Strawberry stress resistance and fruit regulation gene facol1 and application thereof

Abstract

The application provides a strawberry stress resistance and fruit quality regulation gene FaCOL1 and application thereof, and belongs to the technical field of plant genetic engineering, wherein the DNA sequence of the gene FaCOL1 is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein of the gene FaCOL1 is shown as SEQ ID NO.2. The application determines the expression level of the FaCOL1 gene in each tissue part of a strawberry "Hongyan" variety, simultaneously carries out low-temperature treatment and dark treatment on the strawberry plant, and determines the gene expression amount of the FaCOL1. The FaCOL1 gene is obtained through gene cloning, a transient silencing vector is constructed, the anthocyanin content, the soluble sugar component and the content change are determined, and it is proved that the FaCOL1 gene has important application value in enhancing the stress resistance of the strawberry and regulating the fruit quality.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically a strawberry stress resistance and fruit quality regulation gene FaCOL1 and its application. Background Technology

[0002] Strawberries are perennial herbaceous plants belonging to the genus *Fragaria* in the family Rosaceae, but are now mostly cultivated as annuals or biennials. The color of strawberry fruit is influenced not only by the variety but also by the combined effects of environmental factors such as temperature, light, and water. Under different light conditions, the color of strawberry fruit exhibits significant differences. For example, in strong light, strawberry fruit is lighter in color and unevenly colored; while under weak light conditions, the content of soluble sugars and soluble solids in the fruit is significantly reduced. Low temperatures slow down strawberry growth and affect flower bud differentiation. Therefore, both low temperature and weak light environments have a certain inhibitory effect on the growth process of strawberries.

[0003] CO (constans) is an important regulator of flowering in plants through the photoperiod pathway. It contains two conserved domains: a B-box domain at the n-terminus and a CCT domain at the c-terminus. In many species, the CONSTANS-like (COL) family is defined by the conserved B-box and CCT domains. Most COL genes promote or inhibit flowering under long-day (LD) or short-day (SD) conditions. For example, in Arabidopsis thaliana, overexpression of the AtCO, AtCOL5, and AtCOL16 genes promotes flowering under LD or SD conditions; in rice, the OsHd1 gene delays flowering under LD conditions and promotes flowering under SD conditions. Some COL genes also have functions related to abiotic stress and plant growth and development. The AtCOL4 transcription factor can enhance plant tolerance to abiotic stress. VvCO and VvCOL1 regulate flowering induction and dormancy in grape buds and the overexpression of CO in tendrils, which may be related to tendril development.

[0004] Currently, no COL gene has been clearly identified in strawberries to regulate fruit quality formation. Research on COL function is mainly limited to flowering regulation in model plants such as rice and Arabidopsis thaliana. However, these plants do not have typical fleshy fruits, nor do they have a post-flowering fruit development and maturation process. Therefore, the role of COL in these plants is necessarily quite different from that in strawberries. Cloning and utilizing the strawberry stress resistance and fruit quality regulation gene COL1 will help elucidate the quality formation process of strawberry fruits after flowering and fruit drop under stress conditions. This will provide molecular tools for strawberry genetic engineering breeding and has potential economic value, such as improving the resistance of strawberries to low temperature and weak light and cultivating high-quality fruits with good color, taste, and sweetness. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a stress-resistance and fruit quality regulation gene, FaCOL1, and its applications to meet usage requirements.

[0006] This invention provides a strawberry stress resistance and fruit quality regulation gene FaCOL1. The nucleotide sequence of gene FaCOL1 is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein of gene FaCOL1 is shown in SEQ ID NO.2.

[0007] This invention provides a silencing vector containing the strawberry stress resistance and fruit quality regulation gene FaCOL1 as described in claim 1, and a method for its application. The silencing vector is used to suppress the expression of the strawberry gene FaCOL1.

[0008] The steps for constructing the silent carrier and instantaneously transforming strawberries are as follows:

[0009] Step (1): Using total RNA from strawberry leaves as a template, reverse transcription was performed. The reverse transcription product was subjected to PCR reaction. The PCR product was ligated with the blunt-zero cloning vector. The ligation product was added to competent E. coli cells for E. coli transformation. After sequencing, E. coli culture containing the FaCOL1 gene was obtained.

[0010] In the PCR reaction, the upstream primer sequence of the FaCOL1 ORF is shown in SEQ ID NO.3; the upstream primer sequence of the ORF is shown in SEQ ID NO.4;

[0011] Step (2): Extract plasmid from E. coli containing the FaCOL1 gene, ligate it into the TRV vector, mix TRAVA1 and TRAV2 and use it as an empty vector to transform the plasmid into Agrobacterium competent cells GV3101 for culture and propagation, and obtain the FaCOL1 gene silencing vector.

[0012] Step (3): After collecting Agrobacterium, resuspend it in the infection solution;

[0013] Step (4): Inject the inoculum into the strawberry ginkgo seeds.

[0014] Beneficial Effects: This invention used the "Hongyan" strawberry variety to determine the expression level of the FaCOL1 gene in various tissues. Simultaneously, the strawberry plants were subjected to low-temperature treatment to determine the FaCOL1 gene expression level. The FaCOL1 gene was obtained through gene cloning, and a transient silencing vector was constructed. Changes in anthocyanin content and the composition and content of soluble sugars were measured, demonstrating its important application value in enhancing strawberry stress resistance and regulating fruit quality.

[0015] The silencing vector inhibits the expression of the FaCOL1 gene, reducing anthocyanin and soluble sugar content. This invention, through VIGS experiments, demonstrates that this gene has a low-temperature resistance effect and also plays a significant role in increasing anthocyanin and soluble sugar content. In agricultural production, utilizing this gene, along with gene editing technology or marker-assisted breeding, can cultivate new varieties that are cold-resistant, have bright fruit color, and sweet flavor. By improving the fruit's low-temperature resistance, it can be adapted for high-latitude or winter-growing regions, thereby expanding the planting area and increasing yield. Simultaneously, it can also be used to enhance the fruit's low-temperature tolerance during post-harvest cold chain transportation, extending its storage time. Attached Figure Description

[0016] Figure 1 This represents the expression level of the FaCOL1 gene in various tissues of strawberries.

[0017] Figure 2 This represents the gene expression level of FaCOL1 in strawberries after low-temperature treatment.

[0018] Figure 3 It includes the phenotype of strawberries after silencing, the content of anthocyanins, and the content of each component of soluble sugars. Detailed Implementation

[0019] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0020] Example 1: Cloning the FaCOL1 gene

[0021] In this embodiment, the FaCOL1 gene was identified and cloned through transcript homology comparison.

[0022] Based on the conserved region of the plant COL gene, the strawberry transcript was identified by homologous comparison of strawberry transcripts. The strawberry FaCOL1 transcript contains a complete open reading frame (ORF). Using total RNA from strawberry leaves as templates, the ORF sequence (cDNA sequence) of FaCOL1 was cloned and cloned into the blunt-zero vector. After positive screening of the inserted fragment, sequencing was performed. The sequencing results were confirmed to be the complete FaCOL1 gene by comparison with the strawberry gene sequence.

[0023] I. Primer Design

[0024] The ORF primer pair for FaCOL1 is:

[0025] ORF-F (SEQ ID NO.3): 5'ATGTTGAAAGAGGAGAGCAAT3';

[0026] ORF-R (SEQ ID NO. 4): 5'CTAGTACGAAGGAACAATGC3'.

[0027] II. ORF sequence cloning, the specific steps are as follows:

[0028] (1) Reverse transcription

[0029] The reverse transcription reaction system consisted of: 1 μg Total RNA, 1 μL dNTP Mix, 1 μL Oligo dT Primer, 4 μL 10X RT Buffer, 0.5 μL RNase Inhibitor, 1 μL M-MLV Reverse Transcriptase, and Nuclease-free Water to a final volume of 20 μL. The reaction program was: 42℃ for 60 min, followed by 70℃ for 15 min to obtain the cDNA template.

[0030] (2) PCR

[0031] The PCR reaction system consisted of: 25 μL of 2×Keypo Master Mix, 2 μL of ORF-F, 2 μL of ORF-R, 1 μL of cDNA, and Nuclease-free ddH2O to a final volume of 50 μL. The reaction program was 98℃, 60℃, 72℃, for 35 cycles to obtain the PCR product.

[0032] (3) Linked cloning vector

[0033] Ligation was performed with the Blunt-Zero cloning vector. The ligation reaction mixture (5 μL) consisted of 4 μL of the recovered PCR product and 1 μL of Blunt-Zero. The reaction conditions were 25 °C for 5 min to obtain the ligation product.

[0034] (4) Escherichia coli transformation

[0035] Take 5 μL of the ligation product and add it to 100 μL of competent E. coli cells. Mix gently, incubate on ice for about 10 min, incubate in a 42°C water bath for 60 sec, and then quickly place on ice for 3-5 min. Add 800 μL of LB liquid medium and incubate at 37°C and 100 rpm for 1 h. Centrifuge at 4000 rpm for 3 min, remove the upper 600 μL of medium, mix the remaining bacterial solution, and spread it evenly on an LB selection plate containing Cb resistance. Incubate upside down at 37°C overnight.

[0036] (5) Positive clone screening and sequencing

[0037] Single colonies were selected from overnight culture plates and inoculated into LB liquid medium, and cultured overnight at 37°C and 250 rpm to obtain bacterial suspension. The bacterial suspension was used as a template for PCR detection of recombinant transformants. The PCR reaction system was: 12.5 μL 2×Rapid Taq MasterMix, 11 μL Primer, 21 μL Primer, 8.5 μL dd H2O, and 2 μL bacterial suspension. The reaction program was: 95°C for 3 min; 95°C for 15 sec, 60°C for 15 sec, 72°C for 10 sc, 35 cycles; 72°C for 5 min. Electrophoresis was used for positive detection. Positive bacterial suspensions were sent to Sangon Biotech (Shanghai) for sequencing.

[0038] FaCOL1 gene DNA sequence (SEQ ID NO.1):

[0039]

[0040] FaCOL1 gene protein amino acid sequence (SEQ ID NO.2)

[0041] MLKEESNGAAAANSWARVCDTCRSAPCTVYCRADSAYLCSGCDATIHAANRVASRHERVSVCEACERAPAAFLCKADAASLCTACDADIHSANPLARRHQRVPILPISGCQIMVGSTPADTTEDGFLSQEGDEEVMDEEDEDEAASWLLLNPVKNSNNHNSNNNNPNNNNNGFLFGVEVDEYLDLVEYNSSD QNQFSGTTATNDQHNYGVPHKISYGGDSVVPVQYGEGKVTQMQMQQKHNFHQLGMEYESSKAAYGYDGSISHTVSVSSMDVGVVPDSTMSEMSVCH PRTPKGTIDLFNGPTIQMPTQLSPMDREARVLRYREKKKTRKFEKTIRYASRKAYAETRPRIKGRFAKRTDIEVEVDQMFSTSLMGETGYGIVPSY

[0042] Example 2: Analysis of FacoL1 gene tissue expression characteristics under low temperature treatment

[0043] Strawberry plants were placed in a 4°C freezer, and samples were taken at 0h, 1h, 3h, 6h, 12h, and 24h. The relative expression levels of the target gene were detected using real-time quantitative PCR (qPCR). Strawberry plant leaves that had undergone low-temperature treatment were frozen in liquid nitrogen and then ground. The relative expression levels of the target gene were detected using qPCR, with the strawberry FaActin gene selected as the internal reference gene. RNA was extracted from the strawberry leaf samples and reverse transcribed. The cDNA stock solution was diluted 10-fold and used as a template for qPCR. Quantitative primer qFaCOL1 was designed based on the cloned gene sequence.

[0044] Fluorescent quantitative primers:

[0045] F(SEQ ID NO.5):AACAGCTGGGCACGTGTCTG

[0046] R(SEQ ID NO.6):CGTCGCAGCCGGAACATAGG

[0047] Expression levels were detected using the SYBR Green fluorescent dye method. A Bio-Rad CFX96 real-time PCR instrument was used. The reaction volume was 10 μL, including 5 μL of quantitative reaction mix and forward and reverse primers (10 μmol·L⁻¹). -1) Add 1 μL of each of the following: 1 μL of cDNA template and 2 μL of water to a final volume of 10 μL. The PCR reaction procedure is a three-step method; refer to the Bimake SYBR Green qPCRMasterMix instruction manual for specific procedures. Calculate the relative expression level using the 2-ΔΔCT method.

[0048] Figure 1 The expression levels of the FaCOL1 gene in various tissues of a normally growing strawberry plant.

[0049] Figure 2 The expression level of the FaCOL1 gene in strawberry leaves after low-temperature treatment is shown, where CK represents strawberry plants placed at room temperature for 24 hours, and low-temperature treatment refers to strawberry plants treated at 4℃ for 24 hours. Figure 2 As shown, FaCOL1 expression levels increased continuously in the first 12 hours with increasing treatment time. Therefore, it can be concluded that FaCOL1 has an anti-stress effect.

[0050] Example 3: Construction of FaCOL1 transient silencing vector and transient silencing via strawberry fruit injection

[0051] I. Construction of FaCOL1 transient silencing vector

[0052] Plasmids were extracted from the bacterial culture prepared above (the bacterial culture prepared in the E. coli transformation step of Example 1) and ligated into the TRV vector. A resuspension solution (10 mmol·L⁻¹) was prepared. -1 MES, 10 mmol·L -1 MgCl2, 250 μmol·L -1 TRV1 and TRV2 cells were resuspended separately with acetylsylcholine to an OD600 of 2, and then mixed in a 1:1 ratio to form an empty vector. The plasmid (containing FaCOL1) was transformed into Agrobacterium competent cells GV3101 for propagation. After collecting the cells, the infection solution was resuspended to an OD600 of 1. The resuspended cells were then used in a solution of 10 mmol / L... -1 MES, 10 mmol·L -1 MgCl2, 250 μmol·L -1 The OD600 of the bacterial suspension was adjusted to 2 using acetylsyleugenol, and then mixed with TRV1 Agrobacterium at a 1:1 ratio to obtain a resuspension of Agrobacterium containing FaCOL1. The bacterial suspension was drawn up with a 1ml syringe and injected into strawberries. The strawberries were then incubated in an incubator (20℃, 16h light) for 7 days, and photographs were taken for record-keeping. The results are shown below. Figure 3 As shown.

[0053] II. Tissue Expression Characteristics Analysis of FaCOL1 Gene

[0054] The strawberry fruits were frozen in liquid nitrogen and then ground. The relative expression levels of the target gene were detected using real-time quantitative PCR, with the strawberry FaActin gene selected as the internal reference gene. RNA was extracted and reverse transcribed from samples of various strawberry tissues, organs, and fruits at different developmental stages. The cDNA stock solution was diluted 10-fold and used as a template for real-time quantitative PCR.

[0055] Quantitative primer qFaCOL1 was designed based on the cloned gene sequence. Quantitative fluorescence primers:

[0056] F(SEQ ID NO.7):AACAGCTGGGCACGTGTCTG

[0057] R(SEQ ID NO.8):CGTCGCAGCCGGAACATAGG

[0058] Expression levels were detected using the SYBR Green fluorescent dye method. A Bio-Rad CFX96 real-time PCR instrument was used. The reaction volume was 10 μL, including 5 μL of quantitative reaction mix and forward and reverse primers (10 μmol·L⁻¹). -1) Add 1 μL of each of the following: 1 μL of cDNA template and 2 μL of water to a final volume of 10 μL. The PCR reaction procedure is a three-step method; refer to the Bimake SYBR Green qPCRMasterMix instruction manual for specific procedures. Calculate the relative expression level using the 2-ΔΔCT method.

[0059] III. VIGS determination of anthocyanins, soluble sugars, and acids in fruit;

[0060] Anthocyanins were determined using the Ph differential method; the components and content of soluble sugars were determined by HPLC using an Agilent 1290 Infinity II ultra-high performance liquid chromatograph. Figure 3 As shown, the anthocyanin, sucrose, glucose, and fructose contents in strawberry fruits treated with the silence treatment were all reduced. This indicates a positive correlation between FaCOL1 and the anthocyanin and soluble sugar contents in strawberries.

[0061] Figure 3 In this context, CK refers to strawberry fruit injected with a resuspension of a mixture of TRV1 and TRV2, and VIGS refers to strawberry fruit injected with a resuspension of Agrobacterium containing FaCOL1. (a) shows a photograph of the infected strawberry fruit; (b) shows the anthocyanin content measured 7 days after infection; (c) shows the sucrose content measured 7 days after infection; (d) shows the glucose content measured 7 days after infection; and (e) shows the fructose content measured 7 days after infection.

[0062] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A gene for stress resistance and regulation of fruit quality in strawberries FaCOL1 Its characteristics are, Gene FaC The nucleotide sequence of OL1 is shown in SEQ ID NO.1, gene. FaC The amino acid sequence of the OL1 expressed protein is shown in SEQ ID NO.2.

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