Application of thermally induced RCA1 gene in improving heat resistance of plants

Abstract

The invention discloses application of a thermal induction type RCA1 gene in improving heat resistance of plants. The nucleotide sequence of the thermal induction type RCA1 gene is shown as SEQ ID NO. 1. The RCA1 gene disclosed by the invention has the function of maintaining the photosynthesis efficiency of plants under high-temperature stress, enriches the heat-resistant molecular theory of the azalea, and has important practical significance in improving the heat-sensitive defect of the azalea, improving the cultivation efficiency and improving the adaptability and attractiveness of the azalea in landscape architecture. In addition, the application of the RCA1 gene is not limited to rhododendron, can also be used for heat resistance improvement of other plants, and is of great significance to plant heat resistance oriented transformation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of a heat-inducible RCA1 gene in improving plant heat tolerance. Background Art

[0002] High temperature stress (HS) is one of the main abiotic stresses faced by plants during their growth and development. It severely restricts important physiological processes such as photosynthesis, protein synthesis, and enzyme activity, thereby significantly reducing plant yield and survival rate.

[0003] Photosynthesis is the basis of plant growth and biomass accumulation, and its efficiency is significantly affected by high temperature stress. Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a core enzyme in the Calvin cycle of photosynthesis, responsible for fixing carbon dioxide into organic compounds.

[0004] Rubisco activity is regulated by Rubisco activase (RCA), which maintains the active conformation of Rubisco by removing inhibitory sugar phosphates. However, RCA activity is highly sensitive to temperature and is easily inactivated at high temperatures, resulting in ineffective activation of Rubisco and inhibiting photosynthesis efficiency (Sage RF, Way DA, Kubien DS. Rubisco, Rubisco activase, and global climate change. J Exp Bot. 2008: 59(7): 1581–1595.).

[0005] There are differences in RCA thermal sensitivity among different plant species. Species native to warm climates generally have lower RCA thermal sensitivity. This may be because they maintain photosynthetic efficiency at high temperatures by improving the thermal stability of RCA or producing high-efficiency variants during their evolution (Crafts-Brandner SJ, Salvucci ME. Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2. Proc Natl Acad Sci US A. 2000: 97(24): 13430–13435.).

[0006] Rhododendrons are woody ornamental plants, but most wild azaleas are native to cool, alpine regions and have poor adaptability to high temperatures. Breeding heat-resistant azalea varieties has practical application value.

[0007] Hainan azalea (Rhododendron hainanense) has a strong adaptability to high temperature environments. Research on its heat shock response mechanism provides an important theoretical basis for the cultivation of heat-resistant azalea germplasm and also provides a reference strategy for the breeding of horticultural plant stress resistance. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of a heat-inducible RCA1 gene in improving the heat tolerance of plants.

[0009] The present invention first provides an application of a heat-inducible RCA1 gene in improving plant heat tolerance. The nucleotide sequence of the heat-inducible RCA1 gene is shown in SEQ ID NO.1.

[0010] Preferably, the heat tolerance of the plant is enhanced by overexpressing the heat-inducible RCA1 gene in the plant.

[0011] Preferably, the overexpression method comprises: cloning the heat-inducible RCA1 gene into a plant expression vector to obtain an overexpression vector, and then introducing the overexpression vector into plant cells. More preferably, the plant expression vector is pCAMBIA3301, and the overexpression vector is first introduced into Agrobacterium, which infects the plant cells to introduce the heat-inducible RCA1 gene into the plant cells for overexpression.

[0012] The present invention further provides a method for improving plant heat tolerance, comprising the following steps: overexpressing a heat-inducible RCA1 gene in a plant to enhance the plant's heat tolerance, wherein the nucleotide sequence of the heat-inducible RCA1 gene is shown in SEQ ID NO.1.

[0013] Preferably, the heat-inducible RCA1 gene is cloned into a plant expression vector to obtain an overexpression vector, and then the overexpression vector is introduced into plant cells.

[0014] More preferably, the plant expression vector is pCAMBIA3301, the overexpression vector is first introduced into Agrobacterium, and the plant cells are infected by Agrobacterium to introduce the heat-inducible RCA1 gene into the plant cells for overexpression.

[0015] In the present application, the plant for improving heat tolerance is a plant of the Ericaceae family or Arabidopsis thaliana. More preferably, the plant is a plant of the genus Rhododendron, particularly some germplasms with poor heat tolerance such as Rhododendron dauricum L. and Rhododendron 'Baiyu'.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention uses qRT-PCR experiments to detect the significant heat-inducible characteristics of the RCA1 gene in Hainan Rhododendron, and constructs an RCA1 subcellular localization vector to prove that it is localized in the chloroplasts of plant cells. It is speculated that the RCA1 gene is a key gene involved in the regulation of photosynthesis under heat stress in Hainan Rhododendron.

[0018] (2) The present invention constructed transgenic Arabidopsis plants that overexpressed the RCA1 gene. By analyzing the phenotype, survival rate, and chlorophyll fluorescence parameters of the transgenic Arabidopsis plants, it was found that the RCA1 gene plays an important role in maintaining plant photosynthesis efficiency and improving plant heat tolerance under heat stress.

[0019] (3) This invention reveals the function of the RCA1 gene in maintaining plant photosynthesis efficiency, enriching the molecular theory of heat tolerance in azaleas. This has important practical significance for improving the heat sensitivity of azaleas, improving cultivation efficiency, and enhancing their adaptability and aesthetics in landscape gardening. Furthermore, the application of the RCA1 gene is not limited to azaleas; it can also be used to improve the heat tolerance of other plants, thus having important implications for the targeted modification of plant heat tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the phylogenetic analysis of the RCAs genes in Hainan Rhododendron in Example 1.

[0021] Figure 2 The expression level changes of RCAs genes in Hainan Rhododendron in Example 2 at different time points under high temperature stress; wherein, Figure 2 A in is the correspondence between time points and sample numbers; Figure 2 B in the figure shows the expression changes of RCA1; Figure 2 C in the figure represents the expression changes of RCA2; Figure 2 D in the figure represents the expression level change of RCA3.

[0022] Figure 3 This figure shows the differential expression of the RCA1 gene in azalea germplasms with different heat tolerance under high temperature stress in Example 2. Blue columns represent gene expression levels after cultivation at 25°C, and red columns represent gene expression levels after heat treatment at 37°C for 1 hour. Significant differences in gene expression (P < 0.05) are indicated with different lowercase letters.

[0023] Figure 4 This is the subcellular localization of the heat-inducible RCA1 in Rhododendron hainanensis in Example 3.

[0024] Figure 5This is the positive line identified in the T1 generation of Arabidopsis plants overexpressing the RCA1 gene in Example 4.

[0025] Figure 6 This is to screen the RCA1 gene high expression strains from the T1 generation positive strains in Example 4.

[0026] Figure 7 The phenotype and survival rate of homozygous Arabidopsis thaliana seedlings overexpressing the RCA1 gene in Example 5 after being treated at 42°C for 7 hours and then restored to normal temperature at 25°C; wherein, Figure 7 A in the figure is the phenotype of the plant after 2 days after treatment (DAT) after recovery at normal temperature; Figure 7 B in the figure is the phenotype of the plant after 5 days of recovery at normal temperature; Figure 7 The C in the figure indicates the partition of different strains on the culture dish; Figure 7 D in the figure is the plant survival rate statistics after 5 days of recovery to room temperature; Col-0 is wild-type Arabidopsis; OE4, OE7, OE8, and OE12 are four homozygous Arabidopsis lines overexpressing the RCA1 gene; with wild-type Arabidopsis Col-0 as the control, if there is a significant difference in survival rate (P<0.05), it is marked with different numbers of asterisks, * indicates P<0.05, and ** indicates P<0.01.

[0027] Figure 8 is the chlorophyll fluorescence parameter of the Arabidopsis thaliana plants overexpressing the RCA1 gene after high temperature treatment in Example 5; wherein, Figure 8 A in the figure is chlorophyll fluorescence imaging; Figure 8 B in the figure is the maximum photochemical efficiency (Fv / Fm) determination; wild-type Arabidopsis Col-0 was used as the control. If there is a significant difference (P<0.05) in the maximum photochemical efficiency (Fv / Fm), it is marked with different numbers of asterisks, ** indicates P<0.01, and *** indicates P<0.001. DETAILED DESCRIPTION

[0028] Example 1

[0029] Construction of the phylogenetic tree of Rhododendron hainanensis and six other accessions.

[0030] The nucleotide sequences of RCAs genes of six accessions, including Arabidopsis thaliana, rice, maize, tomato, grape, and camellia, were obtained from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). Meanwhile, RCAs transcripts of Rhododendron hainanensis were extracted from the Iso-seq database. Cluster analysis was performed on the RCAs genes of Rhododendron hainanensis and the other six accessions. Finally, the RCAs genes of Rhododendron hainanensis were divided into three categories: RCA1, RCA2, and RCA3. Figure 1The phylogenetic tree was generated using MEGA11 software.

[0031] Example 2

[0032] 1. Changes in the expression of RCAs genes in Hainan Rhododendron under high temperature stress

[0033] The nucleotide sequence of the RCA1 gene of Rhododendron hainanensis is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. The accession numbers of the RCA2 and RCA3 gene sequences on GenBank are MN729590 and MN729593, respectively.

[0034] The specific operations are as follows:

[0035] RNA was extracted from the test samples (young leaves of three-year-old cuttings of Hainan Rhododendron after high-temperature treatment at 42°C for 0 min, 10 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h) using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.). The integrity was detected by agarose gel electrophoresis, and the RNA concentration was determined by Nanodrop.

[0036] Take 1 μg RNA and use PrimeScript TM cDNA was reverse transcribed using the RT reagent kit with gDNA Eraser (Perfect RealTime) (TaKaRa) and diluted to serve as a template for qRT-PCR. TB Green dye (TaKaRa) was used for qRT-PCR experiments using a BIO-RAD CFX Connect instrument. TM Real-Time System, using 18S rRNA as internal reference, -ΔΔCt The relative expression of genes was analyzed by the IBM SPSS Statistics software combined with the Duncan method, and significant differences were analyzed.

[0037] The specific primers for detecting RCAs genes are:

[0038] RCA1-rtF: 5'-AGTGTTCGATGAGTCCGACG-3';

[0039] RCA1-rtR: 5'-CCGAATCCATGGGAGCTTGA-3'.

[0040] RCA2-rtF: 5'-AGAACAGGAGAATGTGAAGAGA-3';

[0041] RCA2-rtR: 5'-TGAGCTGCTTGACCATAGAA-3'.

[0042] RCA3-rtF: 5'-GACGGCTAGCAAGTACCTGTG-3';

[0043] RCA3-rtR: 5'-AAAGAGCAAGTGTCATCCCTCC-3'.

[0044] The primers for detecting the 18S internal reference gene are:

[0045] 18S-F: 5'-CGCATTCCCCACTGTATTAGAC-3';

[0046] 18S-R: 5'-CGTAACAAGGTTTCCGTAGGTG-3'.

[0047] The reaction system of qRT-PCR was as follows: 4 μL of cDNA template, 0.5 μL of upstream and downstream primers, and 5 μL of TB Green dye mixture.

[0048] The reaction program of qRT-PCR was as follows: 95°C, 2 min; 95°C, 5 s, 58°C, 30 s, 39 cycles; 95°C, 5 s; 65°C-95°C, 5 s.

[0049] The results showed that the RCA1 gene was almost not expressed at room temperature, but its expression level was significantly upregulated after 0.5 hours of high temperature treatment, reaching a peak at 2 hours and gradually decreasing from 2 to 10 hours, showing a clear heat-inducible pattern. In contrast, RCA2 and RCA3 showed constitutive expression ( Figure 2 ).

[0050] 2. The specific procedures for investigating the expression differences of the RCA1 gene in azalea germplasms with different heat tolerance under high temperature stress are as follows:

[0051] RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd.) from young leaves of three-year-old cuttings of Rhododendron latoucheae Franch., Rhododendron molle (Blume) G. Don, Rhododendron 'Elsie Lee', Rhododendron 'Baiyu', Rhododendron farrerae Sweet, Rhododendron hainanense Merr., and Rhododendron dauricum L., treated at room temperature (25°C) or at 37°C for 1 hour. RNA integrity was assessed by agarose gel electrophoresis, and RNA concentration was determined by Nanodrop. Reverse transcription and qRT-PCR were performed as described above.

[0052] The primers for detecting the RCA1 gene in different Rhododendron germplasms are RCA1-rtF and CA1-rtR.

[0053] The primers for detecting the 18S internal reference gene were 18S-F and 18S-R.

[0054] The reaction system and procedure of qRT-PCR were the same as above.

[0055] The results showed that, except for Rhododendron serrata, the expression levels of the RCA1 gene in the other six rhododendron accessions after heat treatment were significantly higher than those in the 25°C normal temperature treatment group, further confirming the heat-inducible pattern of this gene in rhododendrons. In addition, after high temperature treatment at 37°C for 1 hour, the expression level of the RCA1 gene in Rhododendron hainanensis was significantly higher than that in other rhododendron accessions ( Figure 3 ), indicating that the RCA1 gene in Hainan Rhododendron has a strong response to high temperature stress. Of the seven species tested, Xing'an Rhododendron and 'Baiyu Rhododendron' exhibit relatively poor heat tolerance. With the continued development of transgenic azalea technology, it may be possible to improve the heat tolerance of heat-sensitive germplasm by introducing the Hainan Rhododendron RCA1 gene in the future.

[0056] Example 3

[0057] Subcellular localization of the heat-inducible RCA1 gene.

[0058] 1. Construction of RCA1 subcellular localization vector

[0059] According to the RCA1 gene and vector pHB-YFP sequence, restriction enzyme sites (PstI and SpeI) were selected and homology arms were designed. The designed primers were:

[0060] RCA1-YFP-F: 5'-CTTGGATCCTCGAGCTGCAGAGATCCCAAACA GACCCTTGA-3';

[0061] RCA1-YFP-R: 5'-CCCTTGCTCACCATACTAGTGTTCAATGTATACA AGCAGCTTCC-3'.

[0062] Using these primers and cDNA from Rhododendron hainanensis leaves, the full-length target fragment was amplified and purified. Using homologous recombination, the pHB-YFP vector was digested with the restriction endonucleases PstI and SpeI and purified. Subsequently, the RCA1 gene fragment was ligated into the linearized pHB-YFP vector using the homologous recombinase ExnaseII (Novagen). After sequencing, the subcellular localization vector RCA1-YFP was obtained.

[0063] Escherichia coli transformed with the RCA1-YFP vector were obtained by heat shock. Competent DH5α E. coli cells were added to 15 mL of LBC liquid medium and incubated at 37°C on a shaker (200 rpm / min) for 14 h. Plasmids were then extracted using the TIANprep MiniPlasmid Kit (Tiangen Biochemical Technology Co., Ltd.).

[0064] 2. Arabidopsis protoplast isolation and transformation

[0065] Following previous research methods (WU FH, SHEN SC, LEE LY, et al., 2009. Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods, 5(1):16.), Arabidopsis mesophyll protoplasts were isolated and counted using a microscope. The RCA1-YFP plasmid was transformed into Arabidopsis protoplasts at room temperature and cultured under light for 16 hours. After culture, the protoplasts were observed and photographed under a laser confocal scanning microscope.

[0066] The results showed that the fluorescence signal of RCA1-YFP fusion protein overlapped with the chlorophyll fluorescence region when wild-type Arabidopsis thaliana without RCA1-YFP vector was used as control. Figure 4 ), indicating that RCA1 in R. hainanensis is located in the chloroplast.

[0067] Example 4

[0068] Obtaining Arabidopsis plants overexpressing the RCA1 gene.

[0069] 1. Construction of RCA1 gene overexpression vector

[0070] According to the sequence of RCA1 gene and overexpression vector pCAMBIA3301, restriction enzyme cutting site (NcoⅠ) was selected and homology arms were designed. The designed primers were:

[0071] P3301-RCA1-F: 5'-GGGGGACTCTTGACCATGGAGATCCCAAACAGACCCTTGA-3';

[0072] P3301-RCA1-R: 5'-TTACCCTCAGATCTACCATGGTGTTCAATGTAT ACAAGCAGCTTCC-3'.

[0073] Using these primers and cDNA from Rhododendron hainanensis leaves, the full-length target fragment was amplified and purified. Using the principle of homologous recombination, the pCAMBIA3301 vector was digested with the restriction endonuclease Nco I and purified. Subsequently, the RCA1 gene fragment was ligated into the linearized pCAMBIA3301 vector using the homologous recombinase Exnase II (Novagen). After sequencing, the overexpression vector pCAMBIA3301-RCA1 was obtained.

[0074] 2. Obtaining Arabidopsis plants overexpressing the RCA1 gene

[0075] Agrobacterium transformed with pCAMBIA3301-RCA1 was obtained by the freeze-thaw method. The competent state of Agrobacterium was GV3101. The Arabidopsis inflorescence was infected by Agrobacterium using the floral dip method. The Arabidopsis inflorescence was infected with Agrobacterium and the Agrobacterium was introduced into the ovules through the pollination and fertilization process, thereby producing transgenic T1 generation seeds containing the target gene.

[0076] The obtained T1 generation seeds were sown on MSB resistance medium for initial screening, and transplanted to V after the seedlings grew four leaves. 草炭土 :V 蛭石 =1:1 culture medium, placed in an artificial culture room for cultivation. When several strong leaves grow from the base of the seedlings, extract the leaf DNA for PCR identification ( Figure 5 ), the designed PCR primers are:

[0077] P3301-F: 5'-GGGGGACTCTTGACCATGG-3';

[0078] P3301-R: 5'-TTACCCTCAGATCTACCATGG-3'.

[0079] The PCR reaction system is: 1 μL DNA template, 1 μL upstream and downstream primers, Max Buffer 12.5 μL was added with ddH2O to make up to 25 μL. The PCR reaction program was: 98°C, 4 min; 98°C, 10 s, 60°C, 15 s, 72°C, 60 s, 30 cycles; 72°C, 5 min. After positive strains were identified, qRT-PCR was used to screen strains with high expression of the RCA1 gene from the positive strains ( Figure 6 ).

[0080] The methods of reverse transcription cDNA and qRT-PCR experiments were the same as in Example 2, with AtActin2 gene in Arabidopsis thaliana as the reference gene. -ΔΔCt The relative expression of genes was analyzed by the method, and high-expression lines were screened and T2 generation seeds were collected for subsequent experiments. The specific primers for detecting the RCA1 gene in Arabidopsis thaliana are RCA1-rtF and RCA1-rtR.

[0081] The primers for detecting the AtActin2 internal reference gene are:

[0082] AtActin2-F: 5'-TGCCAATCTACGAGGGTTTC-3';

[0083] AtActin2-R: 5'-TTCTCGATGGAAGAGCTGGT-3'.

[0084] The T2 generation seeds were sown on MSB solid culture medium, and the germination was observed. The strains with a green and strong positive seedling ratio of about 75% were selected for subsequent experiments. The seedlings of these strains were transplanted to the nutrient medium for further cultivation, and the T3 generation seeds were collected after two months.

[0085] The T3 generation seeds were sown on MSB solid culture medium and the germination was observed. The lines with all germinated and strong seedlings were the homozygous lines of RCA1 transgenic Arabidopsis thaliana and were used for subsequent functional research experiments.

[0086] Example 5

[0087] 1. Observation of growth phenotype and survival rate of RCA1 gene overexpressing Arabidopsis plants under high temperature stress

[0088] The specific operations are as follows:

[0089] Wild-type Arabidopsis Col-0 and four RCA1 gene-overexpressing Arabidopsis lines were used as experimental materials. Seeds were sown on MS solid medium, vernalized at 4°C for 2 days, and then incubated at room temperature in an artificial incubator. After the seedlings had four leaves, they were subjected to a 42°C heat treatment for 7 hours. After the heat treatment, the plants were returned to a normal temperature of 25°C. Phenotypes were photographed at 2 and 5 days after the initial growth, and survival rates were calculated.

[0090] The results showed that the phenotype and survival rate of transgenic Arabidopsis lines were significantly better than those of wild-type plants ( Figure 7 After being exposed to 42°C for 7 hours and then returned to normal temperature for 5 days, the RCA1 overexpressing strain maintained a survival rate of 80%, significantly higher than that of wild-type plants. This indicates that the RCA1 gene transfer enhances Arabidopsis heat tolerance and significantly reduces heat damage to plant leaves and roots.

[0091] 2. Determination of chlorophyll fluorescence parameters in Arabidopsis plants overexpressing the RCA1 gene after high temperature treatment

[0092] The specific operations are as follows:

[0093] The Arabidopsis plants after high temperature treatment were kept in the dark for half an hour, and chlorophyll fluorescence was measured using the Imaging PAM fluorescence imaging system (IMAG-MAX / L, Germany) to obtain the maximum photochemical efficiency (Fv / Fm) value.

[0094] The results showed that, compared with the control plant Col-0, the RCA1 gene overexpression Arabidopsis plants showed significantly improved resistance to high temperature stress, as shown by an increase in the maximum photochemical efficiency (Fv / Fm) of the plants ( Figure 8 ), indicating that its photosynthesis efficiency was effectively maintained under high temperature stress.

Claims

1. Use of a heat-inducible RCA1 gene in improving plant heat tolerance, characterized in that: The nucleotide sequence of the heat-inducible RCA1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The plant is a plant of the Ericaceae family or Arabidopsis thaliana.

3. The use according to claim 1, characterized in that The heat tolerance of the plant is enhanced by overexpressing the heat-inducible RCA1 gene in the plant.

4. The use according to claim 1, characterized in that The overexpression method comprises: cloning the heat-inducible RCA1 gene into a plant expression vector to obtain an overexpression vector, and then introducing the overexpression vector into plant cells.

5. The use according to claim 4, characterized in that The plant expression vector is pCAMBIA3301. The overexpression vector is first introduced into Agrobacterium, and the plant cells are infected by Agrobacterium to introduce the heat-inducible RCA1 gene into the plant cells for overexpression.

6. A method for improving heat tolerance of plants, characterized in that: The following steps are involved: The heat-inducible RCA1 gene is overexpressed in plants to enhance the heat tolerance of the plants. The nucleotide sequence of the heat-inducible RCA1 gene is shown in SEQ ID NO.

1.

7. The method for improving plant heat resistance according to claim 6, characterized in that: The heat-inducible RCA1 gene is cloned into a plant expression vector to obtain an overexpression vector, and then the overexpression vector is introduced into plant cells.

8. The method for improving plant heat resistance according to claim 7, characterized in that: The plant expression vector is pCAMBIA3301. The overexpression vector is first introduced into Agrobacterium, and the plant cells are infected by Agrobacterium to introduce the heat-inducible RCA1 gene into the plant cells for overexpression.

9. The method for improving plant heat resistance according to claim 6, characterized in that: The plant is a plant of the Ericaceae family or Arabidopsis thaliana.

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