CN113501868A - Application of SPL7 in regulation and control of plant drought resistance - Google Patents

Abstract

The invention discloses application of SPL7 in regulation and control of plant drought resistance. The invention discovers that the SPL7 gene knockout or inactivation can delay the growth of plants, but can improve the drought resistance of the plants. The research shows that SPL7 inhibits transcription of NCED3 and AAO3 in ABA biosynthesis pathway by recognizing GTAC motif on promoter, thereby reducing ABA accumulation and balancing growth and stress resistance of plants. Namely, a potential conservative mechanism exists in terrestrial plants, and the new biosynthesis of ABA is regulated through SPL7-GTAC, so that the growth and stress response of the plants are balanced, and a method is provided for manually regulating the drought resistance of the plants.

Description

Application of SPL7 in regulation and control of plant drought resistance

Technical Field

The invention relates to plant genetic engineering, in particular to a regulatory factor for enhancing the drought resistance of a plant and potential application thereof in breeding new varieties of transgenic plants.

Background

Plants are often exposed to harsh environments because they cannot move on their own. Plants have evolved a complex set of mechanisms to regulate their own metabolism and growth to suit the environment. Drought, changes in soil salinity, and cold all cause water resource deficiency, and are main factors limiting plant habitation and agricultural development. In view of its importance for plant science and agricultural development, extensive research has been conducted over the past decades on how plants balance their own growth and survival. Previous studies have shown that the physiological and molecular responses to water deficiency are mainly coordinated by the classical plant hormone abscisic acid (ABA). Under the severe conditions of drought, salt stress and the like, the ABA content in the plant body is increased, and ABA induces the oscillation of cytoplasmic calcium signals of guard cells, so that stomata closing is promoted, and water loss is reduced. ABA causes drastic changes in the transcriptome level, leading to alterations in the metabolism and cellular processes to adapt to drought tolerance. Furthermore, the plant growth inhibiting effect of ABA has been known for a long time, but the mechanism thereof is not yet clear.

The endogenous ABA content of plants is regulated by complex homeostatic mechanisms including biosynthesis, catabolism, reversible glycosylation and long-range transport. De novo synthesis of ABA proceeds through the carotenoid pathway, which involves a series of enzyme-catalyzed reactions, starting in the plastid and ending in the cytoplasm. The first oxygen-containing carotenoid, zeaxanthin, is converted to violaxanthin in arabidopsis by an ABA 1-encoded zeaxanthin epoxidase. 9-cis epoxidized carotenoid dioxygenases (NCEDs) cleave the cis isomers of violaxanthin and neoxanthin to generate the first cytoplasmic ABA precursor flavonal, a limiting step in the biosynthesis of ABA. The NCED gene belongs to a multigene family, and NCED3 in Arabidopsis is the major stress-induced subtype in leaves. The final step of ABA synthesis is to catalyze the oxidation of abscisic aldehyde by Abscisic Aldehyde Oxidase (AAO) to generate ABA. AAO3 is considered to be the only AAO gene involved in ABA synthesis in arabidopsis thaliana. In arabidopsis, ABA catabolism proceeds through cytochrome P450707A family member-mediated hydroxylation and glycosyltransferase-mediated glucose binding. The inactive glucose-binding form of ABA can be hydrolyzed by β -glucosidase, supplementing ABA in the cytoplasm. Although earlier studies have largely elucidated the molecular framework of ABA homeostasis, it is unclear how ABA homeostasis is coordinated with other important physiological processes during plant growth and development.

Copper (Cu) is a trace transition metal in plants and acts as a cofactor for many important copper proteins with electron transfer and redox reaction functions. In higher plants, Cu is essential for growth, because the copper protocyanin, which requires Cu as a cofactor, is abundantly present in the thylakoid space and is an indispensable electron carrier in photosynthesis Z-scheme. There have been several studies suggesting that Cu homeostasis may be involved in stress responses. First, Cu is involved in the synthesis or perception of hormones such as ethylene and ABA. Secondly, Cu participates in the formation of structures with protective functions, such as lignification secondary cell walls, Kjeldahl belts, seed coats and the like. Third, the expression of many copper homeostatic genes is influenced by the environment. Thus, copper homeostasis is involved in balancing plant growth and stress response, but its underlying molecular mechanisms remain to be further elucidated.

SPL7 and its homologous genes are highly conserved copper homeostatic regulators in green plants with a unique Squamosa promoter binding protein (SBP) domain comprising a nuclear localization signal sequence and a zinc finger domain that has been shown to bind to GTAC-containing DNA motifs. The plant is in a copper-deficient environment, SPL7 is activated, on one hand, expression of a COPT gene family is up-regulated, absorption of copper ions is promoted, and on the other hand, expression of Cu-miRNA is up-regulated, and the Cu-miRNA targets Cu/Zn superoxide dismutase (CSDs), laccase and other copper blue proteins, degrades and inhibits expression of the unnecessary copper proteins, and promotes Cu to be transported to a path necessary for growth such as photosynthesis. In addition, SPL7 interacts with other transcription regulatory factors, such as ELONGATED HYPOCOTYL 5(HY5), participates in light signal transduction, and interacts with Cu-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1(CITF1), and promotes jasmonic acid accumulation during reproductive development. These studies indicate that SPL 7-mediated copper homeostasis is involved in a series of vital biological processes for growth development and environmental response.

Disclosure of Invention

The invention aims to research the function of the plant SPL7, focuses on the regulation and control of the plant SPL7 on balanced plant growth and drought response, provides an effective method for directionally creating drought-resistant plant varieties, and aims to provide a certain thought for solving the drought problem.

The research shows that the SPL7 gene knockout or inactivation can delay the growth of plants but can improve the drought resistance of the plants. This study focused on the resolution of the molecular mechanisms behind it. Subsequent studies found that SPL7 inhibits transcription of NCED3 and AAO3 in ABA biosynthetic pathway by recognizing GTAC motif on promoter, thereby reducing ABA accumulation and balancing plant growth and stress tolerance. This study shows that there is a potential conserved mechanism in terrestrial plants to balance plant growth and stress response by modulating the new biosynthesis of ABA via SPL 7-GTAC.

In the first aspect of the invention, the protein for regulating and controlling the drought resistance of the plant is provided, is a highly conserved copper steady-state regulation factor SPL7 in the plant, and has an amino acid sequence shown as SEQ ID No: 1 or a homologous protein thereof.

SEQ ID No: 1 shows the amino acid sequence of Arabidopsis SPL7, which consists of 818 amino acid residues, and the SBP structural domain at the 136-215 amino acid residues.

The expression level and/or activity of SPL7 can affect the drought resistance of plants, wherein, the drought resistance of plants can be improved by knocking out or inactivating SPL7 gene.

The genome sequence of the coding arabidopsis SPL7 is shown as SEQ ID No: 2, and the coding sequence (CDS) is shown as SEQ ID No: 3, respectively.

In a second aspect of the present invention, a method for improving drought resistance of a plant is provided, wherein a plant with improved drought resistance is obtained by knocking out or mutating and inactivating the SPL7 gene of the plant.

The SPL7 gene of a plant can be knocked out by adopting a gene editing technology, for example, the gene knocking out is carried out by utilizing a CRISPR-Cas9 system, and the method comprises the following steps:

1) selecting a coding region sequence of an SPL7 gene and a flanking sequence thereof as a template to design sgRNA required by CRISPR-Cas9 system knockout;

2) inserting the sgRNA target sequence into a Cas9 vector to construct an expression vector knocked out by an SPL7 gene;

3) introducing the expression vector constructed in the step 2) into plant cells, tissues or organs, and culturing the transformed plant cells, tissues or organs into plants to obtain transgenic plants;

4) and screening and identifying the offspring of the transgenic plant to obtain a homozygous strain with the knocked-out SPL7 gene.

The sgrnas designed in step 1) above include sgRNA1 and sgRNA2, and target sequences of the sgrnas are respectively as set forth in SEQ ID nos: 4 and 5.

The Cas9 vector in the step 2) is preferably a binary expression vector which can be used for agrobacterium transformed plants, such as a pCAMBIA1300-35S-Cas9 vector, and the SPL7 gene knockout binary expression vector pCAMBIA1300-AtU6-26-sgRNA1-AtU6-26-sgRNA2-35S-Cas9 is obtained by inserting target sequences of sgRNA1 and sgRNA 2; in step 3), the constructed SPL7 gene knockout binary expression vector is transferred into agrobacterium, and then plant floral organs are transfected by the agrobacterium to obtain a transgenic plant.

Methods for mutational inactivation of plant SPL7 genes include, but are not limited to, T-DNA insertion mutations, for example, insertion into the sixth exon of the Arabidopsis thaliana SPL7 gene using the T-DNA vector pBIN-ROK2, resulting in inactivation of the SPL7 protein by failure to properly encode, resulting in Arabidopsis thaliana T-DNA insertion mutant SPL 7-1. The detection by real-time fluorescent quantitative PCR (qRT-PCR) showed that the expression level of SPL7 could not be detected in the SPL7-1 mutant (see C in FIG. 1).

Expression vectors, strains and transgenic plant lines with sequence knockout of the coding region of the SPL7 gene are all within the scope of the present invention.

In the embodiment of the invention, 200bp of the coding region sequence of an arabidopsis thaliana SPL7 gene and the left and right of a flanking sequence thereof are selected as templates to design sgRNA required by CRISPR-Cas9 technical knockout, the sgRNA is inserted into a pCAMBIA1300-35S-Cas9 vector, a binary expression vector pCAMBIA1300-AtU6-26-sgRNA1-AtU6-26-sgRNA2-35S-Cas9 knocked out of the SPL7 gene is constructed, the vector is transferred into agrobacterium GV3101, then an arabidopsis thaliana floral organ with a col-0 background is infected by a soaking method, a transgenic plant is obtained, and a homozygous arabidopsis thaliana strain (SPL7-ko) with a SPL7 gene coding region completely knocked out is screened and identified by progeny.

Furthermore, the self promoter and the protein coding sequence (CDS) of the SPL7 gene are selected to be inserted into a pCAMBIA1381 vector, and a binary expression vector pCAMBIA1381-SPL7pro-SPL7 complemented by the SPL7 gene is constructed. Plasmid pCAMBIA1381-SPL7pro-SPL7 is transferred into agrobacterium GV3101, then an arabidopsis flower organ with SPL7-ko background is infected by a soaking method to obtain a transgenic plant, and progeny is screened and identified to obtain an arabidopsis thaliana strain (pSPL7: SPL7/SPL7-ko) supplemented with SPL7 gene.

The real-time quantitative RT-PCR method is used for detecting the expression quantity of SPL7 in SPL7-ko Arabidopsis thaliana, and the result shows that the expression quantity of SPL7 is hardly detected, while the expression quantity of SPL7 in the anaplerotic material SPL7pro, SPL7/SPL7-ko Arabidopsis thaliana is restored to the wild type level (figure 1), which indicates that the invention successfully obtains an Arabidopsis thaliana plant with SPL7 completely knocked out by the criprpr-cas 9 technology, and successfully obtains the SPL7 anaplerotic material by the transgenic technology.

Firstly, the growth conditions of the SPL7 knockout and complementation Arabidopsis lines obtained by the invention are analyzed, and the results show that the phenotype is consistent with the reported SPL7-1 mutant, and the SPL7-ko plants grow weakly, wherein the length of a main root when the plant grows for 5 days, the area and fresh weight of a leaf when the plant grows for 7 days, and the area and fresh weight of the leaf when the plant grows for 1 month are all obviously lower than that of a wild type. It can be seen that SPL7 affected vegetative growth of plants (fig. 2).

Furthermore, the invention carries out drought treatment on related plant materials, and determines drought parameters in the four aspects of survival rate, water loss rate of single leaf, leaf temperature and electric conductivity. Wherein the leaf temperature represents the transpiration of the plant, and the lower the leaf temperature, the stronger the transpiration and the higher the water loss rate. The conductivity represents the cellular integrity of the plant, and the higher the conductivity, the lower the integrity of the cell membrane, and the higher the damage of the cell membrane after the drought stress of the plant. The results show that after 21 days of drought treatment, spl7-1 and spl7-ko plant material have significantly increased survival rates relative to wild type (about 98%, figure 3). And the water loss rate of individual leaves and leaf temperature parameter measurements showed that the water loss rate of spl7-1 and spl7-ko plant materials was much lower than that of wild type (FIG. 4). It can be seen that SPL7 has a significant impact on the drought resistance of plants.

Furthermore, the expression quantity and ABA content of the ABA synthetic gene in the wild type and the spl7-ko mutant are detected, and the result shows that the ABA synthetic gene in the spl7-ko mutant is obviously reduced and the ABA content is obviously reduced compared with the wild type (figure 5). This demonstrates that SPL7 can down-regulate key genes ABA1, NCED3, and AAO3 in the ABA synthesis pathway, thereby reducing ABA synthesis and balancing plant growth and drought resistance.

In addition, the present invention performed alignment analysis of the sequence of the SBP domain of the SPL7 homologous protein in different species, and the results showed that the SBP domain in the SPL7 protein is highly conserved in terrestrial plants (including dicotyledonous plants, monocotyledonous plants, and early landing bryophytes) (fig. 6). In view of the application value of the gene and the huge application prospect of the utilization potential of the gene, the gene should be protected by a patent.

Drawings

FIG. 1 shows the construction process and results of SPL7 knockout plants in the examples, wherein: panel A is a schematic structural representation of the coding region of SPL7 with triangles indicated as insertion sites for the SPL7-1 mutant; the sequence diagrams of the SPL7 gene in wild type WT and knockout mutant SPL7-ko plants are arranged below; b is an electrophoretogram of the identification result of the spl7-ko plant, and asterisks respectively correspond to PCR bands corresponding to WT and spl 7-ko; c is the expression quantity of SPL7 in different gene type plants; d is the growth of seedlings of different genotypes (7 days).

Fig. 2 shows that SPL7 affects vegetative growth of plants, wherein: a is a seedling of a 5-day old different genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL 7-ko); b is different genotype seedlings growing for 7 days; c is different genotype seedlings growing for 11 days; d is the statistical analysis of the leaf area (7 days), fresh weight (7 days) and root length (5 days) of seedlings with different genotypes; e is the growth condition of different 1 month old plants with different genotypes; f is a statistical analysis of leaf area and fresh weight of 1 month old plants of different genotypes.

Fig. 3 shows that knockout SPL7 enhances drought resistance in plants, wherein: a is the growth conditions of nodes of different genotypes in three time periods of normal growth for 14 days, drought treatment for 21 days and rehydration for 3 days; b is the survival rate statistical analysis of different genotypes after the normal growth for 14 days, the drought treatment for 21 days and the rehydration for 3 days; c is the expression mode of drought response genes in plants with different genotypes.

Fig. 4 shows that SPL7 knock-out reduces blade water loss rate, where: a is phenotype before and after dehydration of single leaves of different genotypes; b is a schematic diagram of leaf temperature of different genotypes; c is the dehydration rate of single leaf with different genotypes; d is the statistical analysis of leaf temperature of different genotypes.

Figure 5 shows that the SPL7 knockout increased ABA content in plants, where: a is ABA anabolic pathway; b is the expression pattern of ABA1, NCED3 and AAO3 in Wild Type (WT) and spl7-ko mutant; c is ABA content in wild type and spl7-ko mutant after drought treatment.

FIG. 6 shows the conservation of the SBP domain of SPL7 in terrestrial plants, and a representative 25 terrestrial plants were selected, and the sequence of the SBP domain of the SPL7 homologous protein is shown for each plant, wherein, represents the key amino acids (C: cysteine; H: histidine) of the zinc finger structure in the SBP domain.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The consumable laboratory reagents used in the following examples were purchased from conventional biochemical reagents companies, unless otherwise specified.

Plant material: arabidopsis thaliana (Col-0).

1. Acquisition of transgenic Arabidopsis with SPL7 Gene knockout

1.1SPL7 knock-out target design

The protein coding sequence (shown as SEQ ID No: 2 in a sequence table) of Arabidopsis SPL7 is obtained according to NCBI database (https:// pubmed. NCBI. nlm. nih. gov) information, and a CRISPR-GE website (http:// skl. scau. edu. cn) is utilized to design a target spot and a vector construction primer sequence on line. The sgRNA required for technical knockout of criprpr-cas 9 is designed by selecting 200bp of the SPL7 coding region sequence and the left and right flanking sequences thereof as templates, and the target sequences and primer sequences thereof are shown in Table 1.

TABLE 1SPL7 Gene knockout target sequences and target primer sequences

1.2 target primer annealing

Dissolving target primers into 10 μ M mother solution with water, adding 10 μ L of each mother solution into 80 μ L of 0.5 × TE (pH 8.0) to obtain a final concentration of 1 μ M; the water can be heated to boiling, the power supply of the heater is turned off, the EP tube containing the target point primer is put into hot water, taken out after 30s, and naturally cooled to room temperature. In addition, the PCR tube can be heated in a PCR instrument at 98 ℃ for 3min, and then immediately taken out, and naturally cooled to room temperature for connection.

1.3 construction of sgRNA cassette

1.3.1BsaI enzyme digestion AtU6-26-sgRNA-SK plant editing plasmid

Mu.g of AtU6-26-sgRNA-SK plasmid is taken, enzyme digestion is carried out for 60min at 37 ℃ by 10U Bsa I-HF in 50 mu L reaction, and the plasmid is subpackaged after being purified by a DNA purification kit and is frozen for storage (can be repeatedly used).

1.3.2 target ligation

The ligation was carried out at room temperature (20-25 ℃ C.) for 30 minutes.

1.3.3 transformation and identification of Positive clones

10 μ L of the ligation product was transformed into E.coli XL1-Blue or DH5 α, coated with Amp⁺And (3) selecting a single clone to perform colony PCR identification and sequencing after 10-12h of an LB plate, and confirming whether a target point is connected with a vector.

Primers for colony PCR identification:

forward primer SK-gRNA-F: 5'-CTCACTATAGGGCGAATTGG-3' (SEQ ID No: 10);

reverse primer: see table 1 for reverse primers for targets.

The PCR fragment was 499bp in length.

Carrying out amplification culture on correct clones, extracting plasmids, carrying out double enzyme digestion by Nhe I and Spe I, carrying out electrophoresis, then tapping and recovering fragments with the size of about 642bp, and recovering sgRNA1 cassette and sgRNA2 cassette.

1.4 binary vector construction

1.4.1SpeI digestion of pCAMBIA1300-35S Cas9 plasmid

Taking 1-2 mu g of pCAMBIA1300-35S Cas9 plasmid, carrying out enzyme digestion for 10-30min at 37 ℃ by using 10U Spe I in a 50 mu L reaction, inactivating for 20min at 80 ℃, adding 0.2 mu L of alkaline phosphatase CIAP to prevent the self-ligation of the vector, carrying out dephosphorylation treatment on the vector at 37 ℃ for 10min, carrying out electrophoresis gel cutting, recovering, subpackaging and freezing for storage (the enzyme digestion product can be recycled).

1.4.2SgRNA cassette ligation reactions

The ligation was carried out at room temperature (20-25 ℃ C.) for 30 minutes.

1.4.3 transformation and identification of Positive clones

10 μ L of the ligation product was transformed into E.coli DH5 α, coated with Kan⁺And (3) selecting a single clone after 14-16h on an LB plate, carrying out colony PCR identification by using a primer sequence on a binary vector, selecting a plasmid (named as pCAMBIA1300-AtU6-26-sgRNA1-35S-Cas9) with correct sequencing, carrying out SpeI enzyme digestion, carrying out a ligation reaction with sgRNA2 cassette, and transforming and sequencing the plasmid (named as pCAMBIA1300-AtU6-26-sgRNA1-AtU6-26-sgRNA2-35S-Cas9) with correct identification into agrobacterium.

Primers identified by colony PCR are positioned at two ends of the Spe I enzyme cutting site, and the sequences of the primers are as follows:

1300-gRNA-F：5’-CCAGTCACGACGTTGTAAAAC-3’(SEQ ID No：11)

1300-gRNA-R：5’-CAATGAATTTCCCATCGTCGAG-3’(SEQ ID No：12)

the length of the PCR fragment after the successful ligation of a single sgNRA cassette is about 750bp, and the length of the cloned PCR fragment without the successful ligation is only about 100 bp.

1.5 Gene transformation of SPL7 knock-out Material

Transferring a correctly sequenced plasmid pCAMBIA1300-AtU6-26-sgRNA1-AtU6-26-sgRNA2-35S-Cas9 into agrobacterium GV3101, soaking an arabidopsis flower organ with a col-0 background in an agrobacterium solution to obtain a transgenic T0 generation plant, screening the resistance of descendant Hygromycin to obtain a T1 generation positive plant, performing PCR (polymerase chain reaction) identification and breeding to a T4 generation SPL7 gene knockout plant (marked as SPL7-ko), screening transgenic arabidopsis thaliana strains which are respectively marked as #21, #39 and #47 and have three SPL7 gene coding regions of 4185bp and are completely knocked out, and referring to B, C and D in figure 1.

2. Acquisition of transgenic Arabidopsis thaliana complemented with SPL7 Gene

2.1CTAB method for extracting Arabidopsis thaliana genome DNA

Firstly, weighing about 200mg of arabidopsis thaliana leaf tissues in a 2mL EP tube, adding 5mm steel balls, freezing by using liquid nitrogen, and then putting the frozen arabidopsis thaliana leaf tissues in a vibration grinder to grind a sample; adding 200 μ L CTAB extraction buffer solution directly into EP tube, reversing and mixing; incubating the mixed sample at 65 ℃ for 15 minutes; adding chloroform with the same volume, and reversing and mixing uniformly; centrifuging at 12,000rpm for 10 minutes at room temperature, and taking the supernatant in a new tube; adding isopropanol with the same volume to the supernatant to precipitate DNA, reversing and mixing evenly, and centrifuging for 10 minutes at room temperature at 12,000 rpm; discarding the supernatant, adding 500. mu.L of 75% ethanol to wash the DNA, reversing and mixing uniformly, centrifuging at 12,000rpm for 10 minutes at room temperature, and discarding the supernatant; after the DNA is air-dried at room temperature, deionized water is added to dissolve the DNA.

2.2 extraction of Arabidopsis thaliana-based RNA by TRIZOL method

Firstly, weighing about 200mg of arabidopsis thaliana leaf tissues in a 2mL EP tube, adding 5mm steel balls, freezing by using liquid nitrogen, and then putting the frozen arabidopsis thaliana leaf tissues in a vibration grinder to grind a sample; adding 1mL of TRIZOL extraction buffer solution directly into an EP tube, reversing and mixing uniformly, and standing for 5 minutes at room temperature; adding chloroform with the same volume, and uniformly mixing for 20 seconds by vortex; centrifuging at 4 ℃ for 15 minutes at 12,000rpm, taking the supernatant in a new tube without RNase; adding isopropanol with the same volume into the supernatant to precipitate RNA, reversing and mixing uniformly, standing for 10 minutes at room temperature, and centrifuging at 4 ℃ for 15min at 12,000 rpm; discarding the supernatant, adding 500 μ L75% ethanol to wash the precipitate, reversing and mixing, centrifuging at 12,000rpm for 1min at room temperature, and discarding the supernatant; after standing at room temperature for 5 minutes, RNase-free deionized water was added to dissolve RNA.

2.3 Total RNA reverse transcription

RNA was reverse transcribed using the FastKing cDNA first strand synthesis Kit (cat # KR113(FastKing RT Kit); TIANGEN Co.) in the following reaction system 1:

after thorough mixing, the mixture was centrifuged briefly and incubated at 42 ℃ for 3 min. Immediately placed on ice to carry out the second reaction, and the reaction system 2 is as follows:

after mixing well, incubation was carried out at 42 ℃ for 60 min. Incubate immediately at 95 ℃ for 3min, followed by storage on ice.

2.4 amplification of Arabidopsis SPL7 Gene promoter and coding sequence fragment

The promoter (1696bp, see SEQ ID No: 13 in the sequence Listing) and the coding sequence (CDS) (2475bp, see SEQ ID No: 3 in the sequence Listing) of Arabidopsis SPL7 were obtained according to the NCBI database (https:// pubmed. NCBI. nlm. nih. gov) information, and primers were designed according to the gene sequence, see Table 2.

TABLE 2 amplification primers for the SPL7 promoter and coding sequence

The promoter and CDS gene fragments of arabidopsis thaliana SPL7 were subsequently amplified using KOD FX high fidelity DNA polymerase in the following reaction system:

the PCR reaction condition is pre-denaturation at 94 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 3min, and 30 cycles; extending for 10min at 68 ℃; pause at 4 ℃. And after the reaction is finished, carrying out DNA agarose gel electrophoresis to detect the size and specificity of the PCR product band.

2.5 enzymatic cleavage of binary vector pCAMBIA1381

The pCAMBIA1381 plasmid was cleaved using a restriction enzyme (Thermofeisher Co.) double-restriction enzyme system as follows:

the above digestion reaction was incubated at 37 ℃ for 3 h. The digested plasmid was recovered using a gel recovery kit (TIANGEN).

2.6Gibson ligation of binary vector pCAMBIA1381 and SPL7 promoter fragments

The SPL7 promoter fragment and pCAMBIA1381 plasmid were ligated using Gibson homologous recombinase as follows:

the ligation reaction system is incubated at 50 ℃ for 30min, and the ligation product is named pCAM1381-SPL7 pro.

2.7 Escherichia coli transformation of pCAM1381-SPL7pro, identification of Positive clones

Taking out Escherichia coli DH5 alpha competent cells from a refrigerator at-80 ℃, placing on ice until the cells are melted, adding the ligation product pCAM1381-SPL7pro into the competent cells, gently mixing uniformly, and placing on ice for 30 min; heat shock is carried out for 90s in a metal bath at 42 ℃; immediately placed on ice for 3 min. 600. mu.L of non-resistant LB medium was added to the culture broth and the mixture was thawed at 37 ℃ and 220rpm for 35 min. Centrifuging at 8000rpm for 1min, taking 100 μ L of supernatant, resuspending the precipitate, uniformly smearing on an LB plate containing 50mg/mL kanamycin, and culturing in an incubator at 37 ℃ for 12-18 h.

And selecting a single colony, carrying out colony PCR to identify positive clones, sequencing, and culturing the colony which is successfully sequenced overnight to extract plasmids for the next step.

2.8 enzymatic cleavage of binary vector pCAMBIA1381-SPL7pro

The pCAMBIA1381-SPL7pro plasmid was cleaved using a restriction enzyme (Thermofeisher Co.) double restriction enzyme system as follows:

the above digestion reaction was incubated at 37 ℃ for 3 h. The digested plasmid was recovered using a gel recovery kit (TIANGEN).

2.9Gibson ligation of binary vector pCAMBIA1381-SPL7pro and fragment of the coding sequence of SPL7

The fragments of the coding sequences pCAMBIA1381-SPL7pro and SPL7 were ligated using Gibson homologous recombinase as follows:

the ligation reaction system is incubated at 50 ℃ for 30min, and the ligation product is named as pCAM1381-SPL7pro and SPL 7.

2.10 transformation of Escherichia coli with pCAM1381-SPL7pro: SPL7, identification of Positive clones

Taking out Escherichia coli DH5 alpha competent cells from a refrigerator at-80 ℃, putting on ice to be melted, adding ligation products pCAM1381-SPL7pro: SPL7 into the competent cells, gently mixing uniformly, and putting on ice for 30 min; heat shock is carried out for 90s in a metal bath at 42 ℃; immediately placed on ice for 3 min. 600. mu.L of non-resistant LB medium was added to the culture broth and the mixture was thawed at 37 ℃ and 220rpm for 35 min. Centrifuging at 8000rpm for 1min, taking 100 μ L of supernatant, resuspending the precipitate, uniformly smearing on an LB plate containing 50mg/mL kanamycin, and culturing in an incubator at 37 ℃ for 12-18 h.

Single colonies are picked for colony PCR to identify positive clones, then sequencing is carried out, the colonies which are successfully sequenced are cultured overnight, and plasmids (pCAM1381-SPL7pro: SPL7) are extracted for later use.

2.11 Gene transformation of the SPL7 complementation Material (SPL7pro: SPL7/SPL7)

Transferring a plasmid SPL7pro with correct sequencing, SPL7 into agrobacterium GV3101, soaking an arabidopsis flower organ with a SPL7-ko background in an agrobacterium solution to obtain a transgenic T0 generation plant, screening resistance of descendant Hygromycin to obtain a T1 generation positive plant, carrying out PCR identification, breeding to a T3 generation SPL7 anaplerosis material (marked as pSPL7: SPL7/SPL7-ko), and screening transgenic arabidopsis thaliana strains marked as #1, #6 and #7, complemented by three SPL7 genes, wherein C and D are shown in figure 1.

3. Expression level detection of SPL7 and ABA synthetic genes ABA1, NCED3 and AAO3 in SPL7 gene knockout and gene complementation transgenic Arabidopsis thaliana

3.1 Total RNA extraction (see details 2.2)

3.2 Total RNA reverse transcription (see details 2.3)

3.3 real-time quantitative RT-PCR

Using the above-mentioned inverted mRNA first strand cDNA as a template, the expression levels of SPL7 gene, ABA1 gene, NCED3 gene, AAO3 gene and reference gene ACTIN were determined using SuperReal Premix Plus fluorescent quantitative detection kit (SYBR Green) (TIANGEN, cat # FP205), and the primer sequences used are shown in Table 3.

TABLE 3 Gene detection primer sequences

The reaction system is as follows:

the PCR system was amplified using a QuantStaudio 3Real-Time PCR amplification Apparatus (ABI) under the following conditions: 95 ℃ for 15 min; 95 ℃ for 10 s; 30s at 60 ℃; 72 ℃ for 30 s; 40 cycles; the reaction is finally followed by a melting curve determination step. The results of the experiment were analyzed using QuantStaudio 3Real-Time PCR software (ABI).

4. Parameter determination of growth phenotype and drought phenotype of SPL7 gene knockout and gene complementation transgenic Arabidopsis material

4.1 measurement of seedling growth phenotype parameters (FIG. 2)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on MS medium and cultured in a Percival plant incubator (CU-36L5) (light cycle: 16h light/8 h dark; temperature cycle: 22 ℃/20 ℃), and leaf area, root length and fresh weight of the seedlings were recorded after 5 days, 7 days and 11 days, respectively, as shown in A to D in FIG. 2.

4.2 measurement of phenotypic parameters of seedling growth (FIG. 2)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on an MS culture medium, cultured in a Percival plant incubator (CU-36L5) (light period: 16h light/8 h dark; temperature period: 22 ℃/20 ℃), and transplanted into small pots filled with vermiculite mixed soil (vermiculite: 1, mass ratio) (5 seedlings were placed in each small pot) after 2 weeks. Culturing in Percival plant incubator (E-36L) (light period: 10 hr light/14 hr dark; temperature period: 22 deg.C/20 deg.C; humidity: 50%). After 2 weeks the leaf area, root length and fresh weight of one month old plants were recorded, respectively, as shown in fig. 2, E and F.

4.3 drought resistance assay (FIG. 3)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on an MS culture medium, cultured in a Percival plant incubator (CU-36L5) (light period: 16h light/8 h dark; temperature period: 22 ℃/20 ℃), and transplanted into small pots filled with vermiculite mixed soil (vermiculite: 1, mass ratio) (9 seedlings were placed in each small pot) after 2 weeks. Culturing in Percival plant incubator (E-36L) (light period: 10 hr light/14 hr dark; temperature period: 22 deg.C/20 deg.C; humidity: 50%). Watering was stopped after 2 weeks of normal culture, and the number of surviving plants (survival rate) was counted after 21 days, and plant phenotype was observed again after 3 days of returning watering, as shown in A and B in FIG. 3.

4.4 drought phenotype-determination of dehydration Rate of Individual leaves (FIG. 4)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on MS medium and cultured in a Percival plant incubator (CU-36L5) (light period: 16h light/8 h dark; temperature period: 22 ℃/20 ℃), 2 ℃ C.)After week, the seedlings were transplanted into small pots filled with vermiculite mixed soil (vermiculite: soil: 1, mass ratio), and 5 seedlings were placed in each small pot in a plant incubator. Culturing in Percival plant incubator (E-36L) (light period: 10 hr light/14 hr dark; temperature period: 22 deg.C/20 deg.C; humidity: 50%). Removing the rosette leaves of plants with different genotypes after 2 weeks, placing in a 60mm culture dish, weighing the weight of the leaves in the period of just separation (t0) and different time periods (t is 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300min) after separation, and calculating the water loss rate (t is 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300min) of each leaf₀-t/t₀). Three replicates were taken, each containing 8 leaves ex vivo per genotype, as shown in a and C in figure 4.

4.5 drought phenotype-leaf temperature determination (FIG. 4)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on an MS culture medium, cultured in a Percival plant incubator (CU-36L5) (light period: 16h light/8 h dark; temperature period: 22 ℃/20 ℃), transplanted into small pots filled with vermiculite mixed soil (vermiculite: soil: 1, mass ratio) after 2 weeks, and 9 seedlings were placed in each small pot in the plant incubator. Culturing in Percival plant incubator (E-36L) for 2 weeks (light cycle: 10 hr light/14 hr dark; temperature cycle: 22 deg.C/20 deg.C; humidity 70%). Subsequently, the plants were transferred to a low humidity environment (70%) for growth. The blade temperature was measured after 3 days using a far infrared camera (model: VarioCAM HD; brand: InfraTec), as shown in B and D in FIG. 4.

4.6 drought phenotype-determination of ABA content (FIG. 5)

Seeds of each genotype (WT, SPL7-1, SPL7-ko, pSPL7: SPL7/SPL7-ko) were spotted on an MS culture medium, cultured in a Percival plant incubator (CU-36L5) (light period: 16h light/8 h dark; temperature period: 22 ℃/20 ℃), and transplanted into small pots filled with vermiculite mixed soil (vermiculite: 1, mass ratio) (9 seedlings were placed in each small pot) after 2 weeks. Culturing in Percival plant incubator (E-36L) (light period: 10 hr light/14 hr dark; temperature period: 22 deg.C/20 deg.C; humidity: 50%). Watering was stopped after 2 weeks of normal culture, and ABA content in plants was measured after 15 days using UPLC-MS/MS (produced by wuhan green sword biology ltd.) as shown in fig. 5, B and C.

4.7 conservation analysis of the SBP Domain of the SPL7 protein (FIG. 6)

14 dicotyledonous plants (Arabidopsis thaliana: Arabidopsis thaliana; Arabidopsis thaliana: Arabidopsis thaliana; Brassica juncea: Brassica juncea; Brassica oleracea; Brassica rapana: Capsella; Capsella capsulata: Glycine max; Brassica vulgaris: Phaseolus vulgaris; Trifolius Trifolium: Trifolius pratense; Fragaria wild strawberry: Fragaria vesca; Prunus hirsutus: Cusica; Cusius sativus: savus; Solanum lycopersicum: Solomonicum; Solanum solanacearum: Solanum nigricans; Solanum solanacearum: Brassica purpurea; Brassica oleracea: Brassica juncea; Solanum solanacearum: Brassica brevis sp: Brassica juncea; Solanum brachyparia japonica: Brassica brevis: Brassica juncea; Solanum brachyparia strain: Brassica Brevictoria; Solanum brachyparia strain: Brassica Brevictoria; Brassica Brevicornyces: Brassica juncea; Solanum strain: Brassica brachyparia: Brassica sp: Brassica var italica; Solanum: Brassica strain: Brassica) and Brassica brachyparia strain (Brassica brachyparia) are used as a. Multiple sequence alignments were performed using the MUSCLE program in MEGA X software under default settings to analyze the conservation of the SPL7 homologous protein in 25 plants.

The alignment results are shown in fig. 6, which shows that the SBP domain in the SPL7 protein is highly conserved in terrestrial plants (including dicotyledonous plants, monocotyledonous plants, and early landing bryophytes).

SEQUENCE LISTING

<110> Beijing university

Application of <120> SPL7 in regulation and control of plant drought resistance

<130> WX2021-03-163

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 818

<212> PRT

<213> Arabidopsis thaliana

<400> 1

Met Ser Ser Leu Ser Gln Ser Pro Pro Pro Pro Glu Met Asp Ile Gln

1 5 10 15

Pro Pro Ala Leu Val Asn Asp Asp Pro Ser Thr Tyr Ser Ser Ala Leu

20 25 30

Trp Asp Trp Gly Asp Leu Leu Asp Phe Ala Ala Asp Glu Arg Leu Leu

35 40 45

Val Asp Gln Ile His Phe Pro Pro Val Leu Ser Pro Pro Leu Pro Pro

50 55 60

Leu Ile Pro Thr Gln Thr Pro Ala Glu Ser Glu Leu Asp Pro Ser Pro

65 70 75 80

Glu Glu Ser Gly Ser Gly Ser Asp Arg Val Arg Lys Arg Asp Pro Arg

85 90 95

Leu Ile Cys Ser Asn Phe Ile Glu Gly Met Leu Pro Cys Ser Cys Pro

100 105 110

Glu Leu Asp Gln Lys Leu Glu Asp Ala Glu Leu Pro Lys Lys Lys Arg

115 120 125

Val Arg Gly Gly Ser Gly Val Ala Arg Cys Gln Val Pro Asp Cys Glu

130 135 140

Ala Asp Ile Ser Glu Leu Lys Gly Tyr His Lys Arg His Arg Val Cys

145 150 155 160

Leu Arg Cys Ala Thr Ala Ser Phe Val Val Leu Asp Gly Glu Asn Lys

165 170 175

Arg Tyr Cys Gln Gln Cys Gly Lys Phe His Leu Leu Pro Asp Phe Asp

180 185 190

Glu Gly Lys Arg Ser Cys Arg Arg Lys Leu Glu Arg His Asn Asn Arg

195 200 205

Arg Lys Arg Lys Pro Val Asp Lys Gly Gly Val Ala Ala Glu Gln Gln

210 215 220

Gln Val Leu Ser Gln Asn Asp Asn Ser Val Ile Asp Val Glu Asp Gly

225 230 235 240

Lys Asp Ile Thr Cys Ser Ser Asp Gln Arg Ala Glu Glu Glu Pro Ser

245 250 255

Leu Ile Phe Glu Asp Arg His Ile Thr Thr Gln Gly Ser Val Pro Phe

260 265 270

Thr Arg Ser Ile Asn Ala Asp Asn Phe Val Ser Val Thr Gly Ser Gly

275 280 285

Glu Ala Gln Pro Asp Glu Gly Met Asn Asp Thr Lys Phe Glu Arg Ser

290 295 300

Pro Ser Asn Gly Asp Asn Lys Ser Ala Tyr Ser Thr Val Cys Pro Thr

305 310 315 320

Gly Arg Ile Ser Phe Lys Leu Tyr Asp Trp Asn Pro Ala Glu Phe Pro

325 330 335

Arg Arg Leu Arg His Gln Ile Phe Gln Trp Leu Ala Asn Met Pro Val

340 345 350

Glu Leu Glu Gly Tyr Ile Arg Pro Gly Cys Thr Ile Leu Thr Val Phe

355 360 365

Ile Ala Met Pro Glu Ile Met Trp Ala Lys Leu Ser Lys Asp Pro Val

370 375 380

Ala Tyr Leu Asp Glu Phe Ile Leu Lys Pro Gly Lys Met Leu Phe Gly

385 390 395 400

Arg Gly Ser Met Thr Val Tyr Leu Asn Asn Met Ile Phe Arg Leu Ile

405 410 415

Lys Gly Gly Thr Thr Leu Lys Arg Val Asp Val Lys Leu Glu Ser Pro

420 425 430

Lys Leu Gln Phe Val Tyr Pro Thr Cys Phe Glu Ala Gly Lys Pro Ile

435 440 445

Glu Leu Val Val Cys Gly Gln Asn Leu Leu Gln Pro Lys Cys Arg Phe

450 455 460

Leu Val Ser Phe Ser Gly Lys Tyr Leu Pro His Asn Tyr Ser Val Val

465 470 475 480

Pro Ala Pro Asp Gln Asp Gly Lys Arg Ser Cys Asn Asn Lys Phe Tyr

485 490 495

Lys Ile Asn Ile Val Asn Ser Asp Pro Ser Leu Phe Gly Pro Ala Phe

500 505 510

Val Glu Val Glu Asn Glu Ser Gly Leu Ser Asn Phe Ile Pro Leu Ile

515 520 525

Ile Gly Asp Ala Ala Val Cys Ser Glu Met Lys Leu Ile Glu Gln Lys

530 535 540

Phe Asn Ala Thr Leu Phe Pro Glu Gly Gln Glu Val Thr Ala Cys Ser

545 550 555 560

Ser Leu Thr Cys Cys Cys Arg Asp Phe Gly Glu Arg Gln Ser Thr Phe

565 570 575

Ser Gly Leu Leu Leu Asp Ile Ala Trp Ser Val Lys Val Pro Ser Ala

580 585 590

Glu Arg Thr Glu Gln Pro Val Asn Arg Cys Gln Ile Lys Arg Tyr Asn

595 600 605

Arg Val Leu Asn Tyr Leu Ile Gln Asn Asn Ser Ala Ser Ile Leu Gly

610 615 620

Asn Val Leu His Asn Leu Glu Thr Leu Val Lys Lys Met Glu Pro Asp

625 630 635 640

Ser Leu Val His Cys Thr Cys Asp Cys Asp Val Arg Leu Leu His Glu

645 650 655

Asn Met Asp Leu Ala Ser Asp Ile His Arg Lys His Gln Ser Pro Ile

660 665 670

Glu Ser Lys Val Asn Pro Pro Ser Ser Gly Cys Cys Cys Val Ser Ser

675 680 685

Gln Lys Asp Ile Pro Ser Arg Ile Leu Asn Phe Asn Lys Asp Pro Glu

690 695 700

Ala Gly Leu Asp Cys Lys Glu Arg Ile Gln Ala Asp Cys Ser Pro Asp

705 710 715 720

Ser Gly Gly Lys Glu Thr Asp Pro Leu Leu Asn Lys Glu Val Val Met

725 730 735

Asn Val Asn Asp Ile Gly Asp Trp Pro Arg Lys Ser Cys Ile Lys Thr

740 745 750

His Ser Ala Leu Ala Phe Arg Ser Arg Gln Thr Met Phe Leu Ile Ala

755 760 765

Thr Phe Ala Val Cys Phe Ala Val Cys Ala Val Leu Tyr His Pro Asn

770 775 780

Lys Val Thr Gln Leu Ala Val Ala Ile Arg Met Arg Leu Cys Ser Thr

785 790 795 800

Glu Leu Ser His Asn Ser Phe Ser Glu Ser Phe Pro Ser Glu Arg Asp

805 810 815

Arg Ile

<210> 2

<211> 4112

<212> DNA

<213> Arabidopsis thaliana

<400> 2

atgtcttctc tgtcgcaatc gccaccaccg ccggagatgg atatccaacc cccggcattg 60

gttaacgatg atccttccac ttattcctcc gctttatggg attggggaga tctccttgac 120

ttcgccgcag acgaacgcct tctcgtcgat caaattcatt tccctcccgt tctctctcct 180

cctctaccgc ctctgattcc gacgcaaact ccggcggaat ctgaattaga tccttctccg 240

gaagaatcgg gttctggttc ggatcgggtt aggaagcgag acccgaggtt gatttgttcc 300

aatttcattg aaggtatgct tccttgttcg tgtcctgagc ttgatcagaa attggaagac 360

gccgagcttc cgaagaagaa acgggttcgc ggcgggtcgg gcgtggctcg atgtcaggtt 420

ccggattgtg aagcggatat tagcgagctc aaagggtacc ataagaggca tagggtttgt 480

ctccgttgcg ctaccgccag ctttgttgtg cttgatggag agaataagag atactgtcaa 540

cagtgtggaa agtatgttac ttttcttggc tttgagaata agaattggat ttgataggtg 600

aatccagcgg gaactgtctt agttaacctc ctatgcatcc tcctgggatt tttgtgtgtg 660

tttgacttag gtgttgttca gatttgattt gcagcacaaa atgatcgaag atcgggatgg 720

gtttctgttg tctgtttgat tgggatagag actttgaatc tattgttaag aaattggata 780

actttgggag agactgttga tcagtttgaa tgatcccacg gaatttactt agtctctttg 840

ttgagcataa gctgagctta acttctgttt agcagtatta ttgtttgtat taattctttc 900

aatctatgct gttatccatc attatagtga gtagtactat ctggcaggtt tcatttgctc 960

ccggactttg atgaaggaaa acgcagctgt cggagaaagc tagagcgtca caacaacaga 1020

cggaaaagga aacctgtaga taaaggaggt gttgctgcag aacaacagca agtgctttca 1080

cagaatgata acagcgtcat tgatgttgag gatggaaaag gtataagtga actgcgcaaa 1140

tctcaaatca tatcaccatg tggtttcttt gttttagttg tttgctttgc atattttgct 1200

agttaccaac gtttagagtt ctcttggaaa ttcaagctga ttcgattgtc aatgctatca 1260

ttttgaagca gatatcacat gctctagtga ccagagagct gaagaagagc cttcattgat 1320

ttttgaagat cggcatatta ccactcaggg ttctgtacct tttacccgaa gcatcaacgc 1380

agacaacttt gtctctgtta caggttcggg tgaagctcaa ccagatgaag gaatgaatga 1440

cacaaaattt gaacgttcac cttctaatgg cgacaacaaa agtgcttact caactgtggt 1500

gagtttctct tcagcctcta acatcaaaaa catcggtgca cttaatgaac taatttccca 1560

tttcattgca gtgtccaact ggtcggatct cttttaagct ctacgattgg aatccagcgg 1620

agttcccacg gagactacgt catcaagtat tctcaagaat tatatattac agtttagtgt 1680

tcaaagacta tgtttacctt ctcagaatct caaagaacta atatcttgcg attaatttga 1740

atttgcagat atttcaatgg ttggccaaca tgcctgttga gctggagggc tatatccgcc 1800

caggatgtac aattttgact gtctttatag caatgccaga gattatgtgg gcgaaggtac 1860

atcctgttat cttttactca tcattttcat gttcatgatg ccgcgacaca acataagaaa 1920

ttgtttattt tcagtaggtt gtagtctcga gatatgctct atttctctat gcaccttacc 1980

ctcttaagta ccatatacac attaaagctc ttacgttttt gtgtgacctg aagttctttt 2040

gttactaaaa attgtccatt atttggaagc ttttgccctg gagggagaaa atgcaaactt 2100

agtatctctt cccgtgaact ctcttttgac tttgcactgt aataaacttt tccaatttgt 2160

tcctgtaaac agttatctaa agatcctgtg gcatatctgg atgaatttat tcttaaacct 2220

ggaaagatgc tgtttggaag aggctcgatg actgtctatt tgaacaacat gattttccgt 2280

cttattaaag gcaagttggt gtgctttact tgttttctct ggattttttc actctatcat 2340

attggataac cataatgtcg tgagctatta atgtgaacta atacttattt gtcttggttt 2400

tgacaggtgg aacaacttta aagagagtcg atgtaaaatt agagtcgccg aaacttcagt 2460

ttgtgtatcc tacatgtttt gaagctggaa aaccaattga actcgttgta tgtggacaaa 2520

accttctgca acctaaatgc cggtattaat gtcaaccctt tttttgagct atgcttcaga 2580

catctctcgc atttctagtt ctgtcttatt tctcatatga ggaagctgat attgcaagaa 2640

ctttcaaaat gttgcatttc gattactacc catcatttgt tggtttgatt tttatcactt 2700

gtgattacta aactgttcca tatacgaaag aaaaacatgc tgcttacaga taatggtttc 2760

attgtaggtt tctcgtgtct ttttctggga agtacttgcc acataactat tctgttgtac 2820

ctgcaccgga ccaggatggg aagcgttctt gtaataacaa gttctacaag atcaatattg 2880

tgaattctga ccctagtctc tttggccctg cctttgtcga ggtacccatt tgtttgatga 2940

tatacagttt agaagtagct catttcaata cgatcctcat ctaatccttt tgtaggttga 3000

aaatgaatct ggcctatcaa atttcatacc tctaattatt ggagatgcag ctgtttgttc 3060

cgaaatgaaa ctaatagagc agaagttcaa tgctacactc tttccagagg gacaagaagt 3120

tactgcttgc tcttctttga cttgctgttg cagggatttt ggggagagac agagcacctt 3180

tagtggcctc ttgttagata ttgcatggtc ggtgaaggta ccctctgcag aacgcactga 3240

gcaacctgtg aaccgatgtc agataaaaag atacaacaga gtgttgaatt atctgataca 3300

gaataactca gcgtcgatct taggaaacgt actgcacaat ttggaaactt tggtgaagaa 3360

aatggagcca gacagtcttg ttcactgtac ctgtgactgc gacgtgaggc ttctacatga 3420

aaatatggat ttggcgagtg atattcacag aaagcatcaa agccctatag agtcaaaggt 3480

gaatcctcct tcgtcaggtt gctgttgtgt gagtagtcag aaggacatac catcaagaat 3540

attaaacttc aataaggttt ttcactatta tctgacttcc cctattttcc tgtttgatct 3600

atactggact ctgcccacaa tctatacttg tcttgcacat ttggtttctt gctccactcc 3660

tgagtgctga acactaactt ttgtgtgttt atggtatatt tggcacattg tgacaatatt 3720

acaggagtct gcgcacaata ttgtgtctgt ttcttggttt atttgcatag tttctgttat 3780

ttaatgtttg ccatttatta tgtttacagg atcctgaagc aggattagat tgtaaagaga 3840

gaatacaggc agactgttca ccagatagcg gcggaaaaga gactgatcct ctgttaaaca 3900

aagaggttgt catgaacgta aatgacatag gagactggcc aaggaagtcg tgtataaaaa 3960

cgcactcagc tttagcattc aggtcccgtc aaactatgtt cttaatcgct acgttcgctg 4020

tctgttttgc tgtctgtgcg gttctctacc atccaaacaa ggtcacacag cttgcagtgg 4080

caatccgaat gagattggta cacaaaattt ga 4112

<210> 3

<211> 2457

<212> DNA

<213> Arabidopsis thaliana

<400> 3

atgtcttctc tgtcgcaatc gccaccaccg ccggagatgg atatccaacc cccggcattg 60

gttaacgatg atccttccac ttattcctcc gctttatggg attggggaga tctccttgac 120

ttcgccgcag acgaacgcct tctcgtcgat caaattcatt tccctcccgt tctctctcct 180

cctctaccgc ctctgattcc gacgcaaact ccggcggaat ctgaattaga tccttctccg 240

gaagaatcgg gttctggttc ggatcgggtt aggaagcgag acccgaggtt gatttgttcc 300

aatttcattg aaggtatgct tccttgttcg tgtcctgagc ttgatcagaa attggaagac 360

gccgagcttc cgaagaagaa acgggttcgc ggcgggtcgg gcgtggctcg atgtcaggtt 420

ccggattgtg aagcggatat tagcgagctc aaagggtacc ataagaggca tagggtttgt 480

ctccgttgcg ctaccgccag ctttgttgtg cttgatggag agaataagag atactgtcaa 540

cagtgtggaa agtttcattt gctcccggac tttgatgaag gaaaacgcag ctgtcggaga 600

aagctagagc gtcacaacaa cagacggaaa aggaaacctg tagataaagg aggtgttgct 660

gcagaacaac agcaagtgct ttcacagaat gataacagcg tcattgatgt tgaggatgga 720

aaagatatca catgctctag tgaccagaga gctgaagaag agccttcatt gatttttgaa 780

gatcggcata ttaccactca gggttctgta ccttttaccc gaagcatcaa cgcagacaac 840

tttgtctctg ttacaggttc gggtgaagct caaccagatg aaggaatgaa tgacacaaaa 900

tttgaacgtt caccttctaa tggcgacaac aaaagtgctt actcaactgt gtgtccaact 960

ggtcggatct cttttaagct ctacgattgg aatccagcgg agttcccacg gagactacgt 1020

catcaaatat ttcaatggtt ggccaacatg cctgttgagc tggagggcta tatccgccca 1080

ggatgtacaa ttttgactgt ctttatagca atgccagaga ttatgtgggc gaagttatct 1140

aaagatcctg tggcatatct ggatgaattt attcttaaac ctggaaagat gctgtttgga 1200

agaggctcga tgactgtcta tttgaacaac atgattttcc gtcttattaa aggtggaaca 1260

actttaaaga gagtcgatgt aaaattagag tcgccgaaac ttcagtttgt gtatcctaca 1320

tgttttgaag ctggaaaacc aattgaactc gttgtatgtg gacaaaacct tctgcaacct 1380

aaatgccggt ttctcgtgtc tttttctggg aagtacttgc cacataacta ttctgttgta 1440

cctgcaccgg accaggatgg gaagcgttct tgtaataaca agttctacaa gatcaatatt 1500

gtgaattctg accctagtct ctttggccct gcctttgtcg aggttgaaaa tgaatctggc 1560

ctatcaaatt tcatacctct aattattgga gatgcagctg tttgttccga aatgaaacta 1620

atagagcaga agttcaatgc tacactcttt ccagagggac aagaagttac tgcttgctct 1680

tctttgactt gctgttgcag ggattttggg gagagacaga gcacctttag tggcctcttg 1740

ttagatattg catggtcggt gaaggtaccc tctgcagaac gcactgagca acctgtgaac 1800

cgatgtcaga taaaaagata caacagagtg ttgaattatc tgatacagaa taactcagcg 1860

tcgatcttag gaaacgtact gcacaatttg gaaactttgg tgaagaaaat ggagccagac 1920

agtcttgttc actgtacctg tgactgcgac gtgaggcttc tacatgaaaa tatggatttg 1980

gcgagtgata ttcacagaaa gcatcaaagc cctatagagt caaaggtgaa tcctccttcg 2040

tcaggttgct gttgtgtgag tagtcagaag gacataccat caagaatatt aaacttcaat 2100

aaggatcctg aagcaggatt agattgtaaa gagagaatac aggcagactg ttcaccagat 2160

agcggcggaa aagagactga tcctctgtta aacaaagagg ttgtcatgaa cgtaaatgac 2220

ataggagact ggccaaggaa gtcgtgtata aaaacgcact cagctttagc attcaggtcc 2280

cgtcaaacta tgttcttaat cgctacgttc gctgtctgtt ttgctgtctg tgcggttctc 2340

taccatccaa acaaggtcac acagcttgca gtggcaatcc gaatgagatt gtgttcaaca 2400

gagctcagcc acaacagctt ctcagagagt ttcccttcag aaagagacag gatatga 2457

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence

<400> 4

ggatatccaa cccccggcat tgg 23

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence

<400> 5

gtttcccttc agaaagagac agg 23

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<400> 6

attggatatc caacccccgg cat 23

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

<400> 7

aaacatgccg ggggttggat atc 23

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

attgtttccc ttcagaaaga gac 23

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

aaacgtctct ttctgaaggg aaa 23

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

ctcactatag ggcgaattgg 20

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<400> 11

ccagtcacga cgttgtaaaa c 21

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<400> 12

caatgaattt cccatcgtcg ag 22

<210> 13

<211> 1696

<212> DNA

<213> Arabidopsis thaliana

<400> 13

tcggagaaag caagaaaatg ccttgtaatg caagattctt acgctgtaag ccctagttga 60

atgtgtatac ggacatcttt tatattattt tcttttgtca gttttgtgct tagaaagcgt 120

catcaagtct caattattat gcaatttcat cttattaaac ttttgtagcc tatgtggtta 180

tgtcgagacg tatttggagt aattcaatac attaagaata agctctcaat cgacaatcag 240

tagtttttat ctccaattga attacagaga tcgaactcgg gttcttaatc ttaccagacc 300

cttgtgtttt tcctcggttt catatgtggg ttaatgttta aattgttcat ggtgtcatca 360

tttatttttg ggcccggact ggcccatccg aaaagtgggc taatctccaa ttttcatagc 420

acatattctt aataccaatc attaaccatg tcatatttct gtctgcgaaa acaaaacaaa 480

ataaagcaca aaagggtata tagtaactta gtaagcacat ggtggatgag acacgtgtct 540

attttctggt ggtgaatggc ttggaatgag aagaccaaaa ttccaccacc gttattattg 600

gagaaaaaaa aaaagcgaga gagagagagc acttggggtc gctcacgtcc caagtaacac 660

tgaaccgacg tcgtattcga taatgattga tgaaccgttg atttcctcaa actcacgtag 720

cttacacgcg aacctccctt caaccaatca tgtgctgcca tcatttagcg cgtgtctctc 780

acgtcccgac acttcctcct acatagctca acacctcctc cttggccact ttttgtttct 840

tttttctttt gctaattata aagtagggac tcccaaatat aaacctatag gctaattata 900

taaccaagtg tcccaaatat tgtctcccaa aatttttttc tgtagttagg gtttttaatt 960

ctaagaattt tcttttgaat tctgtttatt atgcaacaaa agaacgtttc tatattacaa 1020

ttttttcaag caatctactt ctggttaatg taaaacataa atacaccttt ttggatatta 1080

tataacaaga tgtaataagt tgaaccatat gcaataacca ttccaaaacg agtcgattgt 1140

gctatcaatt atcaaactac ttttagttgt gttaagttgt catctgtcag aatatttcac 1200

atcgtaaatc aaaaaatgat tttaacattt tcattatgca aagataatgg atctttcagg 1260

atttttatca attaattttg aattaaaaca ttcttttaaa aaaaaaaaat agtattagtg 1320

ctatttgtga aatgatgaat tttcggattt ttgtcttttg ccgcaaagaa tttttgaatt 1380

atataagtta ggcaagaaaa aaaaagaata tgtgaataaa ctattaaagc acgattaact 1440

aatcatgatt gtgtttatta gttcaacgag ttctctgttg attgagtcat tccacagata 1500

aaaaaaaaat gtagactatt ttagataatt tattttattt agttccataa aataactcaa 1560

gaaataaaaa gttttgagat gtcaaaaaaa aaaagtaatg taaaaatgca ttattcggag 1620

atgttgatga ctttgtgcaa aattacctaa aaccttcaac caaaatcacc aacttcctca 1680

acaaccgatg gacctt 1696

<210> 14

<211> 41

<212> DNA

<213> Artificial sequence

<400> 14

gcgccgaatt cccggggatc ctcggagaaa gcaagaaaat g 41

<210> 15

<211> 40

<212> DNA

<213> Artificial sequence

<400> 15

tcctcttaaa gcttggctgc agaaggtcca tcggttgttg 40

<210> 16

<211> 40

<212> DNA

<213> Artificial sequence

<400> 16

catggtagat ctgactagta tgtcttctct gtcgcaatcg 40

<210> 17

<211> 42

<212> DNA

<213> Artificial sequence

<400> 17

gaaattcgag ctggtcacct catatcctgt ctctttctga ag 42

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

gagctggagg gctatatccg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence

<400> 19

acagtcatcg agcctcttcc 20

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<400> 20

gatgcagcca aatatgggtc aagg 24

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<400> 21

gccattgcat ggataatagc gactc 25

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

aggtcgcaag attcgggatt 20

<210> 23

<211> 22

<212> DNA

<213> Artificial sequence

<400> 23

gcggatttca gacaggacac tc 22

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence

<400> 24

tcgatgtgca ggtcaaagaa ttgc 24

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence

<400> 25

aacatcggat gaacctcgaa aaagc 25

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence

<400> 26

ggtgtcatgg ttggtatggg tc 22

<210> 27

<211> 23

<212> DNA

<213> Artificial sequence

<400> 27

cctctgtgag tagaactggg tgc 23

Claims (13)

1. The application of the SPL7 protein or the gene thereof in regulating and controlling the drought resistance of plants.
2. 2. The use of claim 1, wherein the SPL7 protein is a protein having an amino acid sequence as set forth in SEQ ID No: 1 or a homologous protein thereof.
3. 3. The use according to claim 1, wherein the drought resistance of a plant is modulated by modulating the expression level and/or activity of SPL7 protein.
4. 4. Use according to claim 3, wherein the drought resistance of a plant is increased by knocking out or inactivating the SPL7 gene of the plant.
5. 5. The use of claim 1, wherein the plant is a terrestrial plant, including a dicot, a monocot, and a bryophyte.
6. 6. A method for improving drought resistance of plants, which is to knock out SPL7 gene of plants or inactivate the mutation to obtain plants with improved drought resistance.
7. 7. The method of claim 6, wherein the SPL7 gene is knocked out in the plant by gene editing techniques.
8. 8. The method of claim 7, wherein the gene knockout is performed using the CRISPR-Cas9 system, comprising the steps of:

   1) selecting a coding region sequence of an SPL7 gene and a flanking sequence thereof as a template to design sgRNA required by CRISPR-Cas9 system knockout;

   2) inserting the sgRNA target sequence into a Cas9 vector to construct an expression vector knocked out by an SPL7 gene;

   3) introducing the expression vector constructed in the step 2) into plant cells, tissues or organs, and culturing the transformed plant cells, tissues or organs into plants to obtain transgenic plants;

   4) and screening and identifying the offspring of the transgenic plant to obtain a homozygous strain with the knocked-out SPL7 gene.
9. 9. The method of claim 8, wherein pairs of sgrnas are designed in step 1), including sgRNA1 and sgRNA2, and target sequences of the sgrnas are shown in SEQ ID nos: 4 and 5.
10. 10. The method of claim 8, wherein the Cas9 vector in step 2) is a binary expression vector that can be used for Agrobacterium transformation of plants, and the SPL7 gene knockout binary expression vector is obtained by inserting paired sgRNA target sequences; in step 3), the constructed SPL7 gene knockout binary expression vector is transferred into agrobacterium, and then plant floral organs are transfected by the agrobacterium to obtain a transgenic plant.
11. 11. The method of claim 6, wherein the plant SPL7 gene is inactivated by means of a T-DNA insertion mutation.
12. 12. The method of claim 6, wherein the plant is a terrestrial plant, including a dicot, a monocot, and a bryophyte.
13. 13. The method of claim 12, wherein the plant is arabidopsis thaliana.

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