CN114106119B - Scoxoist gene of erythropsis pinicola and application thereof - Google Patents

Abstract

The invention discloses a scoliosis gene and application thereof. The present invention provides any one of the following proteins: the amino acid sequence is shown as SEQ ID No.1 or a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues or has more than 80 percent of homology or a fusion protein obtained by connecting a label at the N end and/or the C end of the protein. The Soloist gene derived from the erythropsis pinicola has the function of resisting verticillium dahliae, not only enriches the theoretical knowledge of Soloist subfamily genes in plants, but also provides candidate genes for crop biological stress resistance, especially molecular breeding of plant verticillium resistance.

Description

Scoxoist gene of erythropsis pinicola and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a ScSoloist gene of erythropsis pinicola and application thereof.

Background

Verticillium wilt (Verticillium wilt) is a plant vascular bundle disease caused by verticillium dahliae, has a wide host range, comprises more than 200 dicotyledonous plants, mainly infects commercial crops such as cotton, flax, tomatoes, eggplants, peppers and the like, wherein the verticillium wilt hazard of cotton is the most serious, and the cotton plant infected with verticillium wilt finally develops into the whole plant to die, so serious yield reduction and even harvest of cotton fields are often caused, and after bollworms in China are basically controlled, verticillium wilt is the first major disease of cotton production. For a long time, people use chemical pesticides to prevent and treat the cotton, so that the ecological environment is destroyed, pathogenic bacteria generate drug resistance, the quantity of natural cotton germplasm with verticillium resistance is small, and cotton varieties with verticillium resistance and high yield are difficult to obtain by a conventional hybridization breeding method at present. Therefore, the related genes of verticillium wilt resistance are separated by utilizing the modern molecular biology means, then the transgenic plants of verticillium wilt resistance are obtained by the transgenic technology, and further the genetic engineering means are utilized to cultivate disease-resistant varieties, so that the method has important significance and wide application prospect.

Plant AP2/ERF (APETALA 2/ethylene-responsive factor) is a large family of transcription factor genes, and is named after the AP2/ERF domain consisting of 60-70 amino acids. The AP2/ERF gene has been isolated from a variety of plants such as Arabidopsis, rice, maize, tomato, etc. Numerous studies have demonstrated that such transcription factors play an important role in plant growth and development and plant resistance to biotic and abiotic stress signaling. The AP2/EREBP genes in Arabidopsis are divided into AP2 subfamilies, DREB subfamilies, ERF subfamilies, RAV subfamilies, and individual subfamily members Soloist, depending on the number of AP2 domains contained and whether or not B3 domains are contained. The Soloist gene also has only one AP2 domain, but has a far-reaching evolutionary relationship with other DREB and ERF family genes, and thus becomes a single subfamily of genes. The current research on the AP2/ERF family is focused on the DREB and ERF subfamilies, and the Soloist subfamilies have little research on genes, and the gene characteristics and functions are poorly known. Up to now, no research report on resistance of Soloist gene to verticillium wilt is seen.

The odontoseisis (Syntrichia caninervis) is a dominant species in the dry-resistant moss crust layer in northwest desert areas of China, has super-strong comprehensive stress resistance and is a good material for resistance gene excavation. The development of the resistance gene clone has important significance for developing and utilizing stress resistance gene resources in depth and directionally cultivating new varieties of resistant plants.

Disclosure of Invention

The invention aims to provide a ScSoloist gene of erythropsis pinicola and application thereof.

In a first aspect, the invention claims a protein.

The protein name claimed by the invention is ScSoloist, derived from erythropsis pinicola. Specifically, any one of the following (A1) to (A4) can be mentioned:

(A1) A protein with an amino acid sequence shown as SEQ ID No. 1;

(A2) A protein having the same function by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined in (A1);

(A3) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (A1) or (A2) and having the same function;

(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the aspect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then the value (%) of the identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Of the above proteins, the scloist may be derived from erythrosin.

In a second aspect, the invention claims nucleic acid molecules encoding the proteins described above.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Further, the nucleic acid molecule (scloist gene) may be any of the following:

(B1) A DNA molecule shown in SEQ ID No.2 (cDNA) or SEQ ID No.3 (genome);

(B2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes said protein;

(B3) A DNA molecule having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the DNA sequence defined in (B1) or (B2) and encoding the protein.

In the above nucleic acid molecules, identity refers to nucleotide sequence identity. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of nucleotide sequences can be searched for by using blastp as a program, setting the aspect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then the value (%) of the identity can be obtained.

In the above nucleic acid molecules, the 90% identity or more may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising a nucleic acid molecule as described hereinbefore.

Wherein the expression cassette refers to a DNA capable of expressing the protein of the first aspect in a host cell, which DNA may include not only a promoter for initiating transcription of the gene of interest, but also a terminator for terminating transcription of the gene of interest. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta-glycin (Beachy et al (1985) EMBO J.4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

Constructing a recombinant expression vector containing the ScSoloist gene expression cassette. The plant expression vector used may be binary Agrobacterium vector, such as pCAMBIA1301, pCAMBIA1302, pCAMBIA2301, pCAMBIA1300, pBin438, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When the ScSoloist is used to construct a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In a specific embodiment of the invention, the promoter in the recombinant vector that initiates transcription of the nucleic acid molecule is a 35S promoter. More specifically, the recombinant vector is a recombinant plasmid obtained by inserting the nucleic acid molecule into the pCAMBIA1301 vector at multiple cloning sites (such as Kpn I and BamH I).

In a fourth aspect, the invention claims the use of a protein as described hereinbefore or a nucleic acid molecule as described hereinbefore or an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line as described hereinbefore in any of the following:

p1, regulating and controlling the resistance of plants to verticillium wilt;

p2, regulating and controlling the resistance of the plant to the verticillium dahliae;

p3, regulating and controlling the scavenging capacity of plant Reactive Oxygen Species (ROS);

p4, regulating and controlling lignin content in plants;

p5, regulating and controlling salicylic acid content in plants;

p6, regulating and controlling the expression of key genes of salicylic acid synthase in plants;

p7, regulating and controlling the expression of lignin synthase key genes in plants;

p8, regulating and controlling the expression of genes related to classical disease course in plants;

p9, plant breeding.

Further, the expression level and/or activity of the protein in the plant is increased, and the resistance of the plant to verticillium wilt is increased.

Further, the expression level and/or activity of the protein in the plant is increased, and the resistance of the plant to verticillium dahliae is increased.

Further, the protein has increased expression level and/or activity in the plant, which has increased Reactive Oxygen Species (ROS) scavenging capacity under stress of verticillium dahliae, increased in vivo lignin content, increased in vivo salicylic acid synthase key gene expression level, increased in vivo lignin synthase key gene expression level, and/or increased in vivo classical disease course related gene expression level.

Further, in such applications, plants expressing the protein (scloist protein) may be crossed with other plants for plant breeding.

In a fifth aspect, the invention claims a method of growing plants.

The method of growing plants claimed in the present invention comprises the step of increasing the expression level and/or activity of the aforementioned protein (scloist protein) in the recipient plant, the plant of interest being obtained with any of the following traits:

(a1) Resistance to verticillium wilt is improved;

(a2) Resistance to verticillium dahliae is improved;

(a3) Increased Reactive Oxygen Species (ROS) scavenging capacity under stress of Verticillium dahliae, increased lignin content in vivo, increased salicylic acid content in vivo, increased expression level of a key gene of salicylic acid synthase in vivo, increased expression level of a key gene of lignin synthase in vivo, and/or increased expression level of a gene associated with classical disease processes in vivo.

The method can be realized by hybridization means or transgenic means.

In a sixth aspect, the invention claims a method of growing a transgenic plant.

The method for cultivating transgenic plants claimed in the present invention may comprise the steps of: introducing the nucleic acid molecule (ScSoloist gene) described above into a recipient plant to obtain a transgenic plant; the transgenic plant has any one of the following traits compared with the recipient plant;

(a1) Resistance to verticillium wilt is improved;

(a2) Resistance to verticillium dahliae is improved;

(a3) Increased Reactive Oxygen Species (ROS) scavenging capacity under stress of Verticillium dahliae, increased lignin content in vivo, increased salicylic acid content in vivo, increased expression level of a key gene of salicylic acid synthase in vivo, increased expression level of a key gene of lignin synthase in vivo, and/or increased expression level of a gene associated with classical disease processes in vivo.

In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In the above method, the nucleic acid molecule (ScSoloist gene) may be modified as follows before being introduced into the recipient plant to achieve better expression:

1) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

3) The expression efficiency of the gene of the invention can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present invention;

4) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

The nucleic acid molecule (ScSoloist gene) may be introduced into plant cells by conventional biotechnological methods using Ti plasmids, plant virus cultivars, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology (2 nd Edition).

In the above aspects, the increased resistance to verticillium wilt and the increased resistance to verticillium dahliae may be embodied as a decrease in disease index, a decrease in disease grade, and/or a decrease in the pathogenic bacterial content in the plant.

In the above aspects, the increased Reactive Oxygen Species (ROS) scavenging ability may be embodied as reduced Reactive Oxygen Species (ROS) accumulation and/or increased peroxidase activity.

Further, the reduced Reactive Oxygen Species (ROS) accumulation may be embodied as reduced Malondialdehyde (MDA) content and/or hydrogen peroxide (H) ₂ O ₂ ) The content is reduced; the increased peroxidase activity can be embodied as increased superoxide dismutase activity (SOD) and/or increased peroxidase activity (POD);

In the above aspects, the salicylic acid synthase key gene may specifically be an ICS1 gene and/or an EDS5 gene. The lignin synthase key gene can be specifically PAL2 gene and/or 4CL2 gene. The classical disease course-related gene may specifically be a PR1 gene, a PR2 gene and/or a PR5 gene.

In the above aspects, the plant may be a seed plant or a bryophyte.

Further, the seed plant may be an angiosperm plant. The bryophyte may specifically be erythropsis pinicola.

Still further, the angiosperm may be a dicot.

In a specific embodiment of the invention, the plant is specifically arabidopsis thaliana.

The invention clones a Soloist gene from the erythropsis pinicola, and the amino acid sequence comparison result of the Soloist gene in non-moss plants such as arabidopsis thaliana, soybean, rice and the like shows that besides the conservation of the amino acid composition of an AP2 structural domain, the non-AP 2 structural domain has extremely low amino acid composition consistency, and the phylogenetic tree analysis also shows that the ScSoloist is gathered together with the sphagnum and the physcomitrella patens and has a far evolutionary distance with the species such as arabidopsis thaliana, rice, soybean, cotton and the like. The structure, sequence alignment and evolutionary relationship of the ScSoloist gene all indicate that ScSoloist from the moss odontoid erythrosis in desert is a unique new gene. In addition, experiments prove that the ScSoloist gene can obviously improve the capability of resisting verticillium dahliae of transgenic Arabidopsis, and comprehensively reveal the disease resistance mechanism of the ScSoloist gene from three layers of morphology, physiology and molecules. The first example proves that the Soloist family genes have the function of resisting verticillium dahliae, not only enriches the theoretical knowledge of Soloist subfamily genes in plants, but also provides candidate genes for crop biological stress resistance, especially molecular breeding of plant verticillium wilt resistance.

The beneficial effects are that:

1. the invention is the report that the Soloist gene can obviously improve the resistance of plants to fungal pathogenic bacteria Verticillium dahliae for the first time, and is the new function of Soloist family genes. And comprehensively reveals the verticillium wilt resistance mechanism of the ScSoloist gene from three aspects of morphology, physiology and molecules, and provides a candidate gene with strong disease resistance for plant disease resistance molecular breeding.

2. Compared with WT Arabidopsis, the stress result of the Verticillium dahliae shows that the transgenic strain has obvious capability of resisting Verticillium dahliae of cotton verticillium, the verticillium wilt degree of the transgenic strain is lighter than that of wild Arabidopsis on leaves of Arabidopsis, and the morbidity degree and morbidity of the wild Arabidopsis are obviously higher than those of the transgenic strain, and the transgenic strain is reflected in more serious disease index and disease grade.

3. The transgenic arabidopsis thaliana can improve the capability of resisting verticillium dahliae of the transgenic arabidopsis thaliana by reducing ROS injury, enhancing ROS scavenging capability, improving the expression quantity of related genes such as lignin, disease course related proteins and the like.

4. And constructing a ScSoloist gene over-expression vector to obtain a ScSoloist gene transgenic plant. The resistance of the transgenic plant to verticillium wilt is obviously enhanced, which proves that the ScSoloist gene improves the defending resistance of the plant to verticillium wilt and has genetic engineering application value.

Drawings

FIG. 1 is a diagram showing the structure of the ScSoloist gene.

FIG. 2 shows the sequence alignment and the treeing analysis of the Soloist gene from Scsoloist and Soloist from other species. Multiple sequence alignment of scloist and 4 representative species Soloist, atSoloist from arabidopsis thaliana (At 4g 13040), ppSoloist from physcomitrella patens (pp3c5_ 19550V3.1), gmSoloist from soybean (glyma.18g159900.1), osSoloist from rice (loc_os02g29550.1); b.10 evolutionary tree analyses representing the species Soloist. The 10 species are Arabidopsis thaliana, rice, soybean, cotton, cowpea, sphagnum, physcomitrella patens, lemna minor, and Picea japonica, respectively.

FIG. 3 shows the gene expression levels of the ScSoloist gene in R.dentalis after hormone treatment and pathogen inoculation. A: scSoloist gene expression levels at different time points during the infection of the tooth rib erythrosine 5d by the Verticillium dahliae; b: plant hormone Salicylic Acid (SA) treatment of the ScSoloist gene expression levels at different time points during 2d of odontoid erythrosis; c: gene expression levels of the phytohormone methyl jasmonate (MeJA) at various time points ScSoloist during 2d treatment of Rib erythrosine.

FIG. 4 is a ScSoloist protein subcellular localization and self-activation activity assay. A: scSoloist subcellular localization analysis; b: scSoloist transcriptional autoactivation Activity assay, esDREB as positive control and P1-P5 as ScSoloist different truncated fragments.

FIG. 5 shows the double restriction enzyme identification of the plant expression vector pCAMBIA 1301-ScSolois. Lane a: pCAMBIA1301-ScSoloist double cleavage results; lane b: pCAMBIA1301 is subjected to no-load double enzyme digestion; lane c: scSoloist gene fragment

FIG. 6 is a PCR identification of the transgenic Arabidopsis thaliana line ScSoloist gene. L1-L11 are transgenic Arabidopsis thaliana, and WT is wild type Arabidopsis thaliana.

FIG. 7 shows the onset of wild-type and different strain transgenic Arabidopsis thaliana (1-3 and 2-18) after stress by Verticillium dahliae. A: phenotype comparison of different strains of arabidopsis after stress; b: disease grade distribution of arabidopsis thaliana of different strains after stress; c: disease indexes of different strains of arabidopsis after stress; d: the verticillium dahliae cell content of different strains of arabidopsis after stress p <0.01 and p <0.001.

FIG. 8 is an analysis of the ROS accumulation and ROS scavenging related enzyme activity of transgenic Arabidopsis after 20d stress treatment of Verticillium dahliae. A: DAB staining and NBT staining of arabidopsis leaves of different strains after stress; b: h of Arabidopsis leaves of different strains after stress ₂ O ₂ Measuring the content; c: measuring Malondialdehyde (MDA) content of leaves of different strains of arabidopsis after stress; d: determination of SOD activity in leaves of different strains of arabidopsis after stress E: POD Activity determination in Arabidopsis leaves of different lines after stress p <0.05，**p<0.01。

FIG. 9 shows the measurement of the expression level of isolated salicylic acid and its synthesis related genes from transgenic Arabidopsis plants under stress of Verticillium dahliae. A: the content of the arabidopsis free salicylic acid of different strains after stress; b: the expression level of AtICS1 at different time points; c: expression level of AtEDS5 at different time points.

FIG. 10 shows the analysis of lignin and gene expression levels related to synthesis of transgenic Arabidopsis plants under stress of Verticillium dahliae, and the analysis of gene expression levels related to protein PR related to disease course. A: lignin content of arabidopsis thaliana of different strains after stress; b: expression level of At4CL2 At different time points; c: the expression quantity of AtPAL2 at different time points; d: the expression quantity of AtPR1 in different time periods; e: the expression quantity of AtPR2 in different time periods; f: expression level of AtPR5 at different time periods.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 cloning of the ScSoloist Gene of Dendrolimus

The erythrosine of the dentate rib in the invention is from the Golgi desert of Xinjiang Golgi.

Gene primers were designed for amplification from information in the mixed transcriptome database of R.dentatus. PCR and RT-PCR amplification were performed using the R.dentatus genome and cDNA as templates, respectively. The method comprises the following specific steps:

the amplification primers P1 and P2 are designed by using a mixed transcriptome database of the physcomitrella, PCR amplification is carried out by using the genome DNA of the physcomitrella as a template, and RT-PCR amplification is carried out by using the first strand of the cDNA of the physcomitrella as a template.

P1：5’-ATGGTTAGTATCAGGAAACGCCG-3’；

P2：5’-TTAGGACGGGCACTTTACAGAC-3’。

The PCR and RT-PCR products are subjected to agarose gel electrophoresis with concentration of 1.0%, fragments are recovered by an agarose gel recovery kit of OMEGA company and are respectively connected to a pMD19-T vector (the obtained recombinant plasmid is named pMD 19-ScSoloist), escherichia coli DH5 alpha is transformed, a single clone is detected by PCR, and positive clones are sequenced and stored for standby after the sequencing is correct.

Sequencing results showed:

the PCR amplified product sequence of the genome DNA of the erythropsis pinicola is shown as SEQ ID No. 3; the RT-PCR amplification sequence of the first strand of the erythrosin cDNA is shown as SEQ ID No. 2. SEQ ID No.2 and SEQ ID No.3 both encode the protein shown as SEQ ID No. 1. The protein is named as ScSoloist protein, and the corresponding coding gene is ScSoloist gene.

Example 2 bioinformatics analysis of the ScSoloist Gene

The gene Structure was mapped using Gene Structure Display Server 2.0.0 (GSDS) online software to the ScSoloist gene sequences (SEQ ID No.2 or SEQ ID No. 3), the conserved domain analysis was performed using NCBI online domain prediction tools (http// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), the ScSoloist was predicted by online software cNLS Mapper (http:// NLS-mapper.iab.ac.jp/cgi-bin/NLS_mapper_form.cgi), and the search for homologous protein alignment was performed using NCBI's blastp to the ScSoloist protein (SEQ ID No. 1). A representative sequence similar to the nucleotide sequence of the ScSoloist gene was found, multiple sequence alignment was performed with Clustal W, multiple sequence alignment was edited with Jalview software, and the evolution tree was constructed with Mega6.0 software.

As shown in fig. 1: the full length of the ScSoloist gene 955bp, containing 6 exons, 5 introns, open reading frame 561bp, predictive coding 186 amino acids, containing a typical and complete AP2 conserved domain, with one and nuclear localization signal peptide at the N-terminus.

By comparison with NCBI blastp, the amino acid consistency of ScSoloist and the physcomitrella patens is highest and reaches 69%, but the function of the gene in the physcomitrella patens is not reported. The amino acid sequence alignment of the homologous Soloist gene in ScSoloist and non-bryophytes such as Arabidopsis, soybean, rice, etc. is shown in FIG. 2A, with the exception of the conserved amino acid composition of the AP2 domain, the non-AP 2 domain portion has very low identity in amino acid composition. Analysis of the evolutionary tree of the ScSoloist gene with other Soloist genes in representative plants showed that ScSoloist was clustered together with the moss plant Physcomitrella patens and evolved far from the species Arabidopsis thaliana, rice, soybean, cotton, etc. (FIG. 2B). The structure, sequence alignment and evolutionary relationship of the ScSoloist gene all indicate that ScSoloist from the moss odontoid erythrosis in desert is a unique new gene.

Example 3 detection of Gene expression level of ScSoloist under pathogen and hormone treatment by qRT-PCR

In the invention, the erythrosine is completely rehydrated from the Sinkiang Guerban Gu Techi rib under normal illumination for 24 hours, and then Salicylic Acid (SA) and jasmonic acid (MeJA) and Verticillium dahliae (V991 strain) are treated. The gene expression level of the ScSoloist gene under various treatments was measured using Tubulin as a reference gene. The specific operation is as follows:

the experimental material treatment method comprises the following steps: the erythrosine was completely rehydrated from galban Gu Techi rib in Xinjiang for 24h under normal light, and then subjected to various stress treatments.

Treatment of phytohormone: SA (1 mM), meJA (50 mu M) and MeJA hormone are respectively treated, so that SA and MeJA hormone treatment liquids added in moss cannot be excessive, preferably moss plants are not submerged (8 mL of stress liquid is added in a 90mM culture dish), and moss samples treated by equal amount of distilled water are taken as control groups, and samples are taken at 0,0.5h,2h,4h,6h,8h,24h and 48h after hormone treatment.

Pathogen treatment: selecting mycelium of Verticillium dahliae (V991 strain) and inoculating the mycelium onto a Charles solid culture medium, and inversely culturing the mycelium for 5 to 7 days at the temperature of 25 ℃; inoculating fresh thalli into a Charles liquid culture medium, and culturing for 5-7 d at 200 rpm; filtering with sterilized four-layer gauze to remove spore, counting cells of cultured liquid Verticillium dahliae (V991 strain) with blood cell counting plate, and diluting with sterile distilled water to obtain 10 ⁶ On the order of magnitude of each ml, the erythromyces dentatus (rehydration for 24 h) was infested, each moss to be infested was pricked lightly with a small wound with a syringe, so that pathogenic bacteria could be infested more easily with the erythromyces dentatus, and a distilled water-treated moss sample was used as a control group (the control group was pricked lightly with the syringe as well). Pathogenic bacteria were treated and sampled for 0,1d,3d,5 d.

3 biological repeats are respectively taken for each treatment, 0.1g of sample is weighed for each biological repeat, immediately placed into liquid nitrogen for quick freezing, and then the sample is transferred to a refrigerator at the temperature of minus 80 ℃ for preservation.

Based on the sequences of the specific gene ScSoloist and the reference gene Tubulin, primer5 was used for Primer design as follows:

qPCR-ScSoloist-1F：5’-GGCGGACTCTCTCCAGGTAAAT-3’；

qPCR-ScSoloist-1R：5’-GAATAACTGATGGTGCAGCTCC-3’。

Sc-α-Tubulin-F：5’-CGGTCATTACACCGTGGGAA-3’；

Sc-α-Tubulin-R：5’-CCTCTCCAGCAACAGCGAA-3’。

RNA extraction and synthesis of first strand of cDNA: the moss samples stored at-80℃were taken out for RNA extraction, and extracted with an RNA extraction Kit (Omega, plant RNA Kit, R6827-02). The extracted RNA was used in reverse transcription kit PrimeScript ^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) (takara, RR 047A) the first strand of cDNA was synthesized.

Real-time fluorescent quantitative PCR experiment: the cDNA obtained by the reverse transcription was subjected to real-time quantitative PCR using TB Green Premix Ex Taq II (Tli RNaseH Plus) (takara, RR 420Q). The system is as follows: 7 mu L ddH ₂ O, 10. Mu. L TB Green Premix Ex Taq (Tli RNaseH Plus) (2X), 0.5. Mu.L forward primer (10. Mu.M)/reverse primer (10. Mu.M), 2. Mu.L cDNA, and a total volume of 20. Mu.L. The procedure is: pre-denaturation at 94 ℃ for 30s; denaturation at 94℃for 5s, annealing at 60℃for 30s and extension for 40 cycles.

Data analysis: relative value of Gene expression was 2 ^-ΔΔt The method was calculated using Sc- α -Tubulin as an internal reference, with three biological replicates per treatment, and three technical replicates per biological replicate.

The real-time fluorescence quantification results are shown in fig. 3: the ScSoloist gene is induced to be up-regulated by verticillium dahliae V991, and simultaneously induced to be expressed by SA and MeJA, and shows the expression trend of ascending and then descending and then ascending after SA treatment, and the ScSoloist gene is descending and then ascending after MeJA treatment.

Example 4 ScSoloist protein subcellular localization

According to the method for bombarding onion cells by using a gene gun, pBI121-ScSoloist-EGFP plasmid with green fluorescent protein is brought into onion cells through tungsten powder for expression, and the specific expression site of the green fluorescent protein is observed through a laser confocal microscope, so that the subcellular localization condition of the ScSoloist gene is verified. The specific operation is as follows:

1. Construction of pBI121-ScSoloist-EGFP plasmid

According to the instructions of In-Fusion HD Cloning Kit, primer design was performed based on the cDNA sequence of the ScSoloist gene (SEQ ID No. 2) and 60 or more base sequences at the multicloning cleavage site of the pBI121-EGFP vector.

The pMD19-ScSoloist (carrying SEQ ID No. 2) constructed in example 1 was used as a template, to which was added a homologous recombination sequence, the upstream primer pBI121 (in-fusion) -ScSoloist-F:5' -TCTAGACTGGTACCCATGGTTAGTATCAGGAAACG-3', the downstream primer pBI121 (in-fusion) -ScSoloist-R:5' -CTAGTCAGTCGACCCTTGGACGGGCACTTTACAG-3' (underlined as vector sequence). Cloning of the target gene ScSoloist was performed. The PCR product was recovered using an Omega gel recovery kit, and after recovery, the fragment size and concentration were detected using 1% agarose gel electrophoresis.

pBI121-EGFP was subjected to single cleavage using SmaI restriction enzyme. The digested products were gel recovered and fragment size and concentration were detected using 1% agarose gel electrophoresis.

The amount of join required for the connection was calculated from the web site http:// www.clontech.com/US/Support/xxclt_onlinetoolsload.jspecimeid=http:// bioinfo.clontech.com/infusion/molar ratio.do & section=16260 & xxheight=750 provided by Clontech. The reaction system was incubated at 50℃for 15min and then placed on ice. The ligation solution allowed transformation of DH 5. Alpha. Competent cells.

2. Gene gun bombarding onion epidermis cell

Soaking fresh onion in clear water for one day, tearing off the epidermis of the middle 2-3 layers of onion flaps, and spreading on a solid 1/2MS flat plate poured in advance.

About 30mg of microcarrier is weighed and put into a centrifuge tube with the volume of 1.5 mL; 1mL of 70% ethanol was added to the centrifuge tube; sufficiently vibrating on a vortex mixer for 3-5 min; standing for 15min after the oscillation is finished; centrifuging the centrifuge tube for about 5s, and gently discarding the supernatant by using a pipette; adding 1mL of distilled water into the centrifuge tube; sufficiently shaking for 1min; standing for 1min after the oscillation is finished; centrifuging at maximum speed for 10s, absorbing and discarding the aqueous solution, and repeating the steps of 6-9 for 2 times; 500 μl of sterilized 50% glycerol was added to the centrifuge tube.

Sucking a proper amount of microcarrier solution to make the microcarrier solution about 3mg, and adding the microcarrier solution into a 1.5mL centrifuge tube; mu.L of pBI121-EGFP empty plasmid and pBI121-ScSoloist-EGFP (1. Mu.g/. Mu.L) were added, respectively; 50. Mu.L of 2.5M CaCl was added separately ₂ The method comprises the steps of carrying out a first treatment on the surface of the Adding 20 mu L of 0.1M spermidine respectively, and continuously oscillating for 2-3 min; standing for 1min, slightly centrifuging in a centrifuge for about 2s, and discarding supernatant; adding 140 mu L of 70% ethanol into the centrifuge tube, centrifuging at high speed for about 10s, and discarding the supernatant; adding 140 mu L of 100% ethanol, centrifuging at high speed for about 10s, and discarding the supernatant; finally, 48 mu L of 100% ethanol is added into the embedded microcarrier, and the microcarrier is oscillated at a low speed and time by time on an oscillator; bombardment was performed using a PDS-1000 bench-top gene gun. After the bombarded onion epidermal cells were dark-cultured for 24 hours, they were placed on a glass slide and photographed by a laser confocal microscope.

As shown in FIG. 4A, the ScSolosit protein was specifically localized to the nuclei of onion epidermal cells, and the positive control pBI121-GFP was distributed throughout onion cells.

Example 5 ScSoloist protein self-activation verification

In the invention, whether the ScSoloist has a transcription regulatory region is verified by a yeast double hybrid system. The specific operation is as follows:

1. construction of the bait vector pGBKT7-ScSoloist

Primers were designed according to the instructions of In-Fusion HD Cloning Kit (underlined as vector sequences).

pGBKT7-(in-fusion)-ScSoloist-F：5’-CATGGAGGCCGAATTCATGGTTAGTATCAGGAAACG-3’；

pGBKT7-(in-fusion)-ScSoloist-R：5’-GCAGGTCGACGGATCCTTAGGACGGGCACTTTACA-3’。

The fragment of the ScSoloist gene was cloned using the pMD19-ScSoloist (carrying SEQ ID No. 2) plasmid constructed in example 1 as a template. pGBKT7 empty plasmid was digested with BamH I and EcoR I. The double digested products of the ScSoloist and pGBKT7 vectors were subjected to gel recovery, the method being referred to Omega gel recovery kit. The amount of join required for the connection was calculated from the Clontech online site. The reaction system was incubated at 50℃for 15min and then placed on ice. The ligation solution allowed transformation of DH 5. Alpha. Competent cells.

The full length of the ScSoloist gene is truncated respectively, 5 primers of truncated short fragments are designed (the primers of each fragment are shown in Table 1), the full length pGBKT7-ScSoloist plasmid is amplified by PCR, 5 truncated fragments are cloned to obtain 5, which are respectively named as P1, P2, P3, P4 and P5, after the connection transformation with the pGBKT7 vector, 5 single colonies growing on LB solid plates containing 50mg/mL of kana are picked respectively, and then the single colonies are shaken for 4 to 5 hours on liquid LB medium containing 50mg/mL at 37 ℃ and 200rpm, and then the universal primers pGBKT7-F of the vector are used: 5'-TAATACGACTCACTATGGGC-3' and pGBKT7-R:5'-TTTTCGTTTTAAAACCTAAGAGTC-3' the bacterial liquid PCR identification is carried out.

TABLE 1 construction and identification of primers for the respective truncated fragments of pGBKT7-ScSoloist (underlined parts are vector sequences)

Primer name	Sequence (5 '-3')
		P1-F	CATGGAGGCCGAATTCATGGTTAGTATCAGGAAACG
P1-R	GCAGGTCGACGGATCCGGCTCGGTCATAGAGATG
		P2-F	CATGGAGGCCGAATTCATGGTTAGTATCAGGAAACG
P2-R	GCAGGTCGACGGATCCCGCTTTTGGGGGATTGAAT
		P3-F	CATGGAGGCCGAATTCGAGAAAGAGCTGAAGAAGAG
P3-R	GCAGGTCGACGGATCCTTAGGACGGGCACTTTACA
		P4-F	CATGGAGGCCGAATTCGAGAAAGAGCTGAAGAAGAG
P4-R	GCAGGTCGACGGATCCGGCTCGGTCATAGAGATG
		P5-F	CATGGAGGCCGAATTCGCTTACTTGTGTGGCAGAG
P5-R	GCAGGTCGACGGATCCTTAGGACGGGCACTTTACA

2. Yeast self-activation verification

pGBKT7-EsDREB recombinant plasmid in the previous publication of the subject group was used as a positive control (reference documentXiaoshuang Li,Daoyuan Zhang, haiyan Li, yuanming Zhang, andrew j.wood.esdreb2b, a novel truncated DREB-type transcription factor in the desert legume Eremosparton songoricum, enhances tolerance to multiple abiotic stresses in yeast and transgenic tobacco.bmc Plant Biology,14:44, 2014), pGBKT7 empty vector as negative control, and pGBKT7-ScSoloist and plasmids with P1-P5 fragments, respectively, transformed yeast competence. The strain was cultured on SD/-Trp plates and SD/-Trp/-His plates, respectively, and self-activation was verified.

As a result, as shown in FIG. 4B, neither the full-length ScSoloist protein nor the various forms of the truncated fragments had transcriptional self-activating activity.

EXAMPLE 6 construction of ScSoloist Gene overexpression vector

Designing a primer, cloning out the full length with an enzyme cutting site by taking pMD19-ScSoloist (carrying SEQ ID No. 2) constructed in example 1 as a template, linking the recovered product with a pCAMBIA1301 vector, transferring into escherichia coli DH5 alpha for amplification, extracting plasmids, sequencing, and preserving the correct plasmids for later use. The method comprises the following specific steps:

KpnI and BamHI cleavage sites are respectively added to the front and back ends of the ScSoloist gene fragment, the forward primer of the ScSoloist is pCAMBIA1301-ScSoloist-F, and the reverse primer is pCAMBIA1301-ScSoloist-R (the underlined label part is the cleavage site sequence).

pCAMBIA1301-ScSoloist-F：5’-GGGGTACCATGGTTAGTATCAGGAAACGCCG-3’；

pCAMBIA1301-ScSoloist-R：5’-CGGGATCCTTAGGACGGGCACTTTACAGAC-3’。

And (3) after electrophoresis of the PCR product added with the enzyme cutting sites, purifying and recycling the PCR product by using a gel recycling kit. Double digestion is carried out at 37 ℃ by KpnI and BamHI enzymes, electrophoresis is carried out after double digestion is completed, and a target gene with the sticky end of the digestion site is purified and recovered by a gel recovery kit and used for connection with a carrier.

The plasmid pCAMBIA1301 of the over-expression vector was digested with KpnI and BamHI enzymes at 37℃and, after electrophoresis, a large fragment with the sticky ends of the digested sites was recovered for ligation with the gene of interest.

The double enzyme digestion products of the target gene are mixed with the double enzyme digestion products of the vector pCAMBIA1301 and are connected at 16 ℃ under the action of T4 ligase. And (3) converting the connection product into escherichia coli DH5 alpha, detecting the monoclonal by PCR, selecting positive monoclonal for shaking, extracting recombinant plasmid, and sequencing and preserving the correct plasmid for later use.

As shown in FIG. 5, the double-enzyme test results show that the pCAMBIA1301-ScSoloist recombinant vector has fragments consistent with the ScSoloist gene in size, which are cut off by double-enzyme test enzymes, and the vector is constructed correctly.

Structural description of pCAMBIA1301-ScSoloist vector: a recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.2 between the cleavage sites KpnI and BamHI of pCAMBIA 1301.

Example 7 obtaining ScSoloist transgenic Arabidopsis plants Using Agrobacterium-mediated transformation

The pCAMBIA1301-ScSolois plasmid is transformed into an agrobacterium strain EHA105 to obtain transgenic agrobacterium, and the agrobacterium-mediated flower dipping method is used for transforming the transgenic arabidopsis. Immersing inflorescence parts of arabidopsis plants cultivated to a flowering phase in agrobacterium suspension impregnating solution for 10s, standing for 10min, then impregnating for 10s again, and carrying out dark moisture culture on the infected plants for 36h by using a black plastic bag; after 48h, the transformed plants were thoroughly washed clean with water spray and then normally cultivated until the seeds were mature. Seeds of arabidopsis plants infected with agrobacterium transformation solution are added into a 1/2MS culture medium to be screened, the arabidopsis plants growing with true leaves on a hygromycin plate are transplanted into soil, and DNA of the leaves is extracted after 2 weeks of culture for PCR identification. The screening was repeated until T2 generation seeds were obtained. The method comprises the following specific steps:

1. preparation of Agrobacterium competence

Streaking the stored EHA105 strain on LB solid medium with rifampicin resistance (50 mg/mL), and culturing for 2-3 d at 28 ℃; single colony is selected and inoculated in 5-10 mL of LB liquid medium with rifampicin resistance (50 mg/mL), and is oscillated at 28 ℃ for overnight; 1mL is taken and is connected with 50mL of LB (50 mg/mL of rifampicin), and is cultivated at 28 ℃ until the OD600 = 0.4-0.5; after the fungus liquid is ice-bathed for 30min, the temperature is 4 ℃, the weight is 5000g, the fungus liquid is centrifugated for 10min, and the supernatant is discarded; addingAdding 30mL of sterile water, centrifuging at 4 ℃ and 5000g for 10min, and discarding the supernatant; adding pre-chilled CaCl containing 20% glycerol ₂ (20 mmol/L), mixing, sub-packaging according to 200 mu L of each tube, quick-freezing with liquid nitrogen, and storing at-80 ℃ for standby.

2. Competent transformation of Agrobacterium

Rapidly placing the agrobacterium tumefaciens competence stored at-80 ℃ on ice for standby; 1. Mu.g of pCAMBIA1301-ScSoloist plasmid constructed in example 6 with correct sequencing is added into 200. Mu.L of agrobacteria competence, and the mixture is lightly mixed and then subjected to ice bath for 30min; quick-freezing with liquid nitrogen for 5min, water-bathing at 37 ℃ for 5min, and ice-bathing for 2min; adding 800 mu L of LB liquid medium, recovering for 4-6 h at 28 ℃ and 100 rpm; 50-200 mu L of bacterial liquid is coated on LB (final concentration is 50 mu g/ml rifampicin+final concentration is 50 mu g/ml kanamycin) solid plate, and the culture is carried out for 2d at 28 ℃ in an inverted way.

3. Identification of transgenic Agrobacterium

Colonies growing on resistant LB plates were picked, transferred to LB liquid medium (50. Mu.g/mL rifampicin+50. Mu.g/mL kanamycin), cultured overnight at 28℃and 200 rpm; performing PCR identification on the bacterial liquid; extracting plasmids from the bacterial liquid, and then carrying out KpnI and BamHI double enzyme digestion verification. The recombinant Agrobacterium identified as correct was designated EHA105-ScSoloist.

4. Sterilizing and planting wild type Arabidopsis seeds.

Placing the seeds of Arabidopsis thaliana into a sterilizing EP pipe with the volume of 1.5 or 2.0mL, adding 1mL of sterilizing water, and sucking out the water after full shaking; adding 1mL of 75% alcohol, controlling the time to be about 30s, and not too long; sucking out alcohol, immediately adding sterilized water, washing, and repeating for 2 times; adding 700 μl of sterilized water and 300 μl of sodium hypochlorite, mixing, and removing liquid after 3 min; repeatedly washing with sterilized water for 7-8 times; adding 1mL of sterilized water, blowing seeds onto a 1/2MS culture medium to uniformly distribute the seeds, and then absorbing and removing redundant water; sealing with sealing film, and vernalizing at 4deg.C in dark place for 2d; after the arabidopsis seedlings are cultured for about 1 week under normal illumination, 4 leaves are grown, at the moment, the seedlings are transplanted into sterilized nutrient soil (black soil: vermiculite: perlite=5:3:2), the seedlings are covered for about 3 days by a preservative film and then are uncovered, and water is poured every 3 days.

5. Agrobacterium infection

Streaking the stored EHA105-ScSoloist strain on LB solid medium (50. Mu.g/mL rifampicin+50. Mu.g/mL kanamycin) and culturing at 28℃for 2-3 d; colonies growing on resistant LB plates were picked, transferred to 5mL LB liquid medium (50. Mu.g/mL rifampicin+50. Mu.g/mL kanamycin), cultured overnight at 28℃and 200 rpm; 1mL of bacterial liquid is inoculated into 100mL of LB (50 mu g/mL rifampicin+50 mu g/mL kanamycin) liquid culture medium, and shake culture is carried out at 28 ℃ until the OD600 = 0.6; inoculating the bacterial liquid into fresh 100mL LB liquid medium, performing shake culture at the temperature of 28 ℃ until the OD600 is about 0.2, and then, impregnating the Arabidopsis; centrifugally collecting the thalli at 5000rpm, and suspending the thalli to the OD600 = 0.8 or so by using the prepared dyeing solution; soaking aerial parts of Arabidopsis thaliana in the bacterial solution for 10s, slightly shaking during the soaking, taking out and standing for 10min, and repeating the steps; the infected plants are subjected to dark and moisture preservation culture for 36 hours by using a black plastic bag; after 48h, the transformed plants were thoroughly washed clean with water spray.

6. Transgenic Arabidopsis screening

Seeds (T0 generation seeds) of the infected arabidopsis plants are screened, hygromycin is added into a 1/2MS culture medium, the arabidopsis plants with true leaves growing on a hygromycin plate are transplanted into soil, and DNA of the leaves is extracted after 2 weeks of culture, so that PCR identification is carried out by using the P1 and P2 primers in the embodiment 1. The screening was repeated until T2 generation seeds were obtained.

The PCR results of the transgenic Arabidopsis are shown in FIG. 6: the transgenic Arabidopsis cDNA is amplified to obtain a target band with the same size as the target gene of the ScSoloist, and the PCR product is sent to be sequenced and identified as the ScSoloist gene, so as to obtain the transgenic Arabidopsis of the transgenic ScSoloist gene.

Example 8 functional analysis of transgenic Arabidopsis thaliana against Verticillium dahliae

1. Preparation of Verticillium dahliae spore suspension and stress treatment of Arabidopsis thaliana

Selecting a small amount of mycelium of Verticillium dahliae (V991 strain) and inoculating the mycelium onto a Charles solid culture medium, and inversely culturing the mycelium at 25 ℃ for 5-7 d; inoculating the strain into a Charles liquid culture medium, and culturing at 200rpm for 5-7 d; with sterilized fourFiltering the gauze layer to remove spore filaments; counting the concentration of spore suspension with a hemocytometer, adding appropriate amount of sterilized water to adjust the concentration of spore suspension to 5.0X10 ⁶ Individual spores/mL; culturing the ScSoloist transgenic Arabidopsis thaliana and the wild Arabidopsis thaliana obtained in the example 7 in soil for about 3 to 4 weeks, and pouring the ScSoloist transgenic Arabidopsis thaliana and the wild Arabidopsis thaliana into spore suspension after the soil is slightly dry; standing for a period of time, and pouring out redundant spore suspension after the soil fully absorbs water; the infected arabidopsis thaliana is continuously cultured under normal culture environment and under watering, and the phenotype of the plant infected by the verticillium dahliae is observed. 3 biological replicates of wild-type WT and rosette leaves of transgenic Arabidopsis (1-3 and 2-18) were taken at different post-infection time periods (0 d,5d,10d,15d,20d,25 d), 0.3g of sample was weighed for each biological replicate, immediately frozen in liquid nitrogen, and the samples were then transferred to a-80℃refrigerator for storage. The experiments were also performed on empty control groups into which pCAMBIA1301 was introduced into wild-type Arabidopsis thaliana (see above for specific procedures).

2. Calculation of grade and index of disease of Arabidopsis after stress

After 20d inoculation with verticillium dahliae, the extent of arabidopsis onset was counted and different levels of disease index were defined as follows: 0: no symptoms, 1: up to 20% of the leaves are sallow; 2: up to 40% of the leaves are sallow; 3: up to 60% of the leaves are sallow; 4: up to 80% of the leaves are sallow; 5: up to 100% of the leaves are sallow. And meanwhile, calculating the disease index according to the following formula.

Disease index (%) =100×Σ (leaf number of each stage×representative value of each stage)/(total leaf number of investigation×representative value of highest stage).

3. Analysis of Verticillium dahliae content in plants after stress of Arabidopsis thaliana

The arabidopsis thaliana inoculated with verticillium dahliae 0,5d,10d,15d,20d,25d was subjected to DNA extraction using a DNA extraction kit (Omega, e.z.n.a.Plant DNA Kit) and using primers for 28S ribosomal RNA-specific gene of verticillium dahliae V991 strain, qPCR-V991-F:5' -CGTTTCCCGTTACTCTTCT-3' and qPCR-V991-R:5'-GGATTTCGGCCCAGAAAC T-3' (Guillaume J. Bilodeau, steven T. Koike, pedro Uribe, et al development of an assay for rapid detection and quantification of Verticillium dahliae in soil. Phytophology, 2012,102 (3): 331) was amplified by real-time quantitative PCR (qPCR) to quantitatively analyze the cell content (copy number) of Verticillium dahlia in plants. The (qPCR) experimental procedure was as follows:

(1) Standard plasmid preparation

The Arabidopsis DNA is used as a template, the primers qPCR-V991-F and qPCR-V991-R are used for PCR amplification, the amplified product is constructed on a pMD18-T vector, and the recombinant plasmid which is verified to be correct by sequencing is named as pMD18-V991. The pMD18-V991 plasmid was transformed into Trans5 alpha E.coli and the plasmid of interest for positive cloning was extracted for use.

(2) Absolute quantitative PCR

The extracted standard plasmid was subjected to gradient dilution by 10 times, 8 gradients were diluted, and a standard curve was prepared. A real-time quantitative PCR system was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (takara, RR 420Q), and the PCR system and procedure were performed as in embodiment 3.

(3) Data analysis

The method is used for calculating by absolute quantification, a standard curve is obtained by standard plasmid PCR, and the copy number of the bacterial DNA in each sample is calculated according to the standard curve of the plasmid. The copy number of the bacterial DNA was:

(6.02×10 ²³ )×(g/ml)/(DNA length×660)＝copies/ml。

three biological replicates per treatment and three technical replicates per biological replicate.

As a result, as shown in FIG. 7, after 20D treatment with Verticillium dahliae, wild-type Arabidopsis had severe verticillium wilt and even died, whereas transgenic Arabidopsis had lighter disease (A in FIG. 7), further analysis showed that the transgenic Arabidopsis had lighter disease grade distribution and lower disease index (B and C in FIG. 7) than the WT Arabidopsis, and in addition, the content of Verticillium dahliae in the transgenic Arabidopsis was significantly lower than that in the WT Arabidopsis (D in FIG. 7). The disease index, the disease grade and the pathogenic bacteria content of the over-expressed plants and the control are extremely obviously different. The no-load control is basically consistent with the phenotype, disease index, disease grade and pathogenic bacteria content of wild plants, and has no statistical difference.

Example 9 ROS accumulation assay and determination of peroxidase Activity

Test plants: example 7 ScSoloist transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were obtained, and an empty control of pCAMBIA1301 was introduced into wild type Arabidopsis thaliana, and stress was applied for 20d using Verticillium dahliae as in example 8.

The method for in vitro staining of plant leaves is as follows: taking the verticillium dahliae (V991 strain) in example 8, placing clean in-vitro leaves after 20d stress in 1mg/mL DAB or 1mg/mL NBT solution, dyeing for more than 12h at room temperature, and using absolute ethyl alcohol: acetic acid: glycerol: the decolorized solution of water=8:1:1:1 (volume ratio) is decolorized at 37 ℃ for 24-48 h, and photographed and observed. After stress of different strains for 2 days, malondialdehyde (MDA) and hydrogen peroxide (H) are measured ₂ O ₂ ) The specific methods for the accumulation of ROS represented by the above and the protection of peroxidase represented by superoxide dismutase (SOD) and Peroxidase (POD) were carried out according to the instructions of the reagent company built in tokyo. Experiments were performed in triplicate. And statistical analysis was performed on the test data using Prism 5 software.

The results are shown in FIG. 8: after 20d treatment of Verticillium dahliae, transgenic Arabidopsis has lower ROS damage compared with wild Arabidopsis, and is reflected in shallower DAB and NBT staining degree and lower H ₂ O ₂ And MDA content (A, B and C in FIG. 8), while transgenic Arabidopsis exhibits a stronger ROS scavenging ability, both antioxidant enzyme SOD and POD activities are significantly higher than that of WT plants (D and E in FIG. 8). DAB, NBT staining, H of empty control and wild type plants ₂ O ₂ The activity of the catalyst is basically consistent with that of MDA content and SOD and POD, and no statistical difference exists.

EXAMPLE 10 determination of free salicylic acid and analysis of the expression level of Critical Gene of salicylic acid synthase

Test plants: example 7 ScSoloist transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were obtained, and an empty control of pCAMBIA1301 was introduced into wild type Arabidopsis thaliana, treated with Verticillium dahliae stress for 2d, and sampled 0,3h,6h,12h,24h after the treatment, respectively, for SA content determination and quantitative analysis of SA synthase key genes.

1. Determination of free salicylic acid

The extraction method comprises the following steps: about 0.1g of the sample was weighed, put into a mortar for grinding, 1mL of a precooled 90% aqueous methanol solution was added, and the mixture was extracted at 4℃overnight. 8000g, extracting for 10min, collecting supernatant, extracting residue with 0.5mL of 90% methanol aqueous solution for 2 hr, centrifuging, collecting supernatant, mixing the two supernatants, evaporating at 40deg.C under reduced pressure until no organic phase is contained (about 0.3mL of aqueous solution), adding 20 μL of 1mg/mL trichloroacetic acid aqueous solution, mixing, and shaking for 1min. Adding 1mL of a mixed solution of ethyl acetate and cyclohexane (1:1 v/v), extracting twice, transferring an upper organic phase to a new EP pipe, blowing and drying by nitrogen, adding 0.5mL of a mobile phase for dissolution, mixing uniformly, filtering by a needle filter, and then testing.

HPLC liquid phase conditions: rigol L3000 high performance liquid chromatograph, kromasil C18 reverse phase chromatography column (250 mm. Times.4.6 mm,5 μm), mobile phase methanol: 1% acetic acid water = 3:2. starting a computer, a detector and a pump, installing a chromatographic column, opening software, setting the sample injection amount to 10 mu L, the flow rate to 0.8mL/min, the column temperature to 35 ℃, the retention time to 30min, a fluorescence detector, the excitation wavelength to 294nm, the emission wavelength to 426nm in the method group, and setting the method group. The mobile phase was passed through the column and loading was started after the baseline was stable.

2. Real-time fluorescence quantitative analysis of salicylic acid synthesis key gene

Selecting a free salicylic acid anabolism related gene AtICS1 and AtEDS5; meanwhile, the expression condition of the gene at different times is analyzed by taking the arabidopsis ubiquitin gene AtUBQ10 as an internal reference gene of a fluorescent quantitative PCR reaction. Meanwhile, the expression condition of the gene at different times is analyzed by taking the arabidopsis ubiquitin gene AtUBQ10 as an internal reference gene of a fluorescent quantitative PCR reaction. The primers used in the fluorescent real-time quantitative PCR experiments are shown in Table 2. The PCR amplification system was referred to the manual of TaKaRa SYBR Premix Ex Taq TM kit (Takara) kit, and the two-step PCR reaction procedure was: the pre-denaturation was carried out for 40 cycles at 95℃for 30s,95℃for 5s, and 60℃for 30 s. PCR amplification in The amplification and dissolution curves were observed after the end of the PCR procedure, the data analysis was performed using the instrument kit software, and the relative value of gene expression was 2 ^-ΔΔt Method calculations, three biological replicates per treatment, three technical replicates per biological replicate.

TABLE 2 real-time fluorescent quantitative experiment RT-qPCR primer information

The salicylic acid content and the expression level of the genes related to the synthesis are shown in FIG. 9: after 2d of pathogenic stress, transgenic arabidopsis thaliana has higher free salicylic acid content relative to WT, especially 2-18 strains, SA content is always in an ascending trend after the pathogenic stress (A in FIG. 9), and meanwhile, the expression quantity of the corresponding salicylic acid synthesis key enzyme genes AtICS1 and AtEDS5 is also obviously higher than that of WT plants (B and C in FIG. 9). The no-load control is basically consistent with the SA content of wild plants and the expression level of AtICS1 and AtEDS5 genes, and has no statistical difference.

EXAMPLE 11 determination of lignin content by ultraviolet spectrophotometry

Test plants: example 7 ScSoloist transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were obtained, and an empty control of pCAMBIA1301 was introduced into wild type Arabidopsis thaliana, and stress was applied for 20d using Verticillium dahliae as in example 8.

Since the complex formed by lignin and bromoacetyl at 70 ℃ has a distinct absorption peak at 280nm, the lignin content in plants is often determined by ultraviolet spectrophotometry.

Taking main stems of stress 20d Arabidopsis above 3cm above the ground, grinding and crushing with liquid nitrogen; drying the ground sample in a 60 ℃ oven until the weight is constant, weighing 10mg of the sample, putting the sample into a 15ml centrifuge tube, adding 10ml of deionized water, and heating and vibrating the sample at 65 ℃ for more than 1 hour; after each sample is filtered by GF/A glass fiber filter paper, the filter residue is rinsed with water, ethanol, acetone and diethyl ether in sequence to remove soluble impurities, and each rinsing is carried out for 1-2 minutes; the filter paper is dried and placed in a 15ml centrifuge tube, 2.5ml of 25% bromoacetyl (dissolved in glacial acetic acid) is added, and the filter paper is heated for 1h by shaking at 70 ℃; after the sample had cooled to room temperature, 0.1ml of 7.5M hydroxylamine hydrochloride, 0.9ml of 2M sodium hydroxide and 3ml of glacial acetic acid were added to terminate the reaction; all the samples are transferred into a 50ml volumetric flask, and the samples are kept stand for more than 1h after being fixed in volume by glacial acetic acid; measuring absorbance at 280nm with a Lumda 35 uv-vis spectrophotometer; lignin content (%) =280 nm absorbance constant volume 100/(dry sample weight) Astandard; wherein astandard=17.2.

The results are shown in fig. 10 a: transgenic arabidopsis plants have a higher lignin content after pathogenic stress than WT plants. No load control was substantially identical to the lignin content of wild type plants, with no statistical differences.

EXAMPLE 12 analysis of expression level of lignin Synthesis-related Gene and plant classical disease resistance Gene

Test plants: example 7 ScSoloist transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were obtained, and an empty control of pCAMBIA1301 was introduced into wild type Arabidopsis thaliana, and stress was applied to 5, 10, 15, 20, 25d using Verticillium dahliae as in example 8.

Classical disease-resistant related genes AtPR1, atPR2 and AtPR5 in arabidopsis are selected, lignin anabolism related genes AtPAL2 and At4CL2 are selected, and meanwhile, an arabidopsis ubiquitin gene AtUBQ10 is used as an internal reference gene of a fluorescent quantitative PCR reaction to analyze the expression condition of the genes At different times. The primers used in the fluorescent real-time quantitative PCR experiments are shown in Table 2. The PCR amplification system was referred to the manual of TaKaRa SYBR Premix Ex Taq TM kit (Takara) kit, and the two-step PCR reaction procedure was: the pre-denaturation was carried out for 40 cycles at 95℃for 30s,95℃for 5s, and 60℃for 30 s. PCR amplification was performed on a Bio-Rad CFX96 Real-Time PCR System Real-Time quantitative PCR apparatus, and after the PCR procedure was completed, amplification and dissolution curves were observed, data analysis was performed using the apparatus kit software, and the relative value of gene expression was 2 ^-ΔΔt Method calculations, three biological replicates per treatment, three technical replicates per biological replicate.

The results are shown in fig. 10 as B, C, D, E and F: compared with the WT plant, the transgenic arabidopsis has remarkably high expression levels of the lignin synthase key genes AtPAL2 and At4CL2 in different time periods of inoculation of the pathogenic bacteria V991 after the pathogenic bacteria are stressed. The expression level of classical disease course related genes PR1, PR2 and PR5 in Soloist transgenic Arabidopsis is also obviously higher than that of WT plants. The empty control was substantially identical to the expression of the aforementioned genes of the wild-type plants, with no statistical differences.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> Xinjiang ecological and geographic institute of academy of sciences in China

<120> Scosoloist gene of odontoid erythrose and application thereof

<130> GNCLN202080

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 186

<212> PRT

<213> Syntrichia caninervis

<400> 1

Met Val Ser Ile Arg Lys Arg Arg His Gly Gly Leu Ser Pro Gly Lys

1 5 10 15

Ser Ile Gln Pro Lys Val Tyr Lys Arg Ala Ala Val Thr Ala Asp Gln

20 25 30

Pro Pro Lys Gln Glu Val Arg Phe Ala Pro Ser Pro Pro Val Pro Thr

35 40 45

Phe Gln Glu Leu His His Gln Leu Phe Asn Pro Pro Lys Ala Glu Lys

50 55 60

Glu Leu Lys Lys Arg Lys Arg Gln Arg Arg Lys His Gln Glu Asn Gln

65 70 75 80

Glu Pro Cys Val Met Arg Gly Val Tyr Phe Lys Asn Met Lys Trp Gln

85 90 95

Ala Ala Ile Lys Val Glu Lys Lys Gln Val His Leu Gly Thr Val Asn

100 105 110

Ser Gln Lys Glu Ala Ala His Leu Tyr Asp Arg Ala Ala Tyr Leu Cys

115 120 125

Gly Arg Glu Pro Asn Phe Glu Leu Thr Glu Ala Glu Lys Gln Glu Leu

130 135 140

Gln Leu Leu Gln Trp Glu Asp Phe Leu Glu Gln Thr Arg Gln Ser Ile

145 150 155 160

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

165 170 175

Ser Ser Gln Thr Ser Val Lys Cys Pro Ser

180 185

<210> 2

<211> 561

<212> DNA

<213> Syntrichia caninervis

<400> 2

atggttagta tcaggaaacg ccggcacggc ggactctctc caggtaaatc aattcagccc 60

aaagtctaca agcgcgccgc tgttacggca gaccaaccac caaagcagga agtgagattt 120

gcaccttcgc cgcctgtgcc cacctttcag gagctgcacc atcagttatt caatccccca 180

aaagcggaga aagagctgaa gaagaggaag cggcaacgga ggaagcacca ggagaatcag 240

gagccttgcg tgatgcgtgg agtgtacttc aagaacatga agtggcaagc tgctatcaag 300

gtggagaaga aacaagtgca cctaggcaca gtgaattccc agaaagaggc ggcgcatctc 360

tatgaccgag ccgcttactt gtgtggcaga gagcccaact ttgagctgac tgaagccgag 420

aagcaggagc tccagctgct gcagtgggag gattttctgg agcaaacacg gcaatcaatc 480

ttaagcaaaa agcgaaaacg tggcaataga gacgtcacgg aagcggcttc ttcgcagacg 540

tctgtaaagt gcccgtccta a 561

<210> 3

<211> 955

<212> DNA

<213> Syntrichia caninervis

<400> 3

atggttagta tcaggaaacg ccggcacggc ggactctctc caggtacttc tcacattaga 60

ttgattggga tgagcgctag tccgatgtta aattactatt gatgttgtta ttattcccta 120

gtacaggtaa atcaattcag cccaaagtct acaagcgcgc cgctgttacg gcagaccaac 180

caccaaagca ggtactactc atgattcttc tctccacctc aatcttatca ttcttccact 240

ccgaactcag cagtggaatg tttcaggaag tgagatttgc accttcgccg cctgtgccca 300

cctttcagga gctgcaccat cagttattca atcccccaaa aggttgtgag cttctctagc 360

attcattaag atgcaggttc tgtctgtctg tatcctgaca ctgtcggtgg atgtgcagcg 420

gagaaagagc tgaagaagag gaagcggcaa cggaggaagc accaggagaa tcaggagcct 480

tgcgtgatgc gtggagtgta cttcaagaac atgaagtggc aagctgctat caaggtggag 540

aagaaacaag tgcacctagg cacagtgaat tcccagaaag aggcggcgca tctctatgac 600

cggtatgttt ctcaccctgg aggtggtttc tattaccact tgacgtctct aaatcgtatt 660

gatttggaat tgttggaagc agagccgctt acttgtgtgg cagagagccc aactttgagc 720

tgactgaagc cgagaagcag gagctccagc tgctgcagtg ggaggatttt ctggagcaaa 780

cacggcaatc aatcttaagc aaaagtatgt ccaatctaat cactagaaaa gcactgtttt 840

tgctattgtt tttcttacat ttcaagattt gttggttgtt tcagagcgaa aacgtggcaa 900

tagagacgtc acggaagcgg cttcttcgca gacgtctgta aagtgcccgt cctaa 955

Claims (9)

1. A protein represented by any one of the following (A1) to (A2):

(A1) A protein with an amino acid sequence shown as SEQ ID No. 1;

(A2) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in (A1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as SEQ ID No.2 or SEQ ID No. 3.

4. An expression cassette comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

6. A recombinant bacterium comprising the nucleic acid molecule of claim 2 or 3.

7. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the expression cassette of claim 4 or the recombinant vector of claim 5 or the recombinant bacterium of claim 6 in any of the following:

p1, regulating and controlling the resistance of plants to verticillium wilt;

p2, regulating and controlling the resistance of the plant to the verticillium dahliae;

p3, regulating and controlling the active oxygen scavenging capacity of plants;

p4, regulating and controlling lignin content in plants;

P5, regulating and controlling salicylic acid content in plants;

p6, regulating and controlling the expression of key genes of salicylic acid synthase in plants;

p7, regulating and controlling the expression of lignin synthase key genes in plants;

p8, regulating and controlling the expression of genes related to classical disease course in plants;

p9, plant breeding;

in P1, the expression level of the protein in the plant is increased, and the resistance of the plant to verticillium wilt is increased;

in P1, the expression level of the protein in the plant is increased, and the resistance of the plant to Verticillium dahliae is increased;

in P3-P8, the expression level of the protein in the plant is increased, the plant has increased active oxygen scavenging capacity under stress of Verticillium dahliae, increased in vivo lignin content, increased in vivo salicylic acid synthase key gene expression level, increased in vivo lignin synthase key gene expression level, and/or increased in vivo classical disease course related gene expression level;

the key genes of the salicylic acid synthase are AtICS1 genes and/or AtEDS5 genes;

the lignin synthetase key genes are AtPAL2 genes and/or At4CL2 genes;

the classical disease course related genes are AtPR1 genes, atPR2 genes and/or AtPR5 genes; the plant is Arabidopsis thaliana.

8. A method of growing a plant having any one of the following traits, comprising the step of increasing the expression level of a protein according to claim 1 in a recipient plant;

(a1) Resistance to verticillium wilt is improved;

(a2) Resistance to verticillium dahliae is improved;

(a3) The active oxygen scavenging capacity is improved, the in-vivo lignin content is improved, the in-vivo salicylic acid synthase key gene expression amount is improved, the in-vivo lignin synthase key gene expression amount is improved, and/or the in-vivo classical disease course related gene expression amount is improved under the stress of verticillium dahliae;

the key genes of the salicylic acid synthase are AtICS1 genes and/or AtEDS5 genes;

the lignin synthetase key genes are AtPAL2 genes and/or At4CL2 genes;

the classical disease course related genes are AtPR1 genes, atPR2 genes and/or AtPR5 genes;

the plant is Arabidopsis thaliana.

9. A method of growing a transgenic plant having any one of the following traits, comprising the steps of: introducing the nucleic acid molecule of claim 2 or 3 into a recipient plant to obtain a transgenic plant; the transgenic plant has any one of the following traits compared with the recipient plant;

(a1) Resistance to verticillium wilt is improved;

(a2) Resistance to verticillium dahliae is improved;

(a3) The active oxygen scavenging capacity is improved, the in-vivo lignin content is improved, the in-vivo salicylic acid synthase key gene expression amount is improved, the in-vivo lignin synthase key gene expression amount is improved, and/or the in-vivo classical disease course related gene expression amount is improved under the stress of verticillium dahliae;

the key genes of the salicylic acid synthase are AtICS1 genes and/or AtEDS5 genes;

the lignin synthetase key genes are AtPAL2 genes and/or At4CL2 genes;

the classical disease course related genes are AtPR1 genes, atPR2 genes and/or AtPR5 genes;

the plant is Arabidopsis thaliana.