Genes for regulating stone cell formation in pear fruit and applications

Abstract

The present application belongs to the field of molecular biological technology, aiming at the problem that the function of ZAT family in the process of stone cell formation is rarely reported, the present application provides genes PbZAT11 and PbZAT12 for regulating stone cell formation, the nucleotide sequences of the genes PbZAT11 and PbZAT12 are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the amino acid sequences of the encoded proteins are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4. The genes PbZAT11 and / or PbZAT12 provided by the present application can regulate the content of lignin and stone cells in pear fruit; the discovery of the genes has important significance for accelerating the improvement of pear fruit quality, provides new gene resources for fruit quality breeding, and is an important candidate gene for future genetic engineering improvement of fruit quality breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to genes that regulate the formation of stone cells in pear fruits and their applications. Background Technology

[0002] Pear (Pyrus spp.) belongs to the Rosaceae family and is the second largest deciduous fruit tree species in my country, as well as one of the major fruits ensuring a year-round supply of high-quality fruit for 1.4 billion Chinese people. The concept of "medicine and food sharing the same origin, eating for nutrition and health" has become a common understanding. Pears are known for their ability to quench thirst, relieve dryness, and alleviate coughs and phlegm. During the dry autumn and spring seasons, when colds are prevalent, the market and consumers require a stable supply of high-quality pears. However, the content and size of stone cell clusters significantly affect the edible quality of pears, impacting not only the content of nutrients such as sucrose but also the firmness and chewiness of the flesh. Therefore, studying the formation mechanism of stone cell clusters is crucial for improving pear fruit quality and promoting the development of the pear industry.

[0003] The accumulation of lignin on the cell wall is a key characteristic of secondary cell wall (SCW) formation. Lignin is a complex phenolic polymer formed by the dehydrogenation polymerization of three lignin monomers: p-coumarol, coniferyl alcohol, and sinapyl alcohol. After formation, the lignin monomers are transported to the cell wall, where they are polymerized by peroxidase (POD) and laccase (LAC). In studies on sclereids, it has been clearly established that sclereid formation is closely related to the biosynthesis, translocation, and deposition of lignin in pear pulp tissue. Cys2 / His2 (C2H2) type zinc finger proteins (ZFPs) are one of the largest transcription factor families in plants. The ZAT family is a specific subclass of the C2H2 zinc finger family in plants, containing a unique conserved QALGGH motif, which can participate in plant stress response and developmental regulation. However, the function of the ZAT family in sclereid formation has been rarely reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a gene that regulates the formation of stone cells in pear fruits and its application. This gene was isolated and cloned from the pear variety 'Shannong Su Pear' and named PbZAT11 and PbZAT12. The discovery of this gene may provide new insights into the quality improvement of pear fruits.

[0005] The first objective of this invention is to select one of the following genes for use in (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; The gene includes gene PbZAT11 and / or gene PbZAT12.

[0006] Preferably, the nucleotide sequence of the gene PbZAT11 is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the gene PbZAT12 is shown in SEQ ID NO.2.

[0008] A second object of the present invention is to provide an application of the above-described gene-encoded protein in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; The gene comprises gene PbZAT11 and / or gene PbZAT12. The nucleotide sequence of gene PbZAT11 is shown in SEQ ID NO.1. The nucleotide sequence of gene PbZAT12 is shown in SEQ ID NO.2.

[0009] Preferably, the amino acid sequence of the protein encoded by the gene PbZAT11 is shown in SEQ ID NO.3.

[0010] Preferably, the amino acid sequence of the protein encoded by the gene PbZAT12 is shown in SEQ ID NO.4.

[0011] A third object of the present invention is to provide an application of a recombinant expression vector and / or transient expression vector containing the above-described genes in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; The gene includes gene PbZAT11 and / or gene PbZAT12.

[0012] Preferably, the recombinant expression vector backbone is pPZP211.

[0013] A fourth object of the present invention is to provide an application of the above-mentioned Agrobacterium recombinant strain in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; The gene includes gene PbZAT11 and / or gene PbZAT12.

[0014] The fifth objective of this invention is to provide a method for regulating the stone cell content in pear fruit, wherein the method increases the stone cell content in pear fruit by overexpressing one of the following genes, or inhibits the stone cell content in pear fruit by inhibiting one of the following genes; The gene includes gene PbZAT11 and / or gene PbZAT12.

[0015] Compared with the prior art, the advantages of this invention are as follows: (1) This invention is the first to discover that the gene PbZAT11 and / or the gene PbZAT12 can regulate the content of lignin and stone cells in pear fruit; (2) The gene PbZAT11 and / or gene PbZAT12 provided by the present invention are overexpressed in pear fruit. The obtained pear fruit is verified by staining results and lignin content detection test. The test results show that the lignin staining effect of the overexpressed gene PbZAT11 and / or gene PbZAT12 is significantly enhanced and the lignin content is increased. The content of stone cells in pear fruit can be inhibited by inhibiting gene PbZAT11 and / or gene PbZAT12. (3) The discovery of the gene PbZAT11 and / or gene PbZAT12 provided by the present invention is of great significance for accelerating the improvement of pear fruit quality, providing new gene resources for fruit quality breeding, and is an important candidate gene for future genetic engineering to improve fruit quality breeding. Attached Figure Description

[0016] Figure 1 The image shows the results of in situ RNA hybridization of PbZAT11 and PbZAT12 in pear fruit 35 days after the peak flowering period. Figure 2 Images showing the lignin staining results of transformed and untransformed pear fruits in each group; Figure 3 The graph shows the lignin content detection results of transformed and untransformed pear fruits in each group; Figure 4 The results show the expression levels of genes PbZAT11 and PbZAT12 in transformed and untransformed pear fruits from each group. Figure 5 An image showing the appearance of a pear transgenic callus sample. Figure 6 Results of the detection of the expression level of the target gene in pear callus tissue; Figure 7Image showing the results of lignin content detection in pear callus tissue; Figure 8 The results show the expression levels of genes related to the biosynthesis of secondary cell walls in sclereids. Figure 9 Subcellular localization results for PbZAT11 and PbZAT12; Figure 10 Figure 1 shows the yeast one-hybrid results of PbZAT11 and PbZAT12 with the promoter of the lignin synthesis gene PbLAC1. Figure 11 Figure 1 shows the results of a luciferase reporter assay of PbZAT11 and PbZAT12 with the promoter of the lignin synthesis gene PbLAC1. Detailed Implementation

[0017] The following will be described in conjunction with embodiments of the present invention. Figures 1 to 11 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Experimental methods in the following examples, unless otherwise specified, are generally performed using methods known in the art. Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0019] Example 1: In situ RNA hybridization of genes PbZAT11 and PbZAT12 in 35 DAFB fruit Pear fruits were collected 35 days after full bloom (35 DAFB), fixed in in situ hybridization fixative at 4℃ for 12 hours, dehydrated, embedded in paraffin, and sectioned to obtain 4 μm paraffin sections. RNA in situ hybridization was performed on the paraffin sections using the RNASweAMI™ Plant In Situ Hybridization (BCIP / NBT) detection kit (service-bio, China) according to the manufacturer's instructions. The RNA probe sequences are shown below: PbZAT11-sense: AUGCUACCAUCAUGCAGCAUGGAGACCAAG; PbZAT11-antisense: CTTGGUCUCCAUGCUGCAUGAUGGUAGCAU; PbZAT12-sense: AAAAGGGUGUCGUGCUUGAAUCUGGACUUG; PbZAT12-antisense: CAAGUCCAGAUUCAAGCACGACACCCUUUU.

[0020] Then, the slides were observed under a microscope and images were acquired and analyzed. The results are as follows: Figure 1 As shown, through Figure 1 The results showed that the mRNAs of genes PbZAT11 and PbZAT12 were identified in immature sclereids and at the edges of sclereid clusters, but not in parenchyma cells.

[0021] Example 2: Isolation, cloning, overexpression, and construction of silencing vectors for PbZAT11 and PbZAT12 genes RNA extracted from the pulp of 'Shannong Crisp Pear' with a sample size of 35 DAFB was used as a template for reverse transcription using a one-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Cloning primers with restriction enzyme sites were designed using CE Design V1.04 software according to general primer design principles. The overexpression cloning primer sequences are as follows: PbZAT11-OE-F: gagaacacgggggacgagctcATGAAGAGAGATATGAGAGGAGAAGAATT; PbZAT11-OE-R: agtgagagatctttggtcgacTGATACAAAAGCATCAACTTTTGGT; PbZAT12-OE-F: gagaacacgggggacgagctcATGAAGAGAAGCGGGCAAGAC; PbZAT12-OE-R: agtgagagatctttggtcgacAGAAATCATCTGCAGCTTCGTCA; The primer sequences for silencing cloning are: PbZAT11-pTRV2-F:gtgagtaaggttaccgaattcGCCACAAGAGGCCGAGAAC PbZAT11-pTRV2-R: gagacgcgtgagctcggtaccTCAAGTTCAAATCCATCTCCAAGC PbZAT12-pTRV2-F:gtgagtaaggttaccgaattcTCTAATGCTTTTTATCCAAGGTAGGG PbZAT12-pTRV2-R: gagacgcgtgagctcggtaccCTATGTCTCCTCATGTGTCCCCC The gene cloning procedure is as follows: ① Pre-denaturation at 95℃ for 5 minutes; ② Denaturation at 95℃ for 30 seconds; annealing (temperature designed according to primer temperature, generally Tm-5℃); extension at 72℃ for 1 minute; cycle this stage 38 times; ③ Extend at 72℃ for 10 min, then store at 4℃. After the PCR reaction is complete, perform gel electrophoresis. If the bands are correct, the gel is recovered. The PCR product is purified and recovered using the EasyPure Quick Gel Extraction Kit (TransGen Biotech).

[0022] The target genes (PbZAT11 and PbZAT12 genes) were ligated into recombinant expression vectors: specifically, pPZP211 was used as the overexpression vector, and pTRV2 was used as the virus-induced silencing vector. Ligation was performed at 37°C for 30 minutes using the recombinase Exnase II (purchased from Vazyme). The total reaction volume was 10 μL, containing 2 μL of 5×CE II Buffer, 1 μL of Exnase II, 4 μL of PCR recovered products of PbZAT11 and PbZAT12 genes, and 3 μL of double digestion recovered product of the pPZP211 vector. Subsequently, the cells were transformed into competent E. coli DH5α cells (Tsingke, China). The E. coli transformation method is as follows: ① Add the above ligation product to 50 μL of DH5α competent cells (thawed on ice), gently mix, and place on ice for 25 min; ② Heat shock at 42℃ for 90 seconds, then immediately place on ice for 2 minutes; ③ Add 250 μL of LB medium (antibiotic-free), incubate at 200 rpm and 37°C for 1 h.

[0023] After transformation with *E. coli*, positive clones were screened on LB agar plates containing 100 mg / L spectinomycin or kanamycin. Plasmids were extracted, digested with enzymes, and identified by PCR. The recombinant expression vector plasmid samples were sent to a biotechnology company for sequencing. Sequencing results showed that the full-length CDS of PbZAT11 and PbZAT12 were 558 bp and 516 bp, respectively. The nucleotide sequences of PbZAT11 and PbZAT12 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The PbZAT11 and PbZAT12 genes encode proteins of 185 and 171 amino acid residues, respectively. The amino acid sequences of the proteins encoded by the two genes are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0024] In this embodiment of the invention, the specific sequence of SEQ ID NO.1 is as follows: ; The specific sequence of SEQ ID NO.2 is as follows: ATGAAGAGAAGCGGGCAAGACGAGACCTTAGACTTGGTCAATTGCCTAATGCTTTTGTCCAGGGAACCCGATTCCTCCCCAATCAAACACGAGAACACGAAAAAAGACCGAGTTTTCGTATGCAAGACGTGTAATCGGGAGTTTCCCTCGTTCCAGGCCCTCGGAGGCCACAGAGCGAGTCACAAGAAGCCGAAGCTGATGCCCGGTGGGGCTGCCAACCTTTTACATTTGGCACAGTCGCCGGGGTCGCTGGTTAAGCCCAAGACGCATGAGTGCCCGATTTGCGGGCTTGAGTTTGCGATTGGGCAGGCGCTTGGGGGACACATGAGGAGACATAGGGACGTCATTCAAGCTGCGGCTGTTCGGACTCAGGCGTCATCGGCGATGCCGGTTTTGAAGAAGTCGAATAGCAGCAAAAGGGTGTCGTGCTTGAATCTGGACTTGAATTTGGATCTACATTTGGCACCACCGGGTTATCACCAATATGATTTGACGAAGCTGCAGATGATTTCTTAA; The specific sequence of SEQ ID NO.3 is as follows: MKRDMRGEELGFGLKGLDMAKCLMLPSCSMETKFRTCSNDVFECKTCNRKFTSFQALGGHRASHKRPRTSMSRDDEDRKDEREKGNDKSMVMNKKAKTHECSICGLEFGMGQALGGHMRKHRASNMDMASFSYAPLAVTSKIPVLTRSSSKRVMSLEMDLNLTPLENDLKLLFGKMAPKVDAFVS; The specific sequence of SEQ ID NO.4 is as follows: MKRSRQDETLDLVNCLMLLSREPDSSPIKHENMKKDRVFVCKTCNREFPSFQALGGHRASHKKPKLMPGGAANLLHLAQSPGSLVKPKTHECPICGLEFAIGQALGGHMRRHRDVIQAAAVRTQASPAMPVLKKSNSSKRVSCLNLDLNLDLHLAPPGYHQYDLTKLQLIS.

[0025] Example 3 Instantaneous transformation of pear fruit 1. Transformation of Agrobacterium with recombinant expression vector (1) Place Agrobacterium GV3101 (pSoup-P19) competent cells on ice until they are completely thawed; (2) Add 5 μL of the recombinant expression vector plasmid containing the target gene to 50 μL of competent cells, mix gently with the pipette tip, incubate on ice for 5 min, then in liquid nitrogen for 5 min, in a 37°C metal bath for 5 min, and then incubate on ice again for 5 min. (3) Add 800 μL of antibiotic-free YEP liquid culture medium to the ultra-clean workbench and incubate at 28℃ and 200 rpm for 3 h; (4) Take 120 μL of the cultured bacterial solution, spread it onto YEP solid medium (containing 100 mg / L spectinomycin (i.e. pZPZ211 vector) or kanamycin (TRV2)), invert it in a constant temperature incubator at 28℃ and incubate for 48 h until a single colony grows. (5) Perform PCR amplification on single colonies and screen to obtain Agrobacterium tumefaciens bacterial suspension; The PCR amplification reaction procedure is as follows: ① Pre-denaturation at 95℃ for 1 min; ② Denaturation at 98℃ for 10 sec; denaturation at 58℃ for 30 sec; extension at 68℃ for 2 min; repeat this cycle 32 times. ③ Extend at 68℃ for 5 min, then store at 4℃; (6) Activate the Agrobacterium bacterial suspension, then add the same volume of 50% glycerol to the Agrobacterium bacterial suspension and mix well to obtain recombinant Agrobacterium. Store in a -80℃ refrigerator for later use. The specific activation method is as follows: Add 20 μL of bacterial culture to 2 mL of YEP liquid medium (containing rifampin and spectinomycin or kanamycin), and incubate overnight at 28°C. On day 2, add 1 mL of bacterial culture to 30 mL of fresh liquid medium (containing the corresponding antibiotic), and continue culturing until the OD600 nm reaches 1.0. Centrifuge to collect the bacterial cells, wash once with ddH2O, and resuspend the strain in infection buffer. Then, mix equal volumes of the bacterial culture according to the intended gene injection combination, and incubate in the dark for 3 h. Next, further activate the Agrobacterium culture until the final OD600 is approximately 1.0, centrifuge to collect the bacterial cells, wash once with ddH2O, and finally resuspend the strain in infection buffer. Mix equal volumes of the bacterial culture according to the intended gene injection combination, and incubate in the dark for 3 h to obtain different groups of Agrobacterium culture. The infection buffer system is as follows: 5 mL of 0.2 M MES (pH: 5.5-5.7), 1 mL of 1 M MgCl2, 150 μL of 100 mM ACE, and ddH2O to a final volume of 100 mL.

[0026] The above-mentioned gene injection combinations are as follows: PbZAT11-pPZP211, PbZAT12-pPZP211, PbZAT11-pPZP211+PbZAT12-pPZP211 overexpression vector combination, PbZAT11-TRV, PbZAT12-TRV, PbZAT11-TRV+PbZAT12-TRV silencing vector combination.

[0027] 2. Target gene transformation of pear fruit To further characterize the functions of genes PbZAT11 and PbZAT12 in pear fruit sclereid formation, this invention injected Agrobacterium tumefaciens suspension into pear fruits, including pPZP211 (control), PbZAT11-pPZP211 and PbZAT12-pPZP211, PbZAT11-pPZP211+PbZAT12-pPZP211 overexpression vector combinations, and PbZAT11-TRV and PbZAT12-TRV, and PbZAT11-TRV+PbZAT12-TRV silencing vector combinations, to transiently transform pear fruits. At least 10 fruits were injected into each group, and the experiment was performed in triplicate. After 24 hours of dark culture, the fruits were placed in an incubator with a 16-hour light / 8-hour dark photoperiod, maintained at 24°C, and cultured for 7 days.

[0028] Example 4: Lignin analysis of pear fruit and detection of PbZAT11 and PbZAT12 expression levels In Example 3, fruits from different groups injected with Agrobacterium tumefaciens bacterial solutions and cultured were sliced, treated with 30% hydrochloric acid solution (V / V) for 1 minute, stained with 10% phloroglucinol for 3 minutes, and finally rinsed with sufficient water to terminate the reaction. The staining results are as follows. Figure 2 As shown, through Figure 2 It can be seen that the lignin staining effect was significantly enhanced in the regions where PbZAT11 and PbZAT12 were overexpressed, while the lignin staining effect was inhibited in the regions where PbZAT11 and PbZAT12 were silenced. The lignin content in the pear fruits of each group was determined according to the manufacturer's instructions, specifically using the Solarbio Lignin Content Assay Kit (micro-method). The lignin content detection results are as follows: Figure 3 As shown, through Figure 3The results of lignin content detection showed that the lignin content in pear fruits overexpressing PbZAT11 and PbZAT12 was significantly increased, while the lignin content in parts where PbZAT11 and PbZAT12 were silenced was significantly decreased, indicating that PbZAT11 and PbZAT12 can regulate the lignin content in pear fruits.

[0029] The expression levels of PbZAT11 and PbZAT12 genes in pear fruit were detected according to the manufacturer's instructions. Total RNA was extracted from the infiltrated region using the RNAprep Pure Plant Plus kit (Tiangen Biotech, Beijing). Following the manufacturer's instructions, the extracted RNA was used as a template to synthesize cDNA using the HiScript II Q RT SuperMix for qPCR (+gDNAwiper) kit (Vazyme, Nanjing, China). qRT-PCR amplification was performed using the iCycler iQ5 system (BioRad, Hercules, CA, USA) and SYBR Green PCR Master Mix, with PbActin as an internal control. The primer sequences were as follows: PbActin-qPCR-F:CCATCCAGGCTGTTCTCTC; PbActin-qPCR-R: GCAAGGTCCAGACGAAGG; In this embodiment of the invention, the qRT-PCR amplification reaction procedure is as follows: ① Pre-denaturation at 94℃ for 30 seconds; ② Denaturation at 94℃ for 5 seconds; extension at 60℃ for 15 seconds; cycle 32 times in this phase. ③Extend at 72℃ for 5 minutes.

[0030] The primer sequences for qRT-PCR analysis of genes PbZAT11 and PbZAT12 are as follows: qPbZAT11-F: ATGAGTCGTGATGATGAAGA; qPbZAT11-R:GCCATGTCCATATTAGAAGC; qPbZAT12-F: GACACATGAGGAGACATAG; qPbZAT12-R:CAAGTCCAGATTCAAGCA; The results of detecting the expression levels of genes PbZAT11 and PbZAT12 in pear fruit are as follows: Figure 4 As shown, through Figure 4It can be seen that the expression levels of PbZAT11 and PbZAT12 in pear fruits that overexpressed PbZAT11 and PbZAT12 increased significantly, while the expression levels of PbZAT11 and PbZAT12 in pear fruits that silenced PbZAT11 and PbZAT12 decreased significantly.

[0031] Example 5: Genetic transformation of pear callus and molecular identification of transformed plants (1) LBA4404 Agrobacterium, which was transformed by PbZAT11 and PbZAT12 expression vectors, was prepared for use. The transformation method was the same as the transformation steps of Agrobacterium GV3101 described in Example 3.

[0032] (2) Activate Agrobacterium, following the Agrobacterium activation steps in Example 3, until the OD value of the Agrobacterium culture reaches 0.6-0.8; (3) Infection: Transfer the activated Agrobacterium bacterial suspension to a 100 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, wash once with deionized water, suspend the bacterial suspension with an equal volume of water, and add ACE (1:1000) to obtain the infection solution. Then, place the pear callus tissue cultured for 15 days into the infection solution with tweezers and infect at 200 rpm for 30 min at room temperature. Among them, pear callus tissue cultured for 15 days was obtained by inducing the peel of Shannong crisp pear, specifically as follows: The peel of Shannong crisp pears was placed in the dark under conditions containing... , , The product was obtained by growing and subculturing D on MS medium for 15 days.

[0033] (4) Blot the infection solution on the surface of the callus with sterile filter paper, and co-culture the infected pear callus on solid MS medium for 48 hours. Then, place the callus in a medium containing 100 mg / L kanamycin and 0.5 mg / L sodium chloride solution. −1 IAA, 0.5 mg l −1 6-BA and 1.0 mg l −1 Transgenic callus samples were obtained by screening on solid MS subculture medium of 2-4 D at 24°C in the dark for at least 1 month. (5) RNA was extracted from the transgenic callus samples. Using the cDNA template obtained from the reverse transcription of callus RNA as a control, the expression level of the target gene in the overexpressed callus was detected. The RNA extraction, cDNA synthesis, and quantitative real-time PCR system and procedures were as described in step 4. PbActin was used as an internal control. The primer sequence for PbActin was: PbActin-qPCR-F:CCATCCAGGCTGTTCTCTC; PbActin-qPCR-R: GCAAGGTCCAGACGAAGG; The primer sequences for qRT-PCR analysis of genes PbZAT11 and PbZAT12 are as follows: qPbZAT11-F: ATGAGTCGTGATGATGAAGA; qPbZAT11-R:GCCATGTCCATATTAGAAGC; qPbZAT12-F: GACACATGAGGAGACATAG; qPbZAT12-R:CAAGTCCAGATTCAAGCA.

[0034] Image of the appearance of the transgenic callus sample as shown in the figure. Figure 5 As shown in the figure. The results of the detection of the expression level of the target gene in the callus tissue are as follows. Figure 6 As shown, through Figure 6 It can be seen that the expression level of the target gene is significantly increased in callus tissue overexpressing PbZAT11 or PbZAT12.

[0035] Example 6: Determination of Lignin Content in Transgenic Callus Three-week-old pear callus tissues overexpressing the empty vector (EV-OE group), PbZAT11 overexpressing (PbZAT11-OE group), and PbZAT12 overexpressing (PbZAT12-OE group) were collected. The lignin content in each group of pear callus tissues was detected according to the method described in Example 4. The detection results are as follows: Figure 7 As shown, through Figure 7 It can be seen that the lignin content in pear callus tissue overexpressing PbZAT11 and PbZAT12 is significantly increased, indicating that PbZAT11 and PbZAT12 positively regulate the lignin content in pear callus tissue.

[0036] Example 7: Detection of expression levels of genes related to secondary cell wall biosynthesis in sclereids RNA extraction, cDNA synthesis, and the system and procedures for quantitative real-time PCR were as described in Example 4. PbActin was used as an internal control; the primer sequence for PbActin was as follows: PbActin-qPCR-F:CCATCCAGGCTGTTCTCTC; PbActin-qPCR-R: GCAAGGTCCAGACGAAGG; The expression levels of genes related to secondary cell wall biosynthesis in sclereids were detected as follows: Figure 8 As shown, through Figure 8It can be seen that transient transformation significantly alters the transcriptional levels of secondary cell wall synthesis genes PbLAC1, PbLAC18, PbCCR1, Pb4CL3, and PbPAL1 in pear fruit by regulating the expression levels of PbZAT11 and PbZAT12. The primer sequences for qRT-PCR analysis of secondary cell wall synthesis genes PbLAC1, PbLAC18, PbCCR1, Pb4CL3, and PbPAL1 are shown below: qPbLAC1-F:AAACTCTCTCAAAGCCCTAA; qPbLAC1-R:AACACTAGCCACTACTCTG; qPbLAC18-F:CGCTGCTTCAATCTCATT; qPbLAC18-R: CAACACCAATTCCACACT; qPb4CL3-F:ATCCTGAATCTGGTGCTT; qPb4CL3-R:CTTCCTTGTCTATGGTTGTC; qPbCCOMT1-F:GCTACGACAACACCCTAT; qPbCCOMT1-R:ATCACCAACAGGAAGCAT; qPbCCR1-F:GCTCACATTCTGGTCTATG; qPbCCR1-R: CTTGTATGATTCTGCTCTGG; qPbPAL1-F:CAGCACAACCAAGATGTT; qPbPAL1-R: CCTCAAGTTCTCCTCCAA; Example 8 Subcellular localization analysis of PbZAT11 and PbZAT12 To further clarify the localization of PbZAT11 and PbZAT12, this invention constructed 35S::PbZAT11-EGFP and 35S::PbZAT12-EGFP fusion vectors, respectively, and expressed them in tobacco leaves. The primer sequences are shown below: PbZAT11-pCAMBIA2300-EGFP-F: gagaacacgggggacgagctcATGAAGAGAGATATGAGAGGAGAAGAATT; PbZAT11-pCAMBIA2300-EGFP-R: gcccttgctcaccatgagctcTGATACAAAAGCATCAACTTTTGGT; PbZAT12-pCAMBIA2300-EGFP-F: gagaacacgggggacgagctcATGAAGAGAAGCGGGCAAGAC; PbZAT12-pCAMBIA2300-EGFP-R: gcccttgctcaccatgagctcAGAAATCATCTGCAGCTTCGTCA; The specific construction method of the above-mentioned fusion carrier is as follows: (1) Expression vector construction: The coding sequences of PbZAT11 and PbZAT12 (with stop codons removed) were ligated into the pCAMBIA2300-EGFP vector, and the resulting recombinant plasmids were co-transformed with the nuclear localization plasmid (mCherry) into GV3101 (pSoup-P19) Agrobacterium competent cells; (2) Preparation of infection solution: The bacterial solution was activated to 0.6-0.8 OD600 value, the bacterial cells were collected, and the bacterial cells were resuspended with an equal volume of infection solution and then allowed to stand in the dark for 3 h. (3) Injecting tobacco: Select tobacco plants with four leaves in good growth condition (about 1 month old), and use a medical syringe to inject the bacterial solution obtained in step (2) into the lower epidermal cells of the tobacco. Note that the entire tobacco leaf should be infected. Then place it in the dark for 2 days. (4) Laser confocal microscopy observation: Tobacco epidermal cells were excised to construct sample slides, which were then placed under a laser confocal microscope (LSM880) to observe the GFP fluorescence signal. The GFP signal was excited using a 488 nm solid-state laser, and fluorescence was detected at 498–540 nm with an intensity of 4.9% and a gain of 800. The mCherry signal was excited using a 543 nm solid-state laser, and fluorescence was detected at 590–640 nm with an intensity of 9.1% and a gain of 800. The pinhole was adjusted to 1 Airy unit per wavelength.

[0037] Laser focusing detection results as follows Figure 9 As shown, through Figure 9 The display shows that, by 35S The promoter-driven EGFP signal overlaps with the mCherry signal, indicating that both PbZAT11 and PbZAT12 are located in the cell nucleus.

[0038] Example 9: Yeast one-hybrid analysis (Y1H) of PbZAT11 and PbZAT12 with PbLAC1 promoters. (1) Construct pPbLAC1-Phis, PbZAT11-pGADT7, and PbZAT12-pGADT7 vectors. The construction method is the same as that in Example 2, and will not be repeated here.

[0039] The primer sequences of the constructed vector are shown below: PbZAT11-pGADT7-F: gtaccagattacgctcatatgATGAAGAGAGATATGAGAGGAGAAGAATT; PbZAT11-pGADT7-R: cagctcgagctcgatggatccTGATACAAAGCATCAACTTTTGGT; PbZAT12-pGADT7-F:gtaccagattacgctcatatgATGAAGAGAAGCGGGCAAGAC; PbZAT12-pGADT7-R:cagctcgagctcgatggatccAGAAATCATCTGCAGCTTCGTCA; PbLAC1-pHIS2-F:gactcactatagggcgaattcAGGGGGAGATTCAAAACTATTACAA; PbLAC1-pHIS2-R: gattcgcgaacgcgtgagctcTTCTCAGTACTCTCTCTCTCTCTCTGATT; (2) 3-AT concentration screening pPbLAC1-Phis and pGADT7 were co-transformed into Y187 Agrobacterium competent cells, and then plated on SD-His / -Leu / -Trp medium with different concentrations of 3-AT to remove the self-activation of pPbLAC1-Phis.

[0040] (3) Re-preparation of Y187 Agrobacterium competent cells pPbLAC1-Phis was co-transformed with pGADT7 and PbZAT11-pGADT7 or PbZAT12-pGADT7 into Y187 Agrobacterium competent cells, respectively. The transformed cells were then plated onto medium containing an appropriate concentration of SD-His / -Leu / -Trp, and the growth of the yeast strains was observed. The results are as follows: Figure 10 As shown. (Through) Figure 10The results showed that yeast cells co-expressing the pPbLAC1-pHIS2 construct grew normally on SD / -L / -T / -H+ 3-AT 120mM plates. However, no growth was observed in yeast cells co-expressing pGADT7. The results also indicated that PbZAT11 and PbZAT12... pPbLAC1 It has a strong ability to combine.

[0041] Example 10: Luciferase reporter assay (LUC) of PbZAT11 and PbZAT12 with PbLAC1 promoters. (1) Construct PbZAT11-62SK, PbZAT12-62SK, respectively pPbLAC1 The -0800-LUC vector was used, and the recombinant vector was transformed into GV3101 (pSoup-P19) Agrobacterium competent cells. The primer sequences for each vector are shown below: PbLAC1-pGreenII 0800-LUC-F: gggccccccctcgaggtcgacAGGGGGAGATTCAAAACTATTACAA; PbLAC1-pGreenII 0800-LUC-R: cgctctagaactagtggatccTTCTCAGTACTCTCTCTCTCTCTCTGATT; PbZAT11-pGreenII 62-SK-F: cgctctagaactagtggatccATGAAGAGAGATATGAGAGGAGAAGAATT PbZAT11-pGreenII 62-SK-R: gtcgacggtatcgataagcttTGATACAAAAGCATCAACTTTTGGT; PbZAT12-pGreenII 62-SK-F: cgctctagaactagtggatccATGAAGAGAAGCGGGCAAGAC; PbZAT12-pGreenII 62-SK-R: gtcgacggtatcgataagcttAGAAATCATCTGCAGCTTCGTCA; (2) Preparation of inoculum Agrobacterium bacterial suspension containing the target plasmids (PbZAT11 and PbZAT12) and the empty vector (pPZP211) was activated until the final OD600 was approximately 1.0. The bacterial cells were collected by centrifugation, washed once with ddH2O, and finally resuspended in infection buffer. The bacterial suspension was then mixed with an equal volume of the injected gene combination according to the target, and incubated in the dark for 3 hours. The above-mentioned gene injection combinations are as follows: Empty vector+LUC、35S::ZAT11+LUC、Empty vector+ proLAC1 ::LUC、35S:: ZAT11+ pro LAC1 ::LUC、Empty vector+LUC、35S::ZAT12+LUC、Empty vector+ proLAC1 ::LUC、35S:: ZAT12+ pro LAC1 ::LUC combination.

[0042] (3) Injecting tobacco: Select healthy tobacco plants and use a disposable medical syringe to inject the bacterial solution obtained in (2) into the lower epidermal cells of the tobacco plants, and then place them in the dark for 2 days. (4) Fluorescence intensity and enzyme activity detection: Fluorescence intensity was qualitatively detected using a live in vivo imaging system (Xenogen), and luciferase activity was quantitatively detected using the Dual-Luciferase® Reporter Assay System (Promega). The fluorescence intensity detection results are as follows: Figure 11 As shown, through Figure 11 The results showed that the fluorescence intensity of PbLAC1 in tobacco overexpressing PbZAT11 and PbZAT12 was enhanced, indicating that PbZAT11 and PbZAT12 can activate the expression of PbLAC1.

[0043] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. Choose one of the following genes and apply it in (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; in, The gene includes the gene PbZAT11 and / or the gene PbZAT12.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the gene PbZAT11 is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The nucleotide sequence of the gene PbZAT12 is shown in SEQ ID NO.

2.

4. The use of a gene-encoded protein according to any one of claims 1-3 in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; in, The gene includes the gene PbZAT11 and / or the gene PbZAT12.

5. The application according to claim 4, characterized in that, The amino acid sequence of the protein encoded by the gene PbZAT11 is shown in SEQ ID NO.

3.

6. The application according to claim 4, characterized in that, The amino acid sequence of the protein encoded by the gene PbZAT12 is shown in SEQ ID NO.

4.

7. The use of a recombinant expression vector and / or transient expression vector comprising the gene according to any one of claims 1-3 in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; in, The gene includes the gene PbZAT11 and / or the gene PbZAT12.

8. The application according to claim 7, characterized in that, The recombinant expression vector backbone is pPZP211.

9. The use of a recombinant Agrobacterium comprising the gene according to any one of claims 1-3 in the following (A1)-(A3): (A1) Application of regulating the content of stone cells in pear fruit; (A2) Application in the preparation of products that regulate the content of stone cells in pear fruits; (A3) Application in breeding to regulate the content of stone cells in pear fruits; in, The gene includes the gene PbZAT11 and / or the gene PbZAT12.

10. A method for regulating the stone cell content of pear fruit, characterized in that, The method increases the stone cell content in pear fruit by overexpressing one of the following genes, or inhibits one of the following genes to inhibit the stone cell content in pear fruit; The gene includes gene PbZAT11 and / or gene PbZAT12.

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