CN114031677B

CN114031677B - Poncirus trifoliata transcription factor PtrAHL and application thereof in plant cold-resistant genetic improvement

Info

Publication number: CN114031677B
Application number: CN202111284972.4A
Authority: CN
Inventors: 刘继红; 巴沙尔; 张杨; 李春龙
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-05-27
Anticipated expiration: 2041-11-01
Also published as: CN114031677A

Abstract

The invention belongs to the field of plant genetic engineering, and discloses a trifoliate orange transcription factor PtrAHL and application thereof in plant cold-resistant genetic improvement,PtrAHLthe gene is from extremely cold-resistant Zhi (Poncirus trifoliata) Two transcription factors separated and cloned from the medium are namedPtrAHL14AndPtrAHL17the sequences are shown in SEQ ID NO.1 and SEQ ID NO. 2. The two genes are respectively constructed into overexpression and interference vectors, and are respectively introduced into the lemon and the trifoliate orange through agrobacterium-mediated genetic transformation, and the obtained transgenic plants are proved to be cloned by biological function verificationPtrAHL14AndPtrAHL17the gene has the function of controlling the cold resistance of plants. The development and utilization of the genetic resources are beneficial to reducing the agricultural production cost and realizing environmental friendliness.

Description

Poncirus trifoliata transcription factor PtrAHL and application thereof in plant cold-resistant genetic improvement

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a Poncirus trifoliata transcription factor PtrAHL and application thereof in plant cold-resistant genetic improvement.

Background

Low temperature stress adversely affects plant growth and development, greatly limiting plant geographical distribution and crop economic yield (Ding et al, 2020). Low temperature can affect the stability of cell membranes, reduce enzyme activity, generate reactive oxygen species, cause metabolic disorders of cells, ultimately delay plant growth, and in severe cases cause plant death (Barnes et al, 2016). Plants have evolved a complex set of adaptation mechanisms to sense low temperature signals and respond by triggering various signal transduction pathways, ultimately helping them maintain cellular homeostasis for better cold tolerance (Nagele et al, 2012). In addition, under low temperature stress, plants can accumulate a series of compatible compounds which can be used as an osmotic protective agent to regulate osmotic balance by regulating in vivo metabolic processes, and can improve the scavenging capacity of active oxygen by enhancing an antioxidant defense system, so that the cold resistance of the plants is enhanced (Chen et al, 2019).

Plant low-temperature response molecules are mainly divided into two types, one type is functional protein (LEA protein and the like) which can directly participate in stress response; the other is regulatory molecules (transcription factors, protein kinases, etc.) that regulate stress signal transduction and expression of related stress-responsive genes, and increase plant cold resistance (Zhu, 2016). The transcription factor is used as a kind of regulation gene, and is combined with cis-acting elements in eukaryotic gene promoter regions to regulate the expression of different resistance genes, so that adversity stress response is generated, and the plant resistance is improved. Thus, the use of transcription factors to improve the cold resistance of plants can be used with half the effort (Nakashima et al, 2014).

The AT hook motif nuclear localization (AHL) family is a plant-specific family of transcription factors that play important roles in growth and development and stress response. The AHL family of transcription factors contain one or two DNA Binding Domains (DBDs) at the N-terminus that recognize A/T rich sequences in MARs, while the C-terminus contains a Plant and Prokaryote Conserved (PPC) domain that promotes nuclear localization and protein-protein interactions (Gallavotti et al, 2011). Related studies have shown that AHLs can participate in a variety of plant growth and development processes, including hypocotyl elongation (Zhao et al, 2013), pollen wall and flower development (Lou et al, 2014; Yun et al, 2012), root development (Zhou et al, 2013), petiole growth, and the like (Favero et al, 2020). AHL also involves hormone signaling pathways including gibberellins (Matsushita et al, 2007), abscisic acid (Wong et al, 2019), jasmonic acid (Vom Endt et al, 2007), and auxins and the like (Favero et al, 2016; Lee and Seo, 2017). Furthermore, the AHL gene was found to be involved in plant response to abiotic or biotic stress (Howden et al, 2017; Jeong et al, 2020; Rayapuram et al, 2021; Zhou et al, 2016). However, the research on the cold resistance of the AHL transcription factor is rarely reported, and the research on the low-temperature resistance action mechanism of the AHL protein has important value on the cold resistance breeding of crops.

The trifoliate orange is a kindred genus of citrus, is often used as a stock in citrus production, is extremely cold-resistant, and is an ideal material for researching the cold resistance of woody plants and exploring important 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 a poncirus trifoliata transcription factor PtrAHL, wherein the poncirus trifoliata transcription factor PtrAHL is PtrAHL14 or PtrAHL17, the nucleotide sequences of the poncirus trifoliata transcription factor PtrAHL are SEQ ID NO.1 and SEQ ID NO.2 respectively, and the encoded protein is SEQ ID NO.3 and SEQ ID NO. 4.

The invention also aims to provide the application of the poncirus trifoliata transcription factor PtrAHL in controlling the cold resistance of plants. The gene is overexpressed or silenced and expressed in plants, and plants with enhanced or weakened cold resistance can be obtained.

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

The applicant clones two new genes PtrAHL14 and PtrAHL17 from trifoliate orange based on a plant gene cloning technology, wherein the protein coded by the PtrAHL14 gene is shown in SEQ ID No.3, the nucleotide is shown in SEQ ID No.1, the gene comprises a 1134bp open reading frame, 377 amino acids are coded, the isoelectric point is 9.07, and the predicted molecular weight is 38.89 kDa. The protein coded by the PtrAHL17 gene is shown in SEQ ID NO.4, the nucleotide is shown in SEQ ID NO.2, the gene comprises an open reading frame of 960bp, codes 319 amino acids, has an isoelectric point of 7.89, and has a predicted molecular weight of 33.31 kDa.

The applicant analyzed the relative expression amount of PtrAHL14 and PtrAHL17 genes after low-temperature treatment and protein content by using qRT-PCR technique and immunoprecipitation technique, and the results showed that the expression amount and protein level of PtrAHL14 and PtrAHL17 were increased with the increase of the low-temperature treatment time. In addition, the phenotype and related physiological indexes of the transgenic plants overexpressing PtrAHL14 and PtrAHL17 before and after low-temperature treatment are analyzed, and the results show that: relative to the fieldThe generative plants, PtrAHL14 and PtrAHL17 overexpression plants have stronger cold resistance. In addition, compared with wild plants, the transgenic plants have significantly higher Fv/Fm and A/N-INV activities, glucose and fructose contents, electrical conductivity, MDA content and H₂O₂The content and the sucrose content are lower. However, the phenotypic and physiological data of PtrAHL14 and PtrAHL17 interfering plants are contrary to them, indicating that PtrAHL14 and PtrAHL17 genes are two genes that positively regulate cold resistance.

The application of the cold-resistant gene PtrAHL in regulating and controlling the cold resistance of plants comprises the steps of carrying out over-expression on PtrAHL14 or PtrAHL17 genes in plants by utilizing a conventional mode in the field, and obtaining cold-resistant transgenic plants; by interfering the expression of PtrAHL14 or PtrAHL17 genes in the plant, the transgenic plant with reduced cold resistance can be obtained.

In the above application, preferably, the plant is lemon or trifoliate orange.

In the above applications, preferably, by constructing a plant overexpression vector of the ptrAHL14 or ptrAHL17 gene, the gene is introduced into a plant by using an agrobacterium-mediated genetic transformation method, and the obtained transgenic plant is subjected to biological function verification, which indicates that the cloned ptrAHL14 or ptrAHL17 gene has the function of improving the cold resistance of the plant.

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

successful cloning of the cold-resistant gene PtrAHL of the trifoliate orange 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 development and utilization of the genetic resource are beneficial to reducing 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 patterns of PtrAHL14 and PtrAHL17 of the invention in response to low temperature stress treatment;

wherein: a is the relative expression quantity of the PtrAHL14 gene under low-temperature (4 ℃) treatment; b is the relative expression quantity of the PtrAHL17 gene under low-temperature (4 ℃) treatment; c is the protein expression level of PtrAHL14 and PtrAHL17 under low temperature (4 ℃) treatment.

FIG. 3 is a schematic representation of the subcellular localization and transcriptional activation activity assays for PtrAHL14 and PtrAHL17 of the invention.

Wherein: a is a schematic diagram of the subcellular localization of PtrAHL14 and PtrAHL17 genes; b is a schematic immunoblot of PtrAHL14 and PtrAHL17 in Total Nuclear Protein (TNP) and Matrix Fraction (MF) before and after cryotreatment; c is a schematic diagram of the construction of the PtrAHL14 and PtrAHL17 gene transcription activation vectors; d is the PtrAHL14 and PtrAHL17 transcriptional activation activity assays of the invention.

FIG. 4 is a schematic diagram of relative expression analysis of PtrAHL14 and PtrAHL17 transgenic lemons;

wherein: a is the relative expression quantity of PtrAHL14 before and after PtrAHL14 transgenic lemon cryotreatment; and B is the relative expression quantity of PtrAHL17 before and after the PtrAHL17 transgenic lemon is subjected to low-temperature treatment.

FIG. 5 is a schematic diagram of the determination of the low-temperature treatment phenotype and physiological indexes of the PtrAHL14 and PtrAHL17 gene-transferred lemon of the invention;

wherein: a is the phenotype of PtrAHL14 transgenic lemons (#14-7, #14-8), PtrAHL17 transgenic lemons (#17-4, #17-6) and wild type lemons before and after low-temperature treatment; b is relative conductivity after lemon treatment; c is the MDA content before and after lemon treatment; d is a chlorophyll fluorescence phenotype graph before and after low-temperature treatment of the lemons; e is the Fv/Fm value before and after low-temperature treatment of the lemons; f is a DAB staining pattern before and after lemon treatment; g is the A/N-INV enzyme activity after lemon treatment; h is the sucrose content before and after lemon treatment; i is the glucose content of the lemons before and after low-temperature treatment; j is the fructose content before and after low-temperature treatment of the lemons.

FIG. 6 is a schematic diagram of relative expression analysis of VIGS silencing material according to the invention;

wherein: a is the content analysis of PtrAHL14 protein in a PtrAHL14 interference material (TRV-PtrAHL14), and B is the content analysis of PtrAHL17 protein in a PtrAHL17 interference material (TRV-PtrAHL 17); c is the expression analysis of PtrAHL14 in the PtrAHL14 interference material; d is the expression analysis of PtrAHL17 in the PtrAHL17 interference material.

FIG. 7 is a schematic diagram of the analysis of cold resistance of plants with Trapa arborescens PtrAHL14 gene silencing (TRV-PtrAHL14) and Trapa arborescens PtrAHL17 gene silencing (TRV-PtrAHL 17);

wherein: a is the phenotype of no-load TRV, interference plant TRV-PtrAHL14 and interference plant TRV-PtrAHL17 before and after low-temperature treatment; b is the relative conductivity of the interference poncirus trifoliata before and after low-temperature treatment; c is the MDA content before and after the interference Zhi low-temperature treatment; d is a chlorophyll fluorescence phenotype image before and after the interference citrus fruit low-temperature treatment; e is the Fv/Fm value before and after the low-temperature treatment of the interfering Hovenia dulcis. F is a DAB staining pattern before and after low-temperature treatment of the interference poncirus trifoliata; g is the activity of A/N-INV enzyme before and after the low-temperature treatment of the interfering poncirus trifoliata; h is the sucrose content before and after the low-temperature treatment of the interfering citrus; i is the glucose content before and after low-temperature treatment of the interfering citrus; j is the fructose content before and after low-temperature treatment of the interfering citrus.

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.

Example 1: cloning of full-length cDNA of Hovenia dulcis PtrAHL14 and PtrAHL17 genes

The method comprises the following steps of (1) taking semen hoveniae cDNA as a template, and adopting high-fidelity enzyme for amplification, wherein the sequence of an amplification primer is as follows: forward primer for PtrAHL14 gene amplification: 5'-ATGGAACCAAATGATACGCAGC-3' and a reverse amplification primer 5'-CTAGTCTGCAATTTGGTCATAGTC-3'; forward primer for PtrAHL17 gene amplification: 5'-ATGAAAAGTGATTATGTAGTAGAACCC-3' and amplified reverse primer 5'-TCAATAAGGCGGTGGTGGTGG-3';

purifying and recycling the amplified product by adopting an AxyPrep-96 DNA gel recycling kit, connecting the purified product to a pEASY-Blunt vector by utilizing a DNA seamless cloning technology, then transforming DH5 alpha competent cells by the connecting product, plating, shaking bacteria, and then carrying out positive identification. After obtaining positive clone, sending the positive clone to Wuhan Pongziaceae biology company for sequencing, and obtaining the full-length gene sequences of PtrAHL14 and PtrAHL17 according to the sequencing result.

The sequencing result shows that the PtrAHL14 sequence ORF is 1134bp, 377 amino acids are coded, the molecular weight of the protein is 38.89kD, the isoelectric point is 9.07, the nucleotide sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 3. The ORF of the PtrAHL17 sequence is 960bp, 319 amino acids are coded, the molecular weight of the protein is 33.31kDa, the isoelectric point is 7.89, the nucleotide sequence is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 4.

Example 2: expression analysis of PtrAHL14 and PtrAHL17 under low-temperature condition treatment

Wild immature bitter orange seedlings with the same growth vigor and 2 months of seedling age are placed in a low-temperature incubator (HP400G-E type, Rehua, China) for low-temperature treatment (4 ℃), and the sampling time points are 0h, 6h, 10h, 12h, 24h, 48h and 72 h. At each time point, the leaves are taken and then are quickly put into liquid nitrogen for freezing, and then are put into a refrigerator at minus 80 ℃ for refrigeration for standby, and the leaves are used for subsequent gene expression pattern analysis and immunoblotting experiments.

The low-temperature expression modes of PtrAHL14 and PtrAHL17 genes are analyzed by adopting a real-time fluorescent quantitative PCR (qRT-PCR) method, an AceQ qPCR SYBR Green Master Mix reagent is adopted in the real-time fluorescent quantitative PCR, and the method refers to the instruction. The prepared reaction system adopts a QuantStaudio TM7Flex Real-Time PCR fluorescent quantitative analyzer to carry out reaction.

Uses Actin in trifoliate orange as internal reference gene (forward primer: 5'-CCGACCGTATGAGCAAGGAAA-3'; reverse primer: 5'-TTCCTGTGGACAATGGATGGA-3'), adopts 2^-ΔΔCtThe algorithm calculates gene expression. The real-time quantitative primer of PtrAHL14 (forward primer: 5'-GAAAGTACGGGACGCCTGAA-3'; reverse primer: 5'-CACCAAGCTGAGACTTCCCC-3') and the real-time quantitative primer of PtrAHL17 (forward primer: 5'-GGGAGGTTTATAGGCAAGC-3'; reverse primer: 5'-ACGGATCTGGGATCTTGC-3') are selected. Immunoblotting experiments were performed using polyclonal antibodies to PtrAHL14 and PtrAHL17, produced by ABclonal Biotechnology.

The experiment result shows that the expression quantity of the PtrAHL14 gene (A in figure 2) and the expression quantity of the PtrAHL17 gene (B in figure 2) are continuously induced by low temperature, the protein levels of the two AHLs are gradually increased along with the increase of the low-temperature treatment time (C in figure 2), and comprehensively, the PtrAHL14 and the PtrAHL17 are cold-induced genes and probably play an important role in the cold stress resistance of plants.

Example 3: PtrAHL14 and PtrAHL17 subcellular localization and transcriptional activation activity assays

The ORF regions of PtrAHL14 and PtrAHL17 (without stop codon) were amplified and fused to the vector pEG104 (containing YFP protein), the expression of which was driven by the CaMV35S promoter. Then, controls 35S: YFP + mCherry, 35S: PtrAHL14-YFP + mCherry, 35S: PtrAHL17-YFP + mCherry were transiently transformed into leaf epidermal cells of Nicotiana benthamiana, respectively, fluorescence of the controls was observed by a laser confocal microscope to find that the fluorescence of the controls filled the entire epidermal cells including cytoplasm and nucleus, while fluorescence of the transformed 35S: PtrAHL14-YFP and 35S: PtrAHL17-YFP was detected only in the nuclear matrix (A in FIG. 3), and in order to confirm this, total nuclear protein and nuclear matrix protein were extracted and immunoblot analysis was performed using PtrAHL14 and PtrAHL17 antibodies. The results showed that PtrAHL14 and PtrAHL17 were detectable in both fractions, but after cryotreatment nuclear matrix protein levels were higher than total nuclear protein levels (B in fig. 3), confirming that PtrAHL14 and PtrAHL17 are nuclear matrix proteins.

In order to detect the transcriptional activation activity of two transcription factors, the full lengths of the PtrAHL14 and PtrAHL17 genes were constructed into pBD vector containing 5 copies of GAL4 binding element, and the expression of the vector was driven by CaMV35S promoter. Successfully constructed effectors and reporters (C in figure 3) were transformed into a. tumefaciens GV3101 strain containing the pSoup helper plasmid. The bacterial suspension was injected into n.benthamiana tobacco leaves and cultured at 22 ℃ for 3 days. D-luciferine reagent was smeared on the proximal side of the leaves and visual analysis of LUC fluorescence was performed using LB983 NightOWL II with indiGO Software. The experimental results show that when the effector containing PtrAHL14 or PtrAHL17 and the reporter are co-injected into tobacco, the leaf blade presents stronger LUC fluorescence signals, and the fact that PtrAHL14 and PtrAHL17 have transcriptional activation activity is confirmed (D in figure 3). Taken together, these results indicate that PtrAHL14 and PtrAHL17 are transcription factors that have transcriptional activation activity and are localized to the nuclear matrix.

Example 4: construction of plant transformation vector, genetic transformation of lemon and identification of positive seedling

1. Construction of plant transformation vectors

The method is characterized in that Poncirus trifoliata cDNA is used as a template, primers are designed to amplify PtrAHL14 and PtrAHL17 genes in full length, and the sequences of the primers are as follows:

pDONR207-PtrAHL14-F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAACCAAATGATACGCAGC-3’；

pDONR207-PtrAHL14-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTtGTCTGCAATTTGGTCATAGTC-3’；

pDONR207-PtrAHL17-F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAAAGTGATTATGTAGTAGAACCC-3’；

pDONR207-PtrAHL17-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTtATAAGGCGGTGGTGGTGG-3’；

after amplification and recovery, BP reaction connection is carried out with a pDONR207 carrier, and the using method is shown in

BP Clonase TM II kit instruction, will be sequenced correctly positive clone bacteria shake bacteria. 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 instruction, then can carry on the competent transformation of Escherichia coli, shake the bacterium after positive identification, extract the plasmid, obtain the final over-expression vector pGWB411-PtrPtrAHL14 and pGWB411-PtrPtrAHL17, wherein the method of the steps such as amplified fragment recovery, positive clone detection and sending out the appearance to sequence refers to example 1, transfer the vector into Agrobacterium competent GV3101 for subsequent use.

2. 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

On an aseptic bench, Agrobacterium pGWB411-PtrAHL14 and pGWB411-PtrAHL17 stored at-80 ℃ were picked up by using a cauterized and sterilized inoculating loop, streaked on a medium containing 50mg/L of 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. A sterilized 100mL 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 formulations of the media used in the experiments are shown in the table below.

Culture medium used in each stage of transformation

3. Positive seedling identification

The expression levels of PtrAHL14 and PtrAHL17 in the transgenic lemons are quantitatively analyzed in real time, and the results show that the relative expression level of the PtrAHL14 gene (A in figure 4) in the PtrAHL14 transgenic lemons (#14-7, #14-8) and the relative expression level of the PtrAHL17 gene (B in figure 4) in the PtrAHL17 transgenic lemons (#17-4, #17-6) are obviously increased relative to wild WT, and the positive transgenic lemons are used for subsequent cold resistance analysis.

Example 5: analysis of transgenic lemon Cold resistance

PtrAHL14 transgenic lemons (#14-7, #14-8), PtrAHL17 transgenic lemons (#17-4, #17-6), and wild type lemon (WT) were used for low temperature resistance identification. There were no significant phenotypic differences between wild-type and transgenic lemons prior to cryo-treatment. However, after acclimation at 4 ℃ for 12h and then reduction by 2 ℃ every 2h until after treatment at-4 ℃ for 12h, the wild type lemons died by substantial withered water browning, while the transgenic lemons were less damaged (a in fig. 5). Before the low-temperature treatment, the relative conductivity and MDA content of the wild-type WT and the transgenic lemon are not obviously different, but after the low-temperature treatment, compared with the over-expressed lemon, the relative conductivity of the wild-type lemon after the low-temperature treatment is higher (B in figure 5, the first from the left of each group is a WT group), and lower MDA content is accumulated (C in figure 5, the first from the left of each group is a WT group), which indicates that the low-temperature damage of the wild-type lemon is more serious. The chlorophyll fluorescence parameter Fv/Fm value is used for representing the conversion efficiency of light energy of a PS II reaction center, when a plant is stressed externally, the parameter is obviously reduced, and compared with wild lemon, the over-expressed lemon shows stronger chlorophyll fluorescence (D in a graph 5) and higher Fv/Fm value (E in a graph 5, the first from the left of each group is a WT group), which indicates thatThe wild type was more damaged under low temperature stress, and it was found that DAB staining was less severe after the leaves of the transgenic plants were cryogenically treated (F in FIG. 5), indicating that H in the transgenic plants₂O₂Has less accumulation amount and better active oxygen scavenging capacity. Furthermore, the alkaline/neutral sucrose invertase a/N-INV enzyme activity was significantly higher in transgenic plants before and after the cold treatment than WT (G in fig. 5, the first from the left of each group was WT group), and the sucrose content (H in fig. 5, the first from the left of each group was WT group) was found to be lower in transgenic plants, whereas the glucose content (I in fig. 5, the first from the left of each group was WT group) and the fructose content (J in fig. 5, the first from the left of each group was WT group) were higher. In conclusion, phenotypic observation and physiological data measurement show that the transgenic lemon has higher cold resistance and freezing resistance due to the overexpression of the PtrAHL14 or PtrAHL17 gene.

Example 6: identification of VIGS interference positive seedlings

Adopting VIGS mediated method to respectively interfere PtrAHL14 and PtrAHL17 genes in the trifoliate orange. The protein levels of the two in VIGS interference plants were first detected by immunoprecipitation using antibodies PtrAHL14 and PtrAHL17, respectively, and the protein band of PtrAHL14 (A in FIG. 6) and the protein band of PtrAHL17 (TRV-PtrAHL17) in the PtrAHL14 interference material (TRV-PtrAHL14) and the protein band of PtrAHL17 (B in FIG. 6) were weaker than those in the control plant (TRV).

In addition, the expression quantity of PtrAHL14 and PtrAHL17 genes in VIGS interference plants is detected by adopting a real-time fluorescent quantitative PCR (qRT-PCR) method. The results showed that the PtrAHL14 gene was inhibited by 10% to 40% relative to the empty load (C in fig. 6) and the PtrAHL17 gene was inhibited by 10% to 50% relative to the empty load (D in fig. 6), with lower expression levels than the control plant (TRV) at all times. The results show that the VIGS has high interference efficiency, the PtrAHL14 and PtrAHL17 genes are successfully interfered in the VIGS plants, and the subsequent cold resistance analysis is carried out on the positive VIGS plants.

Example 7: identification of cold resistance of VIGS interfering Hovenia dulcis

Selecting plants with good interference effect for low temperature resistance identification, acclimating two-month-old VIGS plants and control plants at 4 deg.C for 12 hr, and directly performing-4 deg.CAnd treating for 24 h. Under normal conditions, the morphology of VIGS and TRV control plants did not differ significantly. However, VIGS plants showed a significant cold-sensitive phenotype (a in fig. 7) compared to the cryoptreated TRV control and contained higher conductivity and MDA content (B in fig. 7, C in fig. 7, the first TRV control from the left of each group). After treatment, the chlorophyll fluorescence signals of VIGS plants are weak (D in figure 7), the maximum photosynthetic rate value Fv/Fm is obviously lower than that of control plants (E in figure 7, the first TRV control group from the left of each group), and the H in leaves₂O₂The level was higher than the control (F in fig. 7). In addition, the low A/N-INV enzyme activity in the interfering plants (G in FIG. 7, the first from the left of each group is the TRV control group) resulted in higher sucrose content than the control (H in FIG. 7), and lower glucose and fructose content than the control (I in FIG. 7, J in FIG. 7, the first from the left of each group is the TRV control group). In conclusion, the interference of the PtrAHL14 or PtrAHL17 gene reduces the scavenging capacity of active oxygen of plants, inhibits the process of converting sucrose into fructose and glucose, and seriously weakens the cold resistance of the plants, thereby further proving the important roles of the PtrAHL14 and the PtrAHL17 in improving the cold resistance of the plants.

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8.Jeong,H.-N.,Sun,H.-J.,Zuo,Z.-F.,Lee,D.H.,Song,P.-S.,Kang,H.-G.,and Lee,H.-Y.Overexpression of ATHG1/AHL23 and ATPG3/AHL20,Arabidopsis AT-hook motif nuclear-localized genes,confers salt tolerance in transgenic Zoysia japonica.Plant Biotech.Reports,2020,14:351-361

9.Lee,K.,and Seo,P.J.Coordination of matrix attachment and ATP-dependent chromatin remodeling regulate auxin biosynthesis and Arabidopsis hypocotyl elongation.PLoS One,2017,12:e0181804

10.Lou,Y.,Xu,X.F.,Zhu,J.,Gu,J.N.,Blackmore,S.,and Yang,Z.N.The tapetal AHL family protein TEK determines nexine formation in the pollen wall.Nat.Commun,2014,5:3855

11.Matsushita,A.,Furumoto,T.,Ishida,S.,and Takahashi,Y.AGF1,an AT-hook protein,is necessary for the negative feedback of AtGA3ox1 encoding GA 3-oxidase.Plant Physiol,2007,143:1152-1162

12.Nagele,T.,Stutz,S.,Hormiller,II,and Heyer,A.G.Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana.Plant J,2012,72:102-114

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

14.Rayapuram,N.,Jarad,M.,Alhoraibi,H.M.,Bigeard,J.,Abulfaraj,A.A.,Volz,R.,Mariappan,K.G.,Almeida-Trapp,M.,Schloffel,M.,Lastrucci,E.,et al.Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.Proc.Natl.Acad.Sci,2021,118:e2004670118

15.Vom Endt,D.,Soares e Silva,M.,Kijne,J.W.,Pasquali,G.,and Memelink,J.Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins.Plant Physiol,2007,144:1680-1689

16.Wong,M.M.,Bhaskara,G.B.,Wen,T.N.,Lin,W.D.,Nguyen,T.T.,Chong,G.L.,and Verslues,P.E.Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10phosphorylation required for stress growth regulation.Proc.Natl.Acad.Sci,2019,116:2354-2363

17.Yun,J.,Kim,Y.S.,Jung,J.H.,Seo,P.J.,and Park,C.M.The AT-hook motif-containing protein AHL22 regulates flowering initiation by modifying FLOWERING LOCUS T chromatin in Arabidopsis.J.Biol.Chem,2012,287:15307-15316

18.Zhao,J.,Favero,D.S.,Peng,H.,and Neff,M.M.Arabidopsis thaliana AHL family modulates hypocotyl growth redundantly by interacting with each other via the PPC/DUF296 domain.Proc.Natl.Acad.Sci,2013,110:E4688-E4697

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Sequence listing

<110> university of agriculture in Huazhong

<120> trifoliate orange transcription factor PtrAHL and application thereof in plant cold-resistant genetic improvement

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1134

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggaaccaa atgatacgca gcaactacaa caactcaact cttacttcca tcaccccacg 60

gccaccacca cctccggcgc cgccgcaacc accggtccct cgccaaccaa tggcctgttg 120

ccatctcagc accagcacca caacaacaac aacaacaata acgacggggg cggcggtggc 180

ggggggatgg tttacccgca ctcggtggcg tcctccgcta tgacgtcgac gctggagccg 240

gcgaagaaga agcgtggcag gcccagaaag tacgggacgc ctgaacaggc cttggccgcc 300

aagaaaacgg cggcgtattc gaattctaag gggaagagag agcagcgaga gctccatcag 360

cagctactgg gttctggcgg ttccggctct tcgtactcgg gggctccggg gaagtctcag 420

cttggtggta ttggcaattt aggacaaggt tttactcctc atgtaattag tgtagctgct 480

ggtgaggatg ttgggcagaa gattatgctg ttcatgcaac aaagtaagcg tgagatatgt 540

attttgtctg catctggttc aatctctaat gcatctctcc ggcagccagc aacatcggga 600

ggcaatatta catatgaggg tcggtttgag attgtttcgc tgtctggatc ttatgtacgc 660

actgaccttg gaggaaggac tggtgggctc agtgtatgtc tatccagtac agatggccag 720

atcattggag gaggggttgg tggaccccta aaagctgctg gcccagttca ggttattgtt 780

ggtacctttc aggttgagtc catgaaggat gttagtgctg gtttaaaagg cgattcttct 840

ggcagcaagt tagcatcacc agttgcaggg gcatctgtat caagtgttgg cttccgctca 900

ccaatcgaat cttacgggag aaatcctgtc aggggaaacg atgattttca aactattgga 960

gggactcatt tcatgattca accatatggt aatcacgtat cacctacgca agccgcagac 1020

tggaggagta gcctagatac cagaagctct gccggttatg acatgacagg aagaacaggt 1080

cgcggaggga atcaatctcc agaaaacgga gactatgacc aaattgcaga ctag 1134

<210> 2

<211> 960

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaaaagtg attatgtagt agaacccaaa accacaaact ctcaaaccat gttctcgaaa 60

cttcatcacc accagcagca acaacaccat cctttctctc atcacttcca gctctcacgt 120

gactctcagg cctccgagga agacaccaac agccataact cccctgttac cactcctcca 180

accaccaacc ccgccgccgc cgccgctgcc gctaaatcaa gacaacaaca gcttcaggaa 240

cccaccacca ccggagggga tggtgctact atcgaagtcg tccgccgccc caggggcaga 300

ccccccggct ccaagaacaa gcccaagcct cctgttttta tcacccgcga gccagaaccc 360

ggcatgagcc cttacattct cgaagtcccc ggcggaaacg acgtcgtgga gaccatctcc 420

aacttctgca ggcgcaagaa catcggtatc tgcgtgctca ccgggtcggg caccgtcgcc 480

aacgttaccc tccgtcagcc ctccgcgacc cctgggtcca ccatcacgtt ccacgggagg 540

ttcgacattc tctcgatctc cgcgacgttc ttgccccaaa acgcagcgta tccgcccttg 600

ccaaacatat tcgcgatatc gctggctggg ccgcaggggc agatcgtggg tggatccgtg 660

gtgggaccgc tgctggctgt tgggacggtg ttcgtggtgg ccgctacgtt taacaacccg 720

tcgtatcacc ggctgccggt gcaggacgag cagcaaagga cttcagtttc agctggcggt 780

gaagggcagt cgcctgtggg atctagtggc ggaggaggag gaggtggagc agagagtgga 840

cacgtggtag gaggagattc atgtgggatg tccatgtata gttgccattt gcctcctggt 900

gccggtggta gtgatgtgat atgggctccg actgcaagac caccaccacc gccttattga 960

<210> 3

<211> 377

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Pro Asn Asp Thr Gln Gln Leu Gln Gln Leu Asn Ser Tyr Phe

1 5 10 15

His His Pro Thr Ala Thr Thr Thr Ser Gly Ala Ala Ala Thr Thr Gly

20 25 30

Pro Ser Pro Thr Asn Gly Leu Leu Pro Ser Gln His Gln His His Asn

35 40 45

Asn Asn Asn Asn Asn Asn Asp Gly Gly Gly Gly Gly Gly Gly Met Val

50 55 60

Tyr Pro His Ser Val Ala Ser Ser Ala Met Thr Ser Thr Leu Glu Pro

65 70 75 80

Ala Lys Lys Lys Arg Gly Arg Pro Arg Lys Tyr Gly Thr Pro Glu Gln

85 90 95

Ala Leu Ala Ala Lys Lys Thr Ala Ala Tyr Ser Asn Ser Lys Gly Lys

100 105 110

Arg Glu Gln Arg Glu Leu His Gln Gln Leu Leu Gly Ser Gly Gly Ser

115 120 125

Gly Ser Ser Tyr Ser Gly Ala Pro Gly Lys Ser Gln Leu Gly Gly Ile

130 135 140

Gly Asn Leu Gly Gln Gly Phe Thr Pro His Val Ile Ser Val Ala Ala

145 150 155 160

Gly Glu Asp Val Gly Gln Lys Ile Met Leu Phe Met Gln Gln Ser Lys

165 170 175

Arg Glu Ile Cys Ile Leu Ser Ala Ser Gly Ser Ile Ser Asn Ala Ser

180 185 190

Leu Arg Gln Pro Ala Thr Ser Gly Gly Asn Ile Thr Tyr Glu Gly Arg

195 200 205

Phe Glu Ile Val Ser Leu Ser Gly Ser Tyr Val Arg Thr Asp Leu Gly

210 215 220

Gly Arg Thr Gly Gly Leu Ser Val Cys Leu Ser Ser Thr Asp Gly Gln

225 230 235 240

Ile Ile Gly Gly Gly Val Gly Gly Pro Leu Lys Ala Ala Gly Pro Val

245 250 255

Gln Val Ile Val Gly Thr Phe Gln Val Glu Ser Met Lys Asp Val Ser

260 265 270

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

275 280 285

Ala Gly Ala Ser Val Ser Ser Val Gly Phe Arg Ser Pro Ile Glu Ser

290 295 300

Tyr Gly Arg Asn Pro Val Arg Gly Asn Asp Asp Phe Gln Thr Ile Gly

305 310 315 320

Gly Thr His Phe Met Ile Gln Pro Tyr Gly Asn His Val Ser Pro Thr

325 330 335

Gln Ala Ala Asp Trp Arg Ser Ser Leu Asp Thr Arg Ser Ser Ala Gly

340 345 350

Tyr Asp Met Thr Gly Arg Thr Gly Arg Gly Gly Asn Gln Ser Pro Glu

355 360 365

Asn Gly Asp Tyr Asp Gln Ile Ala Asp

370 375

<210> 4

<211> 319

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Lys Ser Asp Tyr Val Val Glu Pro Lys Thr Thr Asn Ser Gln Thr

1 5 10 15

Met Phe Ser Lys Leu His His His Gln Gln Gln Gln His His Pro Phe

20 25 30

Ser His His Phe Gln Leu Ser Arg Asp Ser Gln Ala Ser Glu Glu Asp

35 40 45

Thr Asn Ser His Asn Ser Pro Val Thr Thr Pro Pro Thr Thr Asn Pro

50 55 60

Ala Ala Ala Ala Ala Ala Ala Lys Ser Arg Gln Gln Gln Leu Gln Glu

65 70 75 80

Pro Thr Thr Thr Gly Gly Asp Gly Ala Thr Ile Glu Val Val Arg Arg

85 90 95

Pro Arg Gly Arg Pro Pro Gly Ser Lys Asn Lys Pro Lys Pro Pro Val

100 105 110

Phe Ile Thr Arg Glu Pro Glu Pro Gly Met Ser Pro Tyr Ile Leu Glu

115 120 125

Val Pro Gly Gly Asn Asp Val Val Glu Thr Ile Ser Asn Phe Cys Arg

130 135 140

Arg Lys Asn Ile Gly Ile Cys Val Leu Thr Gly Ser Gly Thr Val Ala

145 150 155 160

Asn Val Thr Leu Arg Gln Pro Ser Ala Thr Pro Gly Ser Thr Ile Thr

165 170 175

Phe His Gly Arg Phe Asp Ile Leu Ser Ile Ser Ala Thr Phe Leu Pro

180 185 190

Gln Asn Ala Ala Tyr Pro Pro Leu Pro Asn Ile Phe Ala Ile Ser Leu

195 200 205

Ala Gly Pro Gln Gly Gln Ile Val Gly Gly Ser Val Val Gly Pro Leu

210 215 220

Leu Ala Val Gly Thr Val Phe Val Val Ala Ala Thr Phe Asn Asn Pro

225 230 235 240

Ser Tyr His Arg Leu Pro Val Gln Asp Glu Gln Gln Arg Thr Ser Val

245 250 255

Ser Ala Gly Gly Glu Gly Gln Ser Pro Val Gly Ser Ser Gly Gly Gly

260 265 270

Gly Gly Gly Gly Ala Glu Ser Gly His Val Val Gly Gly Asp Ser Cys

275 280 285

Gly Met Ser Met Tyr Ser Cys His Leu Pro Pro Gly Ala Gly Gly Ser

290 295 300

Asp Val Ile Trp Ala Pro Thr Ala Arg Pro Pro Pro Pro Pro Tyr

305 310 315

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaaccaa atgatacgca gc 22

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctagtctgca atttggtcat agtc 24

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgaaaagtg attatgtagt agaaccc 27

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaataaggc ggtggtggtg g 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgaccgtat gagcaaggaa a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttcctgtgga caatggatgg a 21

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaaagtacgg gacgcctgaa 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caccaagctg agacttcccc 20

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gggaggttta taggcaagc 19

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

acggatctgg gatcttgc 18

<210> 15

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggggacaagt ttgtacaaaa aagcaggctt aatggaacca aatgatacgc agc 53

<210> 16

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggggaccact ttgtacaaga aagctgggtt gtctgcaatt tggtcatagt c 51

<210> 17

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggggacaagt ttgtacaaaa aagcaggctt aatgaaaagt gattatgtag tagaaccc 58

<210> 18

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggggaccact ttgtacaaga aagctgggtt ataaggcggt ggtggtgg 48

Claims

1. The transcription factor separated from trifoliate orange, and the protein coded by the transcription factor is shown in SEQ ID NO.3 or SEQ ID NO. 4.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein the gene encoding SEQ ID NO.3 is represented by SEQ ID NO. 1; the gene of the code SEQ ID NO.4 is shown as SEQ ID NO. 2.

4. The use of the transcription factor of claim 1 for enhancing cold resistance in plants, such as Citrus limonum and Citrus aurantium.

5. The application of claim 4, wherein the application process comprises: constructing a plant over-expression vector containing a gene shown by SEQ ID NO.3 or SEQ ID NO.4, and introducing the recombinant vector of the component into the lemon or trifoliate orange by utilizing an agrobacterium-mediated genetic transformation method.