Gene ClPHL6 for regulating and controlling sugar content of clausena lansium fruits and application of gene ClPHL6

Abstract

The invention belongs to the technical field of bioengineering, discloses a gene ClPHL6 for regulating and controlling the sugar content of clausena lansium fruits and application of the gene ClPHL6, and particularly discloses application of ClPHL6 in regulating and controlling the soluble sugar content of plant leaves / fruits. According to the invention, the clausena lansium ClPHL6 gene is screened for the first time, and experiments prove that the ClPHL6 gene can significantly enhance the accumulation of soluble sugar in tobacco leaves and tomato fruits. The gene can be used as a target gene for controlling the content of soluble sugar in the clausena lansium fruits and improving the flavor of the fruits for in-depth research, and has high application and research values.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene ClPHL6 for regulating the sugar content of yellow peel fruits and an application thereof. Background Art

[0002] my country is the country with the most genetic resources, the largest cultivated area and the highest yield of Clausena lansium (Lour.) Skeels in the world, and Guangdong Province ranks first in my country in terms of cultivated area of ​​Clausena lansium. There are nearly 200,000 mu of Clausena lansium in Yunan County alone. The fruit of Clausena lansium is rich in nutrition and has the effects of anti-oxidation, anti-aging and anti-tumor. It is a rare fruit that can be used as both medicine and food and is deeply loved by consumers. The mature and marketed period of Clausena lansium is staggered with the market period of the major fruit in the south, lychee and longan. It is a high-economic-value crop worthy of promotion. However, there are very few varieties of Clausena lansium that can be promoted and there is a problem of single fruit taste. The main cultivated varieties are mainly sour Clausena lansium, mainly 'Yunan seedless Clausena' and 'Jixin Clausena', and sweet Clausena, mainly 'Congcheng sweet Clausena' and 'Yunan rock sugar Clausena'. The sour Clausena tastes sour, while the sweet Clausena tastes pure and sweet with extremely low acid content, resulting in serious polarization of the taste of the Clausena varieties on the market. Fruit flavor is a key factor in determining the public acceptance of fruit tree varieties and directly determines whether they can achieve commercial success. The sugar content of kumquat fruit is a key factor affecting fruit flavor. Therefore, it is urgent to further explore the regulatory mechanism of kumquat fruit sugar accumulation and metabolism and promote the creation and improvement of high-quality kumquat varieties.

[0003] Transcription factors affect fruit quality through a variety of mechanisms, including developmental regulation, flavor and aroma formation, pigment synthesis, regulation of the ripening process, and interaction with hormone signals. In apples, researchers found that MdMYB305 can promote sugar accumulation in red-fleshed apple offspring. MdMYB305 binds to sugar-related genes (MdCWI1 / MdVGT3 / MdTMT2), promotes or inhibits their activity, and ultimately regulates sugar accumulation in the fruit. In addition, this study also found that MdMYB305 and MdMYB10 balance the sugar content in the fruit by competing with the binding site of MdbHLH33. In melons, the transcriptional repressor CmMYB44 regulates sucrose accumulation in oriental melon fruits. CMMYB44 affects sucrose accumulation by inhibiting the transcriptional activation of the sucrose phosphate synthase gene (CMSPS1). MDWRKY126 is a transcription factor. Overexpression can upregulate the activity of sucrose synthesis-related genes (SPS) and reduce the expression of sucrose decomposition-related genes.

[0004] In summary, the transcription factor family plays an important role in regulating fruit quality. In-depth research on the functions and mechanisms of transcription factors is of great significance for improving fruit quality. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide the application of ClPHL6.

[0006] The second aspect of the present invention aims to provide applications of biomaterials related to ClPHL6.

[0007] The third aspect of the present invention aims to provide the use of a reagent for targeted upregulation of the expression level of ClPHL6.

[0008] The fourth aspect of the present invention aims to provide a method.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides the use of ClPHL6 as described in any one of (1) to (5):

[0011] (1) Regulate the soluble sugar content of plant leaves;

[0012] (2) preparing a product for regulating the soluble sugar content of plant leaves;

[0013] (3) Regulate the quality of plant fruits;

[0014] (4) preparing products for regulating the quality of plant fruits;

[0015] (5) Genetically engineered plants that produce high-quality fruit;

[0016] The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:2.

[0017] In some embodiments of the present invention, the plants include tobacco and / or fruits and vegetables.

[0018] In some embodiments of the present invention, the fruits and vegetables include at least one of kumquat, tomato, and citrus.

[0019] In some embodiments of the present invention, regulating the quality of plant fruits includes regulating the content of soluble sugars in plant fruits.

[0020] The second aspect of the present invention provides a use of a biomaterial related to ClPHL6 as described in any one of (1) to (5):

[0021] (1) Regulate the soluble sugar content of plant leaves;

[0022] (2) preparing a product for regulating the soluble sugar content of plant leaves;

[0023] (3) Regulate the quality of plant fruits;

[0024] (4) preparing products for regulating the quality of plant fruits;

[0025] (5) Genetically engineered plants that produce high-quality fruit;

[0026] The biological material carries ClPHL6, and the amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:2.

[0027] In some embodiments of the present invention, the biological material is any one of a1) to a12):

[0028] a1) a nucleic acid molecule encoding ClPHL6;

[0029] a2) an expression cassette containing the nucleic acid molecule described in a1);

[0030] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0031] a4) a recombinant vector containing the expression cassette described in a2);

[0032] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0033] a6) a recombinant microorganism containing the expression cassette described in a2);

[0034] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0035] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0036] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0037] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0038] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0039] a12) A transgenic animal cell line containing the recombinant vector described in a4).

[0040] In some embodiments of the invention, the transgenic animal cell line does not contain reproductive material.

[0041] In some embodiments of the present invention, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:1.

[0042] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0043] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0044] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0045] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0046] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0047] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0048] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0049] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0050] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0051] In some embodiments of the present invention, the recombinant expression vector uses super1300 as the original expression vector.

[0052] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0053] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

[0054] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0055] In some embodiments of the present invention, the plants include tobacco and / or fruits and vegetables.

[0056] In some embodiments of the present invention, the fruits and vegetables include at least one of kumquat, tomato, and citrus.

[0057] In some embodiments of the present invention, regulating the quality of plant fruits includes regulating the content of soluble sugars in plant fruits.

[0058] The third aspect of the present invention provides a use of a reagent for targeting upregulating the expression of ClPHL6 as described in any one of (1) to (5):

[0059] (1) Regulate the soluble sugar content of plant leaves;

[0060] (2) preparing a product for regulating the soluble sugar content of plant leaves;

[0061] (3) Regulate the quality of plant fruits;

[0062] (4) preparing products for regulating the quality of plant fruits;

[0063] (5) Genetically engineered plants that produce high-quality fruit;

[0064] The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:2.

[0065] In some embodiments of the present invention, the plants include tobacco and / or fruits and vegetables.

[0066] In some embodiments of the present invention, the fruits and vegetables include at least one of kumquat, tomato, and citrus.

[0067] In some embodiments of the present invention, regulating the quality of plant fruits includes regulating the content of soluble sugars in plant fruits.

[0068] A fourth aspect of the present invention provides a method for overexpressing ClPHL6 in a target plant;

[0069] The method comprises any one of 1) to 3):

[0070] 1) A method for increasing the soluble sugar content of plant leaves;

[0071] 2) A method for improving the quality of plant fruits;

[0072] 3) A method for cultivating genetically modified plants with high quality plant fruits;

[0073] The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:2.

[0074] In some embodiments of the present invention, the method comprises the following steps:

[0075] (1) Construction of ClPHL6 gene overexpression vector;

[0076] (2) The overexpression vector is transferred into Agrobacterium and then infects plants.

[0077] In some embodiments of the present invention, the backbone vector of the overexpression vector is super1300.

[0078] In some embodiments of the present invention, the method for constructing the overexpression vector includes the steps of cloning the ClPHL6 gene, connecting the recovered product with the super1300 vector, and constructing the overexpression vector (ie, super1300-ClPHL6 overexpression vector).

[0079] In some embodiments of the present invention, when the plant is a tomato, the overexpression vector is transferred into Agrobacterium in step (2) and then infects a tomato explant. The infected tomato explant is screened to grow callus tissue, and further cultivated to obtain a ClPHL6 stably overexpressing tomato plant (i.e., a tomato plant whose tomato fruit contains a higher soluble sugar content).

[0080] That is, the ClPHL6 gene overexpression vector super1300-ClPHL6 was used to carry out genetic transformation with tomato explants by Agrobacterium-mediated method, and a ClPHL6 stable overexpression tomato strain was obtained through molecular identification.

[0081] The beneficial effects of the present invention are:

[0082] Based on the flavor of kumquat fruit, the present invention divides kumquat into two subgroups, sweet kumquat and sour kumquat. Based on population selection analysis and transcriptomics analysis, the gene ClPHL6 encoding the ALMT protein family is found in the significantly differentially expressed genes. The RT-qPCR result also confirms this result. The gene is further studied in depth, and the kumquat ClPHL6 gene is cloned for the first time. ClPHL6 is transiently overexpressed in tobacco leaves in a tobacco transient expression system, and it is verified that the gene can significantly enhance the soluble sugar content of tobacco leaves. At the same time, ClPHL6 is heterologously overexpressed in tomatoes using transgenic technology, and the function of the ClPHL6 gene to accumulate soluble sugar content is further verified. The scheme of the present invention effectively overcomes the blank of the research on sugar accumulation regulation of kumquat fruit in the prior art. The kumquat ClPHL6 gene can be used as a target gene for regulating the soluble sugar content of kumquat fruit and improving the fruit flavor for in-depth research, and has high application and research value.

[0083] In the method provided by the present invention, the transient gene expression technology used has simple steps and is effective, which can effectively shorten the time for studying gene function. Heterologous gene expression can play an important role in the study of gene function in species that are not easy to achieve genetic modification, and is of great significance for revealing the function of yellow peel genes, molecular breeding, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 is the discovery of ClPHL6; wherein, A is the detection of candidate divergent regions (CDRs) based on the high genetic differentiation (top 5% of Fst) in sweet kumquat and sour kumquat germplasm in the whole genome; B is the transcriptome analysis of the expression level of ClPHL6 in sour kumquat and sweet kumquat; C is the qPCR analysis of the expression level of ClPHL6 in sour kumquat and sweet kumquat germplasm, respectively; D is the correlation analysis of ClPHL6 expression level and kumquat fruit sugar content; in the figure, ** represents p < 0.01, *** represents p < 0.001.

[0085] Figure 2 The figure shows the cloning of ClPHL6 and the structure of its encoded protein; A is the agarose gel electrophoresis of PCR-amplified ClPHL6, B is the schematic diagram of the ClPHL6 protein structure; C is the homology comparison and domain analysis of ClPHL6 and 8 MYB protein sequences from other species.

[0086] Figure 3 The figure shows the construction and identification of ClPHL6 overexpression vector; A is the colony PCR identification after transformation into DH5α competent cells; B is the colony PCR identification after transformation into GV3101 competent cells.

[0087] Figure 4The figures are phenotypic analysis of tobacco leaves transiently overexpressing ClPHL6 and changes in ClPHL6 expression levels; A is the agarose gel electrophoresis diagram of ClPHL6 expression level detection in tobacco leaves 3 days after ClPHL6 transient overexpression; B is the changes in total soluble sugar content in tobacco leaves 3 days after injection of engineered bacteria containing an empty vector (super1300) and a ClPHL6 overexpression vector (super1300-ClPHL6); in the figure, ** represents p < 0.01.

[0088] Figure 5 The figure shows the process of obtaining stable plants overexpressing ClPHL6 in tomatoes; A is co-cultivation; B is screening and cultivation; C is differentiation and cultivation; D is rooting to form resistant seedlings; E is the fruiting traits of transgenic tomatoes; F is extraction of transgenic tomato genomic DNA and PCR detection of marker gene HPT (hygromycin gene) using HPT-F / HPT-R primers; G is RT-PCR detection of ClPHL6 expression in overexpressing strains; H is detection of total soluble sugar content in fruits of ClPHL6 stably expressing transgenic tomato strains. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below through specific examples.

[0090] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0091] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0092] In the following embodiments of the present invention, the materials involved include:

[0093] Tobacco material was Nicotiana benthamiana, and tomato material was Micro-TOM;

[0094] The primers and sequencing used in the experiment were completed by Sangon Biotech (Shanghai) Co., Ltd.;

[0095] HindIII and KpnI were purchased from Thermo Fisher Scientific;

[0096] High-fidelity enzymes, IIOne Step Cloning Kit, RNA extraction kit, reverse transcription kit, and DNA purification kit were purchased from Nanjing Novogene Biotech Co., Ltd.;

[0097] Escherichia coli competent cells DH5α and Agrobacterium competent cells GV3101 were purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0098] The remaining molecular reagents were mainly purchased from Nanjing Novozymes Biotech Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd.

[0099] The method for determining the total soluble sugar content in the embodiment is as follows: weigh an appropriate amount of fruit and vegetable samples and grind them into a homogenate in a mortar, add a small amount of dd H2O to rinse the mortar several times, transfer to a test tube to the corresponding scale, and shake well. Seal with plastic wrap, poke a hole, and boil in water for 40 minutes (shake well several times during the period); centrifuge at 12000rpm for 15 minutes, and collect the supernatant. Add in the order of 1.5mL ddH2O-5mL concentrated sulfuric acid-(cooled)-0.5mL anthrone ethyl acetate-0.5mL extract, shake well and place in a boiling water bath for 1 minute. After taking out and cooling to room temperature, measure the absorbance at a wavelength of 630nm, and repeat three times. Prepare a standard curve in the same way; according to the absorbance value of the solution, find the corresponding sucrose mass in the standard curve, and calculate the soluble sugar content in the tissue.

[0100] Pre-culture medium: MS+1mg / L IAA+1.75mg / L ZT;

[0101] Co-culture medium: MS+1mg / L IAA+1.75mg / L ZT;

[0102] Screening medium: MS+1mg / L IAA+1.75mg / L ZT+10mg / L hyg+500mg / L Cb;

[0103] Differentiation medium: MS+1mg / L IAA+1.75mg / L ZT+5mg / L hyg+500mg / L Cb;

[0104] Rooting medium: MS+5mg / L hyg+250mg / L Cb;

[0105] Among them, MS was purchased from BioRun with the catalog number RFA01; IAA was purchased from Bio-Tech with the catalog number A600723-0025; ZT was purchased from Bio-Tech with the catalog number A411990-0025; Cb was purchased from BioRun with the catalog number RJD00; and hygromycin B was purchased from BioRun with the catalog number RJA00-1g.

[0106] The information of the Huangpi germplasm resources involved in the embodiment is shown in Table 1, all of which are from the Huangpi germplasm resource garden of the Ministry of Agriculture and Rural Affairs.

[0107] Table 1 Information on Huangpi germplasm resources

[0108]

[0109]

[0110] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0111] Example 1 Discovery of ClPHL6 gene

[0112] The flavor of fruit trees is a scientific issue of common concern to all fruit tree breeders, and kumquat is no exception. Based on the taste, kumquat varieties can be clearly divided into two subgroups: sweet kumquat and sour kumquat. Sweet kumquat has a sweet taste without sourness. The sour kumquat has a different taste of sweet and sour, with a distinct sour taste, and there is a clear difference in sweet and sour taste between the two from the young fruit. In order to study the genetic basis of the taste of kumquat fruit, this example used the previous kumquat resource resequencing data to compare the genomes of sweet kumquat and sour kumquat (Table 1) to find significant selection signals. Candidate divergent regions (CDRs) were detected based on high genetic differentiation in the whole genome (the top 5% of Fst), and a 10Kb sliding window method was used. 1716 genes were screened out from 68 candidate divergent regions (CDRs), of which a MYB transcription factor was found in Chr7 and named ClPHL6 ( Figure 1 Subsequently, the inventors also found in transcriptome analysis of sour and sweet kumquats that the expression level of ClPHL6 in sour kumquats was significantly lower than that in sweet kumquats ( Figure 1 (middle B).

[0113] Furthermore, the inventors selected 5 samples of sweet and sour yellow peel varieties, and analyzed the expression level of ClPHL6 by qPCR. The results showed that the expression level of ClPHL6 in the sweet yellow peel variety was significantly higher than that in the sour yellow peel variety, and the correlation analysis showed that the expression level of ClPHL6 was significantly positively correlated with the total sugar content of the fruit ( Figure 1 C and D).

[0114] The above results suggest that ClPHL6 can regulate the accumulation of sugar in fruits. Since the molecular biology research of yellow peel has just started, there is still a blank in the transient transformation system and stable transgenic system. In order to verify the function of ClPHL6 in yellow peel in regulating the accumulation of total sugar content in fruits, heterologous overexpression is selected for verification.

[0115] Example 2: Cloning and bioinformatics analysis of the CDS region of the C1PHL6 gene

[0116] Referring to the reference genome of Wampee "JingFeng" assembled by our research group (chen et al. 2024. Identification of Key Genes Controlling Sugar and Organic Acid Accumulation in Wampee Fruit (Clausena lansium) via Genome Assembly and Genome-wide Association Analysis), the ClPHL6 sequence was extracted, and the primers ClPHL6-F / ClPHL6-R were designed using Premier 5.0 (Table 2). The total RNA of Wampee leaves was extracted using an RNA extraction kit (FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics–rich), Novazon, China), and reversed into cDNA using the HiScriptIV 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Novazon, China).

[0117] The coding sequence CDS was cloned using cDNA as template and ClPHL6-F / ClPHL6-R as primers. The PCR product was subjected to 1.5% agarose gel electrophoresis and the results were detected and recorded using a gel imaging system (Bio-Rad). Figure 2 A), the PCR product was recovered and connected to BlunT3, transformed into DH5α competent cells, single colonies were picked for colony PCR, and the positive bacteria were sent to Sangon Biotechnology (Guangzhou) Co., Ltd. for sequencing to obtain the CDS sequence of ClPHL6, with a sequence size of 1503 bp. The CDS sequence of the ClPHL6 gene was determined, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0118] ClPHL6 gene CDS sequence:

[0119]

[0120] Table 2 Cloning, identification and real-time fluorescence quantitative (RT-qPCR) primers used in the experiment

[0121]

[0122] ExPaSy was used to analyze the conserved domains and physicochemical properties of ClPHL6: The ClPHL6 gene encodes 500 amino acids (the amino acid sequence of ClPHL6 is shown in SEQ ID NO: 2), with a theoretical molecular weight of 54.8 kD and a theoretical isoelectric point of 6.36. The protein contains 60 negatively charged residues (Asp+Glu) and 54 positively charged residues (Arg+Lys), an instability coefficient of 50.93, an aliphatic index of 69.86, and an average hydrophilicity of -0.604, indicating that ClPHL6 is a hydrophilic and stable protein. According to the protein structure prediction of the SMART website (http: / / smart.embl.de / smart / set_mode.cgi?NORMAL=1), ClPHL6 contains a MYB-DNAbinding domain located at positions 269-320 of the amino acid sequence; and a MYB-CC domain located at positions 356-403 of the amino acid sequence (the protein structure diagram is shown in Figure 2 (middle B).

[0123] The amino acid sequence encoded by the ClPHL6 gene: MNHSITSVTKNESNKGVSQSCCTALSPVHNFLSVETEGQSLSTGECPPFPHPSPFKRKESLSSPKHMQASTVVPQKNGLISTPDSPISPGSHFQHSKGGFSRSSVFCTSLYLSSS ASSETHRQIGNFPFLPHPPTYNRSVSAVDSTMSSLLFSEDIGNPYQEEHSESLMKDFLNLPRDASDASFHGVTCMGERLGLNEQLELQFLSDELDIAITDHGENPRLDEIYDASKPSLKPPMGLLCNQNY VSSAPPVDALSSHTSPGSATAHKPRMRWTPELHECFVEAVSKLDGAEKATPKGVLKLMNVEGLTIYHVKSHLQKYRLAKYMPEKKEEKKACSSEEKKAASSIESDGRKKGSIQITEALRMQMEVQKQLHE QLEVQRALQLRIEEHARYLQKILAEQQKAGSAMVLPQAQSLSTITNGSKASEQQPSSPSFTVTASAILSPEQPAESKTESSSTSLLSKHKATDGRESKPDACLKRIRLENKPEIASDEAVVENPVQ*(SEQ ID NO:2).

[0124] Example 3 Construction of ClPHL6 overexpression vector and phenotypic analysis

[0125] 1. Construction of ClPHL6 overexpression vector

[0126] According to the ClPHL6 coding sequence, a forward primer ClPHL6-F1 containing a homology arm of the Hind III restriction site (CCAAATCGACTCTAGAAAGCTT, SEQ ID NO: 13) and a reverse primer ClPHL6-R1 containing a homology arm of the Kpn I restriction site (CCTCGCCCTTGCTCACCATGGTACC, SEQ ID NO: 14) were designed (primer sequences are shown in Table 2). The cDNA was used as a template for amplification and recovered by gel running. The recovered product and the overexpression vector super1300 were digested and recovered using restriction endonucleases Hind III and Kpn I (Thermo Fisher Scientific), respectively. After that, DH5α competent cells were transformed, and single colonies were picked for colony PCR (such as PCR) using vector universal primers Super1300-F / Super1300-R (primer sequences are shown in Table 2). Figure 3The positive colonies were shaken and sent to the company for sequencing. After the sequencing was correct, the plasmid was extracted to obtain the overexpression vector super1300-ClPHL6.

[0127] 2. Molecular identification and phenotypic analysis after transient overexpression of ClPHL6

[0128] The plasmids containing ClPHL6 overexpression vector (super1300-ClPHL6) and empty vector (super1300) were transformed into Agrobacterium GV3101, and colony PCR was performed for identification ( Figure 3 (B), positive single clones were picked to prepare engineered bacteria, and the engineered bacteria containing ClPHL6 overexpression vector (super1300-ClPHL6) and empty vector (super1300) were injected into 4-6 week-old Nicotiana benthamiana leaves, and RT-PCR primers qClPHL6-F / qClPHL6-R were designed (primer sequences are shown in Table 2), and the expression of ClPHL6 was detected after 3 days.

[0129] The results showed that the expression of ClPHL6 could be detected in tobacco leaves injected with super1300-ClPHL6 overexpression vector ( Figure 4 Then, the soluble total sugar content of tobacco leaves injected with empty vector (super1300) and injected with overexpression vector (super1300-ClPHL6) was detected. Compared with tobacco leaves injected with empty vector, the soluble total sugar content of tobacco leaves injected with overexpression vector (super1300-ClPHL6) was significantly increased ( Figure 4 (middle B).

[0130] 3. Molecular identification and phenotypic analysis after heterologous overexpression of ClPHL6

[0131] Take the germinated sterile seedlings of tomato (Micro-TOM), wait for the cotyledons to fully expand, cut off the cotyledon petiole and cotyledon tip with a scalpel, keep the middle part and cut it into 2-3 sections and inoculate it into the pre-culture medium, and pre-culture it at 23±2℃ for 2-3 days. Pick the identified positive Agrobacterium (obtained from the molecular identification and phenotypic analysis after transient overexpression of 2.ClPHL6) and put it into the infection solution to prepare the OD 600 = 0.1 Agrobacterium resuspension; infect for 10-15 min, inoculate the dried explants in co-cultivation medium, and culture in the dark at 23±2℃ for 2 days ( Figure 5 The callus after recovery culture was inoculated into the screening medium and cultured at 23°C with 16h / 8h light / darkness for 15 to 30 days ( Figure 5 The selected calli were inoculated into differentiation medium and cultured at 23°C with 16h / 8h light / darkness for 30-40 days ( Figure 5C). When the seedlings to be differentiated grow to about 2-3 cm, they are cut off from the callus and inoculated into rooting medium. They are cultured at 23°C and 16h / 8h light / dark for 10-15 days to obtain resistant regenerated plants ( Figure 5 D). Tomato genomic DNA was extracted and PCR detection of the marker gene HPT (hygromycin gene) was performed. The detection primers were HPT-F / HPT-R (primer sequences are shown in Table 2). The PCR detection results of the marker gene HPT are shown in Figure 5 Middle F.

[0132] RNA was extracted from the mature fruits of the positive plants and the untransformed wild-type tomato plants, respectively, and reverse transcribed into cDNA. The cDNA was amplified using qClPHL6-F / qClPHL6-R, and the wild-type was used as a control. The expression of ClPHL6 in the regenerated plants was detected by agarose gel electrophoresis. The plants that could amplify the target fragment band were the plants with stable ClPHL6 heterologous overexpression ( Figure 5 Middle E).

[0133] The results are as follows Figure 5 As shown in G to H, the total soluble sugar content of the fruits of the wild type and two overexpression tomato lines was detected respectively. The results showed that the fruit sugar content of the overexpression lines was significantly higher than that of the wild type, proving that the improvement method of the present invention for the total soluble sugar content of fruits based on the ClPHL6 gene can significantly increase the total soluble sugar content of fruits, and has high application and research value.

[0134] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of ClPHL6 in any one of (1) to (5): (1) Regulate the soluble sugar content of plant leaves; (2) preparing a product for regulating the soluble sugar content of plant leaves; (3) Regulate the quality of plant fruits; (4) preparing products for regulating the quality of plant fruits; (5) Genetically engineered plants that produce high-quality fruit; The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:

2.

2. Use of the biomaterial related to ClPHL6 as described in any one of (1) to (5): (1) Regulate the soluble sugar content of plant leaves; (2) preparing a product for regulating the soluble sugar content of plant leaves; (3) Regulate the quality of plant fruits; (4) preparing products for regulating the quality of plant fruits; (5) Genetically engineered plants that produce high-quality fruit; The biological material carries ClPHL6, and the amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that: The biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding ClPHL6; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) A transgenic animal cell line containing the recombinant vector described in a4).

4. The use according to claim 3, characterized in that: The nucleic acid molecule includes the sequence shown in SEQ ID NO:

1.

5. Use of a reagent for targeting upregulation of ClPHL6 expression as described in any one of (1) to (5): (1) Regulate the soluble sugar content of plant leaves; (2) preparing a product for regulating the soluble sugar content of plant leaves; (3) Regulate the quality of plant fruits; (4) preparing products for regulating the quality of plant fruits; (5) Genetically engineered plants that produce high-quality fruit; The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:

2.

6. The use according to any one of claims 1 to 5, characterized in that: The plants include tobacco and / or fruits and vegetables; preferably, the fruits and vegetables include at least one of kumquat, tomato and citrus.

7. The use according to any one of claims 1 to 5, characterized in that: The regulating the plant fruit quality includes regulating the content of soluble sugar in the plant fruit.

8. A method comprising overexpressing ClPHL6 in a target plant; The method comprises any one of 1) to 3): 1) A method for increasing the soluble sugar content of plant leaves; 2) A method for improving the quality of plant fruits; 3) A method for cultivating genetically modified plants with high quality plant fruits; The amino acid sequence of ClPHL6 includes the amino acid sequence shown in SEQ ID NO:

2.

9. The method according to claim 8, characterized in that The method comprises the following steps: (1) Construction of ClPHL6 gene overexpression vector; (2) transferring the overexpression vector into Agrobacterium and then infecting plants; Preferably, the backbone vector of the overexpression vector is super1300.

10. Application of the reagent for detecting ClPHL6 in distinguishing sweet and sour yellow peel varieties; Preferably, the reagent includes a primer sequence for detecting ClPHL6; Preferably, the primer sequences are shown in SEQ ID NO:3 to SEQ ID NO:4.

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