Poncirus trifoliate cold-resistant gene CtrBBX32 and application thereof in cold-resistant genetic improvement of plants

Abstract

The invention discloses a trifoliate orange cold-resistant gene CtrBBX32 and application thereof in cold-resistant genetic improvement of plants, the CtrBBX32 gene is a gene of a subfamily of a zinc finger structural protein family separated and cloned from a very cold-resistant citrus material trifoliate orange, and the sequence of the CtrBBX32 gene is as shown in SEQ ID No.1. The gene is used for constructing an interference vector and an overexpression vector, and the interference vector and the overexpression vector are introduced into trifoliate orange through agrobacterium tumefaciens and agrobacterium rhizogenes mediated genetic transformation to obtain a transgenic plant. Biological function verification shows that the CtrBBX32 gene cloned by the invention has the function of regulating and controlling the cold resistance of plants. The discovery of the gene provides a new gene resource for plant stress resistance molecular design breeding and provides a new genetic resource for implementation of green agriculture and water-saving agriculture, and development and utilization of the genetic resource are beneficial to reduction of agricultural production cost and realization of environmental friendliness.

Description

Technical Field

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

[0002] Extreme low temperature stress 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 the winter growth cycle is particularly critical. Cold stress can generally be divided into two levels: cold (0-15°C) and freezing (<0°C) (Ding et al., 2019). Cold stress reduces the membrane fluidity of 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 studies have shown that the membrane system of the cell is the main site of freezing injury in plants (Steponkus, 1984). In addition, it is well known that freezing-induced membrane damage is mainly caused by the severe dehydration associated with freezing (Steponkus, 1984). The accumulation of sugars and other low sugars that usually occurs in cold acclimation may also contribute to membrane stability, because these molecules can protect membranes from freezing-induced damage in vitro (Anchordoguy et al., 1987; Strauss & Hauser, 1986). Although freezing injury is considered to be mainly due to membrane damage caused by cellular dehydration, other factors may also contribute to freezing-induced cell damage. Freezing-induced reactive oxygen production can cause membrane damage (Tao et al., 1998), and intercellular ice can form adhesions with cell walls and membranes and cause cell rupture (Olien & Smith, 1977). In addition, plants undergo protein denaturation at low temperatures (Guy et al., 1998), which can cause cell damage.

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

[0005] Hauser, 1986). During cold acclimation, plants perceive cold signals through potential cold sensors and activate a set of regulatory networks. For example, the signaling pathway that depends on the key transcription factor CBF enhances the ability to resist cold stress (Islam et al., 1981; Monroy et al., 1993). Plants then improve their cold tolerance by accumulating many protective substances such as soluble sugars and proline (Crowe et al., 1988; Tarchevsky & Egorova, 2022).

[0006] The expression level of genes is 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 overexpressing plants showed reduced heat tolerance.

[0007] (Wang et al., 2013). Chrysanthemum Cm BBX19 negatively regulates chrysanthemum drought tolerance 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 resistance has not been reported yet. Studying the mechanism of action of BBX on low temperature resistance is of great value to crop cold resistance breeding.

[0008] Poncirus trifoliata is often used as a rootstock for citrus production. It is an important resource for citrus rootstocks due to its strong cold resistance, resistance to epidemics, and hybridization affinity with citrus. Therefore, cloning genes related to cold resistance in Poncirus trifoliata is the key and foundation of 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 separated from Poncirus trifoliata, and its amino acid sequence 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 plants, thereby reducing the cold resistance of plants.

[0037] A method for improving the cold resistance of plants, the method 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-resistance gene CtrBBX32 of Poncirus trifoliata, wherein 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 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 through the transcriptome data of trifoliate orange treated in the laboratory and the transcriptome data of different tissue parts of trifoliate orange (roots, stems, leaves). The results showed that the expression of CtrBBX32 increased with the extension of low-temperature treatment time, and it was highly expressed in the roots of trifoliate orange. CtrBBX32 overexpression and transient silencing strains were constructed, and the cold resistance 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 resistance of CtrBBX32 overexpression transgenic trifoliate orange was reduced, while the conductivity, MDA content and ROS content increased, and the soluble sugar content was significantly reduced. The cold resistance phenotype of the trifoliate orange plants in the CtrBBX32 silenced line 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, leaves).

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

[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 positive identification and relative expression analysis of the VIGS silencing material of the present invention;

[0051] Wherein: A is the positive plant identified by TRV1 specific primers for CtrBBX32 gene of the present invention (TRV2-CtrBBX32); B is the positive plant identified by TRV2 specific primers for CtrBBX32 gene of the present invention (TRV2-CtrBBX32); C is the expression level analysis of CtrBBX32 in CtrBBX32 interference material. 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 of trifoliate orange;

[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 positive identification and relative expression analysis of hairy root transformation materials of the present invention;

[0055] Wherein: A is the positive plant (OE-CtrBBX32) of the CtrBBX32 gene of the present invention identified by specific primers; 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 content of fructose, glucose and sucrose 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 Prunus 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 recovery kit (Axygene, USA), and then the purified product was connected to the pEASY-Blunt vector (Quanshijin, China). The connection system is shown in Table 3. After incubation at room temperature for 15 minutes, the competent Escherichia coli DH5α was transformed. The plate was coated, inverted and cultured 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. The positive clone was then sent to Wuhan Qingke Biological Company for sequencing. According to the sequencing results, the full-length gene sequence of CtrBBX32 was obtained.

[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] The open reading frame (ORF) prediction revealed that the gene contained one ORF, was 732 bp in length, encoded a protein of 243 amino acids, had a molecular weight of 26.30 kDa, and an isoelectric point pI of 8.50. The gene was named CtrBBX32, the nucleotide sequence was shown in SEQ ID No.1, and the amino acid sequence was shown in SEQ ID No.2.

[0072] Embodiment 2:

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

[0074] The ORF region of CtrBBX32 (without the stop codon) was amplified with primer sequences of 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 the 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 control 35S:YFP+mCherry-VirD2NLS were transiently transformed into leaf epidermal cells of Nicotiana benthamiana. Laser confocal fluorescence observation revealed 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 (A).

[0075] In order to study and analyze the transcriptional activation activity of CtrBBX32, the full-length gene was amplified and connected to 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', and then transformed into yeast competent cells AH109, ​​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 cDNA of Prunus trifoliata as template, specific primers were designed to amplify a 225 bp fragment (SEQ ID No. 3) in the CDS of CtrBBX32 gene. TMSoSoo Cloning Kit (Qingke, China) was used to insert the construct between the two restriction sites of BamH I and SmaI on the pTRV2 vector in one step. The constructed vector was transformed into Agrobacterium tumefaciens GV 3101 competent cells after sequencing. 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 off the seeds from the fruits of the orange tree stored in a refrigerator at 4°C, soak them in 1mol / L NaOH solution for 15 minutes to remove the pectin, rinse them with distilled water 2-3 times, bury the seeds with vermiculite, keep the vermiculite moist, and place them in a 28°C incubator in the dark to germinate. After about 4 weeks, when the seed buds germinate to 1-2cm in length, they can be used for VIGS infection. The operation is as follows:

[0081] 1) TRV1, TRV2, TRV2-CtrBBX32 and other glycerol Agrobacterium stored at -80°C were streaked onto LB solid medium (containing 50 mg / L Rif and 50 mg / L Kan) and cultured inverted at 28°C for 2 days to obtain single clones;

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

[0083] 3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a ratio of 1:100, and cultured at 28°C, 220 r / min, for 6-8 h, and then centrifuged at 6000 r / min to collect the cells, and MES buffer (10 mmol / LMES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7) was added to suspend the cells, and the OD 600 Adjust to 0.6;

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

[0085] 5) Use a syringe needle to lightly poke some small holes on the germinating seedlings, completely immerse them in the prepared Agrobacterium infection solution, vacuum for 10 minutes, quickly release the air to allow the Agrobacterium to penetrate the germinating seeds, and repeat 3 times. Then let it stand for 15 minutes, take out the infected seeds and air them on dry filter paper, let it stand for 2-3 minutes, then spread them flat on a large dish soaked with filter paper with sterile water, and place them in a dark incubator at room temperature for 2-3 days;

[0086] 6) Rinse the seeds after dark culture with clean water to remove the residual bacterial solution, 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] The positive plants with high expression inhibition of TRV2-CtrBBX32 silenced plants were selected and treated at -4℃ for 4-6 hours. Before the treatment, there was no significant difference in the growth of the empty plants and the silent plants. After the low temperature treatment, the leaves of the TRV2-CtrBBX32 silent plants showed slight curling and waterlogging, while the empty plants had serious leaf curling and even death ( Figure 5 A), 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 empty plants ( Figure 5(B), indicating that the photosynthetic response of the TRV2-CtrBBX32 silenced line plants under low temperature stress was less damaged. 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 At the same time, the soluble sugar content of the interference line was higher than that of the empty-loaded plants ( 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 cDNA of Prunus trifoliata as template, specific primers were designed to amplify CDS of CtrBBX32 gene. TM SoSoo Cloning Kit inserted the CtrBBX32 gene CDS without the terminator into the designated restriction site of the pENTR1A vector. Subsequently, the LR cloning enzyme in the Gateway technology (Thermo Fisher, USA) was used to transfer it into the pK7WG2D vector with the CaMV35S promoter to obtain the final overexpression vector. After the constructed vector was sequenced correctly, it was transferred into the Agrobacterium rhizogenes competent MSU440. 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 wild-type trifoliate orange seedlings that are 3-4 months old and have the same growth potential for infection.

[0098] 1) The pK7WG2D-CtrBBX32 glycerol Agrobacterium stored at -80°C was streaked onto LB (50 mg / L Kan) solid medium and incubated at 28°C for 2 days to obtain a single clone;

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

[0100] 3) The activated Agrobacterium culture liquid was inoculated into the same LB liquid medium at a ratio of 1:100, and cultured at 28°C, 220 r / min, for 6-8 h, and then centrifuged at 6000 r / min to collect the cells, and MES buffer (10 mmol / LMES, 10 mmol / L MgCl2, 150 μmol / L AS, pH = 5.6-5.7) was added to suspend the cells, and the OD 600 Adjust to 0.6;

[0101] Then, incubate in a dark incubator at 28°C for 2-4 hours to prepare the infection solution;

[0102] 4) Cut the trifoliate orange into stem segments with four to five leaves each, and puncture a small hole at the lower end with a syringe needle. Completely immerse the punctured area in the prepared Agrobacterium infection solution, vacuum evacuate for 10 minutes, and quickly deflate to allow the Agrobacterium to penetrate into the germinated seeds. Repeat 3 times. Then let it stand for 15 minutes, take out the infected seeds and air them on dry filter paper, then insert them into a white plastic dish of pure vermiculite. Keep the vermiculite moist, cover with a lid and keep 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 roots that do not fluoresce, and then carry out hydroponic culture. After a month of growth, many positive roots will grow on the roots, which can be identified using a handheld fluorescent lamp again;

[0103] 3. Identification of positive materials

[0104] To identify the positive plants of trifoliate orange, the DNA of trifoliate orange root 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℃ for 4-6 hours. Before the treatment, there was no significant difference in the growth of the empty-loaded plants and the silent-loaded plants. However, after the low-temperature treatment, the leaves of the OE-CtrBBX32 plants had already shown severe leaf curling or even death, while the empty-loaded plants showed slight curling and waterlogging ( Figure 7A in the middle), indicating that OE-CtrBBX32 trifoliate orange was more 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 empty-load 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 Middle E). Root activity was also 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 plants without any growth factor ( 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 protein isolated from Poncirus trifoliata, the amino acid sequence of which is shown in SEQ ID No.

2.

2. A gene encoding the protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. A recombinant vector containing the gene according to claim 2 or 3.

5. A method for reducing the cold resistance of plants, characterized in that: The method comprises overexpressing the gene according to claim 2 or 3 in a plant, thereby reducing the cold resistance of the plant.

6. A method for improving the cold resistance of plants, characterized in that: The method is to knock out, inhibit or silence the gene shown in SEQ ID No. 1 in the plant, thereby improving the cold resistance of the plant.

7. The method according to claim 5 or 6, characterized in that: The plant is Poncirustrifoliata.

8. A specific fragment for silencing the cold-resistance gene CtrBBX32 of Poncirus trifoliata, characterized in that: The nucleotide sequence of the specific fragment is shown in SEQ ID No.

3.

9. A recombinant vector, characterized in that: The recombinant vector is composed of a pTRV2 vector and the specific fragment according to claim 8 connected to the pTRV2 vector.

Images