CN118308374A - Citrus CsAP2-16 gene and application thereof in regulating and controlling fruit ripening - Google Patents

Abstract

The invention discloses a citrus transcription factor CsAP-16 gene and application thereof in regulating and controlling fruit ripening. The gene belongs to an AP2/ERF family member, the CDS sequence is shown as SEQ ID NO.1, the length of the CDS sequence is 1290bp, 429 amino acids are encoded, and the encoded amino acids are shown as SEQ ID NO. 2. The primer is designed to amplify genes CsAP-16 on the vone navel orange, the genes are introduced into Mic-Tom tomatoes and kumquats by using an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are verified by phenotype observation statistics and gene expression analysis, so that the cloned CsAP-16 genes have the function of remarkably promoting citrus fruit maturation, and the invention provides new gene resources for molecular design and breeding in the fruit maturation period.

Description

Citrus CsAP2-16 gene and application thereof in regulating and controlling fruit ripening

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a gene CsAP-16 related to Citrus fruit maturation, which is isolated and cloned from Citrus fruit (Citrus sinensis), and application of the gene in regulating and controlling fruit maturation, wherein the gene is introduced into a plant, and the obtained transgenic plant is earlier than wild fruit maturation.

Background

Citrus is a evergreen tree (except for hovenia dulcis) in the Rutaceae (Rutaceae) citrus subfamily (Aurantioidease), and has 33 genera and more than 200 species, and is one of the most important fruit crops in the world. The citrus 90% of China is used for fresh food, the mature period of the citrus in China is mainly concentrated in 11 months to 2 months next year, the variety of the super-early-maturing and late-maturing high-quality citrus is still relatively less, the variety of the citrus is still quite different from the annual balanced supply of fresh fruits, the unbalanced market supply still cannot meet the requirements of consumers on the fresh fruits, particularly in the period of the citrus concentrated marketing, the difficult fruit selling is still happened, the structural yield of the market is easy to be excessive, and the planting benefit of the citrus is greatly influenced. Therefore, the method analyzes the mechanism of citrus fruit maturation regulation and digs the maturation period regulation genes, and has great significance for promoting the breeding of new varieties of citrus in different maturation periods, promoting the structural perfection of the maturation period of the citrus industry and the like.

Fruit ripening is a complex process that is mainly manifested by changes in color, texture, aroma, sugar to acid ratio, hormonal levels, etc. Fruit ripening is a process commonly regulated by a number of factors, the core of which is the gene regulation network inside, in addition to environmental factors affecting fruit ripening such as temperature, light, moisture. With the development of sequencing technology and bioinformatics, a great deal of important crop genomes are continuously decrypted, and the research on the mechanism of transcription factors is gradually increased. Transcription factors serve as important gene expression control elements and play a vital role in controlling all factors of fruit ripening. In the fruit ripening process, various transcription factors are found to participate in the regulation and control process, and the expression quantity of different transcription factors in different development and ripening stages of the fruit is different, so that the regulation and control direction is also different. Studies have shown that CsMYB77 modulates abscisic acid (ABA) signaling by inhibiting the expression of downstream gene SINAT, while activating the expression of downstream gene PIN5, thereby modulating auxin signaling, thereby delaying fruit ripening (Zhang et al, 2023); crNAC036 and CrMYB synergistically inhibit ABA biosynthesis in citrus fruits by regulating expression of CrNCED5, thereby controlling fruit ripening (Zhu et al 2020); in studying the mechanism of papaya maturation, cpMYB and CpMYB2 were found to play a role in papaya fruit softening and carotenoid accumulation by regulating cell wall degradation and carotenoid biosynthesis-related genes (Fu et al 2020); in tomato, the promoter activity of transcription factor SlbHLH95 is regulated by RIN in vivo, inhibiting SlbHLH, which reduces the sensitivity of fruits to ethylene, reduces the accumulation of carotenoids, reduces glutathione content, and inhibits the expression of fruit maturation-related genes and glutathione metabolism-related genes (Zhang et al 2020).

The AP2/ERF family is one of the largest families of transcription factors in plants and plays an important role in the growth and maturation process of plants. The AP2/ERF transcription factor has a binding domain of about 60 amino acids that binds directly to the target gene promoter (Okamuro et al, 1997), and in Arabidopsis, the AP2/ERF family is primarily divided into four subfamilies: dehydration response element binding protein (DREB), ethylene response element binding protein (ERF), AP2, and RAV (Feng et al, 2005). All members of the AP2/ERF family contain at least one DNA binding domain, termed the AP2 domain (MAGNANI ET al, 2004). The AP2/ERF transcription factor plays an important role in fruit ripening, and DkERF and DkERF in persimmons promote the conversion of persimmon acid-soluble pectin into water-soluble pectin and the release of ethylene, promote the softening speed of fruits and promote the fruit ripening (He Yiheng 2020); in tomato SIERF, inhibition of carotenoid and ethylene synthesis, revealing the role of SIERF6 in fruit ripening (Lee et al 2012); in apples MdERF negatively affects ethylene biosynthesis and fruit ripening by inhibiting the transcription of MdACS (Li et al 2016).

Because the citrus has long juvenile period and the conventional breeding means has low efficiency, the improvement of the mature period of the citrus by utilizing the molecular technology becomes an important breeding means. Through analysis of the expression pattern of the Fengjie '72-1' navel orange AP2/ERF family, csAP2-16 genes are screened. On the basis, carrying out carrier construction and genetic transformation on CsAP-16, verifying the function of CsAP-16 in the fruit ripening process, and having important research significance and application value for analyzing the citrus fruit ripening regulation molecular mechanism and the development of the citrus industry.

Disclosure of Invention

The invention aims to provide a citrus fruit maturation control related gene CsAP-16, the CDS sequence of which is shown as SEQ ID NO.1, and primers for amplifying cDNA sequences are shown as SEQ ID NO.3 and 4. The CsAP-16 gene codes 429 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention also aims to provide an application of the related gene CsAP2-16 of citrus fruit maturation in regulating fruit maturation, the gene is transformed into tomato and kumquat by an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are proved to have the functions of regulating fruit maturation by phenotypic observation statistics and genetic expression analysis verification, wherein the average time of fruit color breaking of the strain of the tomato of the CsAP2-16 gene is 28 days and 15 days earlier than that of wild type, and the strain of the kumquat is obviously advanced compared with that of the strain of the tomato of the T2 generation, and the CsAP2-16 gene identified by the invention has the function of regulating fruit maturation.

Compared with the prior art, the invention has the following technical effects and advantages:

according to the invention, the gene cloning technology is utilized to separate and clone the citrus fruit ripening gene CsAP-16 from the vone navel orange, the gene is transformed into tomatoes and kumquats by an agrobacterium-mediated genetic transformation method, the gene is identified to have a function of obviously promoting fruit ripening, and excellent gene resources are provided for molecular design and breeding in the citrus ripening period.

Drawings

FIG. 1 is a schematic flow chart of cloning, isolation and functional verification of citrus fruit development mature genes CsAP-16 in the present invention.

FIG. 2 shows the subcellular localization of citrus fruit maturation genes CsAP-16 according to example 2 of the present invention.

FIG. 3 shows the results of the identification of CsAP-16 gene transcriptional activation activity in example 3 of the present invention.

FIG. 4 is a diagram showing the genetic transformation of CsAP-16 genes into Mic-Tom tomato and regeneration process in example 4 of the present invention.

FIG. 5 is a DNA positive identification gel chart of tomato transgenic positive seedlings in example 4 of the present invention.

FIG. 6 shows the result of the quantitative detection of CsAP-16 genes of tomato transgenic positive seedlings in example 4 of the present invention.

FIG. 7 is a graph showing the statistical results of color change and days of fruit breaking of tomato fruits over-expressing CsAP-16 genes in example 4 of the present invention.

FIG. 8 shows the expression level of CsAP gene in kumquat transformed fruit in example 5 of the present invention. PK7 represents pK7WG2D vector transformed kumquat, TRV represents pTRV2 vector transformed kumquat, PK7-CsAP2-16 represents CsAP-16 gene super-surface strain, TRV-CsAP2-16 represents CsAP2-16 gene silencing strain (the same applies hereinafter).

FIG. 9 shows the phenotype observation of CsAP-16 gene after transiently transforming kumquat in example 5 of the present invention, and the contents of carotenoid and chlorophyll in fruits of transiently transformed kumquat and control group.

Detailed Description

The technical scheme of the invention is further explained in detail below by combining with the embodiment: the present embodiment is carried out on the basis of the technical scheme of the present invention, and from the following description and embodiments, one skilled in the art can determine the essential features of the present invention and make various changes and modifications of the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention.

Example 1: cloning of full-length cDNA of citrus fruit development mature Gene CsAP-16

This example obtained its CDS sequence in the orange genome database (http:// citrus. Hzau. Edu. Cn/orange /), and designed primers in the 5 'non-coding region and 3' non-coding region of the sequence, with forward primers: csAP2-16F1:5'-AAAAAGCAGGCTCCATGATGGCGTCTTCTTCATCG-3', reverse primer: csAP2-16-R1:5'-AGAAAGCTGGGTTTCACTCCTCTGGCCGAAAG-3'. Then, using wild type vone navel orange cDNA as a template, and using high-fidelity enzyme to carry out amplification reaction, wherein the kit is Phanta Max Super-Fidelity DNAPolymerase.

Taking wild vone navel orange leaves frozen at-80 ℃, extracting RNA by using a Reuzan RNA reagent FastPure Plant Total RNA Isolation kit, wherein the specific RNA extraction method comprises the following steps:

1) Taking a proper amount of plant tissue ground by liquid nitrogen, immediately adding 600 μl Buffer EL or 600 μl Buffer PSL, vigorously vortex oscillating for 30sec, fully and uniformly mixing the sample with the lysate, centrifuging at 12,000rpm (13,400×g) for 5min, and immediately performing subsequent operation;

2) Taking about 500. Mu.l to FastPure gDNA-Filter Columns III of the supernatant (FastPure gDNA-Filter Columns III in the collection tube), centrifuging at 12,000rpm (13,400 Xg) for 30sec, discarding FastPure gDNA-Filter Columns III, and collecting the filtrate;

3) Adding 0.5 times of absolute ethyl alcohol (about 250 μl, adjusted according to the actual condition of the supernatant) with the filtrate volume into a collecting pipe, shaking and mixing for 15sec;

4) The above mixture was transferred to FastPure RNA Columns V (FastPure RNA Columns V was placed in a collection tube), centrifuged at 12,000rpm (13,400×g) for 30sec, and the filtrate was discarded;

5) 700 μl Buffer RWA was added to FastPure RNA Columns V, centrifuged at 12,000rpm (13,400Xg) for 30sec, and the filtrate was discarded;

6) To FastPure RNA Columns V was added 500. Mu.l Buffer RWB (please check if 48ml absolute ethanol had been added before use), centrifuged at 12,000rpm (13,400 Xg) for 30sec, and the filtrate was discarded;

7) Repeating the step 6;

8) FastPure RNA Columns V was returned to the collection tube and centrifuged at 12,000rpm (13,400 Xg) for 2min;

9) FastPure RNA Columns V is transferred into a new RNase-free Collection Tubes 1.5.5 ml centrifuge tube, 30-100 μl of RNase-free ddH ₂ O is suspended and added into the center of the adsorption column membrane, and the mixture is centrifuged at 12,000rpm (13,400 Xg) for 1min;

10 The extracted RNA can be directly used for downstream experiments or stored at-85 to-65 ℃.

And (3) reverse transcription is carried out to synthesize cDNA by taking the vone navel orange RNA as a template. cDNA synthesis RNA reverse transcription was performed using reverse transcription kit Hiscript III RT SuperMix for qPCR. The specific operation steps of the synthesis method are carried out according to the specification. The resulting cDNA was used for PCR amplification of CsAP-16 genes. The PCR amplification was carried out using CsAP-16-F1 and CsAP-16-R1 designed as described above as primers. The detailed steps of PCR amplification are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min,35 cycles, extension at 72℃for 5min after the cycle is completed. After amplification, a single-band PCR product is generated, after 1% agarose gel electrophoresis, the gel product obtained by amplification is purified and recovered by adopting an Omega gel recovery kit, and the purified product is connected with an entrance carrier pDOR, wherein the total volume of a reaction system is 10 mu l. E.coli competent cells DH 5. Alpha. Were transformed after 5min incubation at room temperature. Then, PCR positive identification of bacterial liquid is carried out by using a target gene sequence primer (the primer refers to CsAP-16-F1 and CsAP-16-R1) and then the bacterial liquid is sent to a company for sequencing, finally, monoclonal bacterial liquid with correct sequence is selected according to the sequencing result, plasmids are extracted by adopting a well-known century CWO500M, then the plasmids are connected with a final vector pK7WG2D, and the connection product is transformed into agrobacterium GV3101. The plasmid contains CDS sequence of gene CsAP-16, its nucleotide sequence is shown as SEQ ID NO.1, and its amino acid sequence of coded protein is shown as SEQ ID NO. 2.

The CDS sequence length of CsAP gene 2-16 is 1290bp, and it includes coding reading frame capable of coding 429 amino acids and isoelectric point is 9.07. After MEGAX analysis (https:// www.megasoftware.net) of the sequence and the evolution relationship of the AP2/ERF family in Arabidopsis, it was found to belong to the AP2 subfamily, which was named CsAP2-16 according to the order of arrangement on the chromosome in the CsAP subfamily.

Example 2: csAP2-16 Gene subcellular localization

Constructing CsAP-16 gene positioning vector by using PRI101-GFP vector, designing primer according to gene sequence, respectively adding SaII/KPnI enzyme cutting site in forward and reverse direction according to multiple cloning site of PRI101-GFP vector, fusing CDS with stop codon removed to PRI101-GFP vector, and constructing fusion protein CsAP-16: GFP, the recombinant plasmid bacterial liquid was subjected to PCR identification. The recombinant plasmid and the empty plasmid are respectively transferred into an agrobacterium strain GV3101, and then transferred into lower epidermal cells of Nicotiana benthamiana for transient expression.

The infection of tobacco epidermis by agrobacterium is performed as follows:

1) Activation of bacterial cells: taking agrobacterium GV3101 bacterial liquid transferred into recombinant plasmid and empty vector PRI101-GFP plasmid stored at-80 ℃, streaking (containing 25mg/L Kana) in LB, culturing at 28 ℃ for 2d, and activating thalli;

2) Small shaking of bacterial liquid: picking monoclone in 5ml LB liquid culture medium, shaking at 28deg.C and 220r/min for 24h;

3) And (3) large shaking of bacterial liquid: on the day of the experiment, 300 μl of the small-shaking bacterial liquid is added into a conical flask containing 30ml of fresh liquid LB, and the bacterial liquid OD ₆₀₀ is 0.7 after shaking for about 10 hours at 28 ℃ and 220 r/min;

4) And (3) thallus collection: loading the large-shaking bacterial liquid into a 50ml sterile centrifuge tube, centrifuging at 4000r/min for 5min, removing the liquid, and completely draining;

5) And (3) cleaning thalli: adding 10ml of cleaning solution (10mM MES,10mM MgCl ₂, in-situ preparation) into thallus, suspending thoroughly, centrifuging at 4000r/min for 5min, adding 5ml of cleaning solution, cleaning again, and dissolving in 3ml of cleaning solution;

6) Determination of OD ₆₀₀ value of suspension: the suspension was diluted 20 times (example: 150. Mu.l of suspension+2850. Mu.l of washing liquid), and the bacterial solution was allowed to stand: the P19 final OD ₆₀₀ ratio was 0.7:0.5, adding 8ml of cleaning solution, adding 8 μl of acetosyringone (50 mg/ml), mixing well, and keeping the temperature in a 30 ℃ incubator for 3 hours;

7) Tobacco leaf injection: 3 tobacco plants with consistent growth vigor and no disease are selected for injection, 2 leaves are injected into each plant, and the back of each leaf is selected during injection (the back of each leaf is more in pores and is easier to inject bacterial liquid);

8) Culturing and observing: fluorescence observations were performed 24h later with laser confocal.

The results are shown in FIG. 2 as fluorescence detection results, csAP-16: fluorescence of the fusion protein after GFP recombination occurs only in the nucleus, whereas empty fluorescence is distributed throughout the whole cell tissue. The results indicate that CsAP-16 gene is located on the nucleus and is a nuclear localization protein.

Example 3: csAP2-16 Gene transcriptional activation assay

Transcriptional activation activity is a fundamental feature of transcription factors, and here we used pGBKT7 vectors for recombinant construction to verify CsAP-16 whether it has transcriptional activation activity. Designing a specific primer according to a gene sequence, respectively adding EcoRI/SmaI restriction sites on forward and reverse primers according to pGBKT7 vector sequence information, amplifying a full-length CDS region of the gene, and connecting the amplified region to pGBKT7 vector to construct a recombinant plasmid, thereby obtaining a fusion expression vector pGBKT7-CsAP2-16. After confirming the sequence by sequencing, the fusion expression vector and the empty vector (pGBKT 7) were transferred into the same yeast strain AH109 (purchased from Kangji). Finally, bacterial solutions which are positive in identification are respectively and uniformly coated on 4 incomplete solid culture mediums of SD/-Trp, SD/-Trp-His-Ade and SD/-Trp-His-Ade-Leu, and the bacterial solutions are respectively cultured on different deletion culture mediums to detect the survival condition of the transformant. The results showed that the empty vector transformed yeast cells could only grow on deletion medium SD/-Trp, and that none of the yeasts transformed with recombinant plasmids pGBKT7-CsAP2-16 could grow on incomplete medium (FIG. 3), indicating that transcription factor CsAP-16 could not bind to GAL4-BD, could not activate transcription of downstream reporter gene, and that the yeasts could not grow normally on incomplete medium, i.e. CsAP2-16 had no transcription activation function.

Example 4: overexpression of genes CsAP-16 in tomato

1. Construction of plant transformation super-expression vector

Constructing a binary expression vector by using a Gateway system, and taking wild vone navel orange cDNA as a template, wherein the primer is designed as follows:

F1:5’-AAAAAGCAGGCTCCATGATGGCGTCTTCTTCATCG-3’

R1:5’-AGAAAGCTGGGTTTCACTCCTCTGGCCGAAAG-3’。

The cDNA obtained by reverse transcription synthesis is used for PCR amplification of CsAP-16 genes, a single-band PCR product is generated after amplification is finished, after 1% agarose gel electrophoresis, the gel product obtained by amplification is purified and recovered by adopting an Omega gel recovery kit, the product and an entry carrier pDOR221 are subjected to BP reaction, and DH5 alpha escherichia coli is transformed. Selecting a monoclonal on the next day for positive identification, selecting a positive monoclonal in LB liquid medium containing kana (Kan) antibiotic resistance, shaking, detecting the positive clone, and carrying out sample feeding sequencing. After the result to be sequenced is correct, the ligation product is transformed into an escherichia coli competent cell DH5 alpha, and plasmids are extracted from positive strains by adopting a CWO500M vector of century, so that the construction of the overexpression vector pK7WG2D-CsAP2-16 is completed.

2. Genetic transformation of tomato

In scientific research, tomatoes are used as a better material for verifying gene functions according to the characteristics of short growth cycle, easy transformation and the like, mic-Tom is a tomato dwarf plant, compared with common tomatoes, the tomato dwarf plant is dwarf and easy to plant and manage in high density, and agrobacterium-mediated tomato genetic transformation comprises the following steps:

2.1 inoculation

1) Screening full seeds, and soaking the seeds in tap water for 1-2 hours;

2) Sterilizing seeds with 75% ethanol for 1min (stirring continuously);

3) Oscillating 50% 84 disinfectant for 15min;

4) Cleaning with sterile water for 3 times, sucking the surface water of the seeds with sterile filter paper, and uniformly sowing on the S1 culture medium;

5) The culture room with 16h illumination/8 h darkness is cultured for about one week.

2.2 Cotyledon cutting and Agrobacterium infection

1) Activating and propagating strains: streaking agrobacterium tumefaciens bacteria liquid containing an overexpression vector pK7WG2D-CsAP2-16 on LB (SPE resistance) solid culture medium, and culturing at 28 ℃ for 48 hours; spreading the activated strain on a new LB (SPE resistance) solid culture medium again, and performing propagation culture at 28 ℃ for 24 hours;

2) Sowing for about 6-10 days, growing two cotyledons of tomato, cutting off the leaf tips and the junction of the leaf blade and the leaf stalk by using a sterile scalpel, dividing the middle section into two parts (about 0.4cm long), picking the back surface of the middle section, placing the middle section in an S2 co-culture medium, and culturing in dark at 25+/-2 ℃ for 24 hours;

3) Scraping the propagated agrobacterium with a sterile blade, re-suspending in 50ml agrobacterium suspension, shake culturing at 28 ℃ for about 30min until OD ₆₀₀ is 0.2-0.6, and adding 50mg/L AS (acetosyringone) for later use;

4) Taking a sterile empty triangular flask, putting cotyledons cultured by a pre-culture medium into the sterile empty triangular flask, pouring a prepared agrobacterium suspension, and carrying out shaking infection for 5min;

5) The suspension was poured off, the cotyledon surface moisture was blotted with sterile filter paper and placed again in S2 co-culture medium (leaf back side facing upwards) and co-cultured in the dark at 25.+ -. 2 ℃ for 2d.

2.3 Screening culture

The leaves are transferred to an S3 screening culture medium, the cotyledons after 48h of preculture are inoculated on the S3 screening culture medium with the right side facing upwards, and the incision is fully contacted with the culture medium for illumination culture at 25+/-2 ℃.

2.4 Rooting culture

After culturing for about 7 days, white callus grows out from the edge of the tomato leaf, the tomato leaf is transferred into an S3 subculture medium after 12 days, the callus is differentiated to bud points after 10-14 days, and the tomato leaf is transferred into an S4 rooting medium (positive can be identified at the moment) after about 30 days of bud growth to root after about 7 days.

TABLE 1 culture medium for tomato seedlings

3. Screening and identification of tomato transgenic positive seedlings

The tomato plants with the CsAP-16 transgenic genes are obtained according to the method, the leaves of the plants are taken to extract genome DNA, forward and reverse primers are designed according to the 35S sequence and the gene sequence on the vector to amplify, and whether the exogenous target genes are inserted into the genome of the transformation material is verified.

3.1 Tomato leaf DNA extraction

1) Preparation of DNA buffer (1L): 100mL of 1M Tris-HCl (pH 8.0); 100mL of 0.5M EDTA (pH 8.0); 5.0M NaCl 300mL; h ₂ O500 mL.

1M Tris-HCl (pH 8.0): weighing 121g of Tris-Base, adding 800mL of water, stirring on a magnetic stirrer, adding concentrated hydrochloric acid to adjust the pH to 8.0 after the water is fully dissolved, adding water to 1000mL of water for constant volume, sterilizing, and preserving at normal temperature. 0.5M EDTA (pH 8.0): weighing 187g of EDTA-Na ₂ salt, adding about 800mL of water, adding solid NaOH while stirring on a magnetic stirrer, when the EDTA and the NaOH are completely dissolved and the solution becomes clear, adjusting the pH to about 8.0, slightly adjusting by using pH test paper, sterilizing and preserving at room temperature. 5M NaCl: weighing 300g of NaCl, adding about 800mL of distilled water, fully stirring on a magnetic stirrer, stopping stirring after about 5min, standing for 2-3min, pouring out supernatant, adding 100mL of distilled water, repeating the previous steps until the NaCl is fully dissolved, fixing the volume to 1000mL, sterilizing, and preserving at room temperature.

Phenol: chloroform: the preparation of isoamyl alcohol (25:24:1) comprises mixing water saturated phenol, chloroform and isoamyl alcohol according to the volume ratio of 25:24:1, and storing in brown bottle; preparing 70% absolute ethyl alcohol: mixing absolute ethanol and water according to the volume ratio of 7:3 for standby.

The specific extraction steps are as follows:

1) Weighing (0.64N multiplied by 1%) g PVP, (0.64N multiplied by 2%) g CTAB and 0.64N mL DNA buffer to prepare 0.64N mL CTAB buffer solution, and dissolving in a 10mL centrifuge tube at 65 ℃ in water bath (N refers to the number of samples);

2) Weighing about 0.1g to 1.5mL of the sample into a centrifuge tube, adding liquid nitrogen into the centrifuge tube, and grinding the mixture;

3) Adding 100 mu L of beta-mercaptoethanol (1% -4%) into the CTAB buffer solution;

4) 640 mu L of the CTAB buffer mixed solution is added into each sample, and the mixture is oscillated up and down (or put on a vortex oscillator for uniform mixing);

5) Water bath at 65 ℃ for 60-90min;

6) 700. Mu.L of phenol was added: chloroform: isoamyl alcohol (25:24:1), oscillating up and down for about 5min, and centrifuging at 13000rpm/min and 20 ℃ for 8min;

7) Absorbing 500 mu L of supernatant (yellow gun head absorbing), adding 60 mu L of 5M NaCl and 1mL of precooled absolute ethyl alcohol, mixing up and down, and carrying out ice water bath at-20 ℃ for 30min;

8) 13000rpm/min, 4 ℃, centrifugation for 6min;

9) Removing supernatant, adding 1mL of 70% absolute ethanol, and standing at-20 ℃ for 2h;

10 10000rpm/min, 4 ℃, centrifuging for 5min;

11 Discarding the supernatant, and blow-drying (not too dry) the supernatant by a super clean bench;

12 100. Mu.L TE, 1.5. Mu.L 10. Mu.M RNase enzyme (super clean bench operation) was added to each centrifuge tube;

13 37 ℃ water bath overnight.

3.2DNA Positive identification

Positive plants were identified using specific primers 35S and gene reverse primers. Among the selected transgenic lines, if any, the transgenic lines were able to amplify fragments of the expected size, indicating that they were positive transgenic lines, 8 positive plants were finally verified (fig. 5).

35S：5’-GACGCACAATCCCACTAT-3’

CsAP2-16-R1：5’-AGAAAGCTGGGTTTCACTCCTCTGGCCGAAAG-3’。

4. Overexpression analysis of tomato transgenic positive seedlings

RNA of the transplanted positive transgenic seedlings (sequentially designated as #1, #2 and … # 8) was extracted and reverse transcribed to cDNA (RNA extraction method was the same as in example 1), and the cDNA obtained by reverse transcription was diluted 5-fold with ddH ₂ O as a template, and quantitative primers were designed at a wire mesh station.

CsAP2-16 quantitative primers are:

CsAP2-16-qPCR-F：5’-TGAAGCTCACCTTTGGGATAAA -3’

CsAP2-16-qPCR-R：5’-GGCATCTGGTATGGTTCAGTAG-3’。

tomato action gene is used as reference gene, and the primer is:

SlActin-F：5’-GTCCTCTTCCAGCCATCCAT-3’

SlActin-R：5’-ACCACTGAGCACAATGTTACCG-3’

the expression level of the CsAP2-16 gene in positive transgenic tomatoes can be judged to be higher by identifying the expression level of the gene CsAP-16 by qRT-PCR (quantitative reverse transcription-polymerase chain reaction) (FIG. 6).

5. Fruit phenotype observation of tomato transgenic positive seedlings

Fruit peel discoloration means the onset of ripening, and is often used to determine the onset of fruit ripening. Phenotypic observations were performed on Wild Type (WT), and overexpressing CsAP-16 tomatoes, and the time required for the material to begin coloring from flowers to fruits, i.e. days of color break, was statistically analyzed. The results show that the fruits of CsAP-16 overexpressing lines had earlier broken color than the wild-type (WT), and that the fruits were shown in FIG. 7, with wild-type fruits starting to be colored around 43DAF (DAYS AFTER flowering) and fruits of CsAP-16 overexpressing lines starting to be colored around 28DAF, respectively. The results showed that tomato fruits overexpressing CsAP-16 gene had a color break day which was about 15 days earlier than the wild-type (WT) (fig. 7).

Example 5: overexpression and silencing of the expression CsAP gene CsAP-16 in kumquat

Vigs silencing vector construction

The vectors used are pTRV1 and pTRV2, ecoRI and SmaI enzymes are selected as forward and reverse enzyme digestion sites according to the characteristics of the pTRV2 vector, a 200bp sequence before CDS of CsAP-16 genes is used as a template to design primers, and wild type vone navel orange cDNA is used as a template, wherein the design primers are as follows:

F2：5’-GTGAGTAAGGTTACCGAATTCATGATGGCGTCTTCTTCATCG-3’

R2：5’-TGCTCGACGACAAGACCCGGGCAAGGAGGCATCCTACTGATG-3’

After PCR amplification is finished, the gel product obtained by amplification is purified and recovered by adopting an Omega gel recovery kit, then the linearized pTRV2 vector subjected to double enzyme digestion and the gel recovery product are subjected to homologous recombination, escherichia coli DH5 alpha is transformed after homologous recombination, then the sequence is sent to a company for sequencing, bacterial liquid plasmids with correct sequencing are extracted, a successfully constructed silencing vector is named pTRV2-CsAP2-16, finally agrobacterium GV3101 is transformed, and bacterial liquid with correct positive verification is placed in glycerol for storage at the temperature of minus 80 ℃.

2. Instant transformation of kumquat

The agrobacterium solution containing the overexpression vector pK7WG2D-CsAP2-16 and the empty load pK7WG2D is activated for 2D in LB (SPE) solid culture medium, the agrobacterium solution containing the silencing vector pTRV2-CsAP2-16 and the empty load pTRV2 is activated for 2D in LB (Kan) solid culture medium, and then secondary activation is carried out for 1D for subsequent genetic transformation. The agrobacterium-mediated instant kumquat transformation comprises the following detailed steps:

1) For fruit injection, after suspension Buffer washing is carried out once, the target bacterial liquid is mixed with the P19 auxiliary plasmid bacterial liquid, and P19 belongs to a gene silencing inhibitor, so that gene silencing after transcription of transgenic citrus fruits can be prevented, high-level expression of target proteins is promoted, and the mixed bacterial liquid OD ₆₀₀ =0.8.

2) A portion of the syringe needle was cut off, leaving a length of about 0.1-0.3cm, and 0.4mL of the infesting solution was injected into each fruit. The injection process needs to be slowly and uniformly advanced, and the part where the infectious microbe liquid reaches the subcutaneous layer of the fruit can be seen visually, and the part where the microbe liquid is injected is marked by marking strokes so as to be convenient for subsequent sampling. Excess agrobacteria were blotted off and the injected fruits were stored at room temperature for 2 days and then bagged. After 5-15 d infection, the kit is used for detecting CsAP-16 gene expression quantity and chlorophyll and carotenoid contents.

Buffer formulation (10 ml): glucose (0.05 g), MES (500 mM,1 ml), na ₃PO₄ (20 mM,1 ml), acetosyringone (1M, 1 μl) and H ₂ O (make up to 10 ml). 1M Acetosyringone (AS): 0.0392g AS was dissolved in 0.2mL DMSO and stored in-20℃aliquots at 10. Mu.L; 20mM Na ₃PO₄: 0.17g of anhydrous Na ₃PO₄ is dissolved in 50mL of water and stored at 4 ℃;500mM MES:4.88g MES was dissolved in 50mL water and stored at 4 ℃.

3. The qRT-PCR method is used for identifying the expression level of the gene CsAP-16 in kumquat transformed fruits (figure 8), the expression level in the super-surface kumquat fruits is obviously higher than that of PK7 control, and the expression level in the silencing fruits is obviously lower than that of TRV empty load, so that the successful transient transformation of the gene CsAP-16 into the fruits can be obtained.

4. Transgenic kumquat fruit phenotype observation

The color changes of fruits of the empty control group, the CsAP2-16 gene overexpression strain and the silencing strain after the injection is counted on the same day and five days, and the result shows that the color breaking speed of fruits of the overexpression strain is faster than that of fruits of the wild type, and the color breaking speed of fruits of the silencing strain is slower than that of fruits of the wild type. After the carotenoid and chlorophyll contents are counted, the carotenoid of the silent strain is found to be significantly lower than empty load, and the chlorophyll is opposite; the carotenoids of the supersurface lines were significantly higher than the wild type, with chlorophyll also reversed (fig. 9).

Main reference

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Claims (9)

1. The citrus transcription factor CsAP-16 gene is characterized in that the CDS sequence of the gene is shown as SEQ ID NO. 1.

2. The protein encoded by citrus transcription factor CsAP-16 according to claim 1, wherein the amino acid sequence is shown in SEQ ID No. 2.

3. A recombinant expression vector comprising the CsAP gene of claim 1.

4. A recombinant expression vector according to claim 3, wherein: a Gateway system was used to construct a binary expression vector comprising an entry vector pDONR221 and an overexpression final vector pK7WG2D.

5. A host bacterium comprising the CsAP gene according to claim 1.

6. A primer for amplifying the CsAP gene as defined in claim 1, wherein the primer sequences are shown in SEQ ID NO.3 and 4.

7. Use of the citrus transcription factor CsAP-16 gene of claim 1 or the encoded protein of claim 2 or the recombinant expression vector of claim 3 or the host bacterium of claim 5 for regulating fruit ripening.

8. Use according to claim 7, comprising citrus or tomato fruits.

9. A method of promoting ripening in citrus or tomato fruits, wherein the CsAP gene of claim 1 is overexpressed in citrus or tomato.