Application of pear transcription factor PbbHLH164 in promoting fruit ripening

Abstract

The application discloses application of a pear transcription factor PbbHLH164 in promoting fruit ripening, screens a transcription factor PbbHLH164 which is separated from pear fruits and has the function of promoting fruit ripening, and the nucleotide sequence of the transcription factor PbbHLH164 is shown in the sequence table SEQ ID NO.1. The gene is transiently expressed in pear fruits and stably expressed in pear pulp callus through genetic transformation mediated by agrobacterium, and the result shows that the gene expression amount of ethylene synthesis enzyme and the content of ethylene significantly increase after overexpression of PbbHLH164, and the ripening of the fruits is promoted. Through biological function verification, it is shown that the PbbHLH164 gene cloned in the application has the function of promoting ethylene synthesis to participate in fruit ripening. The discovery of the gene provides a new gene resource for molecular breeding of promoting ethylene synthesis, and development and utilization of the resource are favorable to improve the commodity value of pear fruits, prolong the shelf life of the fruits, and are favorable to reduce agricultural cost and realize environment friendliness.

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 PbbHLH164. Specifically, it relates to the application of the PbbHLH164 gene, a member of the bHLH family related to pear fruit ripening, isolated and cloned from pear, in promoting fruit ripening. Technical Background

[0002] Fruit ripening is a complex and orderly process involving a series of physiological and biochemical reactions, including changes in color, flavor, aroma, texture, and hormones (Yin et al., 2009; Giovannini, 2004). Ethylene, as an endogenous plant hormone, plays a crucial role in fruit growth, development, and ripening. Ethylene biosynthesis involves two key steps: ① 1-aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the formation of ACC from S-adenosine-L-methionine; ② ACC oxidase (ACO) catalyzes the formation of ethylene from ACC. Among these, ACC synthase (ACS) and ACC oxidase (ACO) are the two key enzymes. According to reports, numerous different types of transcription factors can regulate ethylene biosynthesis, including NAM / ATAF1 / 2 / CUC2 (NAC), MADS-box, basic helix-loop-helix (bHLH), homeobox HB-box, auxin response factor, basic leucine zipper (bZIP), Apalata2 / ethylene response factor (AP2 / ERF), MYB, and zinc finger proteins (Li et al., 2021; Liang et al., 2020; Guo et al., 2021; Wang et al., 2023). Furthermore, protein phosphatase 2C, mitogen-activated protein kinase, and E3 ligase participate in ethylene biosynthesis through interactions with transcription factors (Ji et al., 2022; Liang et al., 2020; Shan et al., 2020).

[0003] The bHLH transcription factor family is a large family widely distributed in plants, animals, and microorganisms (Hong et al., 2019; Duek et al., 2005). bHLH transcription factors have been reported to participate in plant development and metabolic processes, including photomorphogenesis, flowering, and stress response (Hao et al., 2021). Currently, tomato SlbHLH22 and SlbHLH95 have been found to induce the upregulation of genes related to fruit ripening, accelerating fruit ripening (Zhang et al., 2020; Waseem et al., 2019). Banana MabHLH6 promotes starch degradation during fruit ripening (Xiao et al., 2018). Apple MdMYC2 responds to jasmonic acid signaling by promoting the expression of genes MdRF3 and MdACS1 and inhibiting MdERF2 to regulate ethylene biosynthesis (Lietal., 2017). Furthermore, overexpression of the MdbHLH3 gene in apple fruit increases the expression levels of MdACO1, MdACS1, and MdACS5A, promoting ethylene biosynthesis and thus accelerating fruit ripening (Hu et al., 2019). The bHLH transcription factor family contains over 100 members in perennial fruit trees (Kou et al., 2020), but only five bHLH transcription factors have been reported to be involved in fruit ripening.

[0004] Pear is one of the world's and China's major economic fruit trees, and a typical climacteric fruit, releasing ethylene during ripening. In previous studies, we completed transcriptome sequencing of pear varieties 'Cuiguan', 'Fengshui', and 'Xueqing' during fruit development and ripening (Wang et al., 2022), and identified the ethylene biosynthesis genes PbACS1b and PblACO1 in pear fruit (Hao et al., 2018). We also found that transcription factors PuERF2, PuERF3, PuBZR1, and PbERF24 regulate ethylene biosynthesis during pear fruit ripening (Ji et al., 2021; Hao et al., 2018). Previous researchers have isolated all bHLH genes in the pear genome (Kou et al., 2020), but it is currently unclear which genes are involved in pear fruit ripening. Research on the role of bHLH genes in fruit ripening is limited, and their functions and mechanisms of action remain unclear. Therefore, based on the above research background, the applicant conducted cloning and functional studies on the pear transcription factor bHLH gene, silenced and overexpressed the bHLH gene in pear fruit through Agrobacterium-mediated genetic transformation, and studied its interaction with ripening-related genes. Summary of the Invention

[0005] The purpose of this invention is to screen a transcription factor, PbbHLH164, that regulates ethylene synthesis in pear fruit and promotes fruit ripening; and to provide the application of transcription factor PbbHLH164 in regulating ethylene synthesis and promoting pear fruit ripening.

[0006] Another objective of this invention is to provide the application of this gene in genetic breeding for improving pear fruit quality or promoting early ripening of pear fruit.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] Application of transcription factor PbbHLH164, as shown in SEQ ID NO.1, or biological materials containing transcription factor PbbHLH164, as shown in SEQ ID NO.1, in regulating ethylene synthesis and promoting pear fruit ripening.

[0009] Application of transcription factor PbbHLH164 as shown in SEQ ID NO.1 or biological materials containing transcription factor PbbHLH164 as shown in SEQ ID NO.1 in genetic breeding for improving pear fruit quality or promoting early ripening of pear fruit.

[0010] 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.

[0011] Further preferably, transient overexpression of the transcription factor PbbHLH164 in pear fruit or stable expression in pear pulp callus can promote ethylene synthesis and accelerate pear fruit ripening.

[0012] This invention isolates and clones the PbbHLH164 gene, a transcription factor that regulates ethylene synthesis and promotes fruit ripening, from pear fruit. It belongs to the bHLH family and its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing. The coding sequence (CDS) is 741 bp in length, encoding 247 amino acids, and its encoded amino acid sequence is shown in SEQ ID NO.2 of the sequence listing. The isoelectric point is 7.55 and the molecular weight is 27.31 KD.

[0013] The invention includes a recombinant expression vector containing the PbbHLH164 gene, a transgenic recombinant bacterium, a transient transgenic pear fruit, or a transgenic callus. The recombinant expression vector is preferably the insertion vector pSAK277 containing the PbbHLH164 gene, with HpaI and Xbal restriction enzyme sites, and the recombinant vector is named pSAK277-PbbHLH164.

[0014] Primer pairs for cloning the cDNA sequence of the PbbHLH164 gene described in this invention: the upstream primer PbbHLH164-F1 nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer PbbHLH164-R1 nucleotide sequence is shown in SEQ ID NO.4.

[0015] This invention studied the dynamic development process of the "Sucui No. 1" and "Cuiguan" pear fruits, finding that the ripening periods of "Sucui No. 1" and "Cuiguan" fruits were 95 days and 110 days after flowering, respectively, with "Sucui No. 1" ripening earlier. Ethylene release from "Sucui No. 1" and "Cuiguan" fruits at different developmental and ripening stages was analyzed. Further research identified the transcription factor PbbHLH164, which regulates ethylene synthesis in pear fruits and promotes fruit ripening. Both transient and stable genetic overexpression of PbbHLH164 increased ethylene synthesis in pear fruits and pulp, thus promoting fruit ripening.

[0016] This invention uses transcriptome analysis to show that PbbHLH164 has the highest expression level during the ripening period of pear fruit.

[0017] This invention utilizes qRT-PCR technology to discover that PbbHLH164 is associated with fruit ripening.

[0018] This invention constructs overexpression and silencing vectors for PbbHLH164, and uses Agrobacterium infection-mediated transient transformation to transfer PbbHLH164 into pear fruits. The results show that overexpression and silencing of PbbHLH164 promote and reduce the expression level of the PpACS1b gene and ethylene production, respectively.

[0019] This invention constructs an overexpression vector for PbbHLH164 and uses Agrobacterium infection-mediated transformation to stably transform the PbbHLH164 gene into callus from pear pulp. Biological function analysis of the obtained transgenic callus shows that overexpression of PbbHLH164 can increase the expression level of the PpACS1b gene and ethylene production in positive callus.

[0020] This invention constructs a PbbHLH164 overexpression vector for stable transformation of pear flesh callus. The recombinant plasmid is pSAk277-PbbHLH164, which is used to infect pear flesh callus via Agrobacterium-mediated transformation, resulting in transgenic callus-positive lines overexpressing the PbbHLH164 gene.

[0021] This invention utilizes a dual-luciferase reporter gene system to analyze the regulatory relationship between PbbHLH164, PbACS1b, and PbACO1b, and the results show that there is a promoter interaction between the PbbHLH164 and PpACS1b genes.

[0022] The binding ability of PbbHLH164 and PbACS1b was detected from a kinetic perspective using biomembrane interference. The results showed that the binding constant was KD = 0.02 ± 0.0046 μM, indicating a very strong binding ability.

[0023] EMSA confirmed that PbbHLH164 can directly interact with the binding element of PbACS1b to promote ethylene synthesis and thus regulate fruit ripening.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] 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 PbbHLH164 gene provides a new gene resource for molecular breeding to promote ethylene synthesis. The development and utilization of this resource will help improve the commercial value of pear fruit, extend its shelf life, reduce agricultural costs, and achieve environmental friendliness. Attached Figure Description

[0026] Figure 1 The images show dynamic fruit development at different stages of the two varieties, "Sucui No. 1" and "Cuiguan," harvested according to this invention.

[0027] Figure 2 This invention relates to the comparative analysis of transcriptome data from 2017 and 2018 for two varieties, “Sucui No. 1” and “Cuiguan”, as well as heatmaps of transcriptome data analysis at the ripening and immature stages of fruits from three varieties, “Cuiguan”, “Xueqing”, and “Fengshui”.

[0028] Figure 3 The data represent the ethylene release at different developmental and maturation stages of the two varieties, "Sucui No. 1" and "Cuiguan," according to this invention.

[0029] Figure 4 This is a graph showing the expression level analysis of the gene PbbHLH164 of this invention at different developmental and maturation stages in two varieties, "Su Cui No. 1" and "Cui Guan". Lowercase letters a, b, and c indicate the significance level when P < 0.05.

[0030] Figure 5 These are the experimental results regarding fruit phenotype, fruit ethylene release, and related gene expression levels after overexpression and silencing of the gene PbbHLH164, as described in this invention.

[0031] A represents the fruit phenotypes of pear fruits after transient transformation with Agrobacterium tumefaciens overexpression and silencing vectors of the gene PbbHLH164 of this invention;

[0032] B represents the analysis of ethylene release from fruit after transient overexpression and silencing of the gene PbbHLH164 in this invention.

[0033] C is a graph showing the expression levels of PbbHLH164, PbACS1b, and PbACO1 genes in pear fruit after transient overexpression and silencing of the gene PbbHLH164 according to this invention; lowercase letters a, b, and c represent significant differences with P values ​​< 0.05.

[0034] Figure 6 This is a schematic diagram illustrating the process of stable genetic transformation of the cloned gene PbbHLH164 of this invention into pear flesh callus via Agrobacterium-mediated transformation.

[0035] Figure 7 This is a graph showing the changes in ethylene release from pear pulp callus after stable genetic transformation of the cloned gene PbbHLH164 of this invention via Agrobacterium-mediated transformation.

[0036] Figure 8 This is a graph showing the expression levels of the PbbHLH164, PbACS1b, and PbACO1 genes after stable genetic transformation of the PbbHLH164 clone gene into pear flesh callus following this invention. Lowercase letters a, b, and c represent significant differences with a p-value < 0.05.

[0037] Figure 9 This is a schematic diagram illustrating the vector construction of the reporter gene and promoter of this invention.

[0038] Figure 10 This invention provides a dual-luciferase reporter gene system analysis of the regulatory effect of the cloned gene PbbHLH164 on the promoter PpACS1b.

[0039] Figure 11 This invention utilizes biomembrane interferometry to analyze the binding constants of elements in the PbbHLH164 and PbACS1b promoters.

[0040] Figure 12 This invention investigates the element-specific binding of PbbHLH164 to the PbACS1b promoter using EMSA experiments. Detailed Implementation

[0041] 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.

[0042] Example 1: Dynamic observation and sampling of fruits of two varieties, “Sucui No. 1” and “Cuiguan”, at different developmental and ripening stages.

[0043] The pear varieties “Sucui No. 1”, “Cuiguan”, “Fengshui”, and “Xueqing” were all collected from the Baima Experimental Base of Nanjing Agricultural University. Fruit samples were collected every 15 days from 60 days after flowering until fruit maturity. Each sample was replicated in triplicate, with each replicate containing at least four fruits. The fruits were photographed for documentation, and the pulp was preserved at -80℃. The results showed that “Sucui No. 1” and “Cuiguan” fruits matured at 95 days and 110 days after flowering, respectively, with “Sucui No. 1” maturing earlier. Figure 1 ).

[0044] Example 2: Analysis and Comparison of Pear Fruit Transcriptome Data and Screening of Candidate Genes

[0045] Transcriptome data from different stages of the 2018 pear varieties “Sucui No. 1” and “Cuiguan” were analyzed. The analysis revealed that 125 bHLH genes were expressed in both developing and mature pear fruits, with 17 bHLH genes showing higher expression in mature fruits than in developing fruits. Figure 2 Furthermore, in fruits collected in 2017, the expression level of PbbHLH164 in mature fruits of the three pear varieties "Cuiguan", "Fengshui", and "Xueqing" was higher than that in fruits during development. Figure 2 Therefore, it is preliminarily determined that PbbHLH164 is involved in the fruit ripening process.

[0046] Example 3: Detection of ethylene release at different developmental and ripening stages of fruits of two varieties, "Sucui No. 1" and "Cuiguan".

[0047] After harvesting fruits from two varieties, the fruits were weighed and sealed in airtight containers for ethylene collection. Five replicates were set up for each group, with 3-4 fruits per replicate. The ethylene release from the fruits was detected using a gas chromatograph (Thermo Trace 1300GC, USA) at the National Key Laboratory of Germplasm Resources platform, and the ethylene release concentration per unit time per fruit weight (μL·g) was calculated. -1 ·h -1 The results showed that the 'Sucui No. 1' and 'Cuiguan' varieties released large amounts of ethylene during their maturity period. Figure 3 ).

[0048] Example 4: qRT-PCR analysis of PbbHLH164 gene and related genes

[0049] 1. Download the nucleotide sequence of the PbbHLH164 gene from the pear genome database (http: / / 202.195.250.6:8080 / src / # / home), and design specific primers using Primer 5.0 software according to general primer design principles.

[0050] 2. qRT-PCR detection was performed using mature fruit pulp from two pear varieties, “Su Cui No. 1” and “Cui Guan”. RNA was extracted from pear pulp using a plant total RNA extraction kit for polyphenols and polysaccharides (purchased from TIANGEN, and operated according to the kit instructions), and cDNA was obtained by 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.

[0051] qPCR reaction system: SYBR Green I Master 10 μL, ddH2O 4 μL, forward primer 2.5 μL, reverse primer 2.5 μL, cDNA 1 μL. Reaction program: 95℃ denaturation for 15 s, 60℃ 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 gene.

[0052] The primer sequences for the internal reference gene are as follows:

[0053] GR-F: GATGGTGCTATGAAGATGCCAAATGT (SEQ ID NO.5);

[0054] GR-R:TCCCGAGCATCACGATAGATTCAC (SEQ ID NO. 6).

[0055] qRT-PCR analysis showed that in the 'Sucui No. 1' and 'Cuiguan' varieties, the expression level of PbbHLH164 during fruit ripening was higher than that during other developmental stages of the fruit. Figure 4 The results showed that PbbHLH164 is associated with pear fruit ripening.

[0056] Example 5: Isolation and Cloning of the PbbHLH164 Gene

[0057] 1. The full-length PbbHLH164 gene was amplified using the cDNA obtained from reverse transcription in Example 4. The primer pair for gene amplification was:

[0058] PbbHLH164-F1:gttccggattacgctgttaacATGGGTTCGCAGGATCCTG (SEQ ID NO.3)

[0059] PbbHLH164-R1:tcattaaagcaggactctagaTTACATTATACTAGGGTCGTTTTCCC (SEQ IDNO.4)

[0060] The 50 μL reaction system includes 2 μL of the above primers, 17 μL of ddH2O, 25 μL of 2×Buffer, 1 μL of dNTP, 2 μL of cDNA template, and 1 μL of high-fidelity enzyme (Phanta Super-Fidelity DNA Polymerase) (purchased from Vazyme).

[0061] The PCR reaction was performed according to the following procedure: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 1 minute, 30 thermal cycles, followed by 72°C extension for 10 minutes and 20°C extension for 5 minutes after the cycle.

[0062] 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 741 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.

[0063] Example 6: Construction of PbbHLH164 gene overexpression and silencing vector

[0064] 1. Based on the multiple cloning site of the PSAK277 vector and the restriction enzyme sites in the coding region of the PbbHLH164 gene, HpaI and XbaI were selected as restriction endonucleases. Primers containing restriction sites were designed using Primer 5.0 software according to general primer design principles, as in Example 5.

[0065] The annealing temperature for PCR amplification was 60℃, and the PCR reaction system and amplification procedure were the same as in Example 1. The PSAK277 vector was double-digested with HpaI and XbaI enzymes (purchased from NEB), with a total digestion volume of 50 μL: 5 μL vector plasmid, 5 μL CutSmart Buffer, 1 μL each of HpaI and XbaI, and 38 μL ddH2O. The digestion was incubated at 37℃ for 3-4 hours, and the digestion products were purified using an AxyPrep cleaning kit (procedure followed according to the manufacturer's instructions). Homologous recombination (purchased from Vazyme) was performed on the PCR product and the double-digested product, following the instructions for a one-step cloning recombination kit. The recombinant product was transformed into Escherichia coli DH5α by heat shock method (referring to the 3rd edition of Molecular Cloning Laboratory Manual, Science Press, 2002). It was evenly spread on LB solid plates containing 100 mg / L kanamycin. Positive clones were screened, and 12 single clone sites were selected for PCR identification and sequencing. The sequencing results confirmed that there were no reading frame mutations, and the recombinant vector containing the insert fragment was obtained. It was named the overexpression vector PSAK277-PbbHLH164.

[0066] 2. The PbbHLH164 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 for the TRV-PbbHLH164 silencing vector were the same as above. The silencing vector plasmid was then transformed into Agrobacterium GV3101 using the heat shock method.

[0067] Primers with restriction enzyme sites were designed, and the primer pair sequences are shown below:

[0068] TRV-bHLH164-F:agaaggcctccatggggatccGTTTGGTCAGATTGAATGCTTACAG(SEQ IDNO.7)

[0069] TRV-bHLH164-R: gagacgcgtgagctcggtaccATACCTAGCATGCGGTTTGCTC (SEQ ID NO. 8).

[0070] Example 7 Instantaneous transformation of pear fruit

[0071] 1. Single colonies of Agrobacterium containing the overexpression vector PSAK-PbbHLH164 and the silencing vector TRV-PbbHLH164 were picked and placed in liquid medium containing the corresponding antibiotics. Agrobacterium containing the empty vector PSAK277 and TRV1+TRV2 were used as controls. Incubate at 28℃ and 220 rpm. Then, expand each Agrobacterium culture in Erlenmeyer flasks containing 40 ml of liquid medium until the OD600 reaches 0.8-1.0. Centrifuge at 5000 rpm for 10 minutes at 4℃, discard the supernatant and collect the bacterial suspension. 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, repeating this process once. Finally, suspend the suspension in the infection solution and incubate on a shaker at 25℃ for 2-4 hours.

[0072] 2. Pears of variety 'Sucui No. 1' should be harvested 7-10 days before market harvest. Select fruits with smooth surfaces and no blemishes. Inject the solution vertically onto the fruit surface using a 1ml sterile syringe, being gentle and slow. Inject 40 pears per group of bacterial solutions and observe phenotypic changes at room temperature. Figure 5 A). After 5-7 days of complete infection and noticeable phenotypic changes, the pear fruits 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. Figure 5 B). The method for detecting ethylene is the same as in Example 3.

[0073] Example 8: qRT-PCR analysis of the expression levels of PbbHLH164, PbACS1, and PbACO1 in transiently transformed pear fruits.

[0074] Bacterial-covered pear pulp was collected for qRT-PCR detection. RNA was extracted from the infected pear pulp in Example 7 using a plant total RNA extraction kit for polyphenols and polysaccharides (purchased from TIANGEN, operated according to the kit instructions). cDNA was obtained by reverse transcription. A LightCycler 480 PCR instrument, 96-well qPCR plates, and LightCycler 480SYBRGreen I Master fluorescent dye were used.

[0075] qPCR reaction system: SYBR Green I Master 10 μL, ddH2O 4 μL, forward primer 2.5 μL, reverse primer 2.5 μL, cDNA 1 μL. The reaction program was: 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 45 cycles. All analyses were performed by three independent biological replicates. Primer sequences used are detailed in Table 1. The pear Pbr041114.1 gene was used as an internal control gene, and its primer sequence was the same as in Example 4. qRT-PCR results showed that the expression levels of PbbHLH164 and PbACS1b increased in pear pulp injected with the PbbHLH164 overexpression vector, while the expression level of PbACO1 did not change significantly. After silencing the PbbHLH164 gene, the expression levels of PbbHLH164, PbACS1b, and PbACO1 in pear pulp decreased (…). Figure 5 C). These results indicate that PbbHLH164 can mediate ethylene biosynthesis by regulating the activity of PpACS1.

[0076] Table 1 Primer sequences

[0077]

[0078]

[0079] Example 9: Genetic transformation of pear pulp callus

[0080] 1. The recombinant plasmid pSAk277-PbbHLH164 was constructed in Example 6. Agrobacterium containing the overexpression vector pSAk277-PbbHLH164 was activated, and single colonies that were verified by PCR were picked and inoculated into LB liquid medium containing spectinomycin resistance. The culture was incubated overnight at 28°C and 200 rpm until OD. 600 =0.8-1.0. Collect bacterial suspension at 5000 rpm for 5 min, discard the supernatant, add MS resuspension, OD = 0.5-0.6. Induce at 25℃ and 120 rpm for 1 h. Pick callus with good growth 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 place it in symbiotic medium for culture. After 2 days, transfer to selection medium and incubate in the dark. Figure 6 The gene PbbHLH164 shown is used to stably transform pear pulp callus through Agrobacterium-mediated genetic transformation.

[0081] 2. Positive callus samples were screened, and the results of ethylene release analysis showed that the ethylene release from callus of the PbbHLH164-OE overexpression line was significantly higher than that from callus of the control group. Figure 6qRT-PCR analysis revealed that in the PbbHLH164-OE overexpression line, the expression levels of PbbHLH164 and PpACS1b were significantly increased, while the expression level of PbACO1 remained largely unchanged. Figure 7 ).

[0082] Symbiotic MS medium

[0083]

[0084] Screening MS medium

[0085]

[0086] Example 10: Determination of Dual-Luciferase

[0087] 1. Promoter isolation and vector construction: DNA was extracted from pear pulp using the FastPure Plant DNA Isolation MiniKit. The promoter sequences of PbACS1b and PbACO1 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 5 and 6. The amplification primer sequences are detailed in Table 1.

[0088] 2. Tobacco preparation: Select tobacco plants that have grown for 3-4 weeks and have 4-6 flat leaves, and water them thoroughly to keep them in good growing condition.

[0089] 3. The pSAk277-bHLH164 expression vector was used as the Effector plasmid, and the recombinant plasmids PbACS1bpro-LUC and PbACO1pro-LUC were used as the Reporter plasmids. Figure 9 ), transferred into Agrobacterium, and single colonies of positive Agrobacterium were picked and cultured on antibiotic-resistant liquid medium until OD. 600 The 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 acetylsylcholine), centrifuge at 5000g for 10 min, discard the supernatant and collect the bacterial suspension, repeat once. Finally, resuspend the bacteria 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, aspirate 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, incubate normally in a culture room. 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 to detect LUC and REN values, and the experiment was repeated 6 times. The instrument used was an MD iD5 microplate reader (Spectramax ID5, Molecular, USA). The results showed that the LUC / REN ratio detected by simultaneously adding the promoter PbACS1bpro and the transcription factor pSAk277-bHLH164 was significantly higher than that of the control group, while there was no significant change after adding the PbACO1 promoter. Figure 10 ).

[0090] Example 11 Gel migration assay analysis (EMSA)

[0091] 1. The probe is a biotin-labeled probe manufactured by Sangon Biotech (Shanghai) Co., Ltd.

[0092] The biotin-labeled PbACS1 probe sequence is: GTATGAGGACCACCAAGAGGTACGCTGCCGGTGTACACGTGGCTGTGC;

[0093] The mutant probe sequence is: GTATGAGGACCACCAAGAGGTACGCTGCCGGTGTAaaaaaaaCTG TGC.

[0094] Dilute the probe and primers to 10 μM using ddH2O, and then dilute a portion to 200 nM. Anneal the total volume to 50 μL, as follows:

[0095]

[0096] 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.

[0097] 2. Prokaryotic protein expression and purification: The full-length PbbHLH164 was constructed into the pCold-TF vector using the same method as in Example 6. The recombinant plasmid was transformed into the BMRosetta(DE3) strain. A correctly identified single clone of the strain was cultured at 37°C and 200 rpm until OD600 = 0.4-0.6. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 15°C and 200 rpm for 24 h. The bacterial culture was collected, and the precipitate was resuspended in pre-cooled PBS buffer. The sample was disrupted using an ultrasonic homogenizer, sonicated on ice for 3 seconds, paused for 7 seconds, until the bacterial culture was clear. The Ni-NTA6FF resin column was activated with 15 ml of PBS buffer, the collected protein was added, and the column was slowly passed through at low speed. Then, a low concentration of 10 mM imidazole was added sequentially. Washing off impurities, high concentration 200mM imidacloprid Elute and collect purified protein. Remove imidium ions through an ultrafiltration tube. First, rinse the ultrafiltration tube with PBS buffer for 5 minutes, centrifuge, add the purified protein, centrifuge at 5000 rpm for 5 minutes, and elute 2-3 times. Finally, replace the PBS buffer. Ultrafiltered proteins were collected and the purified proteins were detected by SDS-PAGE gel electrophoresis.

[0098] 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 the 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. The results are as follows: Figure 12 As shown, co-incubation of PbbHLH164-His and PbACS1b probes can detect DNA-protein complexes. However, as the concentration of the cold probe increases, fewer complexes are detected; the highest concentration of the cold probe can 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 12 Mutating elements of the PbACS1b probe revealed that no binding was detected when the mutant probe was co-incubated with the recombinant protein PbbHLH164-His; only the probe remained free. This indicates that PbbHLH164 and the PbACS1b promoter can specifically bind in vitro.

[0099] Example 12 Biomembrane Interference Detection (BLI)

[0100] 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 a DNA probe carrying the promoter PbACS1b and the protein PbbHLH50 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, add 200 μL of 1×PBS dilution buffer 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-PbbHLH50 protein dilution buffer to column 4 (AF). Add dilution buffer to column G as a negative control. Before opening the instrument door, ensure the instrument is in a ready state (check the instrument status window). Open the operating software (make sure the instrument door is closed) and click in the wizard. Set the experimental program and sensor positions, then run it. Analyze the raw data using DataAnalysis9 software to obtain the results. Figure 12 As shown, the binding affinity between PbbHLH164 and PbACS1b increases with increasing concentration of recombinant protein PbbHLH164, with a binding constant of KD = 0.02 ± 0.0046 μM, indicating a very strong binding affinity. These results suggest that PbbHLH164 regulates the upregulation of PbACS1b by binding to the promoter elements of PbACS1b, thereby promoting ethylene synthesis.

[0101] As can be seen from the above implementation examples, the present invention provides the pear bHLH family gene PbbHLH164, which specifically has the function of regulating ethylene synthesis and thus promoting fruit ripening, and can be applied to promote the early ripening of pear fruits.

[0102] 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.

[0103] SEQ ID NO.1 PbbHLH164 gene nucleotide sequence

[0104] ATGGGTTCGCAGGATCCTGTGCCAGCAGCTGGCAAGGGTAATGTTGCAACGGAGGCATC

[0105] AGTGCGCAAGGCTTGTCCTGGAATAAAGAAACAGGGGAAAATTCCGAAAAGAATCCAC

[0106] AAGTCTGAGAGGGAGAGAAAGAAGCGTGAGCGTTTTAACGAGCATTTTTTGGAGCTGG

[0107] CTGATGCTCTTGAACTGAATCAGCAGAACAGTGGAAAGGCCTCCATACTAAGTGAAGCT

[0108] ACTCGACTGTTGAAGGACTTGTTTGGTCAGATTGAATGCTTACAGAAGGAAAATGCATC

[0109] CCTGTTGTCTGAATCAAATTATATGACCCTTGAGAAGAATGAGCTGAGAGACGATAATTC

[0110] TGCCTTAGAAACTCAGATCGAGAAATTGCAAAGTGAGATACAAGAGAGGGTGGTACAG

[0111] TCTAAACGGGATCTAAATGCACCACCTTGCACAGAATTGAGACCAGAAGTTGCCTCCCA

[0112] TTTTACCGGAAATTGCATCGGATTTACCACTCAAGAACCCGGCTTGCAGCAACCACCTG

[0113] CTGTCTTTGTTATGCCCGTCGGCCCTGATCTCCAAGCTTATCCACAGCCGGATGTCGCCC

[0114] ACCTCACATCTAACACCACCTCACATGTGAGCAAACCGCATGCTAGGTATCCAACCTCG

[0115] GTGGATTCTTGGCCGTCCCAACTTCTCGGTGAGAAACCAACCGCAGGCAAAGGGTTTC

[0116] GACAAATCCGGGAAAACGACCCTAGTATAATGTAASEQ ID NO.2 Transcription factor PbbHLH164 gene encoded protein sequence

[0117] MGSQDPVPAAGKGNVATEASVRKACPGIKKQGKIPKRIHKSERERKKRERFNEHFLELADA

[0118] LELNQQNSGKASILSEATRLLKDLFGQIECLQKENASLLSESNYMTLEKNELRDDNSALETQ

[0119] IEKLQSEIQERVVQSKRDLNAPPCTELRPEVASHFTGNCIGFTTQEPGLQQPPAVFVMPVGPDLQAYPQPDVAHLTSNTTSHVSKPHARYPTSVDSWPSQLLGEKPTAGKGFRQIRENDPSIM*PbbHLH164 - F1: TCTGAGAGGGAGAGAAAGAAGCGTG(SEQ ID NO.3)

[0120] PbbHLH164 - R1: CCTTCTGTAAGCATTCAATCTGACCAA(SEQ ID NO.4)

[0121] GR - F: GATGGTGCTATGAAGATGCCAAATGT(SEQ ID NO.5)

[0122] GR - R: TCCCGAGCATCACGATAGATTCAC(SEQ ID NO.6)

[0123] TRV - bHLH164 - F: agaaggcctccatggggatccGTTTGGTCAGATTGAATGCTTACAG(SEQ IDNO.7)

[0124] TRV - bHLH164 - R: gagacgcgtgagctcggtaccATACCTAGCATGCGGTTTGCTC(SEQ IDNO.8)References

[0125] [1] Yin Xueren, Zhang Bo, Li Xian, Chen Kunsong. 2009. Research progress on ethylene signal transduction and fruit ripening and senescence. Journal of Horticulture 36:133-140.

[0126] [2] Duek PD,Fankhauser C.2005.bHLH class transcription factors takecentre stage in phytochrome signaling.Trends in Plant Science 10,51–54.

[0127] [3]Giovannoni JJ.2004.Genetic regulation of fruit development andripening.The Plant Cell 16,S170–S180.

[0128] [4]Gu C, Wu RF, Yu CY, Qi KJ, Wu C, Zhang HP, Zhang SL. 2021. Spatio-temporally expressed sorbitol transporters cooperatively regulate sorbitolaccumulation in pear fruit. Plant Science 303,110787.

[0129] [5]Guo ZH, Zhang YJ, Yao JL, Xie ZH, Zhang YY, Zhang SL, Gu C. 2021. The NAM / ATAF1 / 2 / CUC2 transcription factor PpNAC.A59 enhances PpERF.A16 expression topromote ethylene biosynthesis during peach fruit ripening. Horticulture Research 8,209.

[0130] [6]Hao PP,Wang GM,Cheng HY,Ke YQ,Qi KJ,Gu C,ZhangSL.2018.Transcriptome analysis unravels an ethylene response factor involvedin regulating fruit ripening in pear.Physiologia Plantarum 163,124-135.

[0131] [7]Hao YQ,Zong XM,Ren P,Qian YQ,Fu AG.2021.Basic helix-loop-helix(bHLH)transcription factors regulate a wide range of functions inArabidopsis.International Journal of Molecular Science 22,7152.

[0132] [8]Hu DG,Yu JQ,Han PL,Xie XB,Sun CH,Zhang QY,Wang JH,Hao YJ.2019.Theregulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruitquality in apple.New Phytologist 221,1966-1982.

[0133] [9]Ji YL,Xu MY,Liu Z,Yuan H,Lv TX,Li HJ,Xu YX,Si YJ,WangAD.2022.NUCLEOCYTOPLASMIC shuttling of ETHYLENE RESPONSE FACTOR 5mediated bynitric oxide suppresses ethylene biosynthesis in apple fruit.New Phytologist234,1714-1734.

[0134]

[10] Kou X,Xiong C,Wang D,Sun Y,Wang P,Wu J,Zhang S.2020.Comparativeanalysis of bHLH transcription factors in five rosaceae species,andexpression analysis of PbbHLHs in response to drought stress in pear.ResearchSquare doi.org / 10.21203 / rs.3.rs-78164 / v1.

[0135]

[11] Li C,Hou X,Qi N,Liu H,Li Y,Huang D,Wang C,Liao W.2021.Insightinto ripening-associated transcription factors in tomato:A review.ScientiaHorticulturae 288,110363.

[0136]

[12] Li T,Xu Y,Zhang L,Ji Y,Tan D,Yuan H,Wang A.2017.The jasmonate-activated transcription factor MdMYC2 regulates ETHYLENE RESPONSE FACTOR andethylene biosynthetic genes to promote ethylene biosynthesis during applefruit ripening.The Plant Cell 29,1316-1334.

[0137]

[13] Liang SM,Chen SC,Liu ZL,Shan W,Chen JY,Lu WJ,Lakshmanan P,KuangJF.2020.MabZIP74 interacts with MaMAPK11-3 to regulate the transcription ofMaACO1 / 4during bananafruit ripening.Postharvest Biology&Technology 169,111293.

[0138]

[14] Shan W,Kuang JF,Wei W,Fan ZQ,Deng W,Li ZG,Bouzayen M,Pirrello J,Lu WJ,Chen JY.2020.MaXB3 modulates MaNAC2,MaACS1,and MaACO1 stability torepress ethylene biosynthesis during banana fruit ripening.Plant Physiology184,1153-1171.

[0139]

[15] Wang G,Guo Z,Wang X,Guan SL,Gao H,Qi K,Gu C,ZhangSL.2022.Identification and testing of reference genes for qRT PCRanalysis.Biologia 31,204-211.

[0140]

[16] Wang G,Guo L,Guo Z,Guan SL,Zhu N,Qi K,Gu C,Zhang S.2023.Theinvolvement of Ein3-binding F-box protein PbrEBF3 in regulating ethylenesignaling during Cuiguan pea fruit ripening.Plant Science 329,111600.

[0141]

[17] Waseem M,Li N,Su D,Chen J,Li Z.2019.Overexpression of a basichelix-loop-helix transcription factor gene,SlbHLH22,promotes early floweringand accelerates fruit ripening in tomato(Solanum lycopersicum L.).Planta 250,173-185.

[0142]

[18] Xiao Y,Kuang J,Qi X,Ye Y,Wu Z,Chen J,Lu W.2018.A comprehensiveinvestigation of starch degradation process and identification of atranscriptional activator MabHLH6 during banana fruit ripening.Plantbiotechnology journal 16,151–164.

[0143]

[19] Yao N,Liu JY,Tian YY,Dong YY,Liu XM,Li HY.2019.Cloning andexpression analysis of CtbHLH transcription factor gene from Carthamustinctorius and construction of plant expression vector.China journal ofChinese materia medica 44,278–282.

[0144]

[20] Zhang L,Kang J,Xie Q,Gong J,Shen H,Chen Y,Chen G,Hu Z.2020.Thebasic helix-loop-helix transcription factor bHLH95 affects fruit ripening andmultiple metabolisms in tomato.Journal of Experimental Botany 71,6311-6327。

Claims

1. Application of overexpression of transcription factor PbbHLH164, as shown in SEQ ID NO.1, in promoting ethylene synthesis and pear fruit ripening.

2. Application of biomaterials containing transcription factor PbbHLH164 (SEQ ID NO.1) in promoting ethylene synthesis and pear fruit ripening.

3. Application of overexpression of transcription factor PbbHLH164, as shown in SEQ ID NO.1, in genetic breeding to promote early ripening of pear fruit.

4. Application of biological materials containing transcription factor PbbHLH164 as shown in SEQ ID NO.1 in genetic breeding to promote early ripening of pear fruit.

5. The application according to claim 2 or 4, characterized in that, The biological materials mentioned are expression cassettes, recombinant vectors, transgenic cells, transgenic recombinant bacteria, transiently transgenic pear fruit, or stable transgenic pear flesh callus.

6. The application according to any one of claims 1-4, characterized in that, Transient overexpression of the transcription factor PbbHLH164 in pear fruit or stable expression in pear flesh callus can promote ethylene synthesis and accelerate pear fruit ripening.

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