Application of OsPHOT1 gene in improving rice tolerance to high temperature and / or darkness

Abstract

The present invention discloses the application of OsPHOT1 gene in improving the high temperature and / or darkness resistance of rice, and belongs to the field of biotechnology. The nucleotide sequence of the CDS region of the OsPHOT1 gene is shown in SEQ ID NO.1 or has more than 70% homology with the sequence shown in SEQ ID NO.1 and the encoded protein function is equivalent. The present invention uses transgenic technology to construct OsPHOT1 gene overexpression plants, and verifies the biological function of OsPHOT1 gene in rice. OsPHOT1 gene overexpression significantly improves the survival rate and fruit setting rate of rice under adverse conditions such as high temperature and darkness, and has no effect on the agronomic traits of rice. The present invention provides a theoretical basis and related genes for rice stress resistance breeding, and provides a feasible technical solution for increasing rice yield under adverse conditions.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of OsPHOT1 gene in improving the high temperature and / or darkness resistance of rice. Background Art

[0002] Rice is one of the most important food crops in the world, and it is the main source of energy for most people around the world. As the greenhouse effect continues to intensify, local areas may experience drastic weather changes in a short period of time, leading to abnormal climate, causing natural disasters such as high temperatures and heat waves. Extreme high temperatures will affect the survival rate of rice, thereby affecting rice yields. In addition, sunlight, as one of the sources of energy for plants, also plays an important regulatory role in plant growth. Therefore, by exploring the genetic resources of rice that are resistant to high temperatures and respond to light signals, and using transgenic technology to improve rice's resistance to high temperatures and photosensitivity, it will help improve the plant's stress resistance.

[0003] Since the 1920s, the study of photomorphogenesis has made people's understanding of the relationship between light and plants no longer limited to the energy supply of photosynthesis, but to regard light as a signal, which stimulates the photoreceptors in the plant body and changes genes, proteins and cell metabolism through certain signal transduction processes. PHOT, as a phototropin, is a serine / threonine kinase that depends on blue light. There are two blue light kinases in Arabidopsis, PHOT1 and PHOT2. Both PHOT1 and PHOT2 contain two conserved domains consisting of two light oxygen voltage (LOV) domains (LOV1 and LOV2) and the photoreceptor region at the N terminal and the Ser / Thr protein kinase region at the C terminal (Goh, 2009). In the dark, each LOV domain non-covalently binds to a flavin mononucleotide (FMN). Blue light triggers the covalent binding of the FMN chromophore to the unchanged cysteine ​​residues in each LOV domain, which will lead to changes in protein conformation and kinase activity (Goh, 2009).

[0004] PHOT will undergo autophosphorylation under blue light irradiation, and the autophosphorylated serine / threonine can activate PHOT. PHOT1 (Phototropin1) has been shown to be a receptor kinase. After sensing blue light, PHOT1 will phosphorylate a guard cell-specific blue light signal 1 (BLUS1) kinase, which can activate H + -ATPase hyperpolarizes the membrane, allowing K + Flows into guard cells and opens stomata. This phosphorylated H +-ATPase is stabilized by the presence of 14-3-3 proteins (Gahir et al., 2020); another phototropin protein PHOT2 (Phototropin2) mediates stomatal opening, chloroplast light-focusing movement, phototropism, and cotyledon expansion and development in a functionally redundant manner with PHOT1.

[0005] In rice, both PHOT1 and PHOT2 are predicted to function as blue light receptors. Studies on the rice genome database show that OsPHOT1 has two copies (OsPHOT1a and OsPHOT1b), while OsPHOT2 has one copy. Interestingly, mutations in osphot1a in rice lead to growth defects, while in Arabidopsis, only mutations in phot1 did not result in impaired growth, and only double mutations in phot1 / phot2 resulted in plant growth being affected (Goh, 2009). Therefore, the role of OsPHOT1a in rice remains to be explored.

[0006] Compared with the extensive research on PHOT1 under light regulation, less is known about the regulatory function of PHOT1 under abiotic stress. Therefore, it is of great significance to conduct in-depth research on the molecular mechanism of OsPHOT1 gene in rice growth and development and resistance to high temperature and darkness. Summary of the invention

[0007] The purpose of the present invention is to provide a gene that can regulate the resistance of rice to high temperature and energy deficiency (darkness), and to improve the survival rate of rice under high temperature and energy deficiency conditions through genetic engineering technology.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention uses sequence comparison analysis in the NCBI genome database to search for homologous genes of the Arabidopsis PHOT1 gene sequence in rice, and finds a homologous gene OsPHOT1 in the rice genome. The gene has two copies, named OsPHOT1a and OsPHOT1b, and the homology between the two copies is as high as 97.6%. Previous studies have found that mutations in osphot1a can cause defects in rice growth. The present invention uses transgenic technology to construct OsPHOT1a overexpression plants. Studies have found that the plants show a phenotype of resistance to high temperature and darkness, indicating that the OsPHOT1a gene has certain application value in improving rice resistance to high temperature and darkness.

[0010] Therefore, the present invention provides the use of OsPHOT1 gene in improving the high temperature resistance and / or darkness resistance of rice, wherein the nucleotide sequence of the CDS region of the OsPHOT1 gene is as shown in SEQ ID NO.1 or has more than 70% homology with the sequence shown in SEQ ID NO.1 and the encoded protein is functionally equivalent.

[0011] The CDS region of the OsPHOT1 gene is 2766 bp in length, and the protein sequence it encodes includes 921 amino acid residues. The amino acid sequence is shown in SEQ ID NO.2.

[0012] Any nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO.1 also falls within the protection scope of the present invention.

[0013] Furthermore, the application includes: using overexpression technology to overexpress the protein encoded by the OsPHOT1 gene in rice plants, thereby obtaining rice plants with enhanced resistance to high temperature and / or darkness.

[0014] The present invention utilizes Agrobacterium-mediated genetic transformation technology to construct OsPHOT1 transgenic rice. Studies have shown that overexpression of the OsPHOT1 gene increases the survival rate and grain yield of rice under high temperature and / or dark conditions.

[0015] Furthermore, the high temperature is 40-45°C.

[0016] The present invention also provides a breeding method for improving the high temperature resistance and / or darkness resistance of rice, comprising: inserting an OsPHOT1 gene fragment into an overexpression vector to construct a recombinant plasmid, then using Agrobacterium-mediated technology to introduce the target gene fragment into a rice receptor, and screening to obtain functional transgenic rice plants.

[0017] Preferably, the overexpression vector is a pCAMBIA1300 overexpression vector, which is obtained by transforming the pCAMBIA1300 vector, that is, inserting a corn-derived ubiquitin (Ubi) gene promoter upstream of the T-border of the pCAMBIA1300 vector. Upstream and downstream primers are designed according to the OsPHOT1 nucleotide sequence and connected to the downstream of the Ubi promoter of the pCAMBIA1300 overexpression vector by homologous recombination. The present invention can also use other plant overexpression vectors.

[0018] Preferably, the host bacteria used in the Agrobacterium-mediated technology is Agrobacterium EHA105. The rice receptor material is callus tissue. The constructed vector is transferred into the rice callus tissue using the Agrobacterium-mediated genetic transformation method to obtain T0 generation transgenic plants. Materials with a higher degree of overexpression in the T0 generation are selected for breeding, and homozygous overexpression plants are identified in the T1 generation by DNA level and RNA level detection.

[0019] Preferably, the rice variety is japonica rice kitaake.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a gene OsPHOT1 involved in regulating the high temperature and energy deficiency (darkness) resistance of rice, obtains OsPHOT1 gene overexpression plants by using transgenic technology, verifies the biological function of OsPHOT1 gene in rice, and finds that overexpression of OsPHOT1 gene significantly improves the survival rate and fruit setting rate of rice under adverse conditions such as high temperature and darkness, and has no effect on the agronomic traits of rice. The present invention provides a theoretical basis and related genes for rice stress resistance breeding, and provides a feasible technical solution for increasing rice yield under adverse conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of rice OsPHOT1a overexpression vector.

[0023] Figure 2 Schematic diagram of the positive detection results of rice OsPHOT1a transgenic materials, where M is DNA marker, CT is wild-type plant material, and 1-21 are transgenic plants with family numbers 1-21.

[0024] Figure 3 Schematic diagram of transcriptional expression detection of rice OsPHOT1a transgenic materials, where WT is the wild-type plant material, and 1-15 are transgenic plants with family numbers 1-15.

[0025] Figure 4 The phenotype of the overexpressing material OsPHOT1a under 45℃ high temperature stress. The left picture shows the phenotype before high temperature stress, and the right picture shows the phenotype after high temperature stress.

[0026] Figure 5 The survival rate statistics of overexpressing material OsPHOT1a under 45℃ high temperature stress.

[0027] Figure 6 The phenotypes of the overexpressing material OsPHOT1a under dark treatment. The left picture shows the phenotype before dark treatment, and the right picture shows the phenotype after dark treatment.

[0028] Figure 7 Survival statistics of OsPHOT1a overexpressing materials under dark treatment.

[0029] Figure 8 The figure below shows the comparison of the fruit setting rate and yield per plant of the overexpressing material OsPHOT1a under normal conditions in rice fields. The left figure shows the fruit setting rate, and the right figure shows the yield per plant.

[0030] Fig. 9 Comparison of the fruit setting rate and single plant yield of overexpressing materials OsPHOT1a under high temperature conditions. The left figure is the fruit setting rate, and the right figure is the single plant yield. ** indicates p < 0.01. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example 1: Obtaining rice OsPHOT1a overexpressing plants

[0034] In order to study the function of rice OsPHOT1a gene, an overexpression vector was constructed.

[0035] 1. Construction of pCAMBIA1300 overexpression vector

[0036] 1.1 Sequence comparison analysis was used in the NCBI genome database to search for homologous genes of the Arabidopsis PHOT1 gene sequence in rice, and a homologous gene OsPHOT1 (LOC_Os12g01140) was found in the rice genome. The gene has two copies, named OsPHOT1a and OsPHOT1b. The present invention takes OsPHOT1a as an example for research, and its CDS nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0037] 1.2 First, the pCAMBIA1300 vector was transformed, and the pCAMBIA1300 vector was double-digested with HindIII and PstI, and the Ubiquitin (UBI) promoter (nucleotide sequence as shown in SEQ ID NO.3) was connected to the vector by homologous recombination; the sequence with HA and flag tags was synthesized by the company (HA tag sequence as shown in SEQ ID NO.4, flag tag sequence as shown in SEQ ID NO.5); and then the vector containing UBI, HA and flag was constructed by T4 ligase ligation, and the target gene required in this article was constructed on this basis.

[0038] According to the nucleotide sequence of OsPHOT1a, 20 bp bases were selected from its upstream and downstream, and 20 bp of vector homology arm sequence was added. The primers were designed as follows: OsPHOT1a-F: 5'-ttccagattacgctggatccATGGCTTCCAAAGGCACAGA-3', OsPHOT1a-R: 5'-tggtctttgtagtcggatccGAACATATCCGTGCGGTTAT-3'. Total RNA of rice kitaake variety was extracted and reverse transcribed into cDNA, which was used as a template for amplification of the CDS region of the target gene.

[0039] At the same time, the modified pCAMBIA1300 vector was digested with BamHI, and then the PCR amplification product and the digested vector were detected by 1% agarose gel electrophoresis, and the target product was recovered and purified by gel. Then, the gene recovery product and the digested vector were connected by homologous recombination and transformed into Escherichia coli DH5ɑ. The positive clones were picked, and the bacterial solution with the target band was sent for detection after PCR verification. The single clone with correct sequencing was expanded and the plasmid was extracted. The plasmid was named pCAMBIA1300-UBI::HA-OsPHOT1a-flag( Figure 1 ).

[0040] 2. Genetically modified rice

[0041] The japonica rice variety kitaake was used as the recipient material for transformation. The transformation method used Agrobacterium EHA105-mediated rice callus genetic transformation. After obtaining the T0 generation transgenic plants, the primers on the promoter of the vector pCAMBIA1300UBI and the gene-specific primers were used to identify the positive detection of overexpression plants at the DNA level. The positive detection primers were designed as follows: F: 5'-GATCCACTGATCGATAGTGAT-3'; R: 5'-TCCTTGTAGTCGATGTCATGA-3'. The target fragment was amplified by PCR, and then the PCR product was verified by gel electrophoresis to see if there was a target band. The results showed that except for the families numbered 4, 11, and 21, which were transgenic negative plants, the rest were transgenic positive plants (see Figure 2 ).

[0042] The transcript levels of OsPHOT1a gene in overexpressed plants were detected by real-time fluorescence quantitative PCR. The primers used for real-time fluorescence quantitative PCR were qPCR-OsPHOT1a-F: 5'-AGAGAAAGGAGCAGCAGCAG-3', qPCR-OsPHOT1a-R: 5'-GAAGGAGGAAGCGGATGAGG-3'. According to the results of real-time fluorescence quantitative PCR, OsPHOT1a-OE1, OsPHOT1a-OE2, OsPHOT1a-OE5, OsPHOT1a-OE6, and OsPHOT1a-OE10 were found to be overexpressed lines (see Figure 3 ).

[0043] After harvesting T0 generation seeds, the next generation was propagated, and five families with higher overexpression levels in the T0 generation, OsPHOT1a-OE1, OsPHOT1a-OE2, OsPHOT1a-OE5, OsPHOT1a-OE6, and OsPHOT1a-OE10, were selected for breeding.

[0044] Taking into account the separation of T1 generation transgenic plants, we detected T1 generation transgenic plants by fluorescence quantitative PCR and screened out the T2 generation of transgenic families OsPHOT1a-OE6 and OsPHOT1a-OE10 with higher expression levels for high temperature treatment and dark stress. At the same time, some plants were planted in the transgenic base for breeding. After reaching maturity, the fruit set rate and single plant yield of pCAMBIA1300-UBI::HA-OsPHOT1a-flag overexpressing plants were statistically analyzed.

[0045] Example 2: Identification of stress resistance of rice pCAMBIA1300-UBI::HA-OsPHOT1a-flag overexpressing material

[0046] 1. In order to explore whether the rice OsPHOT1a gene responds to high temperature, the families with higher overexpression multiples, OsPHOT1a-OE6 and OsPHOT1a-OE10, were selected for phenotypic identification.

[0047] The wild type (WT) and T2 generation seeds of overexpression material OsPHOT1a were germinated respectively. After the seeds germinated, the buds with the same growth were selected and sown in hydroponic boxes. Each hydroponic box was planted with 32 wild type control plants and 32 overexpression materials. Three biological replicates were made for each family. Stress treatment was performed after the seedlings grew for 10-15 days. The wild type and transgenic materials sown in the hydroponic boxes were placed in a 45℃ light culture room and subjected to stress treatment until most of the leaves were wilted. After the treatment, they were moved to normal conditions for recovery and growth, and the survival rate was calculated after recovery.

[0048] The results are as follows Figure 4 and Figure 5 As shown, the survival rate of OsPHOT1a overexpressing materials was higher than that of wild type, showing a high temperature resistance phenotype.

[0049] 2. In order to explore whether the rice OsPHOT1a gene responds to darkness, the families with higher overexpression multiples, OsPHOT1a-OE6 and OsPHOT1a-OE10, were selected for phenotypic identification.

[0050] The wild type (WT) and T2 generation seeds of the overexpression material OsPHOT1a were germinated separately. After the seeds germinated, the buds with the same growth were selected and sown in hydroponic boxes. Each hydroponic box was planted with 32 wild type controls and 32 overexpression materials. Three biological replicates were made for each family. Stress treatment was performed after the seedlings grew for 10-15 days. The wild type and transgenic materials sown in the hydroponic boxes were placed in a light-shielded cardboard box and subjected to stress treatment until most of the leaves turned yellow and wilted. After the treatment, they were moved to normal conditions for recovery and growth, and the survival rate was calculated after recovery.

[0051] The results are as follows Figure 6 and Figure 7 As shown, the survival rate of OsPHOT1a overexpressing materials was higher than that of wild type, showing a darkness-resistant phenotype.

[0052] 3. In order to study whether OsPHOT1a overexpression materials have an impact on rice agronomic traits under normal growth conditions, transgenic materials (OsPHOT1a-OE6, OsPHOT1a-OE10) and wild type (WT) were planted in the field and grown normally to maturity. The rice ears were cut off after fruiting to count the number and rate of fruiting.

[0053] The results are as follows Figure 8 As shown, there was no significant difference between the OsPHOT1a overexpressing materials and the wild type.

[0054] The above results indicate that overexpression of the OsPHOT1a gene can increase the survival rate of rice under high temperature and dark conditions and improve the yield of rice under adverse conditions.

[0055] 4. In order to study the fruit setting rate and yield of OsPHOT1a after high temperature treatment at the booting stage, we planted the transgenic materials (OsPHOT1a-OE6, OsPHOT1a-OE10) and the wild type (WT) in large barrels. After normal growth until the booting stage, they were moved to a 45°C plant growth box for 3 days. After the fruit was set, the ears were cut off to count the number of fruits and the fruit setting rate.

[0056] The results are as follows Fig. 9 As shown, the fruit setting rate and single plant yield of the overexpressing material OsPHOT1a were significantly higher than those of the wild type.

[0057] These results indicate that overexpression of the OsPHOT1a gene can improve the resistance of rice to high temperature conditions and increase the yield of rice under high temperature conditions.

Claims

1. Application of OsPHOT1 gene in improving the high temperature resistance and / or darkness resistance of rice, It is characterized in that The nucleotide sequence of the CDS region of the OsPHOT1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, It is characterized in that The amino acid sequence of the protein encoded by the OsPHOT1 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, It is characterized in that The application includes: using overexpression technology to overexpress the protein encoded by the OsPHOT1 gene in rice plants, thereby obtaining rice plants with enhanced resistance to high temperature and / or darkness.

4. The use according to claim 1, It is characterized in that Overexpression of the OsPHOT1 gene improves rice survival and grain yield under high temperature and / or dark conditions.

5. The use according to claim 1, It is characterized in that The high temperature is 40-45°C.

6. A breeding method for improving the high temperature resistance and / or darkness resistance of rice, It is characterized in that include: The OsPHOT1 gene fragment is inserted into an overexpression vector to construct a recombinant plasmid, and then the target gene fragment is introduced into a rice recipient using Agrobacterium-mediated technology, and functional transgenic rice plants are screened. The nucleotide sequence of the OsPHOT1 gene is shown in SEQ ID NO.

1.

7. The breeding method for improving the high temperature resistance and / or darkness resistance of rice according to claim 6, It is characterized in that The overexpression vector is a pCAMBIA1300 overexpression vector, which is constructed by inserting a Ubi promoter derived from corn into the upstream of the T-border of the pCAMBIA1300 vector.

8. The breeding method for improving the high temperature resistance and / or darkness resistance of rice according to claim 6, It is characterized in that The host bacteria used in the Agrobacterium-mediated technology is Agrobacterium EHA105.

9. The breeding method for improving the high temperature resistance and / or darkness resistance of rice according to claim 6, It is characterized in that The rice receptor material is callus tissue.

10. The breeding method for improving the high temperature resistance and / or darkness resistance of rice according to claim 6, It is characterized in that The rice variety is japonica rice kitaake.

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