The cold-resistant gene CtrBBX32 of trifoliate orange and its application in genetic improvement of plant cold resistance

Abstract

The present invention discloses a trifoliate orange cold-resistant gene CtrBBX32 and its application in genetic improvement of plant cold resistance. The CtrBBX32 gene is a gene of a subfamily of the zinc finger structural protein family isolated and cloned from the extremely cold-resistant citrus material trifoliate orange, and its sequence is shown in SEQ ID No. 1. Interference vectors and overexpression vectors are constructed with this gene, and it is introduced into trifoliate orange through genetic transformation mediated by Agrobacterium tumefaciens and Agrobacterium rhizogenes to obtain transgenic plants. Biological function verification shows that the cloned CtrBBX32 gene of the present invention has the function of regulating plant cold resistance. The discovery of this gene provides a new gene resource for plant stress resistance molecular design and breeding, and provides a new genetic resource for the implementation of green agriculture and water-saving agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural production costs and achieving environmental friendliness.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and in particular relates to a trifoliate orange cold-resistant gene CtrBBX32 and its application in genetic improvement of plant cold resistance. Background Art

[0002] Extreme cold weather has a serious economic impact on agricultural production, especially on tropical and subtropical crops. As the world's largest fruit, citrus' tolerance to cold stress during its winter growing cycle is particularly critical. Cold stress can generally be divided into two levels: chilling (0-15°C) and freezing (<0°C) (Ding et al., 2019). Cold stress reduces membrane fluidity in plant cells, disrupts protein stability, inhibits enzyme activity, and affects gene expression and protein biosynthesis. Freezing stress causes ice crystals to form in the apoplast, leading to irreversible damage such as cell dehydration and cell membrane rupture (Dowgert & Steponkus, 1984; Pearce, 2001).

[0003] Extensive research has shown that the cellular membrane system is the primary site of plant freezing injury (Steponkus, 1984). Furthermore, it is well known that freezing-induced membrane damage is primarily due to the severe dehydration associated with freezing (Steponkus, 1984). The accumulation of sugars and other low sugars that typically occurs during cold acclimation may also contribute to membrane stability, as these molecules can protect membranes from freezing-induced damage in vitro (Anchordoguy et al., 1987; Strauss & Hauser, 1986). Although freezing injury is considered primarily due to membrane damage caused by cellular dehydration, other factors may also contribute to freezing-induced cell damage. Freezing-induced production of reactive oxygen species can lead to membrane damage (Tao et al., 1998), and intercellular ice can form adhesions with cell walls and membranes and cause cell rupture (Olien & Smith, 1977). Furthermore, plants undergo protein denaturation at low temperatures (Guy et al., 1998), which can also lead to cell damage.

[0004] Temperate plants can increase their cold tolerance after prior exposure to non-lethal low temperatures, a process known as cold acclimation (Strauss &

[0005] During cold acclimation, plants perceive cold signals through potential cold sensors and activate a set of regulatory networks. For example, signaling pathways dependent on the key transcription factor CBF enhance resistance to cold stress (Islam et al., 1981; Monroy et al., 1993). Plants then increase their cold tolerance by accumulating a number of protective substances, such as soluble sugars and proline (Crowe et al., 1988; Tarchevsky & Egorova, 2022).

[0006] The expression levels of genes are finely regulated by various transcription factors, enabling organisms to respond to external stimuli. A large number of zinc finger proteins involved in gene regulation have been identified in plants, playing a vital role in plant stress resistance (Agnieszka, 2012; Puentes-Romero et al., 2022). BBX (B-box type) proteins are a class of zinc finger proteins widely present in plants, playing an important role in photomorphogenesis (Datta et al., 2007), flowering process (Tiwari et al., 2010) and shade avoidance response (Crocco et al., 2015). BBX proteins are also involved in the signaling pathways of abiotic stress. For example, AtBBX18-RNAi transgenic plants showed enhanced heat tolerance, while overexpression plants showed reduced heat tolerance.

[0007] (Wang et al., 2013). Chrysanthemum Cm BBX19 negatively regulates drought tolerance in chrysanthemum by affecting the expression of genes related to the ABA signaling pathway (Xu et al., 2020). Although BBX family transcription factors have been widely reported, their role in citrus cold tolerance has not yet been reported. Studying the mechanism of action of BBX in cold tolerance is of great value to crop cold tolerance breeding.

[0008] Poncirus trifoliata is frequently used as a rootstock for citrus production. Due to its exceptional cold tolerance, resistance to droughts, and hybrid compatibility with citrus, it is a valuable resource for citrus rootstocks. Therefore, cloning genes associated with cold tolerance in trifoliate orange is crucial and fundamental for cold-resistance genetic engineering.

[0009] References:

[0010] 1.Anchordoguy TJ,Rudolph AS,Carpenter JF,Crowe JH.Modes ofinteraction of cryoprotectants with membrane phospholipids duringfreezing.Cryobiology.1987;24(4):324-331.

[0011] 2.Crocco CD,Locascio A,Escudero CM,Alabadí D,Blázquez MA,Botto JF.Thetranscriptional regulator BBX24 impairs DELLA activity to promote shadeavoidance in Arabidopsis thaliana.Nat Commun.2015;6:6202.

[0012] 3.Crowe JH,Crowe LM,Carpenter JF,et al.Interactions of sugars withmembranes.Biochim Biophys Acta.1988;947(2):367-384.

[0013] 4.Datta S,Hettiarachchi C,Johansson H,Holm M.SALT TOLERANCE HOMOLOG2,a B-box protein in Arabidopsis that activates transcription and positivelyregulates light-mediated development.Plant Cell.2007;19(10):3242-3255.

[0014] 5.Ding Y,Shi Y,Yang S.Advances and challenges in uncovering coldtolerance regulatory mechanisms in plants.New Phytol.2019;222(4):1690-1704.

[0015] 6.Dowgert MF,Steponkus PL.Behavior of the Plasma Membrane of IsolatedProtoplasts during a Freeze-Thaw Cycle.Plant Physiol.1984;75(4):1139-1151.

[0016] 7.Guy,C.,Haskell,D.,and Li,Q.B.Association of proteins with thestress 70molecular chaperones at low temperature:Evidence for the existenceof cold labile proteins in spinach.Cryobiology.1998;36:301-314.

[0017] 8.Islam,A.K.M.R.,Shepherd,K.W.,&Sparrow,D.H.B.Isolation andCharacterization of Euplasmic Wheat-Barley Chromosome AdditionLines.Heredity.1981;46:161-174.

[0018] 9.Agnieszka -Matuk.Involvement of plant CH-type zinc fingertranscription factors in stress responses.Plant Sci.2012;185:78-85.

[0019] 10.Monroy AF,Castonguay Y,Laberge S,Sarhan F,Vezina LP,Dhindsa RS.Anew cold-induced alfalfa gene is associated with enhanced hardening atsubzero temperature.Plant Physiol.1993;102(3):873-879.

[0020] 11.Olien CR,Smith MN.Ice adhesions in relation to freeze stress.PlantPhysiol.1977;60(4):499-503.

[0021] 12.Pearce,R.S.Plant freezing and damage.Ann Bot.2001;87:417-424.

[0022] 13.Pearce,R.S.Plant freezing and damage.Ann Bot.2001;87(4):417-424.

[0023] 14.Puentes-Romero AC,González SA,González-Villanueva E,Figueroa CR,Ruiz-Lara S.AtZAT4,a C2H2-Type Zinc Finger Transcription Factor fromArabidopsis thaliana,Is Involved in Pollen and Seed Development.Plants(Basel).2022;11(15):1974.

[0024] 15.Steponkus P L.Role of the Plasma Membrane in Freezing Injury andCold Acclimation[J].Annu.rev.plantPhysiol.1984;35(1):543-584.

[0025] 16.Strauss G,Hauser H.Stabilization of lipid bilayer vesicles bysucrose during freezing.Proc Natl Acad Sci US A.1986;83(8):2422-2426.

[0026] 17. Tao DL, Oquist G, Wingsle G. Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance. Cryobiology. 1998;37(1):38 - 45.

[0027] 18. Tarchevsky, I.A., & Egorova, A.M. Participation of Proline in Plant Adaptation to Stress Factors and Its Application in Agrobiotechnology (Review). Appl Biochem Microbiol. 2022;58:347 - 360.

[0028] 19. Tiwari SB, Shen Y, Chang HC, et al. The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis - element. New Phytol. 2010;187(1):57 - 66.

[0029] 20. Wang Q, Tu X, Zhang J, Chen X, Rao L. Heat stress - induced BBX18 negatively regulates the thermotolerance in Arabidopsis. Mol Biol Rep. 2013;40(3):2679 - 2688.

[0030] 21. Xu Y, Zhao X, Aiwaili P, et al. A zinc finger protein BBX19 interacts with ABF3 to affect drought tolerance negatively in chrysanthemum. Plant J. 2020;103(5):1783 - 1795. Summary of the Invention

[0031] The technical problem to be solved by the present invention is to provide a trifoliate orange cold-resistant gene CtrBBX32 and its application in genetic improvement of plant cold resistance.

[0032] The technical solution of the present invention is: a protein isolated from Poncirus trifoliata, the amino acid sequence of which is shown as SEQ ID No.2.

[0033] Genes encoding the above-mentioned proteins.

[0034] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0035] A recombinant vector containing the above-mentioned gene.

[0036] A method for reducing the cold resistance of plants, the method comprising overexpressing the above-mentioned gene in the plants, thereby reducing the cold resistance of the plants.

[0037] A method for improving the cold resistance of plants, comprising knocking out, inhibiting or silencing the gene shown in SEQ ID No. 1 in the plant, thereby improving the cold resistance of the plant.

[0038] Furthermore, the plant is Poncirus trifoliata.

[0039] A specific fragment for silencing the cold-resistant gene CtrBBX32 of trifoliate orange (Poncirus trifoliata), the nucleotide sequence of the specific fragment is shown in SEQ ID No.3.

[0040] A recombinant vector is composed of a pTRV2 vector and the above-mentioned specific fragment connected to the pTRV2 vector.

[0041] The applicant isolated and cloned a sugar transporter protein from the extremely cold-resistant trifoliate orange (Poncirus trifoliata) based on plant gene cloning technology. The applicant named it CtrBBX32, its sequence is shown in SEQ ID No.1, and its corresponding amino acid sequence is shown in SEQ ID No.2; open reading frame (ORF) prediction found that the gene contains one ORF, is 732bp in length, and encodes a protein of 243 amino acids. The molecular weight of the protein is 26.30kDa, and the isoelectric point pI is 8.50.

[0042] The applicant analyzed the relative expression and tissue specificity of the CtrBBX32 gene after low-temperature treatment using the transcriptome data of trifoliate orange treated at low temperature in the laboratory and the transcriptome data of different tissue parts of trifoliate orange (roots, stems, and leaves). The results showed that the expression of CtrBBX32 increased with the extension of low-temperature treatment time, and was highly expressed in the roots of trifoliate orange. CtrBBX32 overexpression and transient silencing strains were constructed, and the cold-resistant phenotype and related physiological indicators of CtrBBX32 transgenic plants before and after low-temperature treatment were analyzed. The results showed that compared with the trifoliate orange plants in the control group, the cold-resistant properties of the transgenic trifoliate orange plants with overexpression of CtrBBX32 were reduced, while the electrical conductivity, MDA content, and ROS content increased, and the soluble sugar content was significantly reduced. The cold-resistant phenotype of the trifoliate orange plants with silenced CtrBBX32 was the opposite, indicating that CtrBBX32 is a potential breeding gene that negatively regulates the cold resistance of trifoliate orange.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The discovery and identification of the CtrBBX32 gene provides new genetic resources for the design and breeding of plant stress resistance molecules, and provides new genetic resources for the implementation of green agriculture and water-saving agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural production costs and achieving environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a technical flow chart of the present invention.

[0046] Figure 2 Schematic diagram of the expression pattern of CtrBBX32 in response to low temperature stress treatment of the present invention;

[0047] Among them: A is a heat map of the expression of CtrBBX32 gene in the transcriptome of trifoliate orange under low temperature treatment; B is a heat map of the expression of CtrBBX32 gene in the transcriptome of trifoliate orange in different tissues (roots, stems, and leaves).

[0048] Figure 3 The results of the subcellular localization and transcriptional activation activity analysis of CtrBBX32 of the present invention are as follows;

[0049] Wherein: A is the subcellular localization detection result of CtrBBX32 of the present invention; B is the growth status of CtrBBX32 of the present invention on SD / -Trp and SD / -Trp / -His / -Ade culture plates after transformation into yeast competent AH109.

[0050] Figure 4 This is a schematic diagram of the positive identification and relative expression analysis of the VIGS silencing material of the present invention;

[0051] Wherein: A is a plant positive for the CtrBBX32 gene of the present invention identified by TRV1-specific primers (TRV2-CtrBBX32); B is a plant positive for the CtrBBX32 gene of the present invention identified by TRV2-specific primers (TRV2-CtrBBX32); C is an analysis of the expression level of CtrBBX32 in CtrBBX32-interference materials. Wherein, "M" represents marker, "P" represents plasmid, and "WT" represents wild-type.

[0052] Figure 5 This is a schematic diagram of the cold resistance analysis of trifoliate orange after silencing the CtrBBX32 gene;

[0053] Among them: A is the phenotype of empty TRV2 and interference plant TRV2-CtrBBX32 before and after low temperature treatment; B is the chlorophyll fluorescence phenotype of interference trifoliate orange before and after low temperature treatment; C is the relative conductivity of interference trifoliate orange before and after low temperature treatment; D is the MDA content of interference trifoliate orange before and after low temperature treatment; E is the determination of active oxygen-related physiological indicators of interference trifoliate orange before and after low temperature treatment; F is the fructose, glucose and sucrose content of interference trifoliate orange.

[0054] Figure 6 This is a schematic diagram of the positive identification and relative expression analysis of the hairy root transformation materials of the present invention;

[0055] Among them: A is the positive plant (OE-CtrBBX32) identified by specific primers for the CtrBBX32 gene of the present invention; B is the expression of GFP fluorescent protein in the root of the OE-CtrBBX32 material of the present invention under a handheld fluorescent lamp.

[0056] Figure 7 This is a schematic diagram of the cold resistance analysis of trifoliate orange with overexpression of CtrBBX32 gene;

[0057] Among them: A is the phenotype of empty load and overexpression plants OE-CtrBBX32 before and after low temperature treatment; B is the chlorophyll fluorescence phenotype of overexpression trifoliate orange before and after low temperature treatment; C is the relative conductivity of overexpression trifoliate orange before and after low temperature treatment; D is the MDA content of overexpression trifoliate orange before and after low temperature treatment; E is the determination of root activity index before and after low temperature treatment of overexpression trifoliate orange; F is the determination of active oxygen-related physiological indexes in the roots of overexpression trifoliate orange after low temperature treatment; G is the fructose, glucose and sucrose contents in the roots of overexpression trifoliate orange. DETAILED DESCRIPTION

[0058] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0059] Example 1: Cloning of the full-length cDNA of the CtrBBX32 gene of Poncirus trifoliata

[0060] Poncirus trifoliata cDNA was used as a template and a high-fidelity enzyme was used for amplification. The amplification system is shown in Table 1, the amplification program is shown in Table 2, and the amplification primer sequences are as follows: forward primer: 5'-ATGAAGAGAGCTTGTGAA-3', reverse primer: 5'-TCAAACATTACACTCAGC-3'.

[0061] The amplified product was purified and recovered using the AxyPrep-96 DNA Gel Extraction Kit (Axygene, USA) and then ligated into the pEASY-Blunt vector (Quanshijin, China). The ligation system is shown in Table 3. After incubation at room temperature for 15 minutes, the product was transformed into competent Escherichia coli DH5α. Plates were spread, incubated upside down at 37°C, and the bacteria were picked and shaken. PCR positive detection was performed. The PCR positive detection procedure is shown in Table 4. Positive clones were then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were the full-length sequence of the CtrBBX32 gene.

[0062] Table 1 Gene amplification system

[0063]

[0064] Table 2 Gene amplification PCR program

[0065]

[0066] Table 3 pEASY-Blunt vector ligation system

[0067]

[0068] Table 4 Colony positive identification reaction system

[0069]

[0070]

[0071] Open reading frame (ORF) prediction revealed that the gene contains a single ORF, 732 bp in length, encoding a 243-amino acid protein with a molecular weight of 26.30 kDa and an isoelectric point (pI) of 8.50. The gene was named CtrBBX32, with the nucleotide sequence shown in SEQ ID No. 1 and the amino acid sequence shown in SEQ ID No. 2.

[0072] Example 2:

[0073] Analysis of subcellular localization and transcriptional activation activity of CtrBBX32 gene

[0074] The ORF region of CtrBBX32 (excluding the stop codon) was amplified using the primer sequences p101YFP-CtrBBX32-F: 5'-GGATCTACTAGT GAATTC ATGAAACGAGCTTGC-3' and p101YFP-CtrBBX32-R: 5'-GGTACCGTCGAC GGATCC TACGTTGCACTCAGC-3'. The target gene was constructed into the pRI101 vector (which was modified in the laboratory to insert a YFP tag after the MCS site). The YFP protein was located at the 3' end of the gene, and expression was driven by the CaMV35S promoter ( Figure 3 (A). 35S:CtrBBX32-YFP+mCherry-VirD2NLS and the control 35S:YFP+mCherry-VirD2NLS were transiently transformed into Nicotiana benthamiana leaf epidermal cells. Laser confocal fluorescence observation showed that the fluorescence of the control was expressed in the cytoplasm and nucleus, while the fluorescence of the transformed 35S:CtrBBX32-YFP was only concentrated in the nucleus. This indicates that CtrBBX32 is a nuclear localized protein ( Figure 3 Middle A).

[0075] In order to study and analyze the transcriptional activation activity of CtrBBX32, the full-length gene was amplified and ligated into the pGBKT7 vector. The primer sequences were pGBKT7-CtrBBX32-F: 5'-ATGGCCATGGAGGCC GAATTC ATGAAACGAG CTTGC-3' and pGBKT7-CtrBBX32-R: 5'-CCGCTGCAGGTCGAC GGATCC TACGTTGCACTC AGC-3'. After that, the competent yeast cells AH109 were transformed and the transcriptional activation activity of CtrBBX32 was tested in the corresponding culture medium. The results showed that all transformants could grow in SD / -Trp medium, while all transformants could not grow in SD / -Trp / -His / -Ade medium ( Figure 3 Middle B). This indicates that CtrBBX32 has no transcriptional activation activity.

[0076] Example 3: VIGS intervention in trifoliate orange and identification of positive seedlings

[0077] 1. Vector Construction

[0078] Using the cDNA of Poncirus trifoliata as template, specific primers were designed to amplify a 225 bp fragment (SEQ ID No. 3) in the CDS of the CtrBBX32 gene. TMThe SoSoo Cloning Kit (Qingke, China) was used for one-step insertion between the BamHI and SmaI restriction sites of the pTRV2 vector. The constructed vector was sequenced and transformed into competent Agrobacterium tumefaciens GV3101. The primer sequences were: pTRV2-CtrBBX32-F: 5'-AGAAGGCCTCCATGG GGATCC GGT GTAGAAGGCTGG-3'; pTRV2-CtrBBX32-R: 5'-TGTCTTCGGGACATG CCCGGG CCGG CACTCCTGATA-3'.

[0079] 2.VIGS infection

[0080] Peel seeds from trifoliate orange fruits stored in a 4°C refrigerator, soak them in 1 mol / L NaOH solution for 15 minutes to remove pectin, rinse them 2-3 times with distilled water, cover the seeds with vermiculite, and keep the vermiculite moist. Place the seeds in a 28°C incubator in the dark to germinate. After about 4 weeks, when the seedlings have germinated to 1-2 cm in length, they can be used for VIGS infection. The procedure is as follows:

[0081] 1) Streak TRV1, TRV2, and TRV2-CtrBBX32 glycerol Agrobacterium stored at -80°C onto LB solid medium (containing 50 mg / L Rif and 50 mg / L Kan) and culture in an inverted manner at 28°C for 2 days to obtain single colonies;

[0082] 2) Pick one single colony from each strain and place it in 5 mL of LB liquid medium containing the same antibiotics. Incubate at 28°C, 220 rpm, and shake gently for 16-24 hours to fully activate the bacteria.

[0083] 3) The activated Agrobacterium liquid was inoculated into the same LB liquid medium at a ratio of 1:100, cultured at 28°C, 220 rpm, and expanded for 6-8 h. The cells were collected by centrifugation at 6000 rpm and suspended in MES buffer (10 mmol / LMES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7). OD 600 Adjust to 0.6;

[0084] 4) Mix the resuspensions of TRV1 and TRV2, and TRV1 and TRV2-CtrBBX32 in a 1:1 ratio, mix well, and incubate in a dark incubator at 28°C for 2-4 hours to prepare the infection solution;

[0085] 5) Use a syringe needle to lightly poke small holes in the germinating shoots. Completely immerse them in the prepared Agrobacterium infection solution. Vacuum the solution for 10 minutes, then quickly release the air to allow the Agrobacterium to penetrate the germinating seeds. Repeat this three times. Then, let the solution stand for 15 minutes. Remove the infected seeds and air them on dry filter paper. After 2-3 minutes, spread them flat on a large dish soaked in sterile water. Incubate them in a dark incubator at room temperature for 2-3 days.

[0086] 6) Rinse the dark-cultured seeds with clean water to remove residual bacterial liquid, sow them in a substrate (soil:vermiculite = 3:1), and grow them in a light incubator at room temperature for about one month before positive identification.

[0087] 3. Identification of positive materials

[0088] The positive plants of trifoliate orange silenced by VIGS were identified. The extracted trifoliate orange DNA was used as a template and two pairs of primers were used for PCR identification of positive plants. One was the forward and reverse primers of TRV1, and the other was the reverse primer constructed by the forward primer of TRV2 and the target gene recombination vector. The primer sequences were: TRV1-F: 5'-ATTGAGGCGAAGTACGATGG-3'; TRV1-R: 5'-CCATCCACAATTATTTTCCGC-3'; TRV2-F: 5'-ATTCACTGGGAGATGATACGCT-3'; pTRV2-CtrBBX32-R: 5'-TGTCTTCGGGACATG CCCGGG GAAGCATCACTGCCGAAACC-3'. The results are as follows. Figure 4 As shown in A and B.

[0089] At the same time, the expression level of CtrBBX32 gene in positive plants was detected by real-time fluorescence quantification ( Figure 4 Middle C).

[0090] Example 4: Interventional identification of cold resistance of CtrBBX32 trifoliate orange

[0091] Positive plants with high expression inhibition in the TRV2-CtrBBX32 silenced line were selected and subjected to -4°C low-temperature treatment for 4-6 hours. Before treatment, there was no significant difference in growth between the empty and silent lines. However, after low-temperature treatment, the leaves of the TRV2-CtrBBX32 silenced line plants showed slight curling and waterlogging, while the empty lines showed severe leaf curling and even death. Figure 5 A in the middle), indicating that TRV2-CtrBBX32 interfered with the damage of trifoliate orange to a low degree under low temperature stress. At the same time, the chlorophyll imaging system showed that the photosynthetic intensity of TRV2-CtrBBX32 silenced plants after low temperature treatment was significantly higher than that of the empty plants ( Figure 5B), indicating that the photosynthetic reaction of TRV2-CtrBBX32 silenced plants was less damaged under low temperature stress. Figure 5 C) and MDA content ( Figure 5 The results of histochemical staining showed that the leaves of TRV2-CtrBBX32 silenced plants accumulated less H2O2 and O2 .- ( Figure 5 Meanwhile, the soluble sugar content of the intervention line was higher than that of the empty-loaded plant ( Figure 5 Middle F).

[0092] The above results prove that silencing CtrBBX32 can significantly improve the cold resistance of trifoliate orange plants, demonstrating the important regulatory role of CtrBBX32 in improving plant cold resistance.

[0093] Example 5: Overexpression of CtrBBX32 by Agrobacterium rhizogenes and identification of positive seedlings.

[0094] 1. Vector Construction

[0095] Using the cDNA of Prunus trifoliata as template, specific primers were designed to amplify the CDS of CtrBBX32 gene. TM The SoSoo Cloning Kit was used to insert the CtrBBX32 gene CDS, minus the terminator, into the designated restriction enzyme sites of the pENTR1A vector. Subsequently, the CDS was transferred into the pK7WG2D vector carrying the CaMV35S promoter using the LR cloning enzyme in Gateway technology (Thermo Fisher, USA) to generate the final overexpression vector. The constructed vector was sequenced and transformed into competent Agrobacterium rhizogenes MSU440 cells. The primer sequences were: pENTR1A-CtrBBX32-F: 5'-AAAGGAACCAATTCA GTCGA CATGAAACGAGCTTGC-3'; pENTR1A-CtrBBX32-R: 5'-TGGAAAAGGGAATTC GGTA CCTACGTTGCACTCAGC-3'.

[0096] 2. Overexpression of Trifoliate Orange Infection

[0097] Take 3-4 month old wild-type trifoliate orange seedlings with the same growth vigor for infection.

[0098] 1) Streak the pK7WG2D-CtrBBX32 glycerol Agrobacterium stored at -80°C onto LB (50 mg / L Kan) solid medium and culture inverted at 28°C for 2 days to obtain single colonies;

[0099] 2) Pick one single colony from each strain and place it in 5 mL of LB liquid medium containing the same antibiotics. Incubate at 28°C, 220 rpm, and shake gently for 16-24 hours to fully activate the bacteria.

[0100] 3) The activated Agrobacterium liquid was inoculated into the same LB liquid medium at a ratio of 1:100, cultured at 28°C, 220 rpm, and expanded for 6-8 h. The cells were collected by centrifugation at 6000 rpm and suspended in MES buffer (10 mmol / LMES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7). OD 600 Adjust to 0.6;

[0101] Then, incubate in the dark at 28℃ for 2-4 hours to prepare the infection solution.

[0102] 4) Cut the trifoliate orange into stem segments with about four to five leaves each, and poke a small hole at the lower end with a syringe needle. Soak the poke hole completely in the prepared Agrobacterium infection solution, vacuum pump for 10 minutes, quickly deflate to allow Agrobacterium to penetrate the germinated seeds, and repeat 3 times. Then let it stand for 15 minutes, take out the infected seeds and air them on dry filter paper, and then cut them into a white plastic dish of pure vermiculite. Keep the vermiculite moist, cover it with a lid and keep it away from light. Many roots will grow for about a month (observe whether the dish remains moist in the middle), use a handheld fluorescent lamp to identify, cut off the non-fluorescent roots, and then carry out hydroponic culture. After growing for a month, many positive roots will grow on the roots, and then use a handheld fluorescent lamp to identify;

[0103] 3. Identification of positive materials

[0104] To identify the positive plants of trifoliate orange, DNA from trifoliate orange roots was extracted as a template and PCR was performed using primers, namely 35S:F forward primer and reverse primer constructed by the target gene recombination vector. The results are as follows: Figure 6 As shown in A.

[0105] At the same time, the GFP fluorescent protein in the positive plants was detected by handheld fluorescent lamp ( Figure 6 Middle B).

[0106] Example 6: Identification of cold resistance of trifoliate orange overexpressing CtrBBX32 by Agrobacterium rhizogenes

[0107] The transformed material was treated at -4°C for 4-6 hours. Before treatment, there was no significant difference in the growth of the empty and silent plants. However, after the low temperature treatment, the leaves of the OE-CtrBBX32 plants showed severe leaf curling and even death, while the empty plants showed slight curling and waterlogging. Figure 7A in the middle), indicating that OE-CtrBBX32 trifoliate orange was more seriously damaged by low temperature stress. At the same time, the chlorophyll imaging system showed that the photosynthetic intensity of OE-CtrBBX32 plants after low temperature treatment was significantly lower than that of the empty plants ( Figure 7 Middle B). Electrical conductivity of OE-CtrBBX32 plants after low temperature ( Figure 7 C) and MDA content ( Figure 7 The results of histochemical staining also showed that the roots of OE-CtrBBX32 plants accumulated more H2O2 ( Figure 7 At the same time, root activity was measured, and the results showed that the root activity of OE-CtrBBX32 plants was lower ( Figure 7 Meanwhile, the soluble sugar content in the roots of OE-CtrBBX32 plants was lower than that of the empty-load plants ( Figure 7 Middle G),

[0108] The above results prove that overexpression of CtrBBX32 in roots can significantly reduce the cold resistance of trifoliate orange plants, demonstrating the important regulatory role of CtrBBX32 in improving plant cold resistance.

Claims

1. A method for improving cold resistance of plants, characterized in that: Knockout, inhibition or silencing of the gene shown in SEQ ID No. 1 in a plant, thereby improving the cold resistance of the plant, wherein the plant is a trifoliate orange ( Poncirus trifoliata ).

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