Application of LPD1 gene in regulating plant resistance to high light stress

Abstract

The present invention discloses the application of LPD1 gene in regulating plant high light stress resistance. The nucleotide sequence of the LPD1 gene is shown in SEQ ID NO.1. The present invention discloses for the first time the effect of the photorespiratory protein encoded by the LPD1 gene on plant high light stress resistance, and the mutation of the LPD1 gene causes the mutant plant to have decreased stress resistance under high light stress. The present invention provides new gene resources and theoretical guidance for deepening the functional research of photorespiration and the research and application of plant high light stress resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of LPD1 gene in regulating plant high light stress resistance. Background Art

[0002] The photorespiration pathway is the second largest metabolic flow in plants after the photosynthetic pathway and is essential for the normal growth and development of plants. The photorespiration pathway starts with ribulose bisphosphate carboxylase / oxygenase (Rubisco), which cannot distinguish between CO 2 and O 2 . When Rubisco undergoes carboxylation reaction, the two molecules of 3-phosphoglycerate (3-PGA) obtained directly enter the Calvin cycle; when Rubisco undergoes oxygenation reaction, one molecule of 3-PGA and one molecule of 2-phosphoethanolate (2-PG) are obtained. The latter inhibits several key enzymes in the photosynthetic process, such as triphosphate isomerase and phosphofructokinase, so it is toxic to plant cells. The photorespiration pathway can metabolize 2-PG into 3-PGA and transport it back to the chloroplasts. This process requires the coordinated cooperation of multiple catalytic enzymes located in chloroplasts, mitochondria, peroxisomes, and cytoplasm.

[0003] Mitochondrial dihydrosulfide dehydrogenase (mtLPD; L-protein) is a structurally conserved homodimeric flavoenzyme of the pyridine nucleotide-disulfide oxidoreductase family, which is ubiquitous in aerobic organisms. In plants, mtLPD protein is a component of several important multienzyme complexes in mitochondria, namely, pyruvate dehydrogenase complex (mtPDHC), 2-oxoglutarate dehydrogenase complex (ODHC), 2-ketoacid dehydrogenase complex (BCDHC) and glycine dehydrogenase complex (GDC). One of the main functions of GDC is to catalyze the conversion of glycine to serine in photorespiration (Dellero, Younès, et al. "Decreased Glycolate Oxidase Activity Leads to Altered Carbon Allocation and Leaf Senescence after a Transfer from High CO2 to Ambient Air in Arabidopsis Thaliana." Journal of Experimental Botany, vol. 67, no. 10, 2016, pp. 3149–63, doi: 10.1093 / jxb / erw054.). In the high-flux photorespiratory metabolic flow in green leaf tissue, GDC occupies 50% of mitochondrial matrix proteins. In previous studies, whether it was silencing the expression of GDC-P protein or overexpressing GDC-H protein, it was shown that the activity of GDC was the determining factor of photosynthetic capacity. Timm further discovered that GDC activity promotes photosynthesis and photorespiration of Arabidopsis Thaliana by changing the photorespiratory metabolic flux (Timm, Stefan, et al. “Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis Thaliana.” Plant Cell, vol. 27, no. 7, 2015, pp. 1968–84, doi: 10.1105 / tpc.15.00105.).

[0004] In Arabidopsis, mtLPD is encoded by two genes, mtLPD1 and mtLPD2, but its genetic function is still unclear. At the genetic level, exploring the function of mtLPD in photorespiration will help improve the application of photorespiration in improving crop quality, yield and stress resistance. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of the LPD1 gene in regulating plant resistance to high light stress. The present invention studies and finds the influence of the photorespiratory protein encoded by the plant LPD1 gene on the plant resistance to high light stress, which can be used to improve the stress resistance of plants under high light stress.

[0006] The present invention first provides an application of the LPD1 gene in regulating plant high light stress resistance, wherein the nucleotide sequence of the LPD1 gene is shown in SEQ ID NO.1.

[0007] The present invention further provides the use of LPD1 protein in regulating plant high light stress resistance, and the amino acid sequence of the LPD1 protein is shown in SEQ ID NO.3.

[0008] The plant is a dicotyledonous plant. Preferably, the plant is Arabidopsis thaliana.

[0009] The present invention further provides a method for improving plant resistance to high light stress, comprising the following steps:

[0010] (1) Gene mutation technology was used to cause the deletion of the LPD1 gene, so that its mRNA could not be expressed, thereby constructing a plant model with abnormal high light resistance;

[0011] (2) Introducing the LPD1 gene into a recipient plant to cultivate a gain-of-function transgenic plant; the recipient plant is a plant that has abnormal high light resistance due to the lack of the LPD1 gene.

[0012] The plant is a dicotyledonous plant. Preferably, the plant is Arabidopsis thaliana.

[0013] The present invention also provides a method for screening plants with strong resistance to high light stress, by detecting the expression level of the LPD1 gene in the plants, and retaining plants with high expression levels as plants with strong resistance to high light stress. Preferably, the plants are Arabidopsis plants.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention discloses for the first time the effect of the photorespiratory protein encoded by the LPD1 gene on the resistance of plants to high light stress. The mutation of the LPD1 gene causes the mutant plants to have reduced resistance to high light stress.

[0016] (2) The present invention provides new gene resources and theoretical guidance for deepening the functional research of photorespiration and the research and application of plant resistance to high light stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The subcellular localization diagram of transient expression in tobacco, where A is the expression of LPD1-YFP alone; B is the mixed expression of LPD1-YFP and Mito-CFP, Mito-CFP is a mitochondrial blue-green fluorescent marker, and LPD1-YFP is LPD1 protein fused to the N-terminus of YFP fluorescent protein, Bar=10μM.

[0018] Figure 2 Schematic diagram of the mutant T-DNA insertion position, where the white box represents the UTR region, the black box represents the exon region, and the black line represents the intron region.

[0019] Figure 3 This is the RT result diagram of the lpd mutant, where ACTINE2 is the internal reference gene of the RT experiment.

[0020] Figure 4 These are the results of trait detection of mutants lpd1-1 and lpd1-2 under normal light and high light, where Figure A is the trait graph under normal light and long day; Figure B is the trait graph under normal light and long and short day; Figure C is the trait graph under high light and long day; Bar=1cm.

[0021] Figure 5 For high CO 2 Complementary lpd1-1 and lpd1-2 trait detection results under high light, among which high CO 2 2000PPM; Bar=1cm.

[0022] Figure 6 P LPD1 -Schematic diagram of the structure of the LPD1 complementation vector.

[0023] Figure 7 Figure 1 is the result of trait detection of complementary strains under high light, where Figure A is the mRNA expression detection of LPD1 in the complementary strains; Figure B is the trait of the complementary strains under high light, Bar=1 cm. DETAILED DESCRIPTION

[0024] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0025] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0027] The nucleotide sequence of the Arabidopsis thaliana LPD1 gene is shown in SEQ ID NO.1, wherein the CDS sequence is shown in SEQ ID NO.2. The amino acid sequence of the Arabidopsis thaliana LPD1 protein is shown in SEQ ID NO.3.

[0028] Example 1

[0029] 1. Construction of LPD1 gene subcellular localization vector

[0030] Primers were designed based on the CDS sequence of the LPD1 genome (as shown in SEQ ID NO.2), and the CDS sequence was amplified using the Columbia wild-type Arabidopsis cDNA of our laboratory as a template. The amplified fragment was connected to the YFP coding gene sequence to express the LPD1 protein in fusion with the YFP protein. The LPD1 protein was connected to the N-terminus of the YFP, that is, the YFP protein was connected to the C-terminus of the LPD1 protein. The primers used were LPD1-YF and LPD1-YR.

[0031] The primer sequences are:

[0032] LPD1-YF:

[0033] 5'-gagaacacgggggactctagaATGGCGATGGCGAGCTTAGCCTAG-3';

[0034] LPD1-YR:

[0035] 5'-ttgctcaccattgttggatccccGATGTGAATAGGCTTGTCATAGGTG-3';

[0036] The PCR reaction conditions were:

[0037] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 min, extension at 72°C for 1 min 30 sec, 35 cycles; extension at 72°C for 5 min.

[0038] The two fragments were recovered by agarose gel electrophoresis.

[0039] For the successfully constructed vector, the pCAMBIA1300-YFP vector was double-digested with XbaI and BamHI, and the target fragment and the linearized vector were connected by homologous recombination to obtain the 35S-LPD1-YFP recombinant vector;

[0040] Take 1 μl of the recombinant product and transform it into E. coli DH5α by freeze-thaw method. The transformation product is spread on LB medium with kanamycin resistance (50 μg / ml). Culture at 37°C overnight, and pick a single clone to extract the plasmid. The constructed vector is stored for future use after sequencing verification.

[0041] 2. Subcellular localization experiment

[0042] The correctly sequenced vector obtained in Example 1 was transformed into Agrobacterium.

[0043] The fusion expression vector was injected into Nicotiana benthamiana leaves by Agrobacterium infiltration method, and the mitochondrial fluorescent marker Mito-CFP was injected as mitochondrial control. The fluorescence signal was observed by laser confocal microscopy after 48 hours.

[0044] The experimental results are shown in Figure 1 YFP protein fused to the C-terminus of the major spliceosomal protein of LPD1 (the mitochondrial localization signal peptide of LPD1 protein was not blocked) was localized to peroxisomes.

[0045] Example 2

[0046] 1. Identification of T-DNA insertion mutants

[0047] Two mutants of LPD1 caused by T-DNA insertion were purchased from the Arabidopsis Biological Resource Center (ABRC, https: / / abrc.osu.edu / ) and were designated as lpd1-1 and lpd1-2, respectively. The positions of T-DNA insertion are shown in Figure 2 .

[0048] The mutant genotype was identified using the three-primer method. The primers were designed by the T-DNA primer design website (http: / / signal.salk.edu / tdnaprimers.2.html). The primers designed by the website were named lpd1-1-LP and lpd1-1-RP, lpd1-2-LP and lpd1-2-RP. The LB primer was LBb1.3 provided by the website.

[0049] The primer sequences are:

[0050] lpd1-1-LP:5'-ctctgttcatctctacaaggag-3';

[0051] lpd1-1-RP:5'-CAAGAGAACGTTGAAACTGC-3';

[0052] lpd1-2-LP:5'-TTAGATGTGAATAGGCTTGTC-3';

[0053] lpd1-2-RP: 5'-ATGGCGATGGCGAGTTTAGCTAG-3';

[0054] LBb1.3: 5'-ATTTTGCCGATTTCGGAAC-3'.

[0055] The PCR reaction conditions were:

[0056] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min.

[0057] RNA was extracted from the identified positive seedlings, and the cDNA obtained after reverse transcription was used as a template for RT-PCR experiments.

[0058] The PCR reaction conditions were:

[0059] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 1 min 30 sec, 35 cycles; extension at 72°C for 5 min.

[0060] RT-PCR Primers:

[0061] ACTINE2-F:5'-ACCCGATGGGCAAGTCATCACG-3';

[0062] ACTINE2-R: 5'-TCCCACAAACGAGGGCTGGA-3';

[0063] LPD1-F: 5'-AACGACGTCGTCATCATCGGCGG-3';

[0064] LPD1-R: 5'-CAATCCTTCTGCATTATCAATAG-3'.

[0065] The lpd1-1 and lpd1-2 homozygous mutants were identified and the RT-PCR results were as follows Figure 3 shown.

[0066] 2. Characteristic analysis of the T-DNA mutant lpd under normal light and high light conditions and high CO 2 Restoration of high light stress resistance defects in lpd mutants under high light

[0067] The normal light intensity is 10K Lux, and the high light intensity is 50K Lux; the light / dark duration is 16h / 8h under long-day conditions, and 8h / 16h under short-day conditions.

[0068] like Figure 4 As shown, the lpd1 mutant had no significant difference from the wild type under normal light (long day and short day) with a light intensity of 10K Lux, where the plants under long day were photographed and recorded at 25 days of cultivation (growth temperature was 23°C, humidity was 65%), and the plants under short day were photographed and recorded at 35 days of cultivation (growth temperature was 23°C, humidity was 65%). However, under high light with a light intensity of 50K Lux, the resistance to high light stress decreased, which was specifically manifested in the stunted development of the plant disc leaves and abnormal disc leaf morphology. The plants under high light were photographed and recorded after four weeks of growth (growth temperature was 23°C, humidity was 65%).

[0069] like Figure 5 As shown, high CO 2 (CO 2 The concentration of 2000PPM) can restore the high light stress resistance defect of the lpd1 mutant under high light (light intensity of 50K Lux), and the plants were photographed after four weeks of growth (growth temperature of 23°C and humidity of 65%). This shows that the photorespiration defect in the lpd mutant leads to its high light stress resistance defect.

[0070] Example 3

[0071] 1. LPD1 complementation vector construction and genetic transformation experiment

[0072] In order to verify that the decreased resistance to high light stress in the T-DNA mutant strain was caused by the mutation of the LPD1 gene, rather than by the unknown T-DNA insertion, a vector expressing endogenous LPD1 was constructed for complementation. The schematic diagram of the complementary vector structure is shown in Figure 6 .

[0073] The vector for endogenous expression of LPD1 consists of an endogenous promoter (SEQ ID NO.4) driving the LPD1 coding region sequence (SEQ ID NO.2). The promoter of LPD1 was first amplified using primers LPD1-pro-F and LPD1-pro-R, and the recovered target fragment was connected to the pCAMBIA1300-YFP vector double-digested with HindIII and XbaI, and after transformation into Escherichia coli DH5α competent cells, a single colony was picked to extract the plasmid. The constructed vector was sequenced and verified and stored for future use, recorded as pCAMBIA1300-LPD1pro-YFP.

[0074] Using Arabidopsis thaliana reverse transcribed cDNA as a template, primers LPD1-cF and LPD1-cR were used to amplify the LPD1 coding region sequence (SEQ ID NO.2), and the recovered target fragment was connected to the pCAMBIA1300-LPD1pro-YFP vector double-digested with XbaI and SalI, and transformed into Escherichia coli DH5α competent cells, and a single colony was picked to extract the plasmid. The constructed vector was sequenced and verified and stored for future use, recorded as pCAMBIA1300-proLPD1-LPD1-YFP.

[0075] The sequenced vector was transformed into Agrobacterium using the following method.

[0076] Add 0.1-1 μg (5-10 μl) of plasmid DNA to 1.50 μl of Agrobacterium competent cells, and then place on ice for 30 minutes;

[0077] 2. Place in liquid nitrogen for 5 min (or 1 min), then immediately place in a 37°C water bath for 5 min;

[0078] 3. Take out the centrifuge tube, add 0.5 ml LB, and culture at 28°C and 220 rpm for 3 to 5 hours;

[0079] 4. Take out the bacterial solution and spread it on the LB plate containing kanamycin antibiotics, and culture it upside down in the incubator at 28°C.

[0080] The above Agrobacterium was transformed into Arabidopsis thaliana using the following inoculation method.

[0081] 1. Three days before transformation, inoculate Agrobacterium containing the binary vector into 5 ml of LB liquid medium containing antibiotics (rifampicin 25 mg / L, kanamycin 50 mg / L) and culture at 28°C with shaking for 2 days.

[0082] 2. Two days later, transfer 1 ml of cultured Agrobacterium into 100 ml of LB liquid medium containing antibiotics and continue shaking culture at 28°C for 24 hours.

[0083] 3. Transfer the Agrobacterium into a centrifuge tube, centrifuge at 6000 rpm / min at room temperature for 10 minutes, and then pour out the supernatant.

[0084] 4. Resuspend the precipitate with 200 ml of infection solution to form a uniform Agrobacterium suspension (OD600 = about 0.8), and transfer the Agrobacterium suspension to an open vessel (500 ml beaker).

[0085] 5. Select healthy plants in the early fruiting stage, place them upside down with the pots on top of the container containing the Agrobacterium suspension, and immerse the entire inflorescence in the Agrobacterium suspension for about 20 to 30 seconds.

[0086] The Agrobacterium into which the target plasmid was transferred was transferred into the lpd mutant by the inflorescence infection method to obtain T0 generation transgenic seeds.

[0087] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 5 min.

[0088] The primer sequences are:

[0089] LPD1-pro-F:

[0090] 5'-aaaacgacggccagtgccaagcttCCTACAACTTCTCGGTACCAAGTG-3';

[0091] LPD1-pro-R:

[0092] 5'-TAGCTAAACTCGCCATCGCCATggatcctgttgttttagagagaaaga-3';

[0093] LPD1-cF:

[0094] 5'-ctttctctctaaaacaacaggatccATGGCGATGGCGAGTTTAGCTAG-3';

[0095] LPD1-cR:

[0096] 5'-aaattcgagctctcaagtcgacTTAGATGTGAATAGGCTTGTCATAGGTG-3'.

[0097] 2. Characteristics of complementary strains under high light

[0098] The T0 transgenic seeds were sown in 1 / 2MS medium containing hygromycin (50 μg / ml) for screening, cultured under a light intensity of 10k Lux for about 1 week, and the positive seedlings were observed and screened. The resistant seedlings were transplanted into nutrient soil, and the positive seedlings were tested for LPD1 gene mRNA using primers LPD1-F and lpd1-1-RP. The internal reference genes detected were ACTINE2-F and ACTINE2-R

[0099] The primer sequences are:

[0100] LPD1-F: 5'-AACGACGTCGTCATCATCGGCGG-3';

[0101] lpd1-1-RP:5'-CAAGAGAACGTTGAAACTGC-3';

[0102] ACTINE2-F:5'-ACCCGATGGGCAAGTCATCACG-3';

[0103] ACTINE2-R: 5'-TCCCACAAACGAGGGGCTGGA-3'.

[0104] The PCR reaction conditions were:

[0105] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 1 min, 32 cycles; extension at 72°C for 5 min.

[0106] The results are as follows Figure 7 As shown, the successful complementation strains were screened and restored their high light stress resistance under the high light condition of 50k Lux (growth temperature was 23°C and humidity was 65%), indicating that the reduction of high light stress resistance of lpd mutants was caused by the loss of LPD1.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. LPD1 The application of genes in regulating plant resistance to high light stress, It is characterized in that Said LPD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

2. Application of LPD1 protein in regulating plant resistance to high light stress, It is characterized in that The amino acid sequence of the LPD1 protein is shown in SEQ ID NO.3, and the plant is Arabidopsis thaliana.

3. A method for improving plant resistance to high light stress, It is characterized in that The following steps are involved: (1) Using gene mutation technology to cause LPD1 The deletion of the gene makes its mRNA unable to be expressed, thus constructing a plant model with abnormal high light resistance; (2) LPD1 The gene is introduced into the recipient plant to cultivate a transgenic plant with gain of function; the recipient plant is a plant that lacks LPD1 A plant having a gene causing abnormal high light resistance, wherein the plant is Arabidopsis thaliana; Said LPD1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for screening plants with strong resistance to high light stress, It is characterized in that By detecting the LPD1 The expression level of the gene is high or low, and the plant with high expression level is retained as a plant with strong high light stress resistance, and the plant is an Arabidopsis plant; Said LPD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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