CN112898391B - Application of cold-resistant gene PtrERF9 of trifoliate orange in genetic improvement of cold resistance of plants - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, and discloses application of a trifoliate orange cold-resistant gene PtrERF9 in plant cold-resistant genetic improvement,PtrERF9is a very cold-resistant Zhihui (a)Poncirustrifoliata) The sequence of the transcription factor separated and cloned is shown in SEQ ID NO. 1. The gene is constructed into an over-expression and interference vector, and is respectively introduced into tobacco, lemon and trifoliate orange through agrobacterium-mediated genetic transformation, and the obtained transgenic plant is proved to be cloned by biological function verificationPtrERF9The gene has the function of improving the cold resistance of plants. The discovery of the gene provides a new gene resource for designing and breeding plant stress-resistant molecules, provides a new genetic resource for implementing green agriculture and water-saving agriculture, and the development and utilization of the genetic resource are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Description

Application of cold-resistant gene PtrERF9 of trifoliate orange in genetic improvement of cold resistance of plants

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a Poncirus trifoliata (Poncirus trifoliata) cold-resistant gene PtrERF9 in plant cold-resistant genetic improvement.

Background

Low temperature is one of the major abiotic stresses affecting yield and distribution of some important cash crops. Low temperature can damage plant cell membrane systems, affect chlorophyll synthesis and photosynthesis processes, and lead to reduced enzyme activity, increased reactive oxygen levels, cell metabolic disorders, and, in severe cases, plant death (chinnusmary et al, 2007). In the long-term evolution process of plants, a complex and efficient stress response mechanism is gradually formed, and when the plants are stimulated by external environment changes, the plants can finally induce the expression of plant-related defense and resistance genes through a series of signal transduction processes in vivo (Pankaj et al, 2016). In addition, in stress response, enhancement of antioxidant defense system and alteration of some metabolites, including soluble sugars, fatty acids, proline and betaine, etc., are also caused (Thomashow, 1999).

Low temperature response genes are mainly classified into two types, one is a regulatory gene which can regulate signal transduction and gene expression, and the other is a functional gene whose encoded product directly exerts a protective effect in cells (Wang et al, 2003; Zhu, 2016). The transcription factor has the function of 'top-down' as a type of regulation gene, can sense low-temperature signals, can specifically interact with cis-acting elements in a eukaryotic gene promoter region to regulate the expression of certain low-temperature response genes, and improves the cold resistance of plants (Goldack et al, 2011; Nakashima et al, 2014). Therefore, finding and utilizing transcription factors is a more effective way to improve plant stress resistance.

The AP2/ERF class of transcription factors are located downstream of the ethylene signaling pathway and contain a highly conserved AP2 region (Riec hmann et al, 2000). The domain consists of about 57 amino acids and can form a beta-sheet structure consisting of 1 alpha-helix and 3 antiparallel strands. The ERF transcription factor family is involved in the response of plants to biotic stress and abiotic stress, and is a large family of transcription factors closely related to plant stress resistance response. In the aspect of low-temperature stress, the tolerance of plants to low-temperature stress can be improved by over-expressing tobacco, the tolerance to low temperature is reduced by tomato plants of the ERF2 through antisense expression, and the tolerance to cold stress is recovered by the tomato plants after exogenous ethylene is sprayed (Zhang et al, 2010). Induced expression of ERF1 and ERF4 in tobacco, ERF1 and ERF5 in arabidopsis and lerf 4 in tomato are related to some cold-resistant genes (Song and Galbraith, 2006). However, the AP2/ERF transcription factor is not known to participate in the low-temperature response of the citrus in the prior art.

Citrus is the fruit with the largest global yield, prefers warm and humid climate, and is mainly distributed in the climate between south latitude 40 degrees and north latitude and in the tropical and subtropical areas where the soil is suitable. Citrus, a perennial woody plant, is subject to low temperature freeze damage, causing significant losses to the citrus industry. Therefore, breeding of low temperature freezing injury resistant varieties is one of the important targets of citrus breeding. However, citrus has polyblast, high heterozygosity, male sterility and other factors, and it is difficult to obtain new resistant varieties by the conventional hybridization method. The trifoliate orange is closely related to citrus, is extremely cold-resistant, can resist the low temperature of minus 26 ℃ after being completely domesticated at the low temperature, is widely applied to stocks of citrus varieties, and is an ideal material for discovering important citrus cold-resistant genes. Therefore, cloning of the cold-resistant related genes of trifoliate orange is the key and the basis of cold-resistant gene engineering.

Disclosure of Invention

The invention aims to provide application of a trifoliate orange cold-resistant gene PtrERF9 in controlling the cold-resistant character of plants. The gene is overexpressed or silenced and expressed in plants to obtain plants with enhanced or weakened cold resistance, the corresponding nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2.

In order to achieve the above object, the present invention adopts the following technical measures

The applicant names a transcription factor which is separated and cloned from Poncirus trifoliata (Poncirus trifoliata) based on a plant gene cloning technology and named as PtrERF9, the sequence of the transcription factor is shown as SEQ ID NO.1, and the corresponding amino acid sequence of the transcription factor is shown as a sequence table SEQ ID NO. 2; open Reading Frame (ORF) predicts that the gene is protein containing one ORF, with length of 696bp, coding 231 amino acids, molecular weight of the protein is 24.85kDa, isoelectric point is 9.58. The applicant analyzes the relative expression quantity of the PtrERF9 gene after treatment under different adverse conditions by using a qRT-PCR technology, and the result shows that the relative expression quantity of the PtrERF9 gene is highest under low-temperature stress. In addition, the phenotype and related physiological indexes of the PtrERF9 transgenic line before and after the low-temperature treatment are analyzed, and the results show that: compared with wild plants, the PtrERF9 overexpression plants have stronger cold resistance, and in addition, the survival rate, Fv/Fm, GST activity and GSH content are obviously higher, and the conductivity, MDA content and H content are higher₂O₂Content and O₂ ^·-The content is low, but the PtrERF9 transient silencing plants are opposite, which indicates that the PtrERF9 gene is a potential cold-resistant breeding gene.

The application of the cold-resistant gene PtrERF9 in plant cold resistance comprises the steps of carrying out overexpression, interference on the expression, knockout or mutation of the PtrERF9 gene in plants by utilizing a conventional mode in the field so as to control the cold resistance of the plants; the plant comprises tobacco, lemon and trifoliate orange.

Compared with the prior art, the invention has the following advantages:

the discovery and identification of the gene provide new gene resources for plant stress-resistant molecule design breeding, provide new genetic resources for implementing green agriculture and water-saving agriculture, and the development and utilization of the genetic resources are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Drawings

FIG. 1 is a technical flow diagram of the present invention.

FIG. 2 is a schematic representation of the expression pattern of PtrERF9 of the present invention in response to various stress treatments;

wherein: in FIG. 2, A is a low temperature (4 ℃ C.) treatment; in FIG. 2B is a dehydration treatment; in FIG. 2C is H₂O₂Processing;

in FIG. 2D is ABA treatment.

FIG. 3 is a schematic diagram showing the subcellular localization of PtrERF9 gene according to the present invention.

FIG. 4 is a schematic diagram of PtrERF9 gene transgenic tobacco identification and relative expression analysis;

wherein: in FIG. 4, A is the PtrERF9 gene specific primer of the invention for identifying positive tobacco; in FIG. 4, B is the relative expression level of PtrER F9.

FIG. 5 is a schematic diagram of the measurement of the low-temperature treatment phenotype and physiological index of PtrERF9 gene-transferred tobacco;

wherein: in FIG. 5, A is the phenotypes of transgenic tobacco (#2, #4) and wild-type tobacco before and after the low-temperature treatment; in FIG. 5B is the survival rate of tobacco after treatment; in FIG. 5C is the relative conductivity before and after tobacco treatment; FIG. 5D is MDA content before and after low temperature treatment of tobacco; FIG. 5E is a chlorophyll fluorescence phenotype plot before and after low temperature treatment of tobacco; in FIG. 5, F is the Fv/Fm value before and after tobacco treatment.

FIG. 6 is a schematic diagram of PtrERF9 gene transgenic lemon identification and relative expression analysis.

FIG. 7 is a schematic diagram of the measurement of the low-temperature treatment phenotype and physiological index of the PtrERF9 gene-transferred lemon;

wherein: in FIG. 7A is the phenotype of the transgenic lines (#15, #18) of lemon before and after treatment with wild type lemon (WT); in fig. 7B is the relative conductivity before and after the lemon treatment; in FIG. 7C is MDA content before and after lemon treatment; FIG. 7D is a chart of chlorophyll fluorescence phenotype before and after lemon treatment; in FIG. 7E is the Fv/Fm values before and after lemon treatment.

FIG. 8 shows the measurement of physiological indicators related to active oxygen before and after the treatment of lemon;

wherein: FIG. 8, wherein A is GST enzyme activity before and after lemon treatment; in fig. 8B is the before and after lemon treatment and GSH content; c in FIG. 8 is H before and after lemon treatment₂O₂Content (c); d in FIG. 8 is before and after lemon treatment O₂ ^·-Content (c); FIG. 8, E is DAB staining pattern after lemon treatment; in FIG. 8F is the NBT staining pattern after lemon treatment.

FIG. 9 is a schematic representation of the identification and relative expression quantification of VIGS silencing material according to the invention;

wherein: in FIG. 9, A is the identification of PtrERF9 interference material (PtrERF9-TRV2), "M" represents marker, "P" represents positive plasmid, and "WT" represents wild type Poncirus trifoliata; in FIG. 8B is the identification of empty material TRV 2; in FIG. 8, C is the expression level of PtrERF9 identified by randomly selecting 11 positive materials.

FIG. 10 is a schematic diagram of cold resistance analysis of Hovenia dulcis-silenced PtrERF9 gene plants (TRV-PtrERF 9 for short);

wherein: FIG. 10, panel A shows the phenotype of the empty TRV and interference plant TRV-PtrERF9 before and after the cold treatment; in FIG. 10B is the relative conductivities of the interferons before and after cryogenic treatment; in FIG. 10, C is the MDA content before and after the low temperature treatment of the interfering Zhi; FIG. 10D is a chlorophyll fluorescence phenotype image of an intervention hovenia dulcis before and after cryo-treatment; in FIG. 10, E is the Fv/Fm value before and after the low temperature treatment of the interfering protein.

FIG. 11 shows the determination of active oxygen-related physiological indexes before and after the PtrERF9 gene intervention treatment;

wherein: FIG. 11A is GST activity of empty TRV and interference plant TRV-PtrERF9 before and after cold treatment; in fig. 11B is GSH content before and after low temperature treatment of the interfering hovenia dulcis; in FIG. 11, C is H before and after low temperature treatment₂O₂Content (c); d in FIG. 11 is O before and after low-temperature treatment₂ ^·-Content (c); in FIG. 11, E is the DAB staining pattern after the low temperature treatment of Zhi interferometer; in FIG. 11, F is the NBT staining pattern after low temperature treatment of Zhi.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. In the embodiment of the invention, the PtrERF9 gene is over-expressed in tobacco and lemon, which shows that the PtrERF9 gene can improve the cold resistance of plants. For example, the ptrref 9 gene is overexpressed in trifoliate orange to obtain transgenic plants with enhanced cold resistance, and thus, the expression is not repeated in the examples.

Example 1:

cloning of full-length cDNA of Poncirus trifoliata PtrERF9 gene

The trifoliate orange cDNA is taken as a template, high-fidelity enzyme is adopted for amplification, an amplification system is shown in a table 1, an amplification program is shown in a table 2, and an amplification primer sequence is as follows: a forward primer: 5'-ctctccaaaacaaaaacagcacac-3' and reverse primer 5'-accgtattaaccggctcatcac-3'.

And (2) purifying and recovering the product obtained by amplification by adopting an AxyPrep-96DNA gel recovery kit (Axygene, USA), connecting the purified product with a pEASY-Blunt vector (all-type gold, China), wherein a connection system is shown in a table 3, and after incubation for 5min at room temperature, transforming escherichia coli competence Trans5 alpha. Plating, shaking, performing positive identification, wherein the positive identification system is shown in Table 4, obtaining positive clones, sending the positive clones to Wuhan engine biology company for sequencing, and obtaining the full-length gene sequence of PtrERF9 according to the sequencing result.

TABLE 1 Gene amplification System

TABLE 2 Gene amplification PCR procedure

TABLE 3 pEASY-Blunt vector ligation System

TABLE 4 Positive identification reaction System

Sequencing results show that the gene contains an ORF, is 696bp in length, codes 231 amino acids, has the molecular weight of 24.85kDa and the isoelectric point of 9.58, is named as PtrERF9, and has the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO. 2.

Example 2: expression analysis of PtrERF9 gene under different stress conditions

The expression pattern of the PtrERF9 gene was analyzed by real-time quantitative fluorescent PCR (qRT-PCR) using AceQ qPCR SYBR Green Master Mix (Novozam, Germany) according to the instructions. The reaction system is shown in Table 5, the prepared reaction system is reacted by a QuantStaudio 7Flex system (applied d Biosystems, USA) fluorescence quantitative analyzer, and the reaction program is shown in Table 6. Relative expression level of the gene 2^-ΔΔCTThe method comprises the steps of selecting citrus Actin as an internal reference gene (forward primer: 5'-ccgaccgtatgagca aggaaa-3'; reverse primer: 5'-ttcctgtggacaatggatgga-3'), selecting a PtrERF9 real-time quantitative primer (forward primer: 5'-ccaacgtagagcgtgaggtc-3'; reverse primer: 5'-caaaacaggaggagacgccc-3'), and selecting citrus Actin as an internal reference gene (forward primer: 5'-catccctcagcaccttcc-3'; reverse primer: 5'-ccaaccttagcacttctcc-3').

TABLE 5 quantitative PCR reaction System

TABLE 6 qPCR reaction procedure

The results of this experiment show that the expression level of PtrERF9 gene is continuously induced by low temperature, and the expression level is highest at 24h, and is increased by about 43 times compared with that before treatment, and then is slowly reduced (A in FIG. 2). At the same time, PtrERF9 is also expressed by drought stress, and the highest induction multiple is 14 times (B in figure 2). PtrERF9 at H₂O₂Under treatment, a first rising and then falling trend is shown (C in fig. 2). When treated with ABA, the gene was inhibited at the early stage and slightly induced at the late stage, up to about 3.2-fold (D in fig. 2). In summary, the PtrERF9 gene is most strongly induced and expressed by low temperature, and may play an important role in cold stress resistance of plants.

Example 3: subcellular localization of PtrERF9 gene

The ORF region of PtrERF9 (without stop codon) was amplified, amplification primers (F: 5'-ggaattcatggcacca aaaga-3' and R: 5'-cgggatcctgcgtcaaccactgg-3') were designed and fused to the vector p101LYFP, the YFP protein was located at the 3 ' end of the gene, and expression was driven by the CaMV35S promoter. Then, controls 35S: YFP + mCherry and 35S: PtrERF9-YFP + mCherry are respectively transformed into leaf epidermal cells of Nicotiana benthamiana transiently, fluorescence observed by a laser confocal microscope shows that fluorescence of the controls fills the whole epidermal cells including cytoplasm and nucleus, and fluorescence of the transformed 35S: PtrERF9-YFP is only concentrated in the nucleus and is coincided with red fluorescence of a nuclear localization marker mChery, which indicates that PtrERF9 is a nuclear localization protein (see figure 3).

Example 4: construction of plant transformation vectors

Designing a primer to amplify the PtrERF9 gene in full length, wherein the sequence of the amplification primer is as follows:

pDONR222-PtrERF9-F:5’-ggggacaagtttgtacaaaaaagcaggcttaatggcaccaaaagagagaac-3’；

pDONR222-PtrERF9-R:5’-ggggaccactttgtacaagaaagctgggtttgcgtcaaccactggtgg-3’；

amplified and recovered and then subjected to BP reaction connection with pDONR222vector (Invitrogen), and the using method is shown in

The specification of the BP Clonase TM II kit, the specific reaction system is shown in Table 7, and the positive clone bacteria subjected to sequencing are shaken. Plasmids were then extracted using AxyPrep plasmid DNA miniprep (Axygen, USA) cassettes and LR reacted with the target vector pGWB411, as described in methods reference

LR Clonase TM II (Invitrogen) kit specification, LR reaction system see Table 8, then can carry on the Escherichia coli competence transformation, shake the bacterium after positive identification, extract the plasmid, obtain the final over-expression vector pGWB411-PtrERF9, wherein the method of the steps such as amplified fragment recovery, positive clone detection and sending the sample to sequence refers to example 1, transfer the vector into Agrobacterium competent GV3101 for subsequent use.

TABLE 7 BP reaction System

TABLE 8 LR reaction System

Example 5: genetic transformation and positive identification of tobacco

1) Strain preparation: taking out the preserved agrobacterium transferred to pGWB411-PtrERF9 vector from-80 deg.c, dipping small amount of agrobacterium liquid with aseptic inoculating loop, streaking on LB solid culture medium containing 50mg/L spectinomycin and 50mg/L rifampicin, and culturing at 28 deg.c for 2-3 days; single clones were picked and restreaked on fresh LB solid medium (containing 50mg/L spectinomycin, 50mg/L rifampicin)And culturing for 2-3 days. Scraping thallus with sterilized surgical blade, culturing in MS liquid culture medium without antibiotic at 28 deg.C and 200r/min for 30min, shaking to disperse thallus, and adjusting OD with MS liquid culture medium₆₀₀The value is 0.6-0.8 for infection;

2) preparing an explant: selecting good sterile tobacco, selecting the largest 2-3 leaves, removing main vein and leaf edge, and cutting into 0.5cm pieces²Placing the square blocks with the left and right sizes into a sterile triangular flask added with a small amount of MS liquid culture medium for infection;

3) infection and co-culture: pouring the bacterial liquid cultured in the first step into a triangular flask filled with explants, and infecting for 10min at the temperature of 28 ℃ and at the speed of 200 r/min. After infection, the bacterial solution carried by the explant is blotted dry with sterilized filter paper. Placing the leaf with the back facing downwards on tobacco co-culture medium (MS +2.25 mg/L6-BA +0.3mg/L NAA) paved with sterile filter paper, and culturing in dark in a culture room for 3 d;

4) screening and culturing: transferring all explants after 3d of co-culture into a sterilized triangular flask, adding sterile water containing 400mg/L Cef to clean for 2-3 times, then cleaning for 2-3 times, finally sucking water on the surface of the explants by sterile filter paper, and culturing on a tobacco screening culture medium (MS +400mg/L Cef +100mg/L Km +2.25 mg/L6-BA +0.3mg/L NAA);

5) rooting culture: resistant buds growing to 1-2cm are cut off and placed in MS +400mg/LCef culture medium for rooting culture.

The culture medium contains 3.0% sucrose and 0.8% agar, and the pH value is adjusted to 5.9-6.0. Sterilizing the culture medium at high temperature under high pressure, cooling to below 60 deg.C, adding filtered and sterilized antibiotic, and packaging.

When the resistant bud grows roots and 2-3 leaves grow, taking a small amount of leaves to carry out DNA extraction, wherein the DNA extraction steps are as follows:

1) placing a small amount of leaves into a 1.5mL centrifuge tube, grinding the leaves into powder by using liquid nitrogen, adding 600 mu L of CATB extracting solution, and preparing a CTAB extracting solution according to a method shown in Table 9;

2) mixing completely, placing into 65 deg.C water bath, water bathing for 90min, and mixing by reversing every 30 min;

3) after completion of the water bath, 700 μ L of 24:1 (chloroform: isoamylol), violently mixing for 10min, centrifuging at 12000r/min at normal temperature for 15min, sucking supernatant (about 500 mu L) and transferring to a new centrifugal tube of 1.5 mL;

4) adding precooled isopropanol with the same volume as the supernatant, reversing the upper part and the lower part, uniformly mixing, and putting the mixture in a refrigerator at the temperature of minus 20 ℃ for precipitation (the precipitation time can be prolonged);

5) taking out after the precipitation is finished, and centrifuging at 12000r/min for 10 min. Pouring off the supernatant, adding 1mL of precooled 75% ethanol, washing for 2-3 times, removing the ethanol, and air-drying in a fume hood;

6) adding 20-30 mu LddH into each tube₂And O, dissolving the DNA, and storing the dissolved DNA in a refrigerator at the temperature of-20 ℃.

Concentration was measured by taking 1. mu.L of each sample and measuring its OD in a NanoDrop2000 ultramicro spectrophotometer (Thermo, USA)₂₆₀/OD₂₈₀When the ratio is in the range of 1.8-2.0, the DNA purity is high. And also detected by gel electrophoresis.

TABLE 9 CTAB extractive solution formula

Multiple positive plants are obtained by PCR identification by using identification primers (the result is shown as A in figure 4), and the sequences of the identification primers for the positive plants are 35S-F: 5'-cccactatccttcgcaagacc-3' and Gene-R: 5'-ggggaccactttgtacaagaaagctgggtttgc gtcaaccactggtgg-3'; the relative expression quantity of the PtrERF9 gene in the positive tobacco plants is quantitatively analyzed through real-time fluorescence, the result shows that the expression quantity of the PtrERF9 is obviously increased relative to WT (B in figure 4), and T2 generation seeds of #2 and #4 over-expression plants are selected for subsequent analysis according to the positive seedling identification result.

Example 6: analysis of transgenic tobacco Cold resistance

21d seedling-old potted transgenic tobacco and wild-type tobacco (WT) were used for low temperature resistance identification. Before the cold treatment, there was no significant phenotypic difference between the wild type and the transgenic lines (#2, # 4). However, after 6h of treatment at-4 ℃, most leaves of wild type tobacco were water-stained, and only part of the tobacco of the transgenic line was water-stained (A in FIG. 5). After recovery, the survival rate is counted, the transgenic plants have higher survival rate, wherein the survival rate of the #2 is 80 percent and the survival rate of the #4 is 83.3 percent, while the survival rate of the wild type plants is only 30 percent (B in figure 5), compared with the over-expression tobacco, the relative conductivity of the wild type tobacco after low-temperature treatment is higher (C in figure 5), which indicates that the cell membranes of the wild type tobacco are seriously damaged. In addition, transgenic tobacco accumulated lower MDA content relative to WT tobacco (D in fig. 5). The chlorophyll fluorescence value can indicate the photosynthetic efficiency of the plant, the damage degree of the plant under stress is reflected from the side, the maximum photosynthetic efficiency of the wild type is obviously inhibited after low-temperature treatment (F in figure 5), and the maximum photosynthetic efficiency Fv/Fm value is obviously lower than that of the transgenic tobacco. In conclusion, phenotypic observation and physiological data determination show that the overexpression of the PtrERF9 gene enables the transgenic tobacco to have higher cold and freeze resistance.

Example 7: genetic transformation and positive seedling identification of lemon

1. Genetic transformation of lemon

1) Plant material preparation

Soaking lemon seed in 1mol/L NaOH for about 15min, removing pectin, washing with water, placing the seed in a clean bench, soaking and sterilizing with 2% NaClO for 15min, pouring off the NaClO, and washing with sterile water for 3-4 times. The sterilized seeds are placed in a sterilized triangular flask into which a little water is added, and finally the seeds are stored in a refrigerator at 4 ℃.

Peeling off episperm and episperm of the seed with tweezers on a clean bench, inoculating on MT solid culture medium, culturing in dark for 4-6 weeks, and placing under light for 7-10 days before transformation until the seedling turns green. During this period, an Agrobacterium solution was prepared.

2) Preparation of Agrobacterium infection liquid

pGWB411-PtrERF9 Agrobacterium stored at-80 ℃ was picked using a cauterized sterile inoculating loop on a sterile console, streaked onto a medium containing 50mg/L Spec antibiotic, and the medium was placed in a 28 ℃ incubator for 2d in the dark. Single clones were picked and inoculated into fresh medium containing 50mg/L Spec antibiotic and placed in an incubator for further 2 days. GetA sterilized 100mL Erlenmeyer flask was poured into 50mL of MT liquid medium containing 20mg/L AS (Acetosyringone). Scraping the well grown Agrobacterium, dissolving in MT liquid culture medium containing 20mg/L AS, shaking at 28 deg.C for 20min at 200r/min (cutting lemon stem segment during the period), adding MT liquid culture medium into the bacterial liquid, adjusting concentration to OD₆₀₀The value is 0.6-0.8.

3) Explant preparation

Taking out the lemon seedlings, placing the lemon seedlings in a sterilized large culture dish paved with filter paper for cutting, and cutting the lemon seedlings into stem sections with the length of about 1.5 cm. A small amount of MT liquid medium was added to the sterilized flask to immerse the cut stem segments for moisture retention.

4) Infection and co-culture

Adding the prepared agrobacterium liquid into the triangular flask with the cut stem segments, and shaking for about 20min to finish infection. And (4) pouring out the bacterial liquid, taking out the stem section, and placing the stem section on sterile absorbent paper to remove the bacterial liquid on the surface of the stem section. And uniformly placing the stem sections with the surfaces being cleaned of the bacteria liquid into a co-culture medium paved with filter paper. Then dark culture is carried out, and the culture is carried out for 3d under the condition of 25 ℃.

5) Screening culture and regeneration

Taking out the stem section cultured in dark for 3d, placing into a sterilized small triangular flask, and soaking and cleaning with sterile water for 3-5 times. Placing the stem segments on sterile absorbent paper until water is absorbed, transferring the stem segments to a screening culture medium by using tweezers, culturing under a dark condition until the regenerated buds are larger than 0.5cm, cutting the regenerated buds, and placing the cut regenerated buds into a bud producing culture medium. When the size of the regeneration bud is 2cm, transferring the regeneration bud to a rooting culture medium. The formulation of the medium used in the experiment is shown in table 10.

TABLE 10 media used at the transformation stages

2. Positive seedling identification

The positive shoot identification method was the same as example 5, and multiple positive plants were identified (FIG. 6). Then selecting #15 and #18 with higher expression level as overexpression lines.

Example 9: identification of cold resistance of transgenic lemon

Wild-type lemons were not significantly different from over-expressed lemons (#15, #18) before the low-temperature treatment, but when the low-temperature treatment was carried out, wild-type lemons were severely damaged, leaves were withered and withered, and died, while transgenic lemons were less damaged (a in fig. 7). The relative conductivity of the transgenic plants was also significantly lower than that of the wild type (B in fig. 7), while the transgenic plants accumulated less MDA (C in fig. 7). In addition, chlorophyll fluorescence imaging results and Fv/Fm of over-expressed lemons were significantly better than wild-type (E in fig. 7 and F in fig. 7).

Meanwhile, the GST enzyme activity and the GSH content of the transgenic lemon and the wild lemon are increased after low-temperature treatment, but the increase range of the enzyme activity and the GSH content of the transgenic lemon is larger (A in figure 8 and B in figure 8). Increased GST enzyme activity promotes scavenging of reactive oxygen species, and therefore less H accumulates in treated transgenic lemons than in wild-type lemons₂O₂And O₂ ^■-(C in FIG. 8, D in FIG. 8). In addition, DAB staining and NBT staining of cryogenically treated lemon leaves showed H in wild-type lemons₂O₂And O₂ ^·-The content was higher (E in fig. 8, F in fig. 8).

Thus, transgenic lemons exhibit a stronger capacity for active oxygen scavenging under low temperature treatment, which may be an important reason for their enhanced low temperature resistance. By using the method, the gene is overexpressed in the plant, so that the cold resistance of the plant can be obviously improved.

Example 10: identification of VIGS interfering Hovenia dulcis and positive seedlings

1. Vector construction

Uses Hovenia dulcis cDNA as a template, designs a specific primer to amplify a PtrERF9 gene 5' end UTR and a segment of a non-conserved region 390bp, and adopts Treidef^TMSoSoSoo Cloning Kit (Ongji, China) is inserted between two enzyme cutting sites BamH I and Sma I connected to pTRV 2vector by one-step method, the specific method is shown in Table 11, and the constructed vector is transferred into GV3101 after being sequenced correctly. The primers for constructing the vector were as follows:

pTRV2-PtrERF9-F(BamH I):

5’-agaaggcctccatggggatccgccatcaacaagaatagc-3’；

pTRV2-PtrERF9-R(Sma I):

5’-tgtcttcgggacatgcccggggttaagaaccctccttgg-3’。

TABLE 11 one-step ligase reaction System

Infection with VIGS

1) Preparing an agrobacterium infection solution:

agrobacterium, such as pTRV1, pTRV2, pTRV2-PtrERF9, was streaked and cultured in LB solid medium (50mg/L rifampicin, 50mg/L kanamycin) at 28 ℃ for 2 days to obtain a single clone, which was then picked up in 5mL LB liquid medium containing the same antibiotic at 28 ℃ and 220r/min to sufficiently activate the cells (24-48 hours). Inoculating the activated agrobacterium liquid into a fresh LB culture medium containing antibiotics according to the proportion of 1:100, culturing at 28 ℃ and 220r/min overnight. Centrifuging at 4000r/min, collecting thallus, adding MES buffer (10mmol/L MES,10mmol/L M gCl)₂200. mu. mol/L AS, pH 5.6-5.7) and OD adjustment₆₀₀To 2.0. Mixing pT RV1 and pTRV2 or pTRV2-PtrERF9 bacterial suspension according to the proportion of 1:1, uniformly mixing, placing in a 28 ℃ incubator, and standing for 2-3h to be used for infection.

2) Infection:

taking out fresh semen Hoveniae from fruit, soaking in 1mol/L NaOH solution for 15min to remove pectin, washing with sterile water, spreading on wet clean gauze, placing in incubator (28 deg.C, dark) for accelerating germination, and allowing germination of seed sprout until the seed sprout grows to 1-2cm long for infection with VIGS. Slightly pricking some small holes on the germinated buds with a syringe needle, completely soaking in the prepared agrobacteria infection solution, vacuum pumping for 10min, rapidly deflating to immerse the agrobacteria in the germinated seeds, and repeating for 3 times. Standing for 15min, taking out the infected seeds, airing on dry filter paper, standing for 2-3min, spreading in a large dish with the filter paper wetted with sterile water, and standing in a room-temperature culture chamber in a dark place for 2-3d in a dark place; and (3) taking out the seeds, washing the seeds with clear water to remove residual bacterial liquid, sowing the seeds in a matrix (soil: vermiculite: 3:1), and carrying out positive identification after the seeds grow for 25d in an incubator.

3) Positive material identification

Identification of VIGS silenced Hovenia dulcis positive plants, wherein the extracted Hovenia dulcis DNA is used as a template, and reverse primer positive plants (A in figure 9 and B in figure 9) are constructed by adopting pTRV1 positive and negative primers, pT RV2 positive and negative primers, pTRV2 positive and pTRV2-PtrERF9 vectors. The expression level of ptrrerf 9 gene in positive plants was also detected by real-time fluorescence quantification (C in fig. 9).

TRV1-F：5’-attgaggcgaagtacgatgg-3’

TRV1-R：5’-ccatccacaattattttccgc-3’

TRV2-F：5’-attcactgggagatgatacgct-3’

TRV2-R:5’-agtcggccaaacgccgatctca-3’

Example 11: interference PtrERF9 Citrus aurantium cold resistance identification

Selecting the plant with the best interference effect to perform low-temperature resistance identification, and treating the idle-load control plant and the interference plant at a low temperature of-4 ℃, wherein after the low-temperature treatment, the leaves of the interference plant are more seriously damaged than the idle-load leaves, show wilting curly appearance (A in figure 10), and contain higher conductivity and MDA content (B in figure 10 and C in figure 10). The results of the chlorophyll fluorescence phenotype and maximum photosynthetic efficiency (Fv/Fm) also indicate that the interfering plants of PtrERF9 were more severely injured by cold temperatures (D in FIG. 10, E in FIG. 10). Meanwhile, after low-temperature treatment, the GST enzyme activity and the GSH content of the interference plants of PtrERF9 are lower than those of unloaded plants (A in figure 11 and B in figure 11), and more H is accumulated₂O₂And O₂ ^■-(C in FIG. 11, D in FIG. 11). In addition, the leaves of the interference plants of PtrERF9 after the low temperature treatment were stained more deeply with DAB and NBT (E in FIG. 11, F in FIG. 11). These results indicate that interference with PtrERF9 gene destroys active oxygen scavenging system in plant body, so that active oxygen scavenging is inhibited under low temperature treatment, and the plant is more vulnerable to low temperature stress. In conclusion, PtrER F9 is a positive regulatory factor of plants for resisting low-temperature stress。

Reference to the literature

1.Chinnusamy V,Ohta M,Kanrar S,Lee B ha,Hong X,Agarwal M,Zhu JK.ICE1:A r egulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes and D evelopment,2003,17:1043-54.

2.Song CP,Galbraith DW.AtSAP18,an orthologue of human SAP18,is involved in the regulation of salt stress and mediates transcriptional repression in Arabidopsis.Plant Mol Biol,2006,60:241–257.

3.Pankaj,T.,Manuel,D.B.,Chanda,T.,Hangwei,H.,Anderson,Ian C.,Jeffries,Thomas C.,Zhou,J.Z.,Singh,B.K.,2016.Microbial regulation of the soil carbon cycle evidence from gene-enzyme relationships.ISME J.10(11),2593-2604.

4.Thomashow MF.Plant cold acclimation:freezing tolerance genes and regulatory mechani sms.Annu Rev Plant Biol,1999,50:571-599.

5.Wang W,Vinocur B,Altman A.Plant responses to drought,salinity and extreme tempe ratures:towards genetic engineering for stress tolerance.Planta,2003,218:1-14.

6.Zhu JK.Abiotic stress signaling and responses in plants.Cell,2016,167:313–324.

7.Golldack D,Lüking I,Yang O(2011).Plant tolerance to drought and salinity:stress reg ulating transcription factors and their functional significance in the cellular transcriptional n etwork.Plant Cell Rep 30:1383-1391.

8.Nakashima K,Yamaguchi-Shinozaki K,Shinozaki K.The transcriptional regulatory netw ork in the drought response and its crosstalk in abiotic stress responses including drought, cold,and heat.Front Plant Sci,2014,5:170.

9.Riechmann JL,Heard J,Yu GL et al.Arabidopsis Transcription Factors:Genome-Wide Comparative Analysis Among Eukaryotes.Science,2000,290:2105-2110.

10.Zhang Z，Huang R.Enhanced tolerance to freezing in tobacco and tomato overexpress ing transcription factor TERF2/LeERF2is modulated by ethylene biosynthesis.Plant Mol Biol,2010,73(3):241-249.

Sequence listing

<110> university of agriculture in Huazhong

Application of <120> cold-resistant gene PtrERF9 in plant cold-resistant genetic improvement

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcaccaa aagagagaac aattagtggc aacaggaaaa acattgacaa caggaacaac 60

aataagaaag aggtgcatta cagaggagtt agaaagaggc cttggggccg atacgctgcc 120

gagataagag accccgggaa gaagagccgc gtgtggcttg gcaccttcga taccgctgag 180

gaagcagcca gggcttacga cgccgcggcg cgtgagtttc gcggctcgaa ggccaaaacg 240

aacttccctt tacccagcga aatcgtggcc atcaacaaga atagcaacaa ccagcagagc 300

ccgagccaga gcagtaccgt ggagtcctcg tccagccctc cgccgatgga gcccaacgta 360

gagcgtgagg tcacgcgcca cgtgggttgt tttggcagtg gaagcggact ggtgggtcgg 420

tttccgtttg tgtatcagca gccgggagtg gtggcgttgg gaggtgggtg cgtgaccggg 480

gcgtctcctc ctgttttgtt tcttaacggg tttgggggat cgaacttgat gggttcggtt 540

tatccggtcc ggtttgattc tgctggaatt gggtttaacg gcggggtacg gaatgagact 600

gaaactgaat catcatcggt tgcagccgtt gattgtaaac caaggagggt tcttaacctc 660

gatcttaatc tggccccacc agtggttgac gcatga 696

<210> 2

<211> 231

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Pro Lys Glu Arg Thr Ile Ser Gly Asn Arg Lys Asn Ile Asp

1 5 10 15

Asn Arg Asn Asn Asn Lys Lys Glu Val His Tyr Arg Gly Val Arg Lys

20 25 30

Arg Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Gly Lys Lys

35 40 45

Ser Arg Val Trp Leu Gly Thr Phe Asp Thr Ala Glu Glu Ala Ala Arg

50 55 60

Ala Tyr Asp Ala Ala Ala Arg Glu Phe Arg Gly Ser Lys Ala Lys Thr

65 70 75 80

Asn Phe Pro Leu Pro Ser Glu Ile Val Ala Ile Asn Lys Asn Ser Asn

85 90 95

Asn Gln Gln Ser Pro Ser Gln Ser Ser Thr Val Glu Ser Ser Ser Ser

100 105 110

Pro Pro Pro Met Glu Pro Asn Val Glu Arg Glu Val Thr Arg His Val

115 120 125

Gly Cys Phe Gly Ser Gly Ser Gly Leu Val Gly Arg Phe Pro Phe Val

130 135 140

Tyr Gln Gln Pro Gly Val Val Ala Leu Gly Gly Gly Cys Val Thr Gly

145 150 155 160

Ala Ser Pro Pro Val Leu Phe Leu Asn Gly Phe Gly Gly Ser Asn Leu

165 170 175

Met Gly Ser Val Tyr Pro Val Arg Phe Asp Ser Ala Gly Ile Gly Phe

180 185 190

Asn Gly Gly Val Arg Asn Glu Thr Glu Thr Glu Ser Ser Ser Val Ala

195 200 205

Ala Val Asp Cys Lys Pro Arg Arg Val Leu Asn Leu Asp Leu Asn Leu

210 215 220

Ala Pro Pro Val Val Asp Ala

225 230

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctctccaaaa caaaaacagc acac 24

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

accgtattaa ccggctcatc ac 22

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccgaccgtat gagcaaggaa a 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttcctgtgga caatggatgg a 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccaacgtaga gcgtgaggtc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caaaacagga ggagacgccc 20

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

catccctcag caccttcc 18

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccaaccttag cacttctcc 19

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggaattcatg gcaccaaaag a 21

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgggatcctg cgtcaaccac tgg 23

<210> 13

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggggacaagt ttgtacaaaa aagcaggctt aatggcacca aaagagagaa c 51

<210> 14

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggggaccact ttgtacaaga aagctgggtt tgcgtcaacc actggtgg 48

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cccactatcc ttcgcaagac c 21

<210> 16

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggggaccact ttgtacaaga aagctgggtt tgcgtcaacc actggtgg 48

<210> 17

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agaaggcctc catggggatc cgccatcaac aagaatagc 39

<210> 18

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgtcttcggg acatgcccgg ggttaagaac cctccttgg 39

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

attgaggcga agtacgatgg 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ccatccacaa ttattttccg c 21

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

attcactggg agatgatacg ct 22

<210> 22

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

agtcggccaa acgccgatct ca 22

Claims (3)

1. The application of the protein shown in SEQ ID NO.2 or the gene for coding the protein shown in SEQ ID NO.2 in improving the cold resistance of plants, wherein the plants are tobacco, lemon or poncirus trifoliata.
2. 2. The use of claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
3. 3. The application of claim 2, wherein the application process comprises: the gene shown in SEQ ID NO.1 is over-expressed in plants by utilizing the conventional mode in the field to improve the cold resistance of the plants.

Images