Application of thp1 mutant in regulation and control of high temperature resistance of crops

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a thp1 mutant in regulation and control of high temperature resistance of crops. The sequence of the thp1 mutant gene provided by the invention is shown as SEQ ID NO.1, and the sequence of protein coded by the thp1 mutant gene is shown as SEQ ID NO.2. It is found that crops can highly express THP1 genes in a high-temperature environment, meanwhile, the abundance of THP1 protein in cells in the high-temperature environment is increased, related rice strains carrying THP1 and allelic thp1 genes of THP1 are further tested, and compared with THP1, the thp1 mutant gene has the advantages that the thp1 mutant gene has the advantages that the thp1 mutant gene can be used as a thp1 mutant gene; a rice strain carrying the thp1 mutant gene has higher survival rate, maturing rate and single plant yield at high temperature, so that the effect of the excellent allelic thp1 gene as a forward regulatory factor in regulating high temperature resistance of crops is verified, and a key molecular tool is provided for developing new varieties of crops with high temperature resistance; the gene has wide application potential and market value in the field of agricultural planting, especially in the aspects of improving the high temperature resistance of crops and cultivating high-temperature-resistant transgenic crops.

Description

Technical Field

[0001] The present invention relates to the technical field of crop genetic engineering, and in particular to a thp1 Application of mutants in regulating crop resistance to high temperature. Background Art

[0003] Further digging into the various changes in morphological characteristics caused by high temperature stress: First, in the dimension of plant growth structure, high temperature is like an invisible hand, pushing the growth rhythm of rice to overspeed, resulting in a significant shortening of the growth cycle, and the number of effective tillers and panicles is also helplessly reduced. At the same time, the leaf size becomes smaller, the leaf area continues to shrink, and photosynthesis and normal growth and development processes are hindered. Secondly, in terms of organ development and fruiting rate, the flowering time becomes irregular, the pollination efficiency decreases sharply, the fruiting rate dives, the panicle length shortens, and the number of panicles continues to decrease. Furthermore, by observing the leaves and chloroplasts, it is found that the chloroplast membrane is severely damaged, the internal structure is damaged, and the leaves then show symptoms of yellowing or even whitening, and photosynthesis is naturally unsustainable. Finally, the root morphology is not immune. The appearance and distribution density of the root hairs become abnormal, the root length shortens, and the overall root volume decreases, which seriously affects the intake of water and nutrients.

[0004] In view of this, the corresponding solution strategy focuses on cultivating new rice varieties that can tolerate high temperatures. On the one hand, we conduct rigorous and orderly trait verification and field practice tests to ensure that the new varieties can significantly improve their ability to resist high temperatures while maintaining stable yields and quality; on the other hand, we skillfully use hybridization and backcrossing techniques to perfectly integrate high temperature resistance with other excellent characteristics, and comprehensively strengthen the overall quality of rice.

[0005] Methods for making rice resistant to high temperatures include the rational use of hormones, fertilizers, and reasonable irrigation, but this will increase agricultural costs and cause environmental pollution. Gene breeding technology can also be used to obtain crop varieties that can withstand high temperatures by separating and screening heat-resistant phenotypes. This method has lower application costs and does not require additional operations, and has greater application prospects. Summary of the invention

[0006] The object of the present invention is to provide a thp1 The application of mutants in regulating the high temperature resistance of crops. The present invention found that high temperature stress induces rice to over-express the THP1 gene and greatly increases the abundance of THP1 protein in cells, while its mutant thp1 It exhibits better high temperature resistance and can significantly improve the survival rate, fruit setting rate and single plant yield of rice under high temperature stress. In a first aspect, the present invention provides an application of a thpl protein in regulating the high temperature resistance of crops, wherein the amino acid sequence of the thpl protein is at least one of the following: (1) As shown in SEQ ID NO.2.

[0007] (2) A protein derived from SEQ ID NO.2 by substitution, deletion, and / or addition of one or several amino acid residues, which retains the function of the amino acid sequence shown in SEQ ID NO.2.

[0008] (3) An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 and having the same function.

[0009] (4) An amino acid sequence obtained by linking a tag, a cleavage site, and / or a linker peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (1)-(3).

[0010] In a second aspect, the present invention provides an thp1 application of a gene in improving the high-temperature resistance of crops, wherein the thp1 nucleotide sequence of the gene is at least one of the following: (1) As shown in SEQ ID NO.1.

[0011] (2) A nucleotide sequence obtained by substituting, deleting, and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

[0012] (3) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses the same functional protein. The stringent conditions are hybridization in a solution of 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS at 65°C, and washing the membrane with this solution.

[0013] (4) A nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

[0014] Preferably, the above application is achieved by regulating the abundance of the thp1 gene in crops through genetic engineering or traditional breeding techniques.

[0015] In a third aspect, the present invention provides a method for improving the high-temperature resistance of crops, specifically, enriching the thp1 gene ratio in crops, and the methods include but are not limited to: (1) Directly inserting the thp1 gene by genetic engineering means; or (2) Cultivating thp1 homozygotes by breeding means; or (3) Mutating the THP1 gene intothp1 ; The nucleotide sequence of the thp1 gene is shown in SEQ ID NO.1.

[0016] Preferably, the above method can continuously screen and enrich thp1 genes by backcrossing technology to finally obtain a homozygous plant type.

[0017] Fourthly, the present invention provides an application of the thp1 gene, or the protein encoded thereby, or a biological material containing the gene, or the above method in improving the high-temperature survival rate of crop seedlings; The nucleotide sequence of the thp1 gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0018] Fifthly, the present invention provides an application of the thp1 gene, or the protein encoded thereby, or a biological material containing the gene, or the above method in increasing the yield of crops under high temperature; The nucleotide sequence of the thp1 gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0019] Sixthly, the present invention provides an application of the thp1 gene, or the protein encoded thereby, or a biological material containing the gene, or the above method in cultivating crops with high temperature tolerance; The nucleotide sequence of the thp1 gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0020] Seventhly, the present invention provides an application of the thp1 gene, or the protein encoded thereby, or a biological material containing the gene, or the above method in the genetic improvement of crop germplasm resources; The nucleotide sequence of the thp1 gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0021] The above crops include wheat, corn, rice, soybean, barley, oat, tomato, pepper, rape, etc., preferably rice.

[0022] Advantages of the present invention: The present invention discovers that high-temperature stress induces the overexpression of the THP1 gene in rice and significantly increases the abundance of THP1 protein in cells. Compared with the THP1 lines in Asian cultivated rice, the near-isogenic line thp1 exhibits better high-temperature tolerance characteristics, and can significantly improve the survival rate, seed setting rate, and yield per plant of rice under high-temperature stress. Specifically, compared with normal temperature, the survival rate of the THP1 line under high-temperature stress is reduced to 32%, the seed setting rate is reduced to 33.7%, and the yield per plant is reduced to 11.9 grams, while the survival rate of the line carrying the allele thp1 is 80%, the seed setting rate is 91.7%, and the yield per plant is 26.8 grams, which is significantly higher than that of THP1. Therefore, by pyramiding the excellent allele thp1 in African cultivated rice, the high-temperature resistance and yield traits of crops can be coordinately regulated, which helps the high-yield genetic improvement of crops under high-temperature stress conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 For Example 1 THP1 Schematic diagram of the transcriptional level of the gene changing with temperature.

[0025] Figure 2 Schematic diagram of the structure of the pCB1300-35S-THP1-Flag vector constructed in Example 2.

[0026] Figure 3 Schematic diagram of the expression level of THP1 protein changing with temperature in Example 2.

[0027] Figure 4 Experimental results of the effect of thp1 protein on the high-temperature survival rate of crop seedlings in Example 3.

[0028] Figure 5 Experimental results of the effect of thp1 protein on the seed setting rate of crops in Example 4.

[0029] Figure 6 Experimental results of the effect of thp1 protein on the yield per plant of crops in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0030] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0031] For those without specific techniques or conditions noted in the examples, they are all conventional methods or are carried out according to the techniques or conditions described in the literature in this field, or according to the product instructions. For reagents, instruments, etc. without the manufacturer noted, they are all conventional products that can be obtained through regular channels.

[0032] In the following examples.

[0033] Example 1 Effect of temperature on THP1 Gene transcription level This example is an experiment on the effect of high-temperature environment on the transcription level of THP1 gene. The steps are as follows: The crop material Dianjingyou 1 (DJY1) was divided into 4 groups, with 15 plants in each group, and they were cultured in a crop incubator with temperature gradients of 28 °C, 32 °C, 35 °C and 38 °C for 24 hours respectively.

[0034] The leaves of each group of crops were harvested to extract total RNA, and using the RNA reverse transcription kit EasyScript® One-Step RT-PCR SuperMix (TransGen Biotech, catalog number: AE411-02), after reverse transcribing into cDNA according to the instructions as the template for PCR, the relative expression level of THP1 gene was detected by the method of Real-time RT-PCR, with actin as the internal reference gene. The primer sequences used are as follows: actin forward primer: 5’-AGCAGCATGAAGATCAAGGTGGTC-3’ (SEQ ID NO.3).

[0035] actin reverse primer: 5’-CCTTGGCAATCCACATCTGCTG-3’ (SEQ ID NO.4).

[0036] THP1 forward primer: 5’- CTACTGCTGGAAGAACGGCT-3’ (SEQ ID NO.5).

[0037] THP1 reverse primer: 5’- GGACCTTCTTGTAGTCGCGG-3’ (SEQ ID NO.6).

[0038] The system of Real-time RT-PCR is: a 20 μL amplification reaction system per tube, including: 0.15 μM forward primer and reverse primer, 2×RealStar Fast dye qPCR premix (Beijing Kangrun Chengye Biotechnology Co., Ltd., catalog number: A301-10), 50 - 100 ng template DNA.

[0039] The reaction procedure of real-time RT-PCR was as follows: denaturation at 95°C for 10 minutes, 40 cycles (95°C for 10 seconds, 58°C for 15 seconds, 72°C for 20 seconds), and then extension at 72°C for 5 minutes.

[0040] The results were as Figure 1 shown. Within a certain range, the higher the environmental temperature, the higher the transcriptional level of the THP1 gene. Therefore, high temperature can induce the expression of the THP1 gene.

[0041] Example 2 Effect of temperature on the expression level of THP1 protein This example was an experiment on the effect of high temperature on the expression level of THP1 protein. The steps were as follows: The CDS sequence of the THP1 gene was cloned by PCR. The primers used for cloning were as follows: Forward primer: 5’- ATGTCGGGCTCCTCTGCCGACCCCTCG-3’ (SEQ ID NO.7).

[0042] Reverse primer: 5’- TCGGCCGGAGGAGTCGCCGCTGCGG-3’ (SEQ ID NO.8).

[0043] The PCR system was: a 50 μL amplification reaction system per tube, including: 0.25 μM forward and reverse primers, 2×KOD OneTM PCR Master Mix -Blue premix (Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KMM-101), and 50-100 ng of template DNA.

[0044] The reaction procedure of PCR was: denaturation at 95°C for 2 minutes, 30 cycles (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 60 seconds), and then extension at 72°C for 5 minutes.

[0045] The amplified product was constructed into the pCB1300-35S-Flag expression vector, and then transformed into Escherichia coli competent cells. After culturing at 37°C for 12 hours, single colonies were picked for expansion and plasmid extraction, followed by sequencing. The plasmid with correct sequencing was named pCB1300-35S-THP1-Flag ( Figure 2). Subsequently, by transforming Agrobacterium competent cells and culturing them at 28 °C for 2 - 3 days, single colonies were picked for expansion and injected into the leaves of Nicotiana benthamiana for transient expression. Two days after injection, the tobacco plants were placed in different incubators for temperature treatments at 28 °C, 32 °C, 35 °C, and 38 °C. After 8 hours of treatment, the tobacco leaves were ground in liquid nitrogen and incubated with Anti - Flag beads (Beijing Bormai Biotechnology Co., Ltd., product number: M185 - 10R) at 4 °C for 4 hours. Finally, the internal reference of the sample was detected with an anti - Actin antibody, and the protein level of THP1 - Flag after treatment at different temperature gradients was detected with an Anti - Flag antibody.

[0046] The results are as Figure 3 shown. Within a certain range, the higher the environmental temperature, the higher the expression level of THP1 protein. Therefore, high temperature can increase the expression level of THP1 protein.

[0047] Example 3 Experiment on the effect of thp1 protein on the survival rate of crop seedlings under high temperature This example is for the construction of THP1 near - isogenic lines and the experiment on the growth of seedlings under high temperature. The steps are as follows: Construction of near - isogenic lines: The present invention utilizes 238 chromosome segment substitution lines CSSL18 constructed by crossing the African cultivated rice variety IRGC101901 (from the International Rice Research Institute) with the Asian cultivated rice variety Dianjingyou 1 (DJY1). By backcrossing CSSL18 with DJY1 generation by generation to the parental strain, near - isogenic line (NIL) materials of THP1 under the background of Dianjingyou 1 were obtained. The material carrying the Dianjingyou 1 fragment under the background of Dianjingyou 1 was named THP1 (its nucleic acid encoding is as shown in SEQ ID NO.9, and the amino acid sequence of the protein it encodes is as shown in SEQ ID NO.10); the material carrying the IRGC101901 fragment was named thp1, and its nucleic acid encoding is as shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is as shown in SEQ ID NO.2).

[0048] Experiment on seedling survival rate: After more than 50 seeds of THP1 and thp1 germinated, they were first cultured at normal temperature. After 21 days of germination, THP1 and thp1 were divided into two parts. One part was placed at normal temperature to continue growing, and the other part was transferred to grow at a high temperature of 45 °C. Three days after the normal temperature and high temperature treatments, THP1 and thp1 were transferred back to normal temperature for recovery. Seven days after the normal temperature recovery treatment, the survival rates of THP1 and thp1 seedlings were counted. The results are as Figure 4 shown.

[0049] As Figure 4As shown, environmental temperature has a great impact on the growth and development of crop seedlings. High temperature can significantly inhibit the growth and development of THP1, and more than half of the plants cannot tolerate high temperature and die. After high temperature treatment, the survival rate of the THP1 line is only 32%; under the same treatment conditions, the survival rate of the thp1 line is as high as 80%, significantly higher than that of THP1. This result indicates that under high temperature stress, compared with the THP1 material, the line carrying the thp1 allele can significantly improve the survival rate.

[0050] Example 4 Experiment on the effect of thp1 protein on crop yield under high temperature This example is an experiment on the effect of thp1 protein on crop yield under high temperature. The steps are as follows: To further study the effect of THP1 protein on crop yield under high temperature. After more than 50 seeds of the THP1 line and the thp1 line were germinated, they were first cultured at normal temperature. After germination and booting stage, THP1 and thp1 were divided into two parts and transferred to an artificial climate incubator. One part was continued to grow at normal temperature 28°C / 25°C (day / night), and the other part was transferred to high temperature 33°C / 30°C (day / night) to grow until the harvest period ended. Finally, the seed setting rate and yield per plant of the THP1 line and the thp1 line at different temperatures were counted. The results are as Figure 5 and Figure 6 shown.

[0051] Figure 5 and Figure 6 The results show that under normal temperature conditions, the seed setting rate of DJY1-THP1 is 91.1%, and the yield per plant is 26.0 grams. The seed setting rate of DJY1-thp1 is 91.7%, and the yield per plant is 29.8 grams. The seed setting rate and yield per plant of DJY1-thp1 are only slightly higher than those of DJY1-THP1; while under high temperature conditions, the seed setting rate of DJY1-THP1 is 33.7%, and the yield per plant is 11.9 grams. The seed setting rate of DJY1-thp1 is 78.3%, and the yield per plant is 26.8 grams, much higher than that of DJY1-THP1. This result indicates that regulating the expression level of the THP1 gene in crops can coordinately regulate the high temperature resistance and yield traits of crops, and contribute to the high-yield genetic improvement of crops under high temperature stress conditions.

[0052] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Application of thp1 protein in regulating high temperature resistance of crops, characterized in that, The amino acid sequence of the thp1 protein is at least one of the following: (1) As shown in SEQ ID NO.2; (2) A protein derived from SEQ ID NO.2 by substitution, deletion, and / or addition of one or several amino acid residues, and having the same function as the amino acid sequence shown in SEQ ID NO.2; (3) An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 and having the same function; (4) An amino acid sequence obtained by linking a tag, a cleavage site, and / or a linker peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (1)-(3).

2. An thp1 application of a gene in improving the high temperature resistance of crops, characterized in that The said thp1 The nucleotide sequence of the gene is at least one of the following: (1) As shown in SEQ ID NO.1; (2) A nucleotide sequence obtained by substitution, deletion, and / or addition of one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein; (3) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses the same functional protein, where the stringent conditions are hybridization in a solution of 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS at 65°C and washing the membrane with this solution; (4) A nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expressing the same functional protein.

3. The application according to any one of claims 1 or 2, characterized in that The above application is achieved by regulating the abundance of the thp1 gene in crops through genetic engineering or traditional breeding techniques.

4. A method for improving the high-temperature resistance of crops, characterized in that, Enriching the proportion of the thp1 gene in crops, the methods include but are not limited to: (1) Direct insertion by genetic engineering means thp1 gene; or (2) Cultivation through breeding methods thp1 Homozygous; or (3) By means of genetic engineering, make THP1 mutate into thp1 ; The said thp1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, wherein Continuously screen and enrich using backcross technology thp1 The gene finally obtains a homozygous plant type.

6. An thp1 application of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the method according to any one of claims 4-5 in improving the high-temperature survival rate of crop seedlings; The said thp1 The nucleotide sequence of the gene is as shown in SEQ ID NO.1; or The amino acid sequence of the protein is as shown in SEQ ID NO.

2.

7. An thp1 application of a gene, or a protein encoded thereby, or a biological material containing the gene, or the method according to any one of claims 4-5 in increasing the yield of crops under high temperature; The said thp1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is as shown in SEQ ID NO.

2.

8. Use of a thp1 gene, or a protein encoded thereby, or a biological material containing the gene, or the method according to any one of claims 4-5 in cultivating a crop with high temperature tolerance characteristics; The said thp1 The nucleotide sequence of the gene is as shown in SEQ ID NO.1; or The amino acid sequence of the protein is as shown in SEQ ID NO.

2.

9. An thp1 application of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the method according to any one of claims 4-5 in the genetic improvement of crop germplasm resources; The thp1 nucleotide sequence of the gene is as shown in SEQ ID NO.1; or The amino acid sequence of the protein is as shown in SEQ ID NO.

2.

10. The application according to any one of claims 1-3, 6-9 or the method according to any one of claims 4-5, characterized in that The crop is rice.

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