Application of pear transcription factor PbTCP10 in promoting fruit ripening

Abstract

The application discloses application of a pear transcription factor PbTCP10 in promoting fruit ripening and belongs to the technical field of plant genetic engineering. The PbTCP10 is a transcription factor with the functions of promoting ethylene synthesis and ABA synthesis, which is separated and cloned from pear fruits, and the nucleotide sequence of the PbTCP10 is shown as SEQ ID NO. 1. Through biological function verification, it is shown that the cloned PbTCP10 gene has the functions of promoting ethylene and ABA synthesis and participating in fruit ripening. The discovery of the PbTCP10 gene provides new insights into the transcriptional regulation of ethylene synthesis and ABA synthesis and the mutual regulation between hormones in the fruit ripening process. The development and utilization of the resource are favorable to improving the commodity value and market competitiveness of pear fruits, enriching early-ripening varieties of pear, prolonging the shelf life of fruits, reducing agricultural costs and accelerating the pace of breeding industry.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and plant genetic engineering, and relates to the application of the pear fruit transcription factor PbTCP10. Specifically, it relates to the application of the PbTCP10 gene, a TCP family member related to pear fruit ripening, isolated and cloned from pear, in promoting fruit ripening. Technical Background

[0002] The ripening process of fruit involves a series of complex and orderly physiological and biochemical reactions, including the accumulation and changes in aroma, sugar, acid, color, softening, and hormones (Gu et al., 2017; Hao et al., 2018; Guo et al., 2021). Ethylene and ABA, as endogenous plant hormones, play important roles in the growth, development, and ripening of fruit. Ethylene synthesis involves two key steps: ① S-adenosyl-L-methionine is catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) to generate ACC; ② ACC is oxidized by ACC oxidase (ACO) to generate ethylene. Among these, ACC synthase (ACS) and ACC oxidase (ACO) are the two key enzymes. The synthesis pathway of abscisic acid (ABA) involves neoxanthin as an important precursor, which is converted to xanthophyll by 9-cis-epoxycarotene dioxygenase (NCED), then catalyzed by short-chain dehydrogenase (SDR) to form abscisaldehyde, which is subsequently oxidized by aldehyde oxidase (AAO) to form ABA. Among them, NCED enzyme is the key rate-limiting enzyme.

[0003] Recent studies have revealed that various transcription factors can regulate the biosynthesis of ethylene and ABA, thereby participating in fruit ripening. These include transcription factors such as NAM / ATAF1 / 2 / CUC2 (NAC), zinc finger proteins, and TCP. Current research indicates that the transcription factors SNAC4 and SNAC9 in tomatoes promote fruit ripening. Silencing SNAC4 and SNAC9 reduces the expression levels of ethylene-related genes and decreases carotene content (Zhu et al., 2014; Kou et al., 2016). NOR-like 1 can directly bind to the promoters of downstream target genes SlACS2 and SlACS4, promoting ethylene synthesis in tomato fruits. Simultaneously, ABA can induce NOR-like 1 gene expression, and NOR, as a target gene of SlAREB1, participates in ABA signal transduction (Gao et al., 2018; Mou et al., 2018; Kou et al., 2021). In figs, FcAAO3 not only participates in ABA biosynthesis but also exhibits downregulated expression upon silencing, promoting ethylene synthesis (Qiao et al., 2021). SlZFP2 regulates ABA biosynthesis genes during fruit development and promotes fruit ripening by inhibiting the transcriptional repression of the ripening regulator CNR (Weng et al., 2015). In strawberries, transient overexpression of the FvTCP9 gene significantly increased the expression levels of ripening-related genes such as FaQR and FaSUT1, indicating that FvTCP9 promotes fruit ripening. Furthermore, the ABA content in FvTCP9-OE strawberries was significantly higher than that in the control group and FvTCP9-RNAi fruits (Xie et al., 2020).

[0004] The TCP family is an important family of transcription factors widely found in plants. Its name comes from the first four characteristic members: Aspergillus flavus branching 1 (TB1) in maize, Aspergillus cytokine resistance (CYC) in Chinese cabbage, and proliferative cytokines (PCFs) in rice (Doebley et al., 1997; Luo et al., 1996; Kosugi et al., 1997). The TCP family is characterized by a conserved N-terminal basic helix-loop-helix (bHLH) structure, known as the TCP domain, which primarily binds to DNA and participates in protein-protein interactions (Song et al., 2018; Wang et al., 2022).

[0005] Pear is one of the world's and China's important economic fruit trees, and a typical climacteric fruit, releasing large amounts of ethylene during ripening. We have identified the ethylene biosynthesis genes PbACS1b and PbACO1 in pear fruit (Hao et al., 2018). We have also found that transcription factors PuERF2, PuERF3, PuBZR1, PbERF24, and PbEBF3 regulate the ripening process of pear fruit (Ji et al., 2021; Hao et al., 2018; Wang et a., 2023). A total of 34 TCP family members have been reported in the pear genome (Zhao et al., 2021), but it is currently unclear which TCP genes are involved in the ripening process, and the function and mechanism of TCP genes in regulating fruit ripening are also unknown. Therefore, based on the above research background, the applicant conducted cloning and functional studies on the pear transcription factor TCP gene. Through Agrobacterium-mediated genetic transformation, the TCP gene in pear fruit was silenced and overexpressed, and its interaction with ripening-related genes was investigated. Summary of the Invention

[0006] The purpose of this invention is to screen a transcription factor, PbTCP10, that simultaneously regulates ethylene and ABA synthesis in pear fruit and promotes fruit ripening; and to provide the application of transcription factor PbTCP10 in regulating ethylene and ABA synthesis and promoting pear fruit ripening.

[0007] Another objective of this invention is to provide a method for promoting pear fruit ripening or for cultivating new early-maturing pear germplasm.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] Application of transcription factor PbTCP10, as shown in SEQ ID NO.1, or biological materials containing transcription factor PbTCP10, as shown in SEQ ID NO.1, in regulating ethylene and / or ABA synthesis and promoting pear fruit ripening.

[0010] Application of transcription factor PbTCP10, as shown in SEQ ID NO.1, or biological materials containing transcription factor PbTCP10, as shown in SEQ ID NO.1, in the breeding of new early-maturing pear germplasm.

[0011] Preferably, the above-mentioned biological materials are expression cassettes, recombinant vectors, transgenic cells, transgenic recombinant bacteria, transiently transgenic pear fruit, or stable transgenic pear pulp callus.

[0012] Further preferably, transient overexpression of the transcription factor PbTCP10 in pear fruit or stable expression in pear pulp callus can promote ethylene synthesis and / or hormone ABA synthesis, thereby promoting pear fruit ripening.

[0013] A method for promoting pear fruit ripening or cultivating new early-ripening pear germplasm is also within the scope of protection of this invention. Transient overexpression of the transcription factor PbTCP10, as shown in SEQ ID NO.1, in pear fruit or stable expression in pear pulp callus can promote ethylene synthesis and / or hormone ABA synthesis, thereby promoting pear fruit ripening or cultivating new early-ripening pear germplasm.

[0014] This invention isolates and clones the PbTCP10 gene, a transcription factor that regulates the synthesis of ethylene and ABA in ripe pear fruit and promotes fruit ripening. It belongs to the TCP family and its nucleotide sequence is shown in SEQ ID NO.1. The coding sequence (CDS) is 1134 bp long and encodes 377 amino acids, as shown in SEQ ID NO.2. The isoelectric point is 5.695 and the molecular weight is 39.38 KD.

[0015] The invention comprises a recombinant expression vector containing the PbTCP10 gene described herein, a transgenic recombinant bacterium, transient transgenic pear fruit, and transgenic fruit pulp callus. The recombinant expression vector is preferably the insertion vector pSAK277 containing the PbTCP10 gene, with HpaI and Xbal restriction enzyme sites; the recombinant expression vector is named pSAK277-PbTCP10.

[0016] The primer pair for cloning the cDNA sequence of the PbTCP10 gene described in this invention is shown in SEQ ID NO.7 for the upstream primer PbTCP10-F1 and in SEQ ID NO.8 for the downstream primer PbTCP10-R1.

[0017] This invention investigated the expression level of the transcription factor PbTCP10 at different developmental stages of the fruits of "Sucui No. 1" and "Cuiguan". It was found that the expression level of the PbTCP10 gene was highest in "Sucui No. 1" 110 days after flowering, significantly higher than at other fruit developmental stages. In "Cuiguan" fruits 120 days after flowering, the expression level of the PbTCP10 gene was significantly higher than at other stages.

[0018] This invention utilizes Agrobacterium-mediated transient infection of "Sucui No. 1" fruit in the pre-ripening stage. Both transient overexpression and stable genetic overexpression of the transcription factor PbTCP10 can increase the ethylene and ABA content in the callus of pear fruit and pulp, thus promoting pear fruit ripening.

[0019] This invention uses VIGS technology to silence the transcription factor PbTCP10, which reduces the ethylene and ABA content in pear fruits and delays fruit ripening.

[0020] This invention utilizes qRT-PCR technology to discover that overexpression of the PbTCP10 gene can increase the expression levels of the ethylene synthase PbACS1b gene and the ethylene oxidase PbACO1 gene. It also detected the same upregulation of the expression levels of zeaxanthin epoxidase PbZEP, short-chain dehydrogenase PbSDR, aldehyde oxidase PbAO1, and 9-cis-epoxycarotenoid dioxygenases PbNCED.A1 and PbNCED.B in the ABA synthesis pathway. Furthermore, it was found that the expression level of the transcription factor PbbHLH164, which regulates ethylene maturation, was also increased, indicating that the transcription factor PbTCP10 is involved in the fruit ripening process.

[0021] This invention constructs an overexpression vector for PbTCP10, with the recombinant plasmid being pSAk277-PbTCP10. The PbTCP10 gene is stably transformed into pear flesh callus using Agrobacterium infection-mediated transformation. Biological functional analysis of the resulting transgenic callus shows that overexpression of the PbTCP10 gene can increase the expression levels of PbTCP10, PbACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbAO7, PbNCED.A1, PbNCED.B, and PbbHLH164 genes, as well as ethylene and ABA production in positive callus.

[0022] This invention constructs a PbTCP10 protein expression vector, with the recombinant plasmid being pCold-PbTCP10, to verify the interaction between DNA and protein.

[0023] This invention describes a method for expressing the fusion protein His-PbTCP10 in vitro using Escherichia coli BL21(DE3).

[0024] This invention utilizes a dual-luciferase reporter gene system to analyze the regulatory relationship between PbTCP10 and the genes PbACS1b, PbSDR, PbNCED.A1, and PbbHLH164. The results show that there are promoter interactions between PbTCP10 and the genes PbACS1b, PbSDR, PbNCED.A1, and PbbHLH164.

[0025] This invention utilizes biomembrane interference to quantitatively detect the binding affinity of PbTCP10 and PbACS1b, PbSDRPbNCED.A1 and PbbHLH164 promoters from a kinetic perspective.

[0026] EMSA confirmed that PbTCP10 can directly interact with the binding elements of the promoters of PbACS1b, PbSDR, PbNCED.A1 and PbbHLH164, promoting the synthesis of ethylene and ABA, and thus regulating fruit ripening.

[0027] Compared with the prior art, the present invention has the following advantages and effects:

[0028] This invention screens a gene related to fruit ripening, breaking through the barriers of traditional breeding, with the aim of selecting early-maturing traits in pear hybrids and greatly improving breeding efficiency. The discovery of the PbTCP10 gene provides a new gene resource for molecular breeding that promotes ethylene synthesis, and offers new insights into the transcriptional regulation of ethylene and ABA synthesis and the inter-hormone regulation during fruit ripening. The development and utilization of this resource will help improve the commercial value of pears, enrich early-maturing pear varieties, reduce agricultural costs, and accelerate the pace of the breeding industry. Attached Figure Description

[0029] Figure 1 This is a graph showing the expression level analysis of the gene PbTCP10 of this invention at different developmental and maturation stages in two varieties, "Sucui No. 1" and "Cuiguan". "*" and "**" represent significant differences at P<0.05 and P<0.01 levels, respectively.

[0030] Figure 2 The present invention demonstrates the phenotypic changes in pear fruits mediated by Agrobacterium-mediated transient overexpression of the PbTCP10 gene.

[0031] Figure 3 The phenotypic changes of pear fruits after the gene PbTCP10 of this invention was silenced using VIGS.

[0032] Figure 4 For the analysis of fruit ethylene release and fruit ABA content after transient overexpression and silencing of gene PbTCP10 in this invention, "*" and "**" represent significant differences at the levels of P<0.05 and P<0.01, respectively.

[0033] Figure 5 This diagram illustrates the expression levels of the genes PbTCP10, PbACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbNCED.A1, PbNCED.B, and PbbHLH164 in pear fruit after transient overexpression and silencing of the PbTCP10 gene, as described in this invention. "*" and "**" represent significant differences at P < 0.05 and P < 0.01, respectively.

[0034] Figure 6 The PbTCP10 clone gene of this invention was stably transformed into pear pulp callus.

[0035] Figure A shows the stable genetic transformation of pear flesh callus by transcription factor PbTCP10 mediated by Agrobacterium, and the positive callus PbTCP10-OE obtained through transformation, screening and subculture; Figure B shows the ethylene content analysis in positive callus PbTCP10-OE and empty control group; Figure C shows the ABA content analysis in positive callus PbTCP10-OE and empty control group.

[0036] Figure 7 This is a graph showing the expression levels of the genes PbTCP10, PbACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbNCED.A1, PbNCED.B, and PbbHLH164 after stable genetic transformation of the PbTCP10 gene into pear flesh callus. "*" and "**" represent significant differences at P < 0.05 and P < 0.01, respectively.

[0037] Figure 8 This invention relates to the regulatory effect of PbTCP10 on the promoter of the PbACS1b gene.

[0038] Figure A shows a schematic diagram of the vector construction of the reporter gene PbTCP10 and promoter PbACS1b of this invention; Figure B shows the regulatory effect of PbTCP10 on promoter PbACS1b in the dual-luciferase reporter gene system analysis of this invention; Figure C shows the binding constants of PbTCP10 and PbACS1b promoter elements analyzed by biomembrane interference analysis of this invention.

[0039] Figure 9 This invention provides a dual-luciferase reporter gene system analysis of the regulatory effects of PbTCP10 on the promoters of PbZEP, PbSDR, PbAO1, PbNCED.A1, and PbNCED.B.

[0040] Figure A shows a schematic diagram of the vector construction of the reporter gene PbTCP10 and promoters PbZEPpro, PbSDRpro, PbAO1pro, PbNCED.A1 pro, and PbNCED.B pro of this invention; Figure B shows the dual-fluorescence LUC / REN analysis of the transcription factor PbTCP10 and promoter of this invention compared with the control group.

[0041] Figure 10 This invention utilizes biomembrane interference and EMSA experiments to investigate and analyze the element-specific binding of PbTCP10 to the promoters of PbSDRpro and PbNCED.A1 pro.

[0042] Figure 11 This invention relates to His-PbTCP10, a protein expressed in vitro by in vitro induction of Escherichia coli BL21(DE3). Detailed Implementation

[0043] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and implementation, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from its spirit and scope to adapt it to various uses and conditions.

[0044] Example 1: Analysis of PbTCP10 gene expression levels at different developmental and maturation stages in two cultivars, “Sucui No. 1” and “Cuiguan”.

[0045] 1. Both the pear varieties “Su Cui No. 1” and “Cui Guan” were harvested from the Baima Experimental Base of Nanjing Agricultural University. From 60 days after flowering to fruit maturity, three biological replicates were set up for each sample, and each replicate contained at least four fruits.

[0046] 2. Download the nucleotide sequence of the PbTCP10 gene from the pear genome database (http: / / 202.195.250.6:8080 / src / # / home). Design specific primers and primers for the internal reference gene using Primer 5.0 software according to general primer design principles. The quantitative PCR primers for the PbTCP10 gene are as follows:

[0047] PbrTCP10-RT-F:CTCCTCCAGTCACAACAGTAACGGC(SEQ ID NO.3)

[0048] PbrTCP10-RT-R:GTTATGATTTTCCGGTCCAACTGGT(SEQ ID NO.4)

[0049] The primer sequences for the internal reference gene are as follows:

[0050] GR-F: GATGGTGCTATGAAGATGCCAAATGT (SEQ ID NO.5);

[0051] GR-R:TCCCGAGCATCACGATAGATTCAC (SEQ ID NO. 6).

[0052] 3. RNA was extracted from pear pulp at different growth stages using a plant total RNA extraction kit for polyphenols and polysaccharides (purchased from TIANGEN, operated according to the kit instructions). cDNA was obtained via reverse transcription. qRT-PCR was performed in a LightCycler 480II / 96 thermal cycle. The kit (LightCycler 480SYBR Green I Master) and 96-well qPCR plates were provided by Roche. The qPCR reaction mixture consisted of 10 μL SYBR Green I Master, 4 μL ddH2O, 2.5 μL forward primer, 2.5 μL reverse primer, and 1 μL cDNA. The reaction program was: 95°C denaturation for 15 s, 60°C annealing for 30 s, 45 cycles. All analyses were performed in three independent biological replicates. The pear Pbr041114.1 gene was used as an internal control.

[0053] The results showed that the expression level of the PbTCP10 gene was highest in the fruit of 'Sucui No. 1' cultivar 1, 110 days after flowering, significantly higher than the expression level at other fruit development stages. The expression level of the PbTCP10 gene was also significantly higher in the fruit of 'Cuiguan' cultivar 120 days after flowering than at other stages. Figure 1 This indicates that the PbTCP10 gene is associated with fruit ripening.

[0054] Example 2: Cloning of the PbTCP10 gene

[0055] 1. Using the cDNA obtained by reverse transcription in Example 1 as a template, the full-length PbTCP10 gene was amplified. The primer pair for gene amplification was:

[0056] PbTCP10-F1: gttccggattacgctgttaacATGACGTCGTACTTGGAGGATCA (SEQ ID NO.7)

[0057] PbTCP10-R1:tcattaaagcaggactctagaTCACTGGGTTTGGTTATGATTTTC (SEQ ID NO.8)

[0058] Add the following components to the PCR tube: 1 μL Phanta Super-Fidelity DNA Polymerase (Vazyme), 2 μL of the above primers, 2 μL cDNA template, 25 μL 2× Buffer, 17 μL ddH2O, and 1 μL dNTP. The total reaction volume is 50 μL.

[0059] PCR reaction program: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 1 minute, 30 thermal cycles, 72℃ extension for 10 minutes after the cycle, 20℃ for 5 minutes.

[0060] 2. After PCR product electrophoresis on a 1% agarose gel, the target band was recovered according to the instructions of the AxyPrep DNA gel recovery kit. The purified PCR product was then sequenced. Sequencing results showed that the target fragment amplified in this invention was 1134 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing. Sequence alignment analysis confirmed that this sequence is the target gene required by this invention.

[0061] Example 3: Construction of PbTCP10 gene overexpression vector and VIGS vector

[0062] 1. Based on the multiple cloning site of the PSAK277 expression vector, HpaI and XbaI were selected as restriction endonucleases for double digestion. Primers containing both restriction sites were designed using CE software. The PSAK277 vector was double-digested with HpaI and XbaI (purchased from NEB). The total digestion volume was 50 μL: 6 μL vector plasmid, 5 μL CutSmart Buffer, 1 μL each of HpaI and XbaI, and 38 μL ddH2O. The digestion was incubated at 37°C for 3-4 hours. The digestion products were purified using an AxyPrep cleaning kit (procedure followed according to the manufacturer's instructions).

[0063] The annealing temperature for PCR cloning was 60℃, and the PCR reaction system and amplification procedure were the same as in Example 2. Homologous recombination was performed on the PCR product and the double enzyme digestion product (purchased from Vazyme). The recombination system consisted of: 3 μL PCR product, 4 μL enzyme digestion product, 1 μL Exnase II enzyme, and 2 μL 5×CE II Buffer. The reaction solution was incubated at 37℃ for 30 min. The recombinant product was transformed into *E. coli* DH5α by heat shock and evenly spread on solid medium containing 100 mg / L spectinomycin. Positive clones were screened, and 12 single clones were selected for PCR identification and sequencing. The sequencing results confirmed the absence of reading frame mutations, yielding a recombinant vector containing the insert fragment, which was named the overexpression vector PSAK277-PbTCP10.

[0064] 2. The PbTCP10 gene was silenced using VIGS technology. Based on the multiple cloning site of the TRV2 vector and the restriction enzyme sites in the coding region of the gene, KpnI and XbaI were selected as restriction enzymes. The primer design and construction methods of the TRV-PbTCP10 silencing vector were the same as above.

[0065] Primers with restriction enzyme sites were designed, and the primer pair sequences are shown below:

[0066] TRV-TCP10-F: gtgagtaaggttaccgaattcGGGGGGCCGGACCTTCAGG (SEQ ID NO.9)

[0067] TRV-TCP10-R: gagacgcgtgagctcggtaccTCACTGGGTTTGGTTATGATTTTC (SEQ IDNO.10)

[0068] Example 4: Transient conversion of transcription factor PbTCP10

[0069] 1. Single colonies of Agrobacterium containing the recombinant plasmids PSAK-PbTCP10 and TRV-PbTCP10 were picked and placed in liquid culture medium containing the corresponding antibiotics. Agrobacterium containing the empty vector PSAK277 and TRV1+TRV2 were used as controls. The cultures were incubated at 28℃ and 220 rpm. Then, each Agrobacterium was expanded in an Erlenmeyer flask containing 40 ml of liquid culture medium to OD. 600 Centrifuge at 5000 rpm for 10 minutes at 4℃, discard the supernatant and collect the bacterial suspension. Then resuspend the suspension in the infection solution (10 mM MgCl2, 10 mM MES, pH 5.7, 200 μM acetylsylcholine) at 5000 g, centrifuge for 10 minutes, discard the supernatant and collect the bacterial suspension, and repeat once. Finally, suspend the suspension in the infection solution and incubate on a shaker at 25℃ for 2-4 hours.

[0070] 2. Pear fruits of the "Sucui No. 1" variety should be harvested 7-10 days before commercial harvest. Select fruits with smooth, undamaged surfaces and inject them vertically onto the fruit surface using a 1ml sterile syringe. The injection process should be gentle and slow. Inject 40 pear fruits per group of bacterial suspension and observe phenotypic changes at room temperature. 5-7 days after infection, fruits overexpressing the PbTCP10 gene showed a noticeable yellowing near the injection well, while the unloaded control group showed no significant change. Figure 2 The area around the injection site of pear fruits silenced by VIGS was green, delaying fruit ripening; the untreated control group showed no significant change. Figure 3 This indicates that the PbTCP10 gene plays a role in fruit ripening.

[0071] Example 5: Detection of ethylene in fruits from instantaneous PbTCP10 conversion

[0072] Pear fruits exhibiting significant phenotypic changes after infection were grouped into 2L beakers, sealed with plastic wrap, and the total weight of each group of pear fruits was recorded, along with the amount of ethylene released. Following the method (Guo et al., 2021), the ethylene released from the pear fruits in the beakers after 24 hours of sealing was measured. 1 ml of gas was extracted using a syringe, and the amount of ethylene released from the fruit was detected by gas chromatography (Thermo Trace 1300GC, USA). The concentration of ethylene released per unit time per unit fruit weight (μL·g) was calculated. -1 ·h -1 The chromatographic conditions were as follows: GS-GasPro column (30m × 0.32mm); hydrogen flow rate 40mL / min; air flow rate 500mL / min; nitrogen pressure 68.6kPa, flow rate 64mL / min, column flow rate 2mL / min, purge flow rate 2mL / min; injection port temperature 300℃; column temperature 60℃ for 3.5min, then increased to 260℃ at a rate of 30℃ / min; detector temperature 260℃. Injection method: splitless injection.

[0073] The results showed that the ethylene release from fruits overexpressing the PbTCP10 gene was significantly higher than that from the control group, while the ethylene release from fruits with the PbTCP10 gene silenced was significantly lower than that from the control group. Figure 4 This indicates that the PbTCP10 gene can influence fruit ripening by regulating ethylene.

[0074] Example 6: Detection of ABA in PbTCP10 fruit after instantaneous conversion

[0075] Remove pear pulp with obvious phenotypic changes after infection. Place the sample (1g dry weight tissue) in a pre-cooled mortar, add 5mL of pre-cooled 50% chromatographic methanol (v / v), grind into a slurry in an ice bath (under weak light), and extract at 4℃ for 12h. Centrifuge at 10,000r / min for 10min at low temperature (4℃), collect the supernatant and store it in a 4℃ refrigerator. First, extract the residue with 5mL of pre-cooled 80% chromatographic methanol, 12h interval. Collect the supernatant and then extract the residue with 5mL of pre-cooled 100% chromatographic methanol for 12h, centrifuge as above, and collect and combine all extracts. Add PVPB to the extract at 0.2g / g FW to adsorb phenolic substances and pigments, shake on a shaker at 4℃ for 60min, mix well, and centrifuge as above. First wash with 5mL of 50% chromatographic methanol, then slowly pass the supernatant through a C18 column, and finally elute with a gradient of 5mL of 40% chromatographic methanol. The eluent was poured into a 25 mL glass beaker and freeze-dried in a freeze dryer in the dark (covered with a garbage bag). It was then dissolved in 50% chromatographic methanol and filtered through a 0.22 μm organic ultrafiltration membrane. The sample was detected by liquid chromatography-mass spectrometry with slightly improved colorimetric analysis. Each sample was tested in triplicate.

[0076] The results showed that the ABA content in fruits overexpressing the PbTCP10 gene was significantly higher than that in the control group, while the ABA content in fruits with silenced PbTCP10 gene was significantly lower than that in the control group. Figure 4 This indicates that the PbTCP10 gene can influence fruit ripening by regulating ethylene.

[0077] Example 7: qRT-PCR analysis of expression levels of PbTCP10, bACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbNCED.A1, PbNCED.B, and PbbHLH164 genes in transiently transformed pear fruits.

[0078] Pear pulp with obvious phenotypic changes from the infected pear pulp in Example 4 was used to extract RNA using a plant total RNA extraction kit for polyphenols and polysaccharides (purchased from TIANGEN). cDNA was obtained by reverse transcription. qRT-PCR was performed using a LightCycler 480 PCR instrument, 96-well qPCR plates, and LightCycler 480 SYBR Green I Master fluorescent dye. The qPCR reaction system consisted of 10 μL SYBR Green I Master, 4 μL ddH2O, 2.5 μL forward primer, 2.5 μL reverse primer, and 1 μL cDNA. The reaction program was: 95°C denaturation for 15 s, 60°C annealing for 30 s, for 45 cycles. All analyses were performed in three independent biological replicates. The primer sequences used are detailed in Table 3. The pear Pbr041114.1 gene was used as an internal control gene, and its primer sequence was the same as in Example 1.

[0079] qRT-PCR results showed that the expression levels of PbTCP10, PbACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbNCED.A1, PbNCED.B, and PbbHLH164 in pear pulp overexpressing PbTCP10 increased. After PbTCP10 gene silencing, the expression levels of PbTCP10, PbACS1b, PbNCED.B, and PbbHLH164 in pear pulp significantly decreased, while the expression levels of PbACO, PbZEP, PbSDR, PbAO1, and PbNCED.A1 genes did not change significantly. Figure 5 These results indicate that PbTCP10 can mediate ethylene biosynthesis by regulating the activity of PbACS1b, mediate ABA synthesis by regulating the activity of PbSDR and PbNCED.B genes, and affect fruit ripening by regulating the expression level of the transcription factor PbbHLH164.

[0080] Example 8: Genetic transformation of pear pulp callus

[0081] 1. The recombinant plasmid pSAK277-PbTCP10 was constructed in Example 3. Agrobacterium containing the overexpression vector pSAK277-PbTCP10 was activated, and a single colony was picked and inoculated into LB liquid medium containing spectinomycin resistance. The culture was incubated overnight at 28°C and 200 rpm until OD500. 600 =0.8-1.0. Collect bacterial suspension at 5000 rpm for 5 min, discard the supernatant, add MS resuspending buffer, OD 600 =0.5-0.6. Induce at 25℃ and 120 rpm for 1 h. Select callus in good growth condition and add it to the invasion staining solution for 30 min. Filter the callus through gauze and gently rinse with sterile water, blot dry with filter paper, and culture in symbiotic MS medium (Table 1). After 2 days, transfer to screening MS medium (Table 2) and incubate in the dark. Figure 6 The process of stable genetic transformation of the PbTCP10 gene into pear pulp callus, as shown in Figure A.

[0082] 2. Positive callus strain PbTCP10-OE was screened, and the ethylene release and callus content were measured. The specific operation method was the same as in Examples 5 and 6. The results showed that the ethylene release from the callus of the PbTCP10-OE overexpression line was significantly higher than that from the control group. Figure 6 B), the ABA content in callus from the PbTCP10-OE overexpression line was significantly higher than that in the control group. Figure 6 C). This indicates that the PbTCP10 gene can regulate ethylene release and ABA accumulation.

[0083] Table 1 Symbiotic MS Culture Medium

[0084]

[0085] Table 2 Screening MS medium

[0086]

[0087] Example 9: qRT-PCR analysis of expression levels of PbTCP10, PbACS1b, PbACO1, PbZEP, PbSDR, PbAO1, PbNCED.A1, PbNCED.B, and PbbHLH164 genes in PbTCP10-OE pear pulp callus.

[0088] RNA was extracted from PbTCP10-OE pear callus and reverse transcribed to obtain cDNA for qRT-PCR detection, using the same method as in Example 7. The qPCR reaction system and procedure were followed as in Example 7. All analyses were performed by three independent biological replicates. Primer sequences used are detailed in Table 3.

[0089] Table 3 Primer sequences

[0090]

[0091]

[0092] Quantitative fluorometry results showed that the expression levels of genes PbTCP10, PbACS1b, PbSDR, PbAO1, PbNCED.A1, and PbbHLH164 were significantly increased in the pear flesh callus PbTCP1-OE. The expression levels of PbACO1 and PbZEP genes did not change significantly, while the expression level of PbNCED.B gene was significantly decreased. Figure 7 These results indicate that PbTCP10 can regulate the ethylene synthase PbACS1b, the genes PbSDR and PbNCED.A1 in the ABA synthesis pathway, and the expression level of the transcription factor PbbHLH164.

[0093] Example 10: Determination of dual-luciferase

[0094] 1. Promoter isolation and vector construction: DNA was extracted from pear pulp using the FastPure Plant DNA Isolation Mini Kit. Promoter sequences for PbACS1b, PbZEP, PbSDR, PbAO1, PbNCED.A1, and PbNCED.B were identified using the pear genome. The upstream 2000bp sequence was cloned and ligated into the pGreenII 0800-LUC vector. The cloning and vector construction methods were the same as in Examples 2 and 3. Amplification primer sequences are detailed in Table 3.

[0095] 2. Tobacco preparation: Select tobacco plants that have grown for 4-5 weeks and have 5-6 flat leaves, and water them thoroughly to keep them in good growing condition.

[0096] 3. The pSAK277-TCP10 expression vector was used as the Effector plasmid, and the recombinant plasmids PbACS1bpro-LUC, PbZEPpro-LUC, PbSDRpro-LUC, PbACO1pro-LUC, PbNCED.A1pro-LUC, and PbNCED.A1pro-LUC were used as the Reporter plasmids. Figure 8 A, Figure 9 A) Select positive Agrobacterium colonies and expand them in resistant liquid culture medium to OD. 600The concentration was 0.6-0.8. Collect 5000g of bacterial suspension, centrifuge for 10 min, discard the supernatant, resuspend in the infection solution (10mM MgCl2, 10mM MES, pH=5.7, 200μM acetylsylgenone), centrifuge at 5000g for 10 min, discard the supernatant and collect the bacterial suspension, repeat once. Finally, resuspend the bacterial cells in the infection solution and place on a shaker, inducing at 25℃ for 2-4 h. Mix the Agrobacterium resuspension carrying the promoter and the Agrobacterium resuspension carrying the transcription factor at a concentration ratio of 1:9. Remove the needle from a 1ml sterile syringe, draw up the resuspension, and gently inject it onto the back of tobacco leaves. Inject 4-6 leaves for each combination. After 12 h in the dark, place in a culture room for normal incubation. Perform dual-luciferase detection 2 days later. This experiment was performed in 3 biological replicates. A dual-luciferase reporter gene assay kit was used. The Reporter Assay System (Cat#E1910, promega) was used with an MD iD5 microplate reader (Spectramax ID5, Molecular, USA). LUC and REN values ​​were measured, and the experiment was repeated six times.

[0097] The results showed that the LUC / REN ratio detected by simultaneously adding the promoter PbACS1bpro and the transcription factor pSAK277-TCP10 was significantly higher than that in the control group. Furthermore, the LUC / REN ratios detected by combinations of PbSDRbpro, PbNCED.Bpro, and pSAK277-TCP10 were significantly higher than those in the control group. No significant differences were observed in the detection results for other combinations. Figure 9 B).

[0098] Example 11 Gel migration assay analysis (EMSA)

[0099] 1. The probe is a biotin-labeled probe manufactured by Sangon Biotech (Shanghai) Co., Ltd.

[0100] The probe sequences and mutant sequences of biotin-labeled PbACS1bpro, PbSDRpro, and PbNCED.Bpro are shown in Primer Table 3. The probe primers were diluted 10 μM with ddH2O, and a portion was diluted to 200 nM. The total annealing volume was 50 μL, as detailed in Table 4 below.

[0101] Table 4 Overall Annealing System

[0102]

[0103] Set the annealing reaction conditions as follows: pre-denaturation at 95℃ for 2 minutes, then slowly decrease the temperature by 1℃ every 30 seconds until it reaches 25℃. The product can be stored at -20℃ for a long time.

[0104] 2. Prokaryotic protein expression and purification: The CDS sequence of PbTCP10 was constructed into the pCold-TF vector using the same method as in Example 3. The recombinant plasmid pCold-PbTCP10 was transformed into the BMRosetta(DE3) strain. A correctly identified single clone was cultured at 37°C and 200 rpm until OD500. 600 =0.4-0.6, add IPTG to a final concentration of 0.1 mM, incubate at 15℃ and 200 rpm for 18 h. Collect the bacterial culture and resuspend the precipitate in pre-cooled PBS buffer. Disrupt the sample with an ultrasonic homogenizer, sonicate on ice for 3 seconds, stop for 7 seconds, until the bacterial culture is clear. First activate the Ni-NTA 6FF resin column with 15 ml PBS buffer, add the collected protein, and slowly pass it through the column at low speed. Then, add a low concentration of 10 mM imidazole to wash away impurities, followed by a high concentration of 200 mM imidazole to elute, and collect the purified protein. Remove the imidazole through an ultrafiltration tube. First, rinse the ultrafiltration tube with PBS buffer for 5 min, centrifuge, add the purified protein, centrifuge at 5000 rpm for 5 min, elute 5 times, and finally replace the imidazole. Collect the ultrafiltered protein and detect the purified protein by SDS-PAGE gel electrophoresis. The results are as follows. Figure 11 As shown, His-PbTCP10 protein was expressed and purified in vitro.

[0105] 3. Polyacrylamide gel electrophoresis: Electrophoresis at 100V for 30-40 min, stopping when bromophenol blue migrates to 2 / 3 or 3 / 4 of the gel's bottom. Cut a nylon membrane to the same size as or slightly smaller than the protein gel, transfer at 380mA, 4℃ for 30-60 min. Crosslink the membrane using a UV-light crosslinker, block the membrane with blocking buffer, and incubate on a shaker for 30 min. Discard the blocking buffer, add Conjugate / Blocking Buffer, and gently incubate for 15 min. Add 30 mL of 4×Wash Buffer to 90 mL of pure water to prepare 1×Wash Buffer. Transfer the membrane to a new container and rinse with 30 mL of 1×Wash Buffer for 5 min. Gently wash the membrane three times with 1×Wash Buffer, 10 min each time. Transfer the membrane to a new container, add 30 mL of equilibration buffer, and gently incubate on a shaker for 5 min. Mix 4 mL of BeyoECL Plus Reagent A and 4 mL of BeyoECL Plus Reagent B thoroughly and protect from light to prepare BeyoECL Plus Reagent Working Buffer. Cover the entire surface of the membrane with the prepared Working Buffer, react in the dark for 5 minutes, and then develop and image the membrane on a ChemiDoc MP molecular imaging system.

[0106] The results showed that co-incubation of PbTCP10-His and PbACS1bpro probes could detect DNA-protein complexes. However, as the concentration of the cold probe increased, fewer complexes were detected; the highest concentration of the cold probe could even completely compete with the binding of the fusion protein to the labeled probe. However, no binding was detected when the PbACS1b promoter probe was co-incubated with the His negative control. Figure 8 D). Mutating elements of the PbACS1b probe revealed that no binding was detected when the mutant probe was co-incubated with the recombinant protein PbTCP10-His; only the probe remained free. This indicates that the PbTCP10 and PbACS1b promoters can specifically bind in vitro. Similarly, it was found that both binding sites on the PbSDR promoter and the site on the PbNCED.B promoter can specifically bind to the recombinant protein PbTCP10-His. Figure 10 ).

[0107] Example 12 Biomembrane Interference Detection (BLI)

[0108] Bio-Layer Interferometry (BLI) can be used to detect and analyze biomolecular interactions, and can be used for qualitative and quantitative measurement of protein-biomolecule interactions. The interaction between the DNA probe of promoter PbACS1b and protein PbTCP50 was verified using a ForteBio OCTET RED96 biomolecular interaction analyzer. The specific steps are as follows: Prepare PBS buffer (pH 7.2–7.4, NaCl 137 mmol / L, KCl 2.7 mmol / L, Na₂HPO₄ 4.3 mmol / L, KH₂PO₄ 1.4 mmol / L) + 0.02% Tween-20. Prepare the sample plate, add buffer, solidify the sample, and bind the sample and regeneration buffer. First, add 200 μL of dilution buffer to the first column of the wet plate (greiner PN655209, 200 μL / well) and place it in the SA sensor for pre-wetting for 10 min. In the second black plate, 200 μL of 1×PBS dilution buffer was added to column 1 (AG), 200 μL of probe dilution buffer to column 2 (AG), 200 μL of dilution buffer to column 3 (AG), and different concentrations of His-PbTCP50 protein dilution buffer to column 4 (AF). G was added as a negative control. The experimental program and sensor positions were set and run. The raw data were analyzed using DataAnalysis 9 software to obtain the results.

[0109] The results showed that the binding affinity between PbTCP10 and the PbACS1b promoter increased with increasing concentration of recombinant protein PbTCP10, with a binding constant of KD = 0.02 ± 0.0046 μM, indicating a very strong binding affinity. Figure 8 C). The results show that PbTCP10 regulates the upregulation of PbACS1b by binding to the promoter element of PbACS1b, thereby promoting the synthesis of ethylene.

[0110] Simultaneously, it was found that the promoter binding affinity of PbTCP10 to PbSDR1 and PbNCED.A1 increased with increasing His-PbTCP10 protein concentration, and the calculated binding constants were KD. SDRpro-1 =0.51±0.082μM, KD SDRpro-2 =1.6±0.083μM, KD NCEDpro-1 =0.33±0.047μM.

[0111] As can be seen from the above implementation examples, the present invention provides the TCP family gene PbTCP10 of pear, which specifically has the function of regulating ethylene synthesis and ABA synthesis, thereby promoting fruit ripening, and can be applied to promote the early ripening of pear fruit.

[0112] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should be considered within the scope of protection of the present invention.

[0113] SEQ ID NO.1 PbTCP10 gene nucleotide sequence

[0114]

[0115] Protein sequence encoded by transcription factor PbTCP10 gene of SEQ ID NO. 2

[0116] MTSYLEDQDDDGGASDLSTSTGDLGDNDHTNRDGVVSKQLHYDETTEFHSLKEEPIDSDPPQPPQAYPIGMVPVAMPMPMSVPSAVSTLTRRSSTTDRHTKVEGRGRRIRIPATCAARIFQLTRELGHKSDGETVRWLLEHAEQAIIEATGTGTVPAIAVSVGGTLKIPTTASTSVEDNSLPPSSTKKRKRPSNSEFVDVKNDAVSQSSGLAPVSPSVPQGLVPVWAVGGTGLMVPANALWIGPVGSGAGPSGQPQIWALSPTMTPVFNMAGAARPISSFVANSGGGGAGVVDDRAPSPALSNSAANTSTVGPRAAMWSSTTMAPSMSSSSHNSNGSGSGATTKSQMLRDFSLQIYDKQELQLMGRPVGPENHNQTQ*.

Claims

1. Application of overexpression of transcription factor PbTCP10 as shown in SEQ ID NO.1 or biological materials containing transcription factor PbTCP10 as shown in SEQ ID NO.1 in promoting ethylene and ABA synthesis and promoting pear fruit ripening.

2. The application of biological materials overexpressing transcription factor PbTCP10 as shown in SEQ ID NO.1 or containing transcription factor PbTCP10 as shown in SEQ ID NO.1 in the breeding of new early-maturing pear germplasm, characterized in that, Transient overexpression of the transcription factor PbTCP10 in pear fruit or stable expression in pear flesh callus can promote ethylene synthesis and hormone ABA synthesis, thus promoting pear fruit ripening.

3. The application according to claim 1 or 2, characterized in that, The biological materials mentioned are expression cassettes, recombinant vectors, transgenic cells, transiently transgenic pear fruits, or stable transgenic pear flesh calluses.

4. A method for promoting pear fruit ripening or cultivating new early-maturing pear germplasm, characterized in that, Transient overexpression of the transcription factor PbTCP10, as shown in SEQ ID NO.1, in pear fruit or stable expression in pear flesh callus can promote ethylene synthesis and hormone ABA synthesis, thus promoting pear fruit ripening or cultivating new early-maturing pear varieties.

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