 14 days (25℃for 12h and 20℃for 12h, simulating diurnal cycling conditions), 30℃/25℃and 35℃/30 ℃. Phenotypic statistical analysis found that the seedling height of the mutant was significantly lower than that of the wild type at high ambient temperature, but similar in length to the wild type at low ambient temperature, indicating that the seedling height of the mutant was hypersensitive to high ambient temperature response (FIG. 1, b). In order to further study whether the elongation of the rice plant height is caused by cell division or cell elongation, resin section analysis is carried out on cells between the wild type and the inverted node of the mutant, and the result shows that the cells of the mutant are obviously reduced, the total number of the cells is obviously reduced, the stalks are obviously thinned, the plants are obviously shortened, the leaf length in the seedling stage is obviously shortened, and the air hole length on leaf epidermis cells is obviously shortened (figures 2, a-c).

Example 2 cytological mapping of high environmental temperature sensitivity control genes in rice

To explore the function of protein OsFIE at the cellular level, we performed a subcellular localization experiment of OsFIE 2. By electron microscopy of root tip cells over-expressing transgenic line pUb: osFIE2-GFP, the expression signal of OsFIE-GFP protein was found to be distributed in the cytoplasm and nucleus of the cells (FIG. 3, a). To further examine whether post-mutation protein togr4 affected subcellular localization, we transiently transformed two plasmids carrying OsFIE-GFP and togr-GFP genes, respectively, into rice protoplasts (FIG. 3, b) and tobacco leaf cells (FIG. 3, c), and electron microscopy showed that the expression signal of the togr4 protein remained distributed in the cytoplasm and nucleus after mutation. The above results indicate that OsFIE2 acts in both the cytosol and nucleus of the cell, and that the localization of protein togr is unchanged after mutation.

Example 3 genetic mapping of high environmental temperature sensitivity control genes in Rice

To investigate the reasons responsible for the molecular level responsible for the OsTOGR4 phenotype, we crossed togr and ZH11 and constructed F1 plants (fig. 4, a), and obtained F2 populations from F1 plants, and counted the number of individual plants in the populations that were similar to the togr and ZH11 phenotypes, respectively. According to the diversity of restriction fragment lengths between japonica rice and indica rice, the candidate mutant gene of togr is initially located by using SSR molecular markers (FIG. 4, b). The map-based clonal population is linked on chromosome 8 of rice. By analyzing the recombination rate of the molecular markers of chromosome 8, it was found that the recombination efficiency at the positions of the molecular markers M2 and M3 was significantly reduced (FIG. 4, c), suggesting that the region in the middle of these two molecular markers was highly linked, thus the candidate mutant gene was initially located in the region between M2 and M3 of chromosome 8. Since M2 and M3 were preceded by 1.96Mb, approximately 291 genes. To confirm the mutated gene, we resequenced the entire genome of togr4 and compared to the wild-type genome, and found that there were only 2 missense mutated loci between the M2 and M3 regions. One is LOC_Os08g05450, and the 176 th amino acid of the gene is mutated from Ala to Thr, but because the encoded protein is a transposon, other chromosomes have the gene fragments, so the gene is not considered as a candidate gene. Another candidate mutant gene is LOC_Os08g04270, and the 419 base of the CDS sequence of the gene is subjected to base substitution, so that the mutation from C to T leads to the mutation from threonine (Thr) to isoleucine (Ile) of the 140 amino acid of the encoded protein. Based on the above results, we treated loc_os08g04270 as a candidate mutant gene for togr, which encodes an endosperm autonomous gene OsFIE2 independent of fertilization (fertilization-INDEPENDENT ENDOSPERM GENE 2).

Construction of the complementing vector and genetic transformation of Rice in example 4OsTOGR4

The complete gene OsTOGR4, which is the nucleotide sequence shown in SEQ ID NO. 1 (including the self promoter sequence, genomic sequence and 3' non-coding region sequence), was PCR amplified from wild-type in flower 11, and then digested at the multiple cloning site of the pCAMBIA1300 plasmid (available from CAMBIA Co.) to yield the complementing vector pUb: osTOGR4 (FIG. 5).

The constructed complementary vector was transformed into E.coli DH5α competent cells, and positive clones were selected using kanamycin. Extracting plasmid and carrying out sequencing identification to obtain OsTOGR positive clone with complete OsTOGR sequence in the cloning vector, and then electrically transforming the plasmid of the positive clone into EHA105 agrobacterium competent cells (refer to plant genetic engineering, wang Guanlin, fang Hongjun, scientific press, 2 nd edition 2004) by a conventional method. The successfully transformed clones were then subjected to a transgenic procedure using the Agrobacterium infection method with OsTOGR's 4 receptor.

The transgenic operation method is as follows:

(1) The seed sterilization comprises weighing 20-30 g oven-dried rice seeds, removing seed hulls by a small sheller in laboratory, soaking the shelled seeds in 70% ethanol for 1 min, soaking in 30% sodium hypochlorite (stock solution is 10% available chlorine, 1 drop of Tween-20 is added to 50: 50 mL) for 30min, slightly shaking on a shaker, washing with sterilized water for 5-6 times, sterilizing with 30% sodium hypochlorite (without Tween-20) once and washing with sterilized water for 5-6 times, and transferring the seeds to filter paper in an ultra-clean workbench for air drying.

(2) Inducing callus, transferring the seeds to N6D culture medium prepared in advance, and culturing in an illumination incubator at 32 ℃ for 24 hours until the seeds grow golden yellow callus.

(3) Agrobacterium infection Agrobacterium EHA105 containing the specific plasmid was streaked on YEB medium containing the antibiotics Rif 25-50 mg/L and kanamycin (Kan 50 mg/L) and then placed in an oven at 28℃for 2-3 days in dark, and the monoclonal strain was picked up to liquid YEB medium containing 5-6 mL with the same resistance and placed in a shaker at 28℃ (220 rpm) overnight with shaking. Inoculating the bacterial solution into AAM culture medium of 50mL at a ratio of 1:100, shaking and culturing overnight in shaking table of 28 ℃ and 220 rpm until OD ₆₀₀ is about 0.1, soaking the well-conditioned callus in the agrobacterium bacterial solution for 2 min, and rapidly taking out the callus with forceps and placing on sterile filter paper for air drying. After the callus is dried, transferring to N6D-As culture medium with sterile filter paper (AAM soaking) laid in advance, packaging the culture dish with callus with sealing film and tinfoil paper, and dark culturing at 25deg.C for 2-2.5 days.

(4) Screening and differentiating, namely rinsing the callus co-cultured with the agrobacterium in sterilized double distilled water for 3-4 times, then washing the callus with the sterilized double distilled water containing the carbenium (500 mg/L) for 2-3 times, then starting soaking, soaking for 30 minutes each time, repeating for 3-5 times, and cleaning the agrobacterium on the surface of the callus as much as possible. The washed calli were blotted with sterile filter paper and air dried, then transferred to N6DS medium containing the antibiotics hygromycin B (50 mg/L) and carboxin (400 mg/L) and incubated for 2-3 weeks in an illumination incubator at 32℃for 24 hours with continuous illumination. The well-grown calli were transferred to regenerated RE medium containing hygromycin B (50 mg/L) and carboxin (250 mg/L), differentiation was induced by culturing in a 32 ℃ incubator for 1 month, RE medium was changed about two weeks until green seedlings appeared, calli differentiated from green seedlings were transferred to MS (sucrose 30 g/L) medium containing hygromycin B (50 mg/L) and carboxin (200 mg/L), rooting was induced, and seedlings were transferred to MS medium without antibiotics after growing to a certain size.

The medium related to the transformation of rice by agrobacterium infection is shown in table 1.

TABLE 1 Agrobacterium infection method for transforming rice-related Medium

Example 5OsTOGR phenotyping of complementation transgenic plants

To examine whether OsTOGR4 can complement the phenotype of togr4, positive plants pUb-OsTOGR-GFP/togr 4 obtained from the transgenic experiments of example 2 were dark cultured with medium flowers 11 (wild type) and togr4 at different temperatures, and analysis found that pUb-OsTOGR4-GFP restored the plant height and seedling height of togr4 (FIGS. 6, a and b). This result demonstrates that OsTOGR4 is a key gene regulating the response of rice plant height to high ambient temperature.

EXAMPLE 6 transcript level analysis of OsTOGR4 at different temperatures

To determine how OsTOGR4 responds to changes in ambient temperature, we analyzed the transcript levels of OsFIE2 at various tissues and different temperatures (fig. 7,a). As shown in the results, osFIE2 was widely expressed in various tissues of rice, and its expression in the above-ground tissues (mature leaf, leaf sheath, internode tissue and seedling leaf) was relatively high, significantly high Yu Yousui and mature ears and roots, suggesting that OsFIE2 might play a major role in the growth and development of the above-ground tissues of rice. To further explore the effect of ambient temperature on OsFIE's 2 transcription, we placed rice at three temperatures for 2 weeks, then sampled seedlings, leaves, leaf sheaths and their entire aerial tissues, extracted RNA and examined the transcriptional expression level of OsFIE gene (FIG. 7, b). The amount of OsFIE2 expression in the leaf decreases with increasing ambient temperature, while the amount of OsFIE2 expression in the leaf sheath does not change significantly with increasing ambient temperature, and the amount of OsFIE2 expression in the whole upper tissue is significantly lower than 25 ℃ under conditions of increasing ambient temperature (30 ℃ and 35 ℃), suggesting that the temperature increase reduces the transcriptional expression level of OsFIE 2. There was no significant difference in gene expression levels in leaf sheath tissue of mutant togr4 compared to wild type, whereas at low ambient temperatures the gene expression levels in leaf and aerial parts of mutant togr4 were significantly lower than ZH11, and at high ambient temperatures there was no significant difference compared to ZH11, suggesting that OsFIE2 expression levels were not affected by the mutation at high ambient temperatures. The above transcriptional expression analysis results indicate that an increase in ambient temperature inhibited the expression of OsFIE gene, whereas the mutation of OsFIE2 had no significant effect on gene expression at high ambient temperatures.

EXAMPLE 7 analysis of protein OsTOGR4 content at different temperatures

To explore how protein OsFIE responds to temperature, we selected two complementary transgenic lines pUb-OsFIE2-GFP-1 and pUb-OsFIE2-GFP-2, cultured and extracted the above ground tissue proteins at different temperatures, and then performed immunoblotting experiments (WB). The results showed that the protein content of OsFIE-GFP at 30℃and 35℃was significantly lower than 25℃suggesting that high ambient temperatures also inhibited the protein level of OsFIE2 (FIG. 7, C). To further confirm the inhibitory effect of ambient temperature elevation on protein OsFIE2, we performed a heat treatment experiment in which pUb-OsFIE2-GFP transgenic seedlings grown at 25℃for 2 weeks were transferred to 35℃and sampled with a time gradient, protein was extracted and WB assay performed (FIG. 7,d). As a result, it was found that the protein level of pUb-OsFIE-GFP was significantly decreased with the increase in the heat treatment time, demonstrating that the increase in the ambient temperature suppressed the protein level of OsFIE 2. In combination with OsFIE transcriptional level analysis results, it was shown that high ambient temperature inhibited the transcription and expression of OsFIE 2.

Rice is one of the most important grain crops in China, and the normal response of the rice to normal environment temperature is the basis for ensuring normal growth, development and maturity. The invention clones the key factors for controlling the sensitivity of the rice to high environmental temperature, and provides important guidance for improving the sensitivity of the rice to the environmental temperature through rice genetic engineering and molecular breeding.

Example 8 reduction of Rice sensitivity to high ambient temperature Using CRISPR/Cas9 technology

To explore the role of OsFIE protein structure in temperature response, we obtained the OsFIE allelic mutant of osfie2CRISPR using CRISPR/Cas9 technology, mutant types were three amino acid substitution versions (fig. 8, a). Wild type ZH11 and osfie2CRISPR were selected and combined with the other two mutants for phenotypic characterization by field and incubator temperature treatments. The plant type of the field allelic mutant was analyzed and found that osfie < 2 > CRISPR showed consistent plant height and tillering with mutant togr, both at high ambient temperature (30 ℃ and 35 ℃) and similar plant height levels at normal temperature (25 ℃) and a high ambient temperature insensitive phenotype (fig. 8, b). Temperature experimental treatment of the incubator found that the phenotype difference results were consistent with the performance in the field, osfie CRISPR seedlings exhibited a high-ambient temperature seedling high-defect phenotype (fig. 8,c).

In this example, the CRISPR/Cas9 original vector used by CRISPR/Cas9 technology was constructed and provided by the hundred gene company under the designation SG11588.

The nucleotide sequence of the sgRNA site of action is 5'-TGGCACACCGCTGTTAG-3'.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Reference is made to:

[1]Baile, F., Gómez-Zambrano, Á., and Calonje, M. (2022). Roles of Polycomb complexes in regulating gene expression and chromatin structure in plants. Plant Communications 3, 100267.

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[3]Cheng, X., Pan, M., E, Z., Zhou, Y., Niu, B., and Chen, C. (2020). Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development. The Plant Journal 104, 124-137.

[4]Goodrich, J., Wangsomnuk, P., Martin, M., Long, D., Meyerowitz, E., and Coupland, G. (1997). A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386, 44-51.

[5]Krishnan, P., Ramakrishnan, B., Reddy, K.R., and Reddy, V.R. (2011). Chapter three - High-Temperature Effects on Rice Growth, Yield, and Grain Quality. In Advances in Agronomy, D.L. Sparks, ed (Academic Press), pp. 87-206.

[6]Liao, Z., Yu, H., Duan, J., Yuan, K., Yu, C., Meng, X., Kou, L., Chen, M., Jing, Y., Liu, G., Smith, S.M., and Li, J. (2019). SLR1 inhibits MOC1 degradation to coordinate tiller number and plant height in rice. Nature Communications 10, 2738.

[7]Liu, X., Wei, X., Sheng, Z., Jiao, G., Tang, S., Luo, J., and Hu, P. (2016). Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice. PLOS ONE 11, e0164748.

[8]Mozgova, I., and Hennig, L. (2015). The Polycomb Group Protein Regulatory Network. Annual Review of Plant Biology 66, 269-296.

[9]Nallamilli, B.R.R., Zhang, J., Mujahid, H., Malone, B.M., Bridges, S.M., and Peng, Z. (2013). Polycomb Group Gene OsFIE2 Regulates Rice (Oryza sativa) Seed Development and Grain Filling via a Mechanism Distinct from Arabidopsis. PLOS Genetics 9, e1003322.

[10]Quint, M., Delker, C., Franklin, K.A., Wigge, P.A., Halliday, K.J., and van Zanten, M. (2016). Molecular and genetic control of plant thermomorphogenesis. Nature Plants 2, 15190.

[11]Sultan, B., Defrance, D., and Iizumi, T. (2019). Evidence of crop production losses in West Africa due to historical global warming in two crop models. Scientific Reports 9, 12834.

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Claims (9)

1. Application of rice high-environmental-temperature response gene OsTOGR4 in regulating and controlling sensitivity of plants to high environmental temperature;

The gene OsTOGR is a gene encoding the following protein (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2, or

(B) And (b) a protein which is derived from (a) and has equivalent functions and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

2. Use according to claim 1, characterized in that said plants comprise plants of the poaceae family, preferably rice.

3. The use according to claim 2, wherein the sensitivity of the rice to high ambient temperature is reduced by modifying the gene OsTOGR for high ambient temperature response of the rice to change the function of the gene.

4. A method for reducing sensitivity of rice to high environmental temperature, which is characterized in that the method comprises weakening or knocking out a rice high environmental temperature response gene OsTOGR4 by using genetic engineering means, wherein the gene OsTOGR is as described in claim 1.

5. The method of claim 4, wherein the gene OsTOGR is used as a target, a CRISPR/Cas 9-based sgRNA sequence is designed, a DNA fragment containing the coding the sgRNA sequence is connected to a carrier carrying CRISPR/Cas9, and rice is transformed or transfected, so that transgenic rice with the gene function deletion is obtained.

6. The method of claim 5, wherein the nucleotide sequence of the sgRNA site of action is 5'-TGGCACACCGCTGTTAG-3'.

7. The method of claim 5 or 6, wherein said transformation or transfection is performed using agrobacterium-mediated or biolistic methods.

8. Use of transgenic rice obtainable according to the method of any one of claims 4-7 in plant breeding.

9. The use according to claim 8, wherein the breeding method comprises transgenesis, crossing, backcrossing, selfing or asexual reproduction.