CN110041414B - Cold-resistant related protein derived from leymus chinensis and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a protein derived from leymus chinensis and related to cold resistance, and a coding gene and application thereof. The protein related to cold resistance provided by the invention is A1) or A2) or A3): A1) a protein with an amino acid sequence of sequence 1; A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 and has the same function and is derived from A1); A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). The protein related to cold resistance provided by the invention can regulate and control the cold stress resistance of plants, is induced by low temperature stress, can participate in the response of the leymus chinensis to the low temperature stress, improves the cold resistance of the plants, can be used for cultivating and identifying new cold-resistant varieties required by crops and important animal husbandry grasses, has higher application value, and has wide application prospects in the fields of agriculture, animal husbandry, green clean energy and the like.

Description

Cold-resistant related protein derived from leymus chinensis and encoding gene and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a protein derived from leymus chinensis and related to cold resistance, and a coding gene and application thereof.

Background

During the growth and development of plants, temperature plays a crucial role as an important environmental factor in the growth and development of plants. Many plants are exposed to low temperature damage, and the plants can be damaged by cold in different degrees under low temperature conditions, and even can die in severe cases. Low temperature stress is divided into cold injury (0-15 ℃) and freeze injury (<0 ℃). Many plants originating in tropical and subtropical regions are not cold-acclimated, and they are sensitive to low temperatures, and their survival ability is greatly reduced even by cold stress. Low temperature stress significantly reduces the yield of plants, particularly crops, with global losses of up to several billions of dollars per year due to low temperature injury.

In higher plants, the best studied to date is the CBF (CRT/DRE binding factor, also known as DREB) low temperature regulatory network. CBF is induced to be expressed under the condition of low temperature, is one of important transcription factors in a low temperature signal pathway, and the downstream target genes mainly regulated by the CBF relate to a series of biological changes related to low temperature, such as phosphoinositide metabolism and transcription, active oxygen scavenger, membrane transport, hormone metabolism, signal pathway and the like. Although some genes related to low temperature stress resistance have been isolated in previous studies, functional verification studies have been carried out using transgenic technology. However, in general, the genes which can be used for plant molecular breeding and have great application value are still extremely deficient. Firstly, the low-temperature regulation network of the plant is complex, and the research on the signal conduction and regulation mechanism of the plant is lack of breakthrough research results. Secondly, most of the researches on the cloning and the function of the genes related to the plant response stress mainly focus on model plants such as arabidopsis thaliana and rice or some important annual cultivated crops, the plants cannot tolerate the cold environment, the damage is caused at 0-12 ℃, and the freezing resistance is limited. This may limit the depth of the disclosure and study of the mechanisms of low temperature response in important perennial crops if only model plants are studied to reveal the mechanisms of low temperature response in plants. Under the long-term environmental pressure, perennial pasture forms resistance to extreme environment and can adapt to winter cold climate (-4 ℃ to-51 ℃). Therefore, the key resistance genes of the perennial pasture are cloned and functionally studied, and the high-quality cold-resistant gene resources are used for improving the freezing resistance of important crops and livestock pasture by using the transgenic technology, so that the transgenic freezing-resistant breeding process of agriculture and animal husbandry in China and the world is certainly accelerated.

In recent years, with the rapid development and wide application of high-throughput sequencing technology, the excavation of excellent stress-resistant plant gene resources is accelerated, and the high-throughput sequencing technology is utilized to perform sequencing analysis on plant materials subjected to specific stress treatment to obtain differential expression genes closely related to the specific stress treatment, so that a new way is developed for discovering new genes in stress response ways. With the continuous development and further functional verification of new low temperature resistant genes, the understanding of plant cold resistance mechanism is expanded. With the continuous progress of modern genetic engineering technology, the cold resistance of plants is improved by utilizing transgenic technology, and the method has important significance for maintaining high and stable yield and sustainable development of agriculture and animal husbandry in the future.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the cold resistance of plants.

In order to solve the technical problems, the invention firstly provides a protein which is derived from Leymus chinensis (Trin.) Tzvel) and has the function of improving the cold resistance of plants, and the protein is marked as Lcfin2, Lcfin2 is A1 or A2) or A3):

A1) a protein with an amino acid sequence of sequence 1;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 and has the same function and is derived from A1);

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 1 in the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The LcFIN2 protein of A2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The Lcfin2 protein in A2) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding LcfIN2 protein in A2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown at positions 90-821 of the sequence No. 2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. The DNA molecule shown in the 90 th-821 th sites of the sequence 2 encodes LcFIN2 protein shown in the sequence 1.

The invention also provides a biomaterial related to Lcfin2, wherein the biomaterial is any one of the following B1) to B14):

B1) a nucleic acid molecule encoding LcFIN 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B10) a transgenic plant cell line comprising the expression cassette of B2);

B11) transgenic plant tissue comprising the nucleic acid molecule of B1);

B12) transgenic plant tissue comprising the expression cassette of B2);

B13) a transgenic plant organ containing the nucleic acid molecule of B1);

B14) a transgenic plant organ containing the expression cassette according to B2).

In the above biomaterial, B1) the nucleic acid molecule may be B1), B2), B3), B4) or B5) as follows:

b1) the coding sequence is cDNA molecule or DNA molecule at 90-821 site of sequence 2 in the sequence table;

b2) the nucleotide sequence is cDNA molecule or DNA molecule at the 90 th to 821 th site of the sequence 2 in the sequence table;

b3) the nucleotide sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b4) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3) and encoding LcFIN 2;

b5) hybridizes with the nucleotide sequence defined by b1) or b2) or b3) under strict conditions and encodes a cDNA molecule or a genomic DNA molecule of LcFIN 2.

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.

The nucleotide sequence encoding Lcfin2 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of the isolated Lcfin2 protein of the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention as long as they encode the Lcfin2 protein and have the function of the Lcfin2 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding LcFIN2 protein (LcFIN2 gene expression cassette) described in B2) refers to DNA capable of expressing LcFIN2 protein in a host cell, and the DNA may include not only a promoter for promoting transcription of LcFIN2 gene, but also a terminator for terminating transcription of LcFIN2 gene. 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: constitutive promoter of cauliflower mosaic virus 35S: the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); fromChemically inducible promoter of tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can 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 terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (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).

The recombinant vector containing the Lcfin2 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be a vector pSN1301 or a pCAMBIA1302 vector.

The recombinant vector can be pSN1301-LcFIN2 or pCAMBIA1302-LcFIN2, the pSN1301-LcFIN2 is a recombinant vector obtained by replacing a DNA fragment between KpnI and SacI recognition sequences of pSN1301 with a DNA fragment shown in the 90 th-821 th position of a sequence 2, and the pSN1301-LcFIN2 can express LcFIN2 protein shown in the sequence 1; the pCAMBIA1302-LcfIN2 is a recombinant vector obtained by replacing a DNA fragment between Bgl II and Spe I recognition sequences of a pCAMBIA1302 vector with a DNA fragment shown in the 90 th-821 th position of a sequence 2, and the pCAMBIA1302-LcfIN2 can express a fusion protein of LcfIN2 fusion GFP.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium EHA 105.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides a plant cold-resistant product, which contains Lcfin2 or the biological material.

The plant cold-resistant product can adopt Lcfin2 or the biological material as an active ingredient, and can also adopt Lcfin2 or the biological material and other substances with plant cold-resistant function as active ingredients.

The invention also provides any one of the following applications of Lcfin2 or the biomaterial:

C1) the application in regulating and controlling the cold resistance of plants;

C2) the application in preparing the product for regulating and controlling the cold resistance of the plant;

C3) the application of the plant cold resistance is improved;

C4) the application in preparing products for improving the cold resistance of plants;

C5) application in cultivating cold resistance enhanced plants.

The present invention also provides a method for breeding a cold resistance-enhanced plant, the method comprising: increasing the activity of Lcfin2 in the target plant, increasing the content of Lcfin2 in the target plant or promoting the expression of a coding gene of Lcfin2 to obtain the cold-resistant plant with enhanced cold resistance compared with the target plant.

In the above method, the cold-resistant plant is a plant obtained by introducing a gene encoding LcFIN2 into the target plant.

In the above method, the gene encoding LcFIN2 may be the nucleic acid molecule.

In the above method, the coding gene of LcFIN2 may be modified as follows, and then introduced into a target plant, so as to achieve a better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the codon of the gene encoding LcFIN2 of the present invention can be changed to conform to the plant preference while maintaining the amino acid sequence thereof according to the preferred codon of the target plant; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

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

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The encoding gene of Lcfin2 can be introduced into a target plant by using a recombinant expression vector containing the encoding gene of Lcfin 2. The recombinant expression vector can be specifically pSN1301-LcfIN 2.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

Said cold-resistant plants are understood to comprise not only the first generation transgenic plants obtained by transforming the plants of interest with the gene coding for said LcFIN2, but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The cold-resistant plants include seeds, callus, whole plants and cells.

Primer pairs for amplifying the full length of the nucleic acid molecule or any fragment thereof are also within the scope of the present invention.

In one embodiment of the present invention, the primer pair is specifically as follows:

F：5’-ATGCCGTGTCTTGGCTCT-3’；

R：5’-TCATTTAATCAAAGTAGTGGTACAG-3’。

in the present invention, the plant may be a dicotyledonous plant (e.g., Arabidopsis) or a monocotyledonous plant; the plant of interest may be a dicotyledonous plant (e.g., Arabidopsis) or a monocotyledonous plant.

In the present invention, the above cold resistance may be embodied as a stress treatment at-8 ℃. The time of the treatment may be 12 hours.

The Lcfin2 provided by the invention can regulate and control the cold stress resistance of plants, is induced by low-temperature stress, can participate in the response of the leymus chinensis to the low-temperature stress, and improves the cold resistance of the plants, and the Lcfin2 and the coding gene thereof have important practical value for cultivating the leymus chinensis with improved cold resistance and other new plant varieties. Experiments show that after the transgenic and wild plants are domesticated at 4 ℃ for 3 days and then stressed at-8 ℃ for 12 hours, the survival rate of the wild arabidopsis thaliana plants is 20%, while the survival rate of the transgenic and wild plants with the Lcfin2 gene is about 80% and is 4 times of that of the wild arabidopsis thaliana. Therefore, the Lc5423 provided by the invention can be used for cultivation and identification of new cold-resistant varieties required by crops and important livestock grasses, and has high application value. The invention has wide application prospect in the fields of agriculture, animal husbandry, green clean energy and the like.

Drawings

FIG. 1 is a relative expression analysis of LcFIN2 gene under low temperature stress (4 ℃). The expression level of LcfIN2 gene at 0h was set to 1.

FIG. 2 shows the expression level analysis of LcfIN2 gene in different tissues. The expression level of LcFIN2 gene in the ear was set to 1.

FIG. 3 is a confocal/TIRF microscope of tobacco epidermal cells transformed with pCAMBIA1302 empty vector. Wherein A is tobacco leaf epidermal cells under fluorescence; b is tobacco leaf epidermal cells under the bright field; c is chloroplast autofluorescence and D is the superposition of A, B and C.

FIG. 4 is a laser confocal/TIRF microscopic image of tobacco epidermal cells transformed with recombinant expression vector pCAMBIA1302-Lc 5423. Wherein A is tobacco leaf epidermal cells under fluorescence; b is tobacco leaf epidermal cells under the bright field; c is chloroplast autofluorescence and D is the superposition of A, B and C.

FIG. 5 shows the results of enzyme digestion identification of the vector containing the recombinant expression vector pSN1301-Lc 5423. Wherein, the lane M is Trans2K Plus DNA Marker (Beijing Quanjin Biotechnology Co., Ltd.); lane 1 shows the restriction of pSN1301-Lc 5423.

FIG. 6 shows the PCR detection results of LcfIN2 transgenic Arabidopsis plants. Wherein lane WT represents a non-transgenic wild type Arabidopsis plant control and the remaining lanes represent different pSN1301-Lc5423 transgenic lines.

FIG. 7 shows the cold resistance test results of LcfIN2 transgenic Arabidopsis thaliana of T3 generation. Wherein a is the growth state before Wild Type (WT) and LcfIN2 gene-transferred Arabidopsis strains (Line1, Line2 and Line4) are not treated; b is the growth state of the wild type and the Lcfin2 gene transferred Arabidopsis thaliana strain which is recovered for 10 days after being treated.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

Example 1 preparation of Leymus chinensis Cold resistance-associated protein Lcfin2

This example provides a cold-resistant related protein from family 1 (described in "XX Li, SL Hou, Q Gao, et al. lcsain1, a novel salt-induced gene from sheet pgras, conjugates salt in transgenic Arabidopsis and rice plant and Cell Physiology,2013,54(7), 1172-1185", publicly available from the plant research institute of chinese academy of sciences) derived from Leymus chinensis (Trin.) Tzvel of variety, named LcFIN2, sequence 1 in the sequence listing, the full-length cDNA sequence of the protein in Leymus chinensis is shown as sequence 2 in the sequence listing, wherein sequence 2 encodes for fln 2 at positions 90-821.

Example 2 expression pattern and tissue specificity analysis of Lcfin2 Gene under stress conditions

Seedlings of family No. 1 of the variety Leymus chinensis growing normally for 4 weeks were subjected to low temperature stress (4 ℃). The stress treatment time was 0, 1, 3,5, 12, 24, 48 hours, respectively. And respectively extracting the total RNA of the processed Chinese wildrye seedlings, and analyzing the expression mode of the Lcfin2 gene under the condition of low-temperature stress by a qRT-PCR method. Wherein the primer sequences for amplifying the LcFIN2 gene are 5'-GCCGTCGTCATCTACTTCGTCT-3' (position 161-182 of the sequence 2) and 5'-GATTCTCACTCCTTGCTCCCC-3' (reverse complementary sequence with the position 308-329 of the sequence 2); taking Actin as an internal reference gene, wherein the primer sequences for amplifying the internal reference Actin are 5'-GTGCTTTCCCTCTATGCAAGTGGT-3' and 5'-CTGTTCTTGGCAGTCTCCAGCTC-3'; the two-step PCR amplification standard procedure was applied: 10s at 95 ℃; 5s at 95 ℃,20 s at 60 ℃ and 40 cycles; 10min at 72 ℃.

As shown in FIG. 1, the transcription level of Lcfin2 gene is induced by low temperature stress, and the expression level of Lcfin2 gene increases rapidly with the increase of low temperature treatment time.

Total RNA of five tissues including ears, leaves, leaf sheaths, stems and roots is respectively extracted by taking the family No. 1 of the 2-year-old Leymus chinensis variety as an experimental material, and the tissue specific expression mode of the Lcfin2 gene is analyzed by utilizing qRT-PCR (see the above for specific operation).

As shown in FIG. 2, the expression of Lcfin2 gene was different in five tissues of tassel, leaf sheath, stem and root of Leymus chinensis, with the highest expression level in leaf.

Example 3 subcellular localization of LcfIN2 Gene

The DNA fragment between the Bgl II and Spe I recognition sequences of the pCAMBIA1302 vector is replaced by LcfIN2 gene shown in the 90 th to 821 th positions of the sequence 2, and the obtained recombinant expression vector with correct sequence is named as pCAMBIA1302-LcfIN 2. pCAMBIA1302-LcfIN2 can express LcfIN2 fusion GFP fusion protein, and the expression of the fusion protein is started by 35S.

The recombinant expression vector pCAMBIA1302-LcfIN2 is introduced into the Agrobacterium tumefaciens EHA105 by a freeze-thaw method, and is transferred into young tobacco leaf cells by a vein injection method, and meanwhile, the tobacco epidermal cells transferred into the pCAMBIA1302 empty vector are used as a control. The transformed tobacco was grown at 23 ℃ for 2-3 days and observed under a confocal/TIRF laser microscope (Leica TCS SP5) and photographed.

The results are shown in FIG. 3 (tobacco transformed with pCAMBIA1302 empty vector) and FIG. 4 (tobacco transformed with recombinant expression vector pCAMBIA1302-LcfIN 2): the protein expressed in the transgenic cell transferred into the vector pCAMBIA1302 is distributed in the whole cell; the protein expressed in the transgenic cells transferred into the recombinant expression vector pCAMBIA1302-Lcfin2 is specifically localized in chloroplasts. This result indicates that the Lcfin2 protein is expressed in chloroplasts of tobacco epidermal cells.

Example 4 acquisition of LcfIN2 Gene transferred Arabidopsis thaliana and Cold resistance test thereof

Firstly, construction of expression vector pSN1301-LcfIN2

The Lcfin2 gene shown at positions 90-821 of the sequence 2 of the coding Lcfin2 is obtained by amplifying cDNA of the family 1 in the variety of leymus chinensis by using the following primers:

F：5’-ATGCCGTGTCTTGGCTCT-3’；

R：5’-TCATTTAATCAAAGTAGTGGTACAG-3’。

the DNA fragment between the KpnI and SacI recognition sequences of the plant expression vector pSN1301(Biovector plasmid vector cell culture Collection) was replaced with LcfIN2 gene indicated at positions 90-821 of SEQ ID NO. 2, and the resulting recombinant expression vector with the correct sequence was named pSN1301-LcfIN 2.

The constructed vector was subjected to enzyme digestion and sequencing (FIG. 5). Wherein, Lane M is the DNA molecular weight standard of DL2000DNA molecular weight standard (Beijing Quanji Biotechnology Co., Ltd.), and Lane 1 is the pSN1301-Lcfin2 plasmid band. And (3) introducing the recombinant vector into the agrobacterium tumefaciens EHA105 by a freeze-thawing method to obtain the recombinant agrobacterium tumefaciens EHA105/pSN1301-Lcfin 2.

Second, obtaining of Lcfin2 transgenic Arabidopsis

The recombinant vector was introduced into Agrobacterium tumefaciens EHA105 by freeze-thawing and the above-obtained recombinant Agrobacterium EHA105/pSN1301-LcFIN2 was transformed into Arabidopsis thaliana variety Columbia (Col-0) (Carolina Biological Supply Company, Arabidopsis: Columbia (Col-0) Seed, Cat.: 177600) by the method of Agrobacterium inflorescence invasion (see: Bechtold N et al, (1993) In planta A grobacterium-medium transfer by infiltration of culture of wild Arabidopsis thaliana plant C.R.Acad.Sci.316: 1194. sup. 1199). Agrobacterium transformed with pSN1301 empty vector was used as a control. After transformation, hygromycin (final content of step 1 of original document: selection with hygromycin, here kanamycin, please check) resistance selection was performed, and seeds of transgenic Arabidopsis thaliana with hygromycin resistance (T1 generation) were collected and cultured on a hygromycin-containing plate. And thirdly, obtaining a transgenic seedling with hygromycin resistance, namely an arabidopsis plant transformed into pSN1301-LcfIN2, through hygromycin resistance screening. Positive lines were screened by PCR identification (fig. 6) using primers F: 5 '-GCCGTCGTCATCTACTTCGTCT 3'; r: 5'-GATTCTCACTCCTTGCTCCCC-3' are provided. Line1, 2, 4, 6 and 7 are positive strains, and 3 homozygote transgenic LcfIN2 gene Arabidopsis strains (T3 generation) are selected for cold resistance test.

Third, cold resistance detection of LcfIN2 gene transferred Arabidopsis thaliana in T3 generation

3 lines of T3 generation homozygotes identified as positive by step two were selected for cold resistance testing (Table 1) and used as controls in Columbia (Col-0) Wild Type (WT) without transgene and Arabidopsis with pSN1301 empty vector. The specific method comprises the following steps:

wild arabidopsis thaliana which normally grows for 2-3 weeks in soil, arabidopsis thaliana which is transferred with pSN1301 empty vector and arabidopsis thaliana which is transferred with LcfIN2 gene (Col-0/pSN1301-LcfIN2) are transferred into a low-temperature incubator with a set specific temperature (4 ℃) to be subjected to low-temperature acclimation for 3 days, then the arabidopsis thaliana is transferred into an incubator with the temperature of-8 ℃ overnight (12 hours) to be subjected to freezing treatment, then the arabidopsis thaliana is taken out, the arabidopsis thaliana is restored to grow for 7-10 days under the normal culture condition of 22 ℃, the phenotype is observed, and the survival rate of each group of plants is counted.

The growth state before the Wild Type (WT) and LcfIN2 transgenic Arabidopsis lines (Line1, Line2 and Line4) were not treated and the growth state after cold-resistant treatment are shown in FIG. 7. The survival rate statistical result shows that the survival rate of the wild type arabidopsis thaliana plant is only 20% after the overnight cold stress at-8 ℃, while the survival rates of the three Lcfin2 gene-transferred lines are respectively 75%, 70% and 80%, which are about 4 times of the survival rate of the wild type arabidopsis thaliana, and the survival rate of the arabidopsis thaliana transferred with the pSN1301 empty vector is not obviously different from that of the wild type arabidopsis thaliana. This indicates that the LcfIN2 gene-transferred strain has a significantly improved cold resistance as compared with WT (wild type) Arabidopsis thaliana used as a blank control.

TABLE 1 survival (%) of LcfIN2 transgenic Arabidopsis thaliana under cold stress (-8 ℃ C.) for T3 generations

Arabidopsis thaliana	Repetition of 1	Repetition 2	Repetition of 3	Repetition of 4	Mean value of
						WT	0	20	40	20	20
Line1	80	80	40	100	75
						Line2	20	60	100	100	70
Line4	60	100	80	80	80

<110> institute of plant of Chinese academy of sciences

<120> a protein derived from leymus chinensis and related to cold resistance, and coding gene and application thereof

<210> 1

<211> 243

<212> PRT

<213> Leymus chinensis

<400> 1

Met Pro Cys Leu Gly Ser Met Pro Leu Gly Phe Gln Pro Pro Glu Leu

1 5 10 15

Ala Ser Ser Ser Arg Trp Thr Leu Pro Ser Ser Ser Thr Ser Ser Pro

20 25 30

Trp Trp Arg Lys Arg Glu Gly Ala Gly Ser Ala Gly Ile Pro Pro Asn

35 40 45

Lys Leu Ala Ala Val Pro Arg Val Glu Ala Asp Leu Val Leu Phe Pro

50 55 60

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

65 70 75 80

His His Gly Phe Val Pro Trp Arg Cys Ala Ser Pro Asp Pro Ser Phe

85 90 95

Arg Arg Arg Leu Tyr Arg Pro Leu Ile Trp Arg Arg Thr Ser Gly Val

100 105 110

Asp Leu Glu Met Asp Asp Gly Asp His Glu Leu Asn Arg Arg Trp Phe

115 120 125

Gln Arg Ser His Gly Ala His Gly Gly Trp Pro Val Ser Ser His Asp

130 135 140

Pro Ser His Leu Arg Ala Glu Trp Arg Pro Ser Ser Phe Leu Leu Ala

145 150 155 160

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

165 170 175

Ser Tyr Cys Gly Gly Leu Ala Gly Pro Ser Gly Pro Ser Pro Ala Pro

180 185 190

Ala Lys Ile Ala Ser Met Ala Lys Gln Phe Gly Pro Asp Cys Val Phe

195 200 205

His Phe Met Phe Gly Val Leu Phe Val Arg Ser Arg Val Trp Ser Val

210 215 220

Ile Phe Ser Phe Phe Arg Val Leu Cys Val Met Leu Cys Thr Thr Thr

225 230 235 240

Leu Ile Lys

<210> 2

<211> 880

<212> DNA

<213> Leymus chinensis

<400> 2

caggacaggt aggagcccgg gatctgcttg tgtatattag cttagctgtc gtggttgtta 60

gtcttggcct tgtgccgtgt cttggctcta tgccgtgtct tggctctatg ccgttgggat 120

tccaaccacc ggagcttgcg agcagcagcc ggtggacgct gccgtcgtca tctacttcgt 180

ctccatggtg gaggaaaagg gagggcgccg gatccgcagg tatcccccct aataagctcg 240

ctgcggtgcc tcgtgttgaa gccgatctgg tgctctttcc tctctctctc cgcagtcgtg 300

gtgacggtgg ggagcaagga gtgagaatcc accacggctt cgtcccttgg cgctgtgcct 360

cgccggatcc cagcttccgg cggcggctgt atcgtcccct gatctggaga aggacctccg 420

gcgtcgatct ggagatggac gatggggatc atgagcttaa ccgacggtgg ttccagagga 480

gccatggcgc ccacggtggc tggccggttt cctctcacga tccgtcgcat cttcgggccg 540

aatggcggcc atcttcattc ctcctggcat gtgtgcctaa tgggaggcag ttctgcacta 600

agatggagtc tgcagcttca tactgcggag gtctcgccgg accaagtggt ccgtccccgg 660

cgccggcgaa gattgcatcc atggcgaagc agtttggacc cgattgcgtt ttccacttta 720

tgttcggggt ccttttcgta agatccaggg tctggtctgt aattttcagt ttctttaggg 780

tcctgtgtgt gatgctctgt accactactt tgattaaatg aagcatctgg ggccttcggg 840

ctcctacctg tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa 880

Claims (8)

1. A protein, as in a1) or a 2):

A1) a protein with an amino acid sequence of sequence 1;

A2) a1) at the N-terminus or/and the C-terminus.

2. The biomaterial related to the protein of claim 1, which is any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

3. The biomaterial of claim 2, wherein: B1) the nucleic acid molecule is b1), b2), b3) or b4) as follows:

b1) the coding sequence is cDNA molecule or DNA molecule at 90-821 site of sequence 2 in the sequence table;

b2) the nucleotide sequence is cDNA molecule or DNA molecule at the 90 th to 821 th site of the sequence 2 in the sequence table;

b3) the nucleotide sequence is a DNA molecule of a sequence 2 in a sequence table;

b4) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes a protein as claimed in claim 1.

4. Plant cold-resistant product comprising a protein according to claim 1 or a biological material according to claim 2 or 3.

5. Use of the protein of claim 1 or the biomaterial of claim 2 or 3 for any of the following applications:

C1) the application in regulating and controlling the cold resistance of arabidopsis thaliana;

C2) the application in preparing the product for regulating and controlling the cold resistance of arabidopsis thaliana;

C3) the application in improving the cold resistance of arabidopsis thaliana;

C4) the application in preparing products for improving the cold resistance of arabidopsis thaliana;

C5) the application in cultivating cold resistance enhanced arabidopsis thaliana.

6. A method of growing cold resistance-enhanced plants comprising: increasing the activity of the protein of claim 1 in a plant of interest, increasing the content of the protein of claim 1 in a plant of interest, or promoting the expression of a gene encoding the protein of claim 1, resulting in a cold-resistant plant with increased cold resistance compared to the plant of interest.

7. The method of claim 6, wherein: the cold-resistant plant is a plant obtained by introducing a gene encoding the protein of claim 1 into the plant of interest.

8. The method of claim 7, wherein: the gene encoding the protein according to claim 1 is the nucleic acid molecule according to B1) in claim 3.

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