CN117304289A - Application of MAX2 in adjusting plant high light adaptability - Google Patents

Abstract

The invention discloses application of MAX2 in regulating plant highlight adaptability, wherein MAX2 protein is protein shown as a sequence 1 in a sequence table. The present invention proposes that NaMAX2 plays a role in highlight tolerance and adaptation enhancement by modulating highlight response independently of the roles in SL and KAR signaling pathways, and has value in studying gene function in complex environments of plant evolution, i.e., in natural environments.

Description

Application of MAX2 in adjusting plant high light adaptability

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a new high-light adaptability function of MAX2 in Nicotiana intensifolia.

Background technique

Plants in nature face complex environments, and phytohormones play a central role in coordinating plant physiological responses to complex environmental stresses. Desert environments are typically characterized by intense sunlight, including photosynthetically active radiation (PAR) and extreme ultraviolet (UV-B) radiation, low water and nutrient availability, extreme heat, wind and dust storms, and intense pest pressure. Nicotiana attenuata is native to the Utah Desert in the United States and has long lived in these abiotic and biotic stress environments. Constructing transgenic plants of Nicotiana intensifolia and planting them in their native habitat to understand the role of genes in plant adaptation to various environmental stresses has proven to be an important means of studying gene functions.

Strigolactones (SLs) are a class of carotenoid-derived hormones that regulate plant structure by inhibiting branch branching. It also serves as a rhizosphere signal between host and parasitic plants and symbiotic fungi. DWARF14 (D14) is an α/β fold hydrolase and a receptor for SLs. Karrikin (KARs) is a class of small molecules produced by burning plants that can stimulate seed germination after fire. The receptor for KARs, KARRIKININSENSITIVE 2 (KAI2), is a paralog of D14. Signaling triggered by both SL and KARs requires the same coreceptor, MOREXILLARYGROWTH2 (MAX2), a leucine-rich F-box protein. max2 mutants in various plants, including Arabidopsis and rice (where they are referred to as dwarf3), are insensitive to both SL and KAR signaling.

SL biosynthesis and signaling mutants are available in Arabidopsis, rice, and petunia, and all exhibit increased branching phenotypes. In addition, the SL pathway is known to regulate other traits that affect yield, such as grain weight in maize and ear development in rice. SL and KAR also play a central role in defense against biological attacks, such as against larvae of the stem-boring weevil (Trichobarus mucorea), as well as nematodes. Both SL and KAR signaling pathways improve plant survival rate under drought and salt stress. These results suggest that the SL and KAR signaling pathways may be desirable targets for improving crop yield. However, most of these studies were conducted in greenhouse or laboratory settings, and the role of SL and KAR signaling pathways in natural environments remains unknown.

To facilitate experimental research, plants are usually grown in greenhouses, and their performance in the field is inferred through their growth and development in the greenhouse. However, there is a big difference between the greenhouse environment and the field environment. For example, light is typically only a fraction of the PAR intensity of full daylight, and often lacks UV-B entirely. Plants growing in the Utah desert are regularly exposed to 2000 μmol/m ² /s of PAR and hundreds of μmol/m ² /s of UV-B, conditions that are difficult to replicate in a controlled environment. Exposure to light levels that exceed photosynthetic capacity can lead to photooxidative stress and chlorophyll damage, and plants have evolved photoprotective measures to minimize damage caused by excess light energy, such as photorespiration, non-photochemical quenching (NPQ), and ultrasonic quenching. The accumulation of oxide dismutase (SOD), as well as antioxidant substances such as carotenoids that prevent chlorophyll from oxidizing. However, whether the SL and KAR signaling pathways play a role in these excess light responses remains unknown.

Contents of the invention

The purpose of the present invention is to provide an application of the MAX2 gene in Nicotiana intensifolia and the protein it encodes in regulating the high-light adaptability of plants. The nucleotide sequence of the MAX2 gene is shown in Sequence 2; the amino acid sequence of the protein encoded by it is shown in Sequence 1.

The present invention claims the use of a protein in regulating the high-light response of plants; the protein is at least one of the following A1)-A3):

A1) The protein shown in sequence 1 in the sequence listing;

A2) A fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in A1);

A3) A protein with the activity of regulating high light response of plants obtained through substitution and/or deletion and/or addition of one or several amino acid residues in A1).

The A3) may be the protein shown in sequence 3 in the sequence listing.

In regulating the high light response of plants, one of the proteins shown in Sequence 1 or Sequence 3 can be used, or they can be used together.

The present invention also claims the application of biological materials related to the protein in regulating the high light response of plants, and the biological materials are any one of the following B1) to B5):

B1) Nucleic acid molecules encoding the proteins described in;

B2) An expression cassette containing the nucleic acid molecule described in B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) Nucleic acid molecules that reduce the expression level of the protein.

Wherein, the nucleic acid molecule described in B1) is the following b11) or b12) or b13) or b14):

b11) The coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;

b12) The cDNA molecule or DNA molecule shown in sequence 2 in the sequence listing;

b13) A cDNA molecule or DNA molecule that hybridizes to the nucleotide sequence defined by b11) or b12) or b13) under stringent conditions and encodes the protein.

Wherein, the high-light response of the plant is regulated by reducing the high-light tolerance of a plant strain that lowers the expression of the protein than that of a plant strain that normally expresses the protein.

The high light refers to high PAR light, which generally refers to PAR light of greater than or equal to 1000 μmol/m ² /s, especially PAR light similar to about 2000 μmol/m ² /s in the Utah desert.

The present invention provides a method for cultivating light-sensitive plants, which includes the step of inhibiting the expression of the protein in the target plant/or silencing the encoding gene. Generally speaking, the coding genes shown in sequence 2 or sequence 4 can be silenced simultaneously to achieve the purpose of light sensitivity.

The present invention provides a method for cultivating plants with strong high light tolerance, which includes the step of overexpressing the protein in the target plant.

Wherein, the light sensitivity refers to being sensitive to high PAR light; the high light tolerance means strong tolerance to high PAR.

The present invention provides a method for cultivating leaf-bleaching plants, which includes expressing/or silencing the encoding gene of the protein in a target plant; and cultivating the target plant under high PAR light conditions.

The application of the above-mentioned proteins or the above-mentioned biological materials in any of the following should also be within the scope of the present invention:

1) Prepare products for cultivating plants with high light tolerance;

2) Cultivate plants with high light tolerance;

3) Prepare products for cultivating plants with low light tolerance;

4) Cultivate plants with weak high light tolerance;

5) Prepare products for cultivating leaf bleaching plants;

6) Cultivate leaf bleaching plants;

7) Preparing products for cultivating plants with reduced chlorophyll content;

8) Cultivate plants with reduced chlorophyll content;

9) Prepare products for cultivating plants with reduced photosynthetic capacity;

10) Cultivate plants with reduced photosynthetic capacity;

11) Preparation of products for cultivating plants with reduced pest resistance;

12) Breed plants with reduced pest resistance.

The beneficial effect of the present invention is that in the lines in which the SL and KAR signaling pathway genes (irMAX2, irD14 and irKAI2 plants) are silenced, irMAX2 and irD14 show the expected increase in branching, but only the irMAX2 plant shows the expected increase in branching when grown in the field. Strong leaf bleaching phenotype. In the field, irMAX2 plants had lower sugar and higher leaf amino acid content, lower life history adaptability and were more susceptible to insect damage compared with wild type. These phenotypes were not observed in greenhouse-grown irMAX2 plants. Transcriptomic analysis showed that irMAX2 leaves in the field responded significantly to high-intensity light; lutein content decreased, and transcriptional responses to high-intensity light, singlet oxygen, and hydrogen peroxide increased. Experiments with PAR and UV-B conducted in the field showed that the irMAX2 bleaching phenotype was reversed by reduced PAR, while UV-B did not affect its phenotype. The present invention proposes that NaMAX2 regulates high light response independently of its role in the SL and KAR signaling pathways, thereby playing a role in high light tolerance and adaptive enhancement. This work provides another example of the value of studying gene function in the complex context of plant evolution—that is, in the natural environment.

Description of the drawings

Figure 1 shows that MAX2 silencing of greenhouse-grown N. attenuata plants enhances growth and fitness without affecting insect resistance. (A) Plant height and number of main branches of 3.5-month-old EV, irMAX2#2 and irD14#2 plants grown in the greenhouse (n=8). (B and C) Phenotype and relative chlorophyll amount at rosette leaf stage (n=8) (B) Chlorophyll a, b content and chl a/b ratio (n=6) (C). (D) Biomass, number of flowers, and number of capsules of EV, irMAX2#2 and irD14#2 plants in the specified months (n=8-12). Data collected at 3.5 months were used for statistical analysis. (E) Lepidopteran pests: generalist insects (Spodoptera littoralis) and specialist insects feeding on EV, irMAX2#1, irMAX2#2 and defenseless (irAOC) plants on the indicated days (n=16-28) (Manduca sexta) larval weight. (F) Levels of JA and JA-Ile in irMAX2#1 and irMAX2#2 leaves without (control) or 1 hour after leaf puncture wound treatment with EV (n=6). Statistical analysis was performed using Turkey’s multiple comparison test (P < 0.05) (A, C, D) or two-tailed t test (n.s. no significant difference) (F) (all values are means ± SE).

Figure 2 shows that MAX2 silencing in N. attenuata plants grown in the field resulted in leaf bleaching and impaired fitness and insect resistance. (A) Representative pictures of 2.5-month-old plants grown on Snow plot in 2018, plant height and primary branch number of WT, irMAX2#1, irD14#2 and irKAI2#2 plants (n=8). (B and C) Phenotype, relative chlorophyll amount (n=6) (B) and chlorophyll a, b, chl a/b (n=8) (C) content of the above plants grown in Lytle plot in 2019. (D) Number of flowers and capsules of 2.5-month-old WT, irMAX2#1, irD14#2 and irKAI2#2 plants in the 2018 field season (n=8-10). (E) Biomass, flower number, and capsule number of 2.5-month-old EV, irMAX2 plants from 2 independent lines during the 2019 field season (n = 8-10). (F) Leaf damage proportions of EV, irMAX2, irD14, irKAI2 and irD14×irKAI2 plants in the 2019 field season. Two lines were included per genotype (mean ± standard error, n = 46-60). Level V: 30-40% blade damage, Level IV: 20-30% blade damage, Level III: 10-20% blade damage, Level II: 5-10% blade damage, Level I: 0-5% blade damage. (G) Levels of JA and JA-Ile in leaves of the above lines without (control) or wound treatment after 1 hour (mean ± SE, n = 8). Statistical analysis was performed using Turkey's multiple comparison test (P < 0.05) (A, C, D, E) or two-tailed Student's t-test (**P < 0.01) (G). All values are: mean ± standard error).

Figure 3 shows changes in primary metabolites in irMAX2 leaves in the field. (A and B) Glucose, fructose, sucrose and starch contents in R6 leaves of EV, irMAX2#2 and irD14#2 plants in greenhouse (A) and 2019 field season (B) (mean ± standard error, n = 6) . (C and D) Proportions of amino acids in R6 leaves of greenhouse (C) and field-grown EV, irMAX2#2 and irD14#2 plants in 2019 (D) Total amounts of amino acids (mean ± SE, n=6). Statistical analysis was performed using Turkey’s multiple comparison test (P<0.05) (A, B) or two-tailed Student’s t-test (**, P<0.01) (D).

Figure 4 shows transcriptomic analysis showing the hypersensitivity of irMAX2 leaves to high-intensity light. (A) Multidimensional scaling (MDS) plot of EV, irKAI2#2, irMAX2#2 and irD14#2 leaf transcriptome data collected during the 2019 field season (mean ± standard error, n = 3). (B) Venn plot showing the overlap between up-regulated and down-regulated genes in EV, irKAI2#2, irMAX2#2 and irD14#2 leaves. (C) Heat map showing specific up- and down-regulated genes in EV and irMAX2#2 leaves. (D) Gene Ontology (GO) enrichment of 640 up-regulated and 683 down-regulated genes from EV and irMAX2#2 leaves (C). Red indicates up-regulated genes, blue indicates down-regulated genes.

Figure 5 shows that reducing PAR but not UV-B can rescue the bleaching phenotype of MAX2 silenced plants. (A) Relative UV-B flux outside and inside UV-blocking cages and UV-transparent cages (n=6). (B) Relative chlorophyll content of EV high UV, irMAX2 high UV (UV transparent cage), and irMAX2 low UV (UV blocking cage) plant leaves (n=6). (C) Schematic of the shade cage and the light intensity (PPFD) and UV-B levels inside and outside the cage. (D) Representative images of irMAX2-control (no shade cage), irMAX2-shade (21 days in shade cage), and irMAX2-reexposed (21 days in shade cage, 5 days cage removed); EV-high light ( Relative chlorophyll amount of leaves without shade cage), irMAX2-high light (without shade cage) and irMAX2-low light (shade cage), shade treatment for 21 days (n=8), irMAX2-low light, irMAX2-high light and irMAX2-removed Relative chlorophyll content of leaves after 5 days in shade cage (n=5-8). (E) Light response curves of EV high light, irMAX2 low light and irMAX2 high light plants (n=3). (F) A-Ci curves of EV-high light, irMAX2-low light and irMAX2-high light plants, each with 2 replicates. (G) Contents of lutein, zeaxanthin and β-carotene in leaves of EV-high light, irMAX2-high light and irMAX2-low light plants (n=6-8). (H) High-light-responsive genes (ELIP1, ELIP2), H ₂ O _2- related genes (SOD, APX2, CAT and ZAT10) and singlet oxygen-responsive genes (WRKY33) in EV-high-light, irMAX2-high-light and irMAX2-low-light plant leaves , WRKY40-1 and WRKY40-2) relative transcript abundance (n=8). All experiments performed in this figure were completed during the 2019 field season. Statistical analysis was performed using Turkey's multiple comparison test (P < 0.05) (G, H) (all values are means ± SE).

Figure 6 is a summary of the functions of MAX2 in greenhouses and fields. (A) When grown at relatively low light levels (200-300 μmol/m ² /s), irMAX2 plants develop more branches, increase aboveground biomass and seed production; insect resistance is not affected. (B) In the Utah desert, where the plant's native environment is characterized by consistently high light intensities (2000 μmol/m ² /s), mature leaves of irMAX2 plants experience bleaching, reduced photosynthesis, H ₂ O ₂ , sugars, and carotenoids, Singlet oxygen levels and protein instead increase, which impairs growth, seed set and resistance to native leaf feeders.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

In the following examples, Nicotiana attenuata was derived from seeds collected from DI Farm (Santa Clara, Utah) in the United States in 1988; plasmid pRESC8 was derived from the laboratory of Ian T. Baldwin of the Max Planck Institute for Chemical Ecology, Germany. Disclosed in the article (Li, et al., 2020, Strigolactone signaling regulates specialized metabolismin tobacco stems and interactions with stem-feeding herbivores).

In the following examples, some of the basic experiments and methods involved are as follows:

1) Insect resistance bioassay: Indoor-reared larvae of the nicotine-resistant Lepidopteran species Nicotiana sexta and Spodoptera litura were used for greenhouse insect resistance assay at a stage when the experimental plants had just begun to transition from vegetative growth to reproductive growth. Single newborn tobacco hornworm larvae are placed on fully expanded leaves of EV or irMAX2 plants and allowed to move and feed freely throughout the plant. Larval biomass was weighed on designated days. For the Spodoptera litura experiment, a second-instar larvae (fed with artificial feed for 3 days after hatching) was placed on a fully expanded rosette leaf in a clamp cage and moved to new leaves every 1-2 d. Insect damage to field-grown plants in 2019 was visually estimated and calculated according to previously described methods. More than 40 plants per genotype were used to quantify the percentage of canopy area being eaten by insects when plants were in early flowering (33 days after planting). Much of the damage is caused by feeding by armyworm larvae, adults and nymphs of tree crickets and grasshoppers, and flea beetle adults.

2) Photosynthesis: Photosynthesis measurements were performed using the LI-6400XT analysis system (Li-Cor Biosciences, USA), with a fluorometer integrated in the leaf chamber. Field measurements were performed on cloud-free days between 10 am and 14 pm. Photosynthetic rate (A) was measured at 400 μmol/m ² /s CO ₂ , 30°C temperature and 2000 μmol/m ² /s light intensity. Photoresponse curve measurements were performed at light intensity levels of 0-2000 μmol/ ^m2 /s using the same reference _CO2 levels and temperatures. A _/ Ci curves were performed at reference CO2 levels at 7 levels from 0 to 900 μmol/ ^m2 /s at a temperature of 25°C and a light intensity of 2000 μmol/ ^m2 /s. In the greenhouse, the photosynthetic rate was measured at 400 μmol/m ² /s CO ₂ , a temperature of 25°C and a light intensity of 0-2000 μmol/m ² /s. Fv/Fm, NPQ, Fv'/Fm' and PhiPSII were measured according to the LICOR 6400XT manual for dark adaptation and light adaptation.

3) Relative chlorophyll and leaf temperature: Use Dualex (FORCE-A) handheld spectrophotometer to measure the relative chlorophyll amount and leaf temperature in the middle layer of fully expanded leaves of plants grown in the field.

4) Determination of chlorophyll and carotenoids: A crushed sample (100 mg) of leaves was extracted with 600 μL of methanol for chlorophyll measurement. After centrifugation, 30 μL of the supernatant sample was separated by HPLC using an Acclaim C30 column (Dionex, 200A, 3.5um, 4.6*250mm) for pigment quantification. Pure chlorophyll a, chlorophyll b and β-carotene were calibrated using external standards. The mobile phase of HPLC is as follows: A: 80% methanol, B: ethyl acetate, flow rate is 1mL/min: 0 minutes, 80% (vol/vol) A, 20% B; 2.5 minutes, 77.5% A, 22.5% B ; 20 minutes, 50% A, 50% B; 22.5 minutes, 50% A, 50% B; 24 minutes, 20% A, 80% B; 26 minutes, 20% A, 80% B; 31 minutes, 0% A, 100% B; 34 minutes, 0% A, 100% B; 42 minutes, 80% A, 20% B; 47 minutes, 80% A, 20% B. Chlorophyll a is detected at 440nm, chlorophyll b and β-carotene are detected at 475nm, and the reference wavelength is 550nm.

Crushed samples of leaves (100 mg) were extracted with 600 μL of methanol for chlorophyll measurement. After centrifugation, 20 μL supernatant samples were separated by HPLC using an Acclaim C30 column (Dionex, 200A, 3.5 μm, 4.6*250 mm) to quantify carotenoids (lutein, zeaxanthin) by an external standard curve of pure standards. . The mobile phase of HPLC is as follows: A: methanol and ethyl acetate (1:1), B: acetonitrile, C: deionized water and 200mM acetic acid, the gradient at a flow rate of 1mL/min is as follows: 0–3.6 minutes, 14.5% (volume /volume) A, 85% B, 0.5% C; 3.6–27 min, gradient phase to 34.5% A, 65% B, 0.5% C; 27–45 min, 34.5% A, 65% B, 0.5% C. Carotenoids are detected at 475nm with a reference wavelength of 550nm.

5) Microarray analysis and RT-qPCR: For transcript abundance quantification, use RNA Plant (Macherey-Nagel) kit extracted RNA from specified leaves. For microarray analysis, RNA extracted from field-grown leaves was labeled and hybridized according to the protocol of the Rapid Amplification Labeling Kit (Agilent, http://www.agilent.com/home). For hybridization, an Agilent Monochrome Technology array (60k) was used. Raw intensity data were normalized using the quantile method and probes with low expression levels were discarded after log2 transformation. Differentially expressed probes were filtered after pairwise comparisons (|fold change| ≥ 2, FDR ≤ 0.05).

For RT-qPCR, cDNA was prepared using PrimeScript RT-qPCR kit (TaKaRa). RT-qPCR was performed on a Mx3005P qPCR machine using SYBR Green reaction mix (Eurogentec, qPCR kit SYBR Green INo ROX). For all RT-qPCR analyses, the N. attenuata IF-5a housekeeping gene was used as an internal reference.

6) Sugar content determination: To determine sugar levels, 100 mg of crushed R6 leaves (see Figure 1B for leaf position determination) leaf samples were extracted with 800 μL of extraction buffer (0.2N formic acid in 80% MeOH). After centrifugation, follow The extract was diluted 200-fold with 500 pmol/μL sorbitol (standard) for HPLC-MS analysis following the procedure described by et al. The analysis was performed on a Bruker Elite EvoQ triple quadrupole MS equipped with a HESI (heated electrospray ionization) ion source.

7) Amino Acid Determination: To determine amino acid levels, 100 mg of crushed leaf leaf samples were extracted with 800 μL of extraction buffer (0.2N formic acid in 80% MeOH). After centrifugation, follow The extracts were diluted 50-fold with a 1 ng/g amino acid standard mixture for HPLC-MS analysis following the procedure described by et al. The analysis was performed on a Bruker Elite EvoQ triple quadrupole MS equipped with a HESI (heated electrospray ionization) ion source.

8) Jasmonic acid measurement: To determine jasmonates (JA and JA-Ile) levels, 100 mg of leaf flake powdered samples were extracted with 800 μL of extraction buffer (0.2 N formic acid in 80% MeOH, isotopically labeled JA internal standard). After centrifugation, follow HPLC-MS analysis of extracts was performed using the procedure described in et al. The analysis was performed on a Bruker Elite EvoQ triple quadrupole MS equipped with a HESI (heated electrospray ionization) ion source.

Example 1 Preparation of Plants

NaMAX2, NaD14 and NaKAI2 of gene-independent lines of N. attenuata were silenced by RNA interference (RNAi) and transformed with empty vector (EV) as a control. The target sequence fragments of the genes to be silenced are connected to plasmid pRESC8 to construct RNAi vectors. The vector was transfected into the hypocotyls of Nicotiana intensifolia to form transgenic plants. The success of fragment silencing was confirmed by detecting the expression of the corresponding genes. Wherein, the nucleotide sequence of the NaMAX2 gene is as shown in Sequence 2 (NaMAX2a) or Sequence 4 (NaMAX2b) of the sequence list, NaMAX2a encodes the protein as shown in Sequence 1, and Sequence 4 (NaMAX2b) encodes as shown in Sequence 3 of protein. The nucleotide sequence of NaD14 and its encoded protein are shown in sequence 6 (NIATv7_g08776) or sequence 8 (NIATv7_g26688) of the sequence list. NIATv7_g08776 encodes the protein shown in sequence 5, and sequence 8 (NIATv7_g26688) encodes the protein shown in sequence 7. The protein; the nucleotide sequence of NaKAI2 and the protein it encodes are shown in sequence 10 or sequence 12 of the sequence listing. Sequence 10 encodes the protein shown in sequence 9, and sequence 12 encodes the protein shown in sequence 11.

1. Preparation of silent plant irMAX2

1) Design the target sequence of RNA interference for NaMAX2a (as shown in Sequence 13); design the target sequence of RNA interference for NaMAX2b (as shown in Sequence 14); connect the DNA fragments as shown in Sequence 13 and Sequence 14 respectively. onto plasmid pRESC8 to obtain the RNAi vector pRESC8-NaMAX2. The pRESC8-NaMAX2 is obtained by replacing the sequence between the SacⅠ and XhoⅠ sites of pRESC8 with the fragment shown in Sequence 15, and replacing the sequence between the PstⅠ and MluⅠ sites with The recombinant vector of the fragment shown in 16.

2) The vector pRESC8-NaMAX2 was transfected into the hypocotyls of Nicotiana angustifolia to form transgenic plants irMAX2, respectively irMAX2#1 and irMAX2#2.

3) Use MAX2a-F (ttgagccttaccgaactagatta) and MAX2a-R (gagtgtgacgcattcttgtag) as primers to detect the expression of NaMAX2a; use MAX2b-F (actatttcaggggtgctcgc) and MAX2b-R (cgattcaagggctggtggta) as primers to detect the expression of NaMAX2b; the results show that irMAX2# 1 and irMAX2#2 plants had the NaMAX2a and NaMAX2b genes silenced.

2. Preparation of irD14 in comparative silenced plants

1) Design RNA interference target sequences for NaD14 (as shown in Sequences 17 and 18); connect the DNA fragments as shown in Sequence 17 and Sequence 18 to plasmid pRESC8, respectively, to obtain RNAi vector pRESC8-NaD14, said pRESC8 -NaD14 is a recombinant vector in which the SacⅠ and XhoI sites of pRESC8 are inserted into the fragment shown in Sequence 19, and the PstⅠ and MluI sites are inserted into the fragment shown in Sequence 20.

2) The vector pRESC8-NaD14 was transfected into the hypocotyls of Nicotiana angustifolia to form transgenic plants irD14 plants, respectively irD14#1 and irD14#2.

3) Use D14-F (gatcggaattctcgcctcca) and D14-R (ccgacagctaacggagcaaa) as primers to detect the expression of NaD14. The results showed that the NaD14 gene was silenced in irD14#1 and irD14#2 plants.

3. Preparation of comparative silenced plants irKAI2

1) Design the target sequence of RNA interference for NaKAI2 (as shown in sequences 21 and 22); connect the DNA fragments as shown in sequence 21 and sequence 22 to the plasmid pRESC8, respectively, to obtain the RNAi vector pRESC8-NaKAI2, the pRESC8 -NaKAI2 is a recombinant vector in which the SacⅠ and XhoI sites of pRESC8 are inserted into the fragment shown in Sequence 23, and the PstⅠ and MluI sites are inserted into the fragment shown in Sequence 24.

2) The vector pRESC8-NaKAI2 was transfected into the hypocotyls of Nicotiana angustifolia to form transgenic plants irKAI2 plants, respectively irKAI2#1 and irKAI2#2.

3) Using KAI2-F (gatcggaattctcgcctcca) and KAI2-R (ccgacagctaacggagcaaa) as primers to detect the expression of NaKAI2, the results showed that irKAI2#1 and irKAI2#2 plants silenced the NaKAI2 gene.

4. Preparation of comparative silenced plants irD14*irKAI2

The irD14*irKAI2 double mutant was prepared by crossing irD14 plants and irKAI2 plants.

KAI2-F and KAI2-R were used as primers to detect the expression of NaKAI2, and D14-F and D14-R were used as primers to detect the expression of NaD14. The results showed that irD14*irKAI2#2 and irD14*irKAI2#2 plants simultaneously silenced NaKAI2. and NaD14 gene.

5. Control plants

The blank vector pRESC8 was transfected into the hypocotyl of Nicotiana intensifolia to obtain plant EV that could be used as a blank control.

Example 2 Silencing NaMAX2 can enhance the adaptability of greenhouse-grown plants

The above plants were cultured in the greenhouse according to the aforementioned method, and the phenotype, chlorophyll content, insect resistance and JA level were measured. The results are shown in Figure 1.

As can be seen from Figure 1, the two lines of irD14 and irMAX2 plants produced more primary branches and the overall plant shape was shorter (as shown in Figure 1A), while the irKAI2 plant did not. The sensitivity of irMAX2 plants to primary root inhibition and the elongation response of hypocotyl to GR24 treatment were lower than those of EV plants. These results indicate that N. attenuata irMAX2 plants exhibit the same phenotype as irMAX2 mutants in other plants, such as Arabidopsis and rice.

As shown in Figures 1B and 1C, the chlorophyll content (including chlorophyll a and b and the a/b ratio in rosette leaves) of irMAX2, irD14, and irKAI2 plants did not differ from EV plants when grown in the greenhouse.

As shown in Figure 1D, after two months of growth, the aboveground biomass and flower number of irD14 and irMAX2 plants increased significantly, and after 2.5 months of growth, the number of capsules was larger.

As shown in Figure 1E, feeding trials with the generalist Spodoptera exigua and the specialist Nicotiana sexta larvae showed that the two species performed similarly when feeding on EV and irMAX2 plants, but not when feeding on defenseless irAOC When fed on plants that silently express the JA biosynthetic gene ALLENE OXIDE CYCLASE, the two insects gained significantly more biomass.

As shown in Figure 1F, the levels of JA and JA-Ile induced and synthesized by irMAX2 leaves after 1 h of wounding treatment were not significantly different from those of EV leaves.

The above results indicate that silencing NaMAX2 expression increases biomass and seed yield without affecting insect resistance, suggesting that silencing MAX2 may be an effective strategy to improve plant fitness.

Example 3 In the field, silencing NaMAX2 causes leaf bleaching and impairs plant fitness

To evaluate whether these results for greenhouse-grown plants can be extended to field growth, EV, irMAX2, irD14, and irKAI2 plants were grown for three years in two field plots in the Great Basin Desert of Utah, USA, as described previously.

After 2.5 months of growth in the field season, the plant phenotype, chlorophyll level and jasmonic acid synthesis level were measured. The results are shown in Figure 2.

As shown in Figure 2A, compared with EV plants of normal plant type, irMAX2 plants are smaller, denser, and have more primary branches;

As shown in Figure 2B and 2C, two independent irMAX2 lines showed severe leaf bleaching during three years of cultivation, and the chlorophyll a and b contents of rosette and stem leaves were significantly reduced, while irD14, irKAI2 and irD14 were different from irKAI2 plants. This situation did not occur in any of the hybrid lines (irD14*irKAI2). Since D14 and KAI2 are receptors for SL and KAR signaling, respectively, these results suggest that MAX2 regulates this bleaching phenotype in a manner independent of SL and KAR signaling.

As shown in Figures 2D and 2E, irMAX2 plants grown in the field in 2018 and 2019 had reduced aboveground biomass, flower and capsule number compared with EV plants, while the other three transgenic plants (irD14, irKAI2 or irD14*irKAI2 plants) This phenotype was not present.

As shown in Figure 2F, the leaves of irMAX2 plants suffered greater biological damage than other transgenic plants and EV plants.

As shown in Figure 2G , the wound-induced jasmonic acid synthesis levels (JA, JA-Ile levels) of irMAX2 leaves were significantly lower than those of EV leaves.

In contrast, in irD14, irKAI2, and irD14*irKAI2 leaves, there were no differences in these insect resistance indicators compared with EV plants. From these results, it can be seen that silencing the NaMAX2 gene in field-grown plants results in leaf bleaching, reduced fitness and insect resistance and is independent of SL and KAR signaling.

Example 4 Changes in leaf nutrients of irMAX2 and irD14

In order to study the changes in leaf nutrients of irMAX2 and irD14, the leaf starch and soluble sugar contents were first analyzed. The results are shown in Figure 3.

As shown in Figures 3A and 3B, soluble sugars, including glucose, fructose, and sucrose, were not altered in the leaves of irMAX2 and irD14 plants grown in the greenhouse. However, starch and soluble sugar contents were significantly reduced in bleached leaves of field-grown plants irMAX2.

Next we analyzed the amino acid content. As shown in Figures 3C and 3D, irMAX2 and irD14 plant leaves contained similar levels to EV leaves under greenhouse conditions. However, among plants grown in the field, the total amino acid content of irMAX2 plants was 2 times higher than that of EV leaves, and the content of some amino acids exceeded these increases, such as asparagine (11 times), tryptophan (10 times). , arginine (7 times), isoleucine (7 times), valine (6 times) and leucine (5 times).

In summary, bleached leaves of field-bleached irMAX2 plants have low sugar content and high amino acid content. These changes may be caused by reduced chlorophyll content, which in turn leads to low photosynthetic capacity, low carbohydrate levels and poor reproduction. poor ability.

Example 5 Transcription reaction in bleached irMAX2 leaves

To explore the cause of irMAX2 bleaching, microarray assays were performed with EV, irMAX2, irKAI2 and irD14 leaves from the field. Multidimensional scaling (MDS) analysis clearly separated irMAX2 samples from EV, irKAI2 and irD14 samples (Figure 4A). Differentially expressed genes (DEGs) (|log2FC|≥1, FDR≤0.05) were identified by Venn plot, including 640 up-regulated and 683 down-regulated DEGs-specific changes in irMAX2 leaves, and presented in heat maps (Figures 4B and 4C ). These DEGs were further used to calculate Gene Ontology (GO) enrichment, which revealed that upregulated genes were enriched in processes related to heat, light intensity, and oxidative stress responses, while downregulated genes were enriched in starvation responses (Fig. 4D).

Example 6 Field PAR shade rescues the bleaching phenotype of irMAX2 plants

To evaluate whether the high UV-B or PAR levels characteristic of the desert light environment are responsible for leaf bleaching, field-grown blank plants and irMAX2 plants were enclosed in cages designed to filter out UV-B, but leave PAR essentially unchanged; Control NaMAX2 gene silenced plants were enclosed in UV-B transparent cages. In the UV-B filter cage, the UV-B flux dropped from 220 μW/cm ² to 30 μW/cm ² at noon, while the flux in the UV-permeable cage was 190 μW/cm ² (Figure 5A). After 21 days of growth, there were no significant differences in chlorophyll levels and leaf color between the plants in the two cages (Figure 5B). From these results, it is concluded that UV-B flux alone is not the cause of irMAX2 leaf bleaching.

To assess whether these high PAR levels were responsible for bleaching of irMAX2 plants, blank and irMAX2 plants were enclosed in PAR shade cages and midday PAR values were reduced 10-fold to levels commonly seen in greenhouses (200 μmol/m ² /s; Figure 5C). After 21 days of growth, compared with irMAX2 enclosed in a metal cage without shading, the leaves of irMAX2 in the sensitive node positions (R1 to R8 are the leaf positions of the rosette leaves, as shown in Figure 2B) were completely re-greened and restored. Chlorophyll a and b levels (Fig. 5D). In addition, greenhouse-grown irMAX2 plants were exposed to 1000 μmol/m ² /s PAR levels for 10 days using LED lights, and the chlorophyll content was monitored. After two days of high light exposure, the chlorophyll content decreased. By the 6th day, the leaves of nodes R1 to R4 Completely bleached. From these results, it was inferred that exposure to high PAR levels could explain the bleaching phenotype.

In order to further characterize the bleaching and greening reactions, infrared gas analysis (IRGA) was performed on field-grown blank plants and the above-mentioned transgenic plants during the 2018 and 2019 field seasons. The photosynthetic rate of bleached rosette leaves and stem leaves of irMAX2 plants was reduced, while the leaves at corresponding positions on irD14, irKAI2 or irD14*irKAI2 plants were not reduced. The light response curve of bleached irMAX2 leaves was attenuated in the initial slope, light saturation rate, and saturated photosynthetic value; but after shading treatment, these had returned to levels close to EV leaves (as shown in Figure 5E). The RuBPCase carboxylation activity inferred from the A/Ci curve calculated by field IRGA basically recovered after 21 days of shading treatment (as shown in Figure 5F). Non-bleached leaves of plants grown at low light levels in the greenhouse (200 μmol/ ^m2 /s) with EV and irMAX2 plants No differences in photosynthetic parameters between plants: light response curve, maximum efficiency of PSII (Fv/Fm), PS II efficiency (Fv'/Fm') and the proportion of absorbed light used for photosynthesis (PhiPSII), and non-photochemical quenching (NPQ). From these results, it can be seen that high PAR light is responsible for the bleaching phenotype and lower photosynthetic capacity of irMAX2 plants in the field. When grown under low PAR light levels in the greenhouse, irMAX2 plants have normal chlorophyll levels and photosynthetic capacity.

Example 7 PAR shading can rescue antioxidant content and ROS response in irMAX2 leaves

High light stress triggers a series of oxidative reactions, in which antioxidant substances, such as lutein, zeaxanthin and β-carotene, protect plant cells from oxidative damage caused by excessive light. The contents of three antioxidant substances, lutein, zeaxanthin and β-carotene, in the leaves of irMAX2 were measured according to the above method. In all three field seasons, the lutein level of irMAX2 leaves was significantly reduced, while the lutein levels of irD14 and irKAI2 leaves were not reduced; the zeaxanthin content was not different from that of EV leaves, both were relatively low; the β-carotene content in the 2019 field season lower than EV leaves, but higher in the 2018 and 2020 field seasons (shown in Figure 5G). After field shading treatment in 2019, the lutein content of irMAX2 leaves returned to WT levels, but the β-carotene level did not (as shown in Figure 5G). In greenhouse-grown plants, the levels of all three antioxidants in irMAX2 leaves did not differ from levels in EV leaves. When these greenhouse-grown plants were exposed to high (1000 μmol/m ² /s) PAR levels for 2 days, the levels of all three antioxidant substances increased in both plant lines and lutein was lower than in EV leaves, but not zeaxanthin and beta-carotene levels. These results indicate that low lutein levels contribute to the sensitivity of irMAX2 leaves to high light stress.

Excess light is often associated with ROS accumulation, such as singlet oxygen, hydrogen peroxide (H ₂ O ₂ ), malondialdehyde (MDA) and related transcription. We detected the transcript abundance of the highly light-responsive genes ELIP1, ELIP2, the singlet oxygen-responsive genes WRKY33, _WRKY40-1 and WRKY40-2, and the _H2O2 catalytic and responsive genes SOD, APX2 and ZAT10. During the 2018 and 2019 field seasons, the transcript levels of these marker genes were elevated in irMAX2 leaves (shown in Figure 5H). After sunshade treatment, the transcription levels of these genes decreased to EV levels (Fig. 5H). Transcript levels of ELIP1, ELIP2 increased significantly (4-fold) in response to 2 days of high PAR exposure. From these results, it is inferred that the light-blocking reversible bleaching phenotype does not involve ROS-mediated cell death responses and eventual senescence, but more detailed kinetic analysis of these responses under high PAR conditions is needed to understand the role of ROS signaling in the bleaching table. role in the model.

Example 8 Silencing of SL signal has opposite effects under field and greenhouse conditions

Blocking SL signaling in N. attenuata under greenhouse conditions will release the inhibition of plant fitness by SLs, thereby enhancing aboveground biomass and seed production. It works mainly by increasing lush branches (Figure 6A). However, in the native habitat of N. attenuata in the Utah desert, high-light stress caused severe leaf bleaching after silencing the MAX2 gene, through a manner that may be independent of the SL and KAR signaling pathways (Fig. 6B). A decrease in the antioxidant lutein may be one of the causes of leaf bleaching. The drastic reduction in photosynthetic capacity leads to a decrease in sugar and changes in amino acids hinder the adaptability and yield of irMAX2 plants. Furthermore, the reduction in JA response resulted in more severe insect damage on leaves (Fig. 6B).

The function of MAX2 in response to strong light stress during evolution in the above examples can be further elucidated in various species, including Coleophyceae, Zygophyceae, bryophytes, ferns, gymnosperms and angiosperms.

However, when grown into the plant's native habitat, Utah's Great Basin Desert, MAX2 was identified as a key gene that regulates high-light responses. Silencing MAX2, but not D14 and KAI2, either alone or together, resulted in leaf bleaching, reduced chlorophyll content, photosynthetic capacity, fitness and pest resistance. The bleaching phenotype of irMAX2 plants was restored by reducing PAR, but not by UV-B shading. The mechanism by which MAX2 regulates responses induced by excess light was investigated through transcriptomic analysis and measurement of sugar, amino acid, antioxidant, and peroxide gene transcript levels. It was inferred from these results that MAX2 enhances plant fitness in nature by regulating high light response and antioxidant accumulation, independent of the SL and KAR signaling pathways.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Claims (9)

1. Use of a protein for modulating plant specular response; the protein is at least one of the following A1) to A3):

a1 Protein shown in a sequence 1 in a sequence table;

a2 Fusion proteins obtained by ligating a tag to the N-terminal and/or C-terminal of the protein of A1);

a3 Protein obtained by substitution and/or deletion of one or more amino acid residues and/or addition of A1) and having plant high photoresponsive activity regulating function.

2. Use of a biological material associated with the protein of claim 1 for modulating plant high light response, said biological material being any one of the following B1) to B5):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A nucleic acid molecule which reduces the expression of the protein of claim 1.

3. The use according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) or b 14) as follows:

b11 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 2 in a sequence table;

b12 A cDNA molecule or a DNA molecule shown in a sequence 2 in the sequence table;

b13 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 11) or b 12) or b 13) and which codes for a protein according to claim 1.

4. Use according to any one of claims 1 to 3, wherein the modulation of plant high light response is such that the plant strain in which the protein expression is reduced is less tolerant to high light than the plant strain in which the protein expression is normally expressed.

5. The use according to claim 4, wherein the high light is high PAR light.

6. A method of growing a light-sensitive plant, comprising the step of expressing and/or silencing the expression of a protein according to claim 1 in a plant of interest, and/or the coding gene according to claim 2.

7. A method for growing a plant with high light tolerance, comprising the step of overexpressing the protein of claim 1 in a plant of interest.

8. The method according to claim 6 or 7, wherein the light sensitivity is to high PAR light; the high light resistance means high PAR resistance.

9. Use of the protein of claim 1 or the biomaterial of claim 2 or 3 in any of the following:

1) Preparing a product for cultivating plants with high light tolerance;

2) Cultivating plants with strong high light tolerance;

3) Preparing a product for cultivating plants with high light tolerance;

4) Cultivating plants with weak high light tolerance;

5) Preparing a product for cultivating plants with increased chlorophyll content;

6) Cultivating a plant having an increased chlorophyll content;

7) Preparing a product for cultivating plants with increased photosynthetic capacity;

8) Cultivating plants with increased photosynthetic capacity;

9) Preparing a product for cultivating plants with increased pest resistance;

10 Plants with increased resistance to pests are grown.