CN117946233A - Environment stress protein CsFKBP and application thereof - Google Patents

Abstract

The invention discloses an environment stress protein CsFKBP which is obtained from tea tree cloud anti-10 (C.sinensis var. Assamica cv. Yunkang10) and the amino acid sequence of which comprises a sequence shown as SEQ ID NO. 1. The invention also provides application of the environmental stress protein CsFKBP to improvement of low-temperature resistance of plants. The invention detects the change of the expression quantity of the cloud antigen No. 10 CsFKBP gene under the low-temperature treatment condition by using a real-time fluorescence quantitative qRT-PCR method, and proves that CsFKBP has obvious forward response condition for low-temperature stress. By transforming CsFKBP53 into arabidopsis, it was verified that overexpression of the CsFKBP53 gene enhances the cold resistance of arabidopsis plants.

Description

Environment stress protein CsFKBP and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an environment stress protein CsFKBP and application thereof.

Background

Tea tree (CAMELLIA SINENSIS var. Assamica) belongs to the genus Camellia (Camellia) of the family Camellia (Theaceae), and research and investigation prove that tea tree originates in southwest area of China, and up to now, more than 3000 years of planting and cultivating history exist. The ancient tea tree refers to an artificially cultivated and wild individual plant of the ancient tea tree with the tree age of more than one hundred years and a community thereof. The old tea tree has longer growth age and deeper rhizome, is more beneficial to absorbing nutrient in deep soil and converting the nutrient into flavor substances, has higher content of tea polyphenol, theanine and other content in fresh leaves of the old tea tree, and ensures that the flavor of the old tea tree product is more fresh and sweet and faster; meanwhile, the wild ancient tea tree resources are scarce and nonrenewable, so that the ancient tea tree has higher economic value. The ancient tea trees are in a growth environment with less human intervention for a long time, so that a plurality of important genes with obvious resistance to biotic stress and abiotic stress are reserved under natural growth conditions, and the excellent resistance genes can provide basis for ascertaining the genomic evolution of the tea trees and the excavation of excellent trait genes, thereby being a precious treasury for the future tea tree germplasm resource innovation and the tea tree variety improvement in China. The genetic evolutionary relationship of the ancient tea tree resources is ascertained, and the excellent resistance genes are screened out, so that the current large-scale planted tea tree varieties have more remarkable stress resistance and generate larger economic benefits, and the method is a hot research direction for protecting and utilizing the ancient tea tree resources.

Temperature is an important factor affecting the growth state of plants. The growth of tea trees extremely depends on proper temperature conditions, the optimal growth temperature range is 20-25 ℃, and the normal growth of tea trees can be limited by long-term high-temperature and low-temperature environments. The temperature of spring bud germination in early growth of tea trees is about 10 ℃, and the temperature below the temperature can be influenced by low-temperature stress. Low temperature stress affects plant yield and quality, mainly by inhibiting plant root development, resulting in plant growth retardation. The tea tree can damage a membrane system when being subjected to low-temperature stress, membrane lipid of a cell membrane is gelled at low temperature, the fluidity and permeability of the cell membrane are reduced, and finally, the cell function of the tea tree is disturbed. The degree of freeze damage of tea trees is positively correlated with the total water content and free water content of leaf cells, and is negatively correlated with the bound water content and the ratio of bound water to free water. After low temperature stress treatment, tea trees typically improve cell membrane permeability by increasing superoxide dismutase (SOD), catalase (CAT) and peroxidase (ROD) activity to cope with low temperature stress damage.

At present, most of the means for coping with low-temperature stress of tea trees in winter and spring are physical and chemical measures, for example: spreading grass in a tea garden, fumigating and smoking in the tea garden, covering for freezing prevention, ditching and watering in the tea garden, pruning and culturing tree crowns, adopting an antifreezing fan, spraying seaweed fertilizer and phosphoric acid mixed solution on leaf surfaces, spraying melatonin, spraying salicylic acid and chitosan mixed antifreezing solution, carrying out frost damage refined early warning by utilizing remote sensing and the like. These measures all require a great deal of labor cost, and related measures not only damage the tea garden environment and the tea quality, but also can lead to final results far from expectations due to the influence of various uncontrollable factors. The cross breeding method is widely applied in the tea tree germplasm resource cultivation process, however, the cross breeding method needs to select excellent character single plants continuously, so that the experimental scale is continuously enlarged, the homozygous varieties are relatively difficult to obtain, and the like, so that the success rate is low and the breeding time is too long. The molecular breeding technology can improve the breeding efficiency and accuracy, avoid the character detection of parent and offspring, reduce the breeding scale through early generation selection, accelerate the plant breeding process and improve the breeding efficiency. Compared with the conventional hybridization breeding method, the blindness of breeding selection is reduced, and the identification of recessive alleles and the overcoming of phenotype identification difficulties are facilitated.

The plant molecular breeding technology mainly comprises molecular marker assisted breeding and transgenic technology, and aims to mainly improve crop quality and enhance stress resistance. In recent years, genome sequencing results of Yunkang No. 10, shucha early, biyun and Longjing 43 are sequentially published, so that identification of cold-resistant genes of tea trees and molecular breeding research are possible. The identification of the cold-resistant character genes of the tea trees and the analysis of the cold-resistant molecular mechanism and the analysis of the cold-resistant gene functions are very important for the cultivation of cold-resistant varieties of the tea trees. FKBP (FK 506 binding protein) family genes play an important role in plant growth and development, regulation of abiotic stress and the like. The study by Breiman et al found that ROF1 (AtFKBP) and ROF2 (AtFKBP 65) respond to high temperature stress by regulating the expression of small heat shock proteins (sHSPs) associated with high temperature stress restoration, and furthermore AtFKBP-1 a was highly expressed in all plant tissues and the expression level was induced by high temperature and drought stress. However, the specific functions of FKBP family genes in tea trees are still to be further studied.

Disclosure of Invention

The invention provides an environment stress protein CsFKBP and application thereof, aiming at the defects of the prior art. Experiments prove that the environment stress protein CsFKBP is applied to improving the low-temperature resistance of plants, especially tea trees.

The specific technical scheme is as follows:

one of the purposes of the present invention is to provide an environmental stress protein CsFKBP, the amino acid sequence of which comprises a sequence as shown in SEQ ID NO. 1;

further, the amino acid sequence of the environmental stress protein CsFKBP is shown as SEQ ID NO. 1.

Specifically, the above-mentioned environmental stress protein CsFKBP is obtained from tea tree (CAMELLIA SINENSIS var. Assamica) which is cloud antigen number 10 (c.sinensis var. Assamica cv.yunkang10).

Through the retrieval and verification of the whole genome data of the cloud antigen No. 10 tea tree, 21 tea tree CsFKBP gene family members are obtained in total. The physicochemical properties of 21 tea trees CsFKBPs were analyzed using bioinformatics websites. The result shows that the protein coded by the family consists of 100-659 amino acids, the CDS length is 306-2429 bp, the protein molecular weight is 10.81-72.43 kDa, and the great difference between the proteins of the tea tree CsFKBP family is proved. The theoretical PI value is between 4.52 and 10.44, wherein 57 percent of the tea CsFKBP family protein members are alkaline.

The real-time fluorescence quantitative qRT-PCR method is used for detecting the change of the expression quantity of 21 cloud anti No. 10 FKBP family genes under the low-temperature treatment condition, and the expression quantity of 4 genes shows an increasing trend. Wherein, csFKBP53 expression quantity is increased more obviously.

The second object of the present invention is to provide the application of the above-mentioned environmental stress protein CsFKBP to the improvement of plant low temperature resistance.

Further, the plant is tea tree.

The invention detects the variation of the expression quantity of the cloud antigen No. 10 CsFKBP gene under the low-temperature treatment condition by using a real-time fluorescence quantitative qRT-PCR method. The expression level of CsFKBP53 showed a general trend of increase in the treatment at 10℃and increased significantly after 24H, and increased significantly to peak after 72 hours treatment, approximately 4 times that of the untreated control group. In the course of the treatment at 4℃the expression level of CsFKBP a was also shown to increase relative to the control. The change of the expression level after 72 hours of treatment at 4 ℃ is less obvious than that of the expression level after 10 ℃ treatment, but the expression level is still increased by 2 times relative to the control expression level, which shows that CsFKBP53 has obvious forward response to low-temperature stress.

Further, the plant is Arabidopsis thaliana.

Through cloning CsFKBP53 and constructing a P30-CsFKBP53-3MYC heterologous expression vector, it is verified that the low temperature tolerance of transgenic Arabidopsis plants is enhanced by over-expression CsFKBP. Under the low temperature stress treatment, the CsFKBP53 over-expression strain has the growth obviously superior to that of the low temperature treatment control group, and is particularly characterized in that the root length is longer and the leaf purple degree is obviously reduced. In addition, the detection results of POD, SOD and CAT show that after the over-expression CsFKBP.sup.53 Arabidopsis is treated for 24 hours at the low temperature of 4 ℃, the activity of the physiological index is obviously improved, which indicates that the over-expression CsFKBP53 enhances the activity of the POD, SOD and CAT in the transgenic Arabidopsis, improves the permeability of cell membranes, and finally leads to the enhancement of the cold resistance of the transgenic plant.

The beneficial effects of the invention are as follows:

The invention screens a gene CsFKBP for responding to low temperature stress from cloud antigen No. 10 (C.sinesis var. Assamica cv. Yunkang10), and has obvious forward response to low temperature stress. By transforming CsFKBP53 into arabidopsis, it was verified that overexpression of the CsFKBP53 gene enhances the cold resistance of arabidopsis plants. The invention provides a reference for promoting the reasonable protection of the ancient tea trees and the innovation of cold-resistant tea tree germplasm resources.

Drawings

FIG. 1 shows the expression analysis (the ordinate indicates the expression level) of CsFKBP gene in Yunkang No. 10 under different low-temperature treatments;

FIG. 2 shows CsFKBP shows the expression analysis of Yunkang No. 10 different tissues and different tea tree varieties (the ordinate shows the expression level; FIG. 2A the abscissa shows that Shoot is bud, root is Root, young Stem is tender Stem, young Leaf is new Leaf, old Leaf is Old Leaf, flower is Flower, bud is bud; FIG. 2B the abscissa shows that YK10 shows Yunkang No. 10, YK8 shows Yunkang No. 8, YK9 shows Yunkang No. 9, ZJ shows Zijuan, TGY shows Tieguanyin, SLX shows Shi Li Xiang, NN shows that Oryza big Leaf tea, LJ43 shows Longjing 43);

FIG. 3 is a DNA verification electrophoresis chart of a positive plant of transgenic Arabidopsis thaliana (lane 1 is 2000bp marker, lanes 2-10 are transgenic Arabidopsis thaliana; lane WT is wild type control WT);

FIG. 4 shows the expression level analysis of CsFKBP transgenic Arabidopsis plants (the ordinate shows the expression level, the abscissa shows wild type plants, and OE-1 to OE-9 show transgenic plants);

FIG. 5 is a CsFKBP transgenic Arabidopsis plant compared to a wild type phenotype;

FIG. 6 is a comparison of CsFKBP transgenic Arabidopsis plants with wild type root length (root length on the ordinate);

FIG. 7 shows the results of activity detection of CsFKBP transgenic Arabidopsis plants and wild type POD, SOD, CAT (POD activity on the ordinate in FIG. 7A; SOD activity on the ordinate in FIG. 7B; CAT activity on the ordinate in FIG. 7C).

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.

In the specific embodiment, the method comprises the following steps: the experimental materials to be tested are two-year-old tea tree variety cloud antigen No. 10 (C.sinensis var. Assamica cv. Yunkang10) and Zijuan (C.sinensis var. Assamica cv. Zijuan) which are planted in a greenhouse of the agricultural college of Yunnan university; 6 different kinds of tea tree (Yunkang No. 8, yunkang No. 9, south glutinous large leaf tea, longjing 43, shilixiang and Tieguanyin) experimental materials are taken from tea planting bases of the university of Yunnan agricultural, longchun Pu tea college; arabidopsis thaliana (Arabidopsis thaliana) T0 generation seed.

In specific embodiments, the amino acid sequence of the environmental stress protein CsFKBP is shown as SEQ ID NO. 1.

Example 1

The real-time fluorescence quantitative qRT-PCR method is used for detecting the change of the expression quantity of CsFKBP genes in cloud antigen No. 10 under the low-temperature treatment condition, and the method and the result are as follows:

S1, treating an experimental material:

The method comprises the steps of selecting tea seedlings of the two-year-old cloud-resistant No. 10, respectively placing the tea seedlings in a low-temperature incubator to be subjected to low-temperature treatment at 4 ℃ and 10 ℃, wherein the culture conditions are that illumination is 16H/8H (day/night), the illumination intensity is set to 240 mu mol m ² s^-1, the relative humidity is kept at 75% +/-5%, simultaneously taking tea trees grown under the normal condition of 25 ℃ as a control, respectively collecting old leaf tissues after 0H, 3H, 6H, 9H, 12H, 24H, 48H and 72H, and sampling and setting three biological repetitions at each time point.

And respectively taking the bud, root, tender stem, flower, bud, new leaf and old leaf tissues of Yunanti No. 10 in a normal growth state, and using the tissues for space-time expression of CsFKBP genes. And carrying out biological repetition for 3 times on each sample by using old leaf tissues of Yunanti No. 8, yunanti No. 9, yunanti No. 10, nannuo big leaf tea, longjing 43, shilixiang, tieguanyin and Zijuan tea tree in a normal growth state, and carrying out expression quantity verification of CsFKBP genes among different varieties.

S2, analysis of low-temperature treatment expression patterns of cloud antigen No. 10 CsFKBP gene:

Extracting RNA of corresponding tissues of the tea tree after low-temperature treatment by using a total plant RNA extraction kit (DP 441) of the biochemical polysaccharide polyphenol of the root of the Chinese flowering plant; using total RNA as template Carrying out reverse transcription on the cDNA by using a PRIMESCRIPT IV-st strand cDNASynthesis Mix kit, finishing reverse transcription by using a PCR instrument, cooling on ice after finishing the reaction, measuring an OD value, and storing in a refrigerator at-20 ℃ for later use; real-time fluorescent quantitative primers were designed using PRIMER PREMIER software, the primer DNA sequences are shown in Table 1, and the internal reference gene selected was CsActin.

TABLE 1 fluorescent quantitative primers

According to Takara TBThe fluorescent quantitative reaction liquid is prepared by the explanation flow of Premix Ex Taq II and reacts. The analysis of the significance difference is completed by using SPSS22.0 software and the graph Pad 8 software is used for drawing an expression level graph by adopting a2 ^-ΔΔCt method for the treatment of the fluorescence quantitative experiment result.

The expression analysis of CsFKBP gene in Yunkang No. 10 under different low temperature treatment is shown in FIG. 1 (FIG. 1A is the expression analysis under 10 ℃ treatment, FIG. 1B is the expression analysis under 4 ℃ treatment), the expression analysis of CsFKBP53 in different tissues and different tea tree varieties of Yunkang No. 10 is shown in FIG. 2 (FIG. 2A is the expression analysis in different tissues, and FIG. 2B is the expression analysis in different varieties). In fig. 1 and 2, vertical lines indicate standard errors. Significance analysis SPSS22 was used for one-way analysis of variance with different lower case letters indicating significant differences (p < 0.05).

Referring to FIG. 1, the amount of CsFKBP expressed generally showed an increasing trend during the 10℃treatment, the difference was not apparent in the 3H treatment compared to the 0H treatment control, but the amount of expression was significantly increased after 24H. The CsFKBP gene expression increased by 1.5-fold after 6 hours of treatment at 10℃and the expression increased significantly to peak after 72 hours of treatment, approximately 4-fold that of the untreated control group. In the treatment at 4℃the amount of CsFKBP53 expressed was also increasing relative to the control, with the highest levels at 24H, 48H and 72H. The change of the expression level after 72 hours of treatment at 4 ℃ is less obvious than that of the expression level after 10 ℃ treatment, but the expression level is still increased by 2 times relative to the control expression level, which shows that CsFKBP53 has obvious forward response to low-temperature stress.

Referring to fig. 2, csfkbp53 has the highest expression level in the root and old leaf tissues of the yunzhan et No. 10, has high tissue expression specificity, and has the expression level of about 3 times that of the tender stem and new leaf tissues and about 5 times that of flowers and buds; compared with different tea tree varieties, the high cold-resistant variety Shilixiang, yunanti No. 8 and Yunanti No. 9 have the highest expression quantity, and CsFKBP gene expression is not detected in Tieguanyin.

Example 2

And verifying the cold-resistant function of the cloud anti-No. 10 tea tree environment stress protein CsFKBP with the base acid sequence shown as SEQ ID NO. 1. The experimental method and results are as follows:

S1.csfkbp53 gene cloning:

The full-length gene sequence and CDS sequence of tea CsFKBP (gene ID: CSASTRANS 084696) were downloaded from TAIR website, and the amplification primers were designed using Oligo 7 software using the cDNA of the tissue of the old leaf of Zijuan and Yunanti No. 10 as templates, respectively. The primer design results are shown in Table 2.

TABLE 2 amplification primers

Reference to the reaction SystemMax DNAPolymerase reagents illustrate the method. After the amplification reaction is completed, the target band is recovered by agarose gel electrophoresis with concentration of 1.2%, the gel recovery product is connected with pEASY-Blunt cloning vector, and the sequence is sequenced after the E.coli is transformed. Sequencing results show that CDS length of the cloud antigen No. 10 CsFKBP gene is 2135bp, 651 amino acids are encoded, the amino acid sequence is shown as SEQ ID No.1, and 202 amino acids are deleted compared with the original sequence. The amino acid sequence of the coding gene of Zijuan CsFKBP is shown as SEQ ID NO. 8.

After the results of the sequence of the cloud antigen No. 10 and the Zijuan CsFKBP gene are converted into protein sequences, the two sequences and the downloaded original sequence are compared by DNAMAN software. Sequence comparison results show that the similarity between the cloud antigen No. 10 CsFKBP and the Zijuan, and the original protein sequence is 97.39% and 78.35%, respectively.

S2, treating an experimental material:

The T0 generation Arabidopsis seeds are soaked in 75% ethanol for 5min, then soaked in 5% sodium hypochlorite for 15min, rinsed with sterile water for 3 times, the sterilized seeds are sown on a 1/2MS solid culture medium, and the seeds are cultured under normal conditions after 24H of low temperature treatment at 4 ℃.

S3, constructing a CsFKBP gene plant expression vector:

the cloud antigen No. 10 tea CsFKBP gene and the P30-3MYC empty vector are subjected to enzyme digestion by a double enzyme digestion method, then a flat end connection reaction is carried out by using T4 ligase, and amplification verification is carried out after connection is completed, wherein the verification primers are shown in Table 3.

TABLE 3 double cleavage ligation verified primer design

After cloning clear target band, connecting the cloning product with pEASY-Blunt vector, sequencing, picking single colony shaking bacteria to extract plasmid with correct sequencing result, and storing in-20 deg.c refrigerator.

S4, agrobacterium is transformed:

Taking 100 mu L of agrobacterium strain GV3101 stored at-80 ℃, adding 1 mu g of the plasmid obtained in the step S2 when the agrobacterium strain GV3101 is in an ice water mixing state, standing on ice for 5min, placing in liquid nitrogen for 5min, placing in a water bath kettle at 37 ℃ for 5min and carrying out ice bath for 5min; then 700 mu L of YEP liquid culture medium without antibiotics is added, shake culture is carried out for 2H at 28 ℃, and bacterial liquid is collected after centrifugation at 6000rpm for 1min on a high-speed centrifuge; finally, 100 mu L of fungus solution is taken to resuspend fungus blocks, the fungus blocks are coated on a YEP solid culture medium containing 50mg/mL spectinomycin, and the fungus blocks are inversely cultured for 48H in a 28 ℃ incubator.

S5, infecting wild arabidopsis plants:

Accelerating germination on a 1/2MS culture medium until arabidopsis seeds growing out of a third leaf grow out, shearing off the main stems of the arabidopsis seeds from the bottom after the main stems grow out, and promoting growth of lateral branches so as to increase inflorescences and promote transformation during infection; 15mL of liquid YEP culture medium containing antibiotics (spectinomycin, 70 mug/mL; rifampin, 100 mug/mL) is added into a 100mL conical flask, and a single colony with correct sequencing result is picked up by a gun head to shake until the concentration reaches 1.6-2.0; centrifuging activated bacterial liquid at 5000rpm for 5min at room temperature, discarding supernatant, adding 20mL of sterilized ddH ₂ O, sucking and beating uniformly mixed bacterial blocks by using a pipetting gun, centrifuging, and retaining agrobacterium precipitation; preparing 300mL of dye-dip solution (sucrose, 15g; surfactant Silwet-77, 150 mu L), and adjusting the concentration of the bacterial solution and the buffer solution to 0.6-0.8; after the concentration is regulated to be normal, the wild arabidopsis inflorescence is soaked in the infection liquid for about 30s, placed in a light incubator for 22H in dark light at 22 ℃, and then taken out to be placed in a long-day incubator for normal culture.

S6, screening positive transgenic arabidopsis plants:

The arabidopsis seeds are disinfected and then sown on a 1/2MS solid medium containing kanamycin for screening, plants with good growth vigor are selected, leaf tissues are taken after the plants grow for two weeks, DNA is extracted by a CTAB method, and CsFKBP genes are cloned and verified according to the method of the step S1.

In the screening of the positive transgenic arabidopsis plants, 9 arabidopsis plants with normal growth vigor are obtained in total, DNA extracted from leaf tissues of the arabidopsis plants is subjected to PCR amplification by CsFKBP gene specific primers, and target strips are detected by 1.2% agarose gel electrophoresis of amplified products, and the result is shown in figure 3, and shows that the target strips of the 9 positive plant DNA are single and clear, and the strip length is correct, so that the successful transfer of the target gene CsFKBP53 into arabidopsis is proved.

In order to confirm the expression level of CsFKBP in transgenic Arabidopsis plants, the expression level of CsFKBP gene in transgenic plants and wild type plants (WT) was detected by qRT-PCR method. The expression level was calculated by 2 ^-ΔΔCt method using tea CsActin as an internal reference gene. Each sample was subjected to 3 biological replicates. The results are shown in FIG. 4, where the vertical lines represent standard error, significance analysis was performed using SPSS 22 for one-way analysis of variance, and the different lower case letters represent significant differences (p < 0.05). The results showed that no fluorescent signal was detected in the wild type control (WT) and that the expression level of CsFKBP genes was high in the over-expressed strains OE-1, OE-6 and OE-9.

S7, detecting physiological indexes:

(1) And (3) respectively sowing and harvesting the seeds of the 3 over-expression strains OE-1, OE-6 and OE-9 obtained in the step (S6) to obtain the T2 generation. 10 transgenic arabidopsis T2 generation seedlings and 10 wild type control (WT) are placed on a 1/2MS culture medium without antibiotics for normal growth, after germination for one week, the seedlings are placed in a 4 ℃ incubator for low-temperature stress treatment for 48H, meanwhile, 10 wild type arabidopsis plants cultured at the normal temperature of 25 ℃ are taken as the control, and are jointly transferred on the 1/2MS culture medium without antibiotics for observation and recording of phenotype changes. The phenotype comparison photograph is shown in FIG. 5. In root length statistics of different strains, 3 repeated measurements are taken for each strain, and the results are shown in fig. 6; in fig. 6, the vertical bars represent standard error, significance analysis single-factor analysis of variance using SPSS22, and the different lower case letters represent significant differences (p < 0.05).

The results show that the phenotype of the Arabidopsis plants is changed more obviously after the low-temperature treatment. Compared with a control line (WT-25 ℃) at 25 ℃, leaf edges of wild arabidopsis thaliana (WT-4 ℃) under the low temperature treatment at 4 ℃ curl, partial plant leaf color is purple and plant root system development is obviously blocked, and over-expression arabidopsis thaliana (OE-4 ℃) OE-1, OE-6 and OE-9 can still grow normally after the low temperature treatment, the root growth condition is that a control plant at 25 ℃ is more than 4 ℃ and an over-expression plant is more than 4 ℃ is used for the control plant, the root system growth development of the wild arabidopsis thaliana is seriously influenced by the low temperature, and the root system growth of a transgenic plant is vigorous after the low temperature treatment.

(2) The T3-generation transgenic Arabidopsis thaliana was subjected to 24H treatment at 4 ℃ and then the change of POD, SOD and CAT index activities was detected, and each sample was subjected to 3 biological replicates, and the results are shown in FIG. 7. The vertical lines in fig. 7 represent standard errors. Significance analysis SPSS22 was used for one-way analysis of variance with different lower case letters indicating significant differences (p < 0.05).

The results showed that POD activity (173.09-318.55 U.g ^-1·min^-1), SOD activity (76.38-140.52 U.g ^-1·min^-1) and CAT activity (132.44-296.04 U.g ^-1·min^-1) were significantly increased in the 3 transgenic lines of OE-1, OE-6 and OE-9 after treatment at low temperature of 4℃relative to activity under normal temperature culture conditions of 25℃and showed a more significant trend of increase compared to the wild-type control.

Plants must regulate various physiological and biochemical processes in order to cope with low temperature stress. Low temperature stress reduces the growth rate of plants, and is manifested in phenotype, mainly in that low temperature hypochondriac influences leaf growth and plant root growth vigor. In terms of the enzyme activity influence of antioxidant enzymes, the excessive accumulation of ROS is caused by low-temperature stress, so that the expression of a series of antioxidant enzyme genes is promoted, and the content of Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in plants is induced to increase to eliminate the accumulation of ROS so as to avoid cell damage. The detection results of POD, SOD and CAT show that after the over-expression CsFKBP a.thaliana is treated for 24H at the low temperature of 4 ℃, the activity of three physiological indexes is obviously improved, which indicates that the over-expression CsFKBP a enhances the activity of POD, SOD and CAT in the transgenic a.thaliana, improves the permeability of cell membranes and finally leads to the enhancement of the cold resistance of the transgenic plants.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (7)

1. An environmental stress protein CsFKBP, characterized in that the amino acid sequence comprises a sequence as shown in SEQ ID No. 1.

2. The environmental stress protein CsFKBP according to claim 1, wherein the amino acid sequence is as shown in SEQ ID No. 1.

3. The environmental stress protein CsFKBP, according to claim 1 or 2, obtained from tea tree.

4. The environmental stress protein CsFKBP according to claim 3, wherein the tea tree is cloud antigen No. 10.

5. Use of an environmental stress protein CsFKBP according to any one of claims 1 to 4 for increasing plant low temperature resistance.

6. The use according to claim 5, wherein the plant is tea tree.

7. The use according to claim 5, wherein the plant is arabidopsis thaliana.