Strawberry stress resistance and fruit regulation gene FaCOL1 and application thereof

Abstract

The invention provides a strawberry stress resistance and fruit quality regulation gene FaCOL1 and application thereof, and belongs to the technical field of plant genetic engineering, the DNA sequence of the gene FaCOL1 is shown as SEQ ID NO.1, and the amino acid sequence of expression protein of the gene FaCOL1 is shown as SEQ ID NO.2. The invention further provides a preparation method of the strawberry stress resistance and fruit quality regulation gene FaCOL1. According to the present invention, the expression level of the FaCOL1 gene in each tissue part is determined by using the strawberry 'Hongyan' variety, and the strawberry plant is subjected to low temperature treatment and dark treatment so as to determine the gene expression level of the FaCOL1; the FaCOL1 gene is obtained through gene cloning, a transient silencing vector is constructed, the anthocyanin content and the component and content change of soluble sugar are measured, and it is proved that the FaCOL1 gene has important application value in the aspects of enhancing strawberry stress resistance and regulating and controlling fruit quality.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular is a strawberry stress resistance and fruit regulation gene FaCOL1 and application thereof. Background Art

[0002] Strawberry is a perennial herb of the genus Fragaria in the Rosaceae family, and is now mostly cultivated as an annual or biennial crop. The color of strawberry fruit is not only affected by the variety, but also regulated by the combined effects of environmental factors such as temperature, light and water. Under different lighting conditions, the color of strawberry fruit shows significant differences. For example, in a strong light environment, the color of strawberry fruit is lighter and unevenly colored; while under weak light conditions, the content of soluble sugar and soluble solids in the fruit is significantly reduced. In a low temperature environment, the growth of strawberries will slow down, affecting the differentiation of flower buds, etc. 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 plant flowering in 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). For example, in Arabidopsis, overexpression of 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 certain functions in abiotic stress and plant growth and development. The AtCOL4 transcription factor can improve plant tolerance to abiotic stress. Vv CO and VvCOL1 regulate flowering induction and dormancy in grape buds and overexpression of CO in tendrils, which may be related to tendril development.

[0004] At present, the COL gene that regulates the formation of fruit quality has not been clearly identified in strawberry, and the research on COL function is mainly limited to the flowering regulation of model plants such as rice and Arabidopsis, which do not have typical fleshy fruits and do not have the development and maturation process of fruits after flowering. Their COL functions must be quite different from strawberry COL. Cloning and utilizing the strawberry stress resistance and fruit regulatory gene COL1 will help to clarify the quality formation process of strawberry fruits under stress environments after flowering and fruit drop, and provide molecular tools for genetic engineering breeding of strawberry. It has potential economic value such as improving the strawberry's resistance to low temperature and weak light, and cultivating high-quality fruits with color, taste and sweetness. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a stress resistance and fruit quality regulation gene FaCOL1 and its application to meet the use requirements.

[0006] The invention provides a strawberry stress resistance and fruit quality regulating gene FaCOL1, the nucleotide 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.

[0007] The present invention provides a silencing vector containing the strawberry stress resistance and fruit quality regulating gene FaCOL1 according to claim 1 and an application method thereof, wherein the silencing vector is used to inhibit the expression of the strawberry gene FaCOL1.

[0008] The steps of constructing the silencing vector and transiently transforming strawberries are as follows:

[0009] Step (1), using total RNA from strawberry leaves as a template for reverse transcription, performing PCR reaction on the reverse transcription product, connecting the PCR product with a blunt-zero cloning vector, adding the connection product to Escherichia coli competent cells for Escherichia coli transformation, and obtaining an Escherichia coli bacterial solution containing the FaCOL1 gene after sequencing;

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

[0011] Step (2), extracting plasmid from the Escherichia coli culture medium containing the FaCOL1 gene, connecting to the TRV vector, mixing TRAV1 and TRAV2 as an empty vector, and transforming the plasmid into the Agrobacterium competent GV3101 for culture and propagation to obtain the FaCOL1 gene silencing vector;

[0012] Step (3), collecting Agrobacterium and resuspending it in infection solution;

[0013] Step (4), injecting the infection solution into the strawberry ginkgo nuts.

[0014] Beneficial effects: The present invention uses the strawberry "Hongyan" variety to determine the expression level of the FaCOL1 gene in various tissues, and at the same time, the strawberry plants are subjected to low temperature treatment to determine the gene expression of FaCOL1. The FaCOL1 gene is obtained by gene cloning and a transient silencing vector is constructed to determine the anthocyanin content and the composition and content changes of soluble sugars, proving that it has important application value in enhancing strawberry stress resistance and regulating fruit quality.

[0015] The silencing vector inhibits the gene expression of the FaCOL1 gene, reduces the anthocyanin content and the soluble sugar content. The present invention proves through VIGS experiments that the gene has the effect of resisting low temperature, and also has a huge effect on increasing anthocyanins and soluble sugars. By using this gene in agricultural production, using gene editing technology or molecular marker-assisted breeding, new varieties with low temperature resistance, bright fruit color and sweet flavor can be cultivated. By improving the low temperature resistance of the fruit, it can be made suitable for high latitudes or winter planting areas, thereby expanding the planting range and increasing yields. At the same time, it can also be used to enhance the low temperature tolerance of the fruit in post-harvest cold chain transportation and extend the storage time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the expression level of FaCOL1 gene in various tissues of strawberry.

[0017] Figure 2 is the gene expression level of FaCOL1 after strawberry was treated with low temperature.

[0018] Figure 3 It is the phenotype, anthocyanin content and the content of each component of soluble sugar of strawberry after silencing. DETAILED DESCRIPTION

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

[0020] Example 1: Cloning of FaCOL1 gene

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

[0022] Based on the conserved region of plant COL genes, homologous comparison of strawberry transcripts was performed and the strawberry FaCOL1 transcript was identified, which contained a complete open reading frame (ORF). The ORF sequence (cDNA sequence) of FaCOL1 was cloned using total RNA from strawberry leaves as a template and cloned into a blunt-zero vector. The inserted fragments were positively screened and sequenced. The sequencing result sequence was compared with the strawberry gene sequence to confirm that it was a complete FaCOL1 gene.

[0023] Ⅰ. Primer Design

[0024] The primer pairs for the ORF of FaCOL1 are:

[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 is: 1μg Total RNA, 1μL dNTP Mix, 1μL Oligo dT Primer, 4μL 10X RT Buffer, 0.5μL RNase Inhibitor, 1μL M-MLV Reverse Transcriptase, Nuclease-free Water to 20μL. The reaction procedure is: 42℃ reaction for 60min, 70℃ reaction for 15min to obtain cDNA template.

[0030] (2)PCR

[0031] The PCR reaction system was: 2×Keypo Master Mix 25 μL, ORF-F 2 μL, ORF-R 2 μL, cDNA 1 μL, Nuclease-free ddH2O to 50 μL; the reaction program was 98°C, 60°C, 72°C, 35 cycles to obtain the PCR product.

[0032] (3) Linked cloning vector

[0033] The ligation reaction system (5 μL) was 4 μL of PCR recovery product and 1 μL of blunt-zero. The reaction conditions were 25°C for 5 min to obtain the ligation product.

[0034] (4) E. coli transformation

[0035] Take 5 μL of the ligation product, add it to 100 μL of E. coli competent cells, mix gently, ice bath for about 10 minutes, 42°C water bath for 60 seconds, and quickly place on ice for 3-5 minutes; add 800 μL of LB liquid culture medium, 37°C & 100 rpm for recovery for 1 hour; centrifuge at 4000 rpm for 3 minutes, aspirate the upper 600 μL culture medium, mix the remaining bacterial liquid, evenly apply it on the LB screening culture plate containing Cb resistance, and invert and culture at 37°C overnight.

[0036] (5) Positive clone screening and sequencing

[0037] Single colonies were selected from the overnight culture plate and inoculated into LB liquid medium. The culture was cultured overnight at 37℃ and 250rmp to obtain bacterial solution. The bacterial solution was used as a template for PCR detection of recombinant transformants. The PCR reaction system was: 2×Rapid Taq MasterMix 12.5μL, Primer 11μL, Primer 21μL, dd H2O 8.5μL, bacterial solution 2μL. The reaction procedure was 95℃ 3min; 95℃ 15sec, 60℃ 15sec, 72℃ 10sc, 35cycles; 72℃ 5min. The positive electrophoresis test was performed, and the positive bacterial solution was 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 the expression characteristics of the FacoL1 gene tissues under low temperature treatment

[0043] The strawberry plants were placed in a 4℃ refrigerator, and samples were taken at 0h, 1h, 3h, 6h, 12h, and 24h, respectively. The relative expression of the target gene was detected by real-time fluorescence quantitative PCR. The leaves of the low-temperature treated strawberry plants were frozen in liquid nitrogen and ground. The relative expression of the target gene was detected by real-time fluorescence quantitative PCR, and the strawberry FaActin gene was selected as the internal reference gene. RNA was extracted and reverse transcribed from the samples of the above strawberry leaves, and the cDNA stock solution was diluted 10 times as a template for fluorescence quantitative PCR. The quantitative primer qFaCOL1 was designed according to the cloned gene sequence.

[0044] Fluorescence quantitative primers:

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

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

[0047] The expression level was detected by SYBR Green fluorescent dye method. The fluorescence quantitative PCR instrument was CFX96 from Bio-Rad. The reaction system was 10 μL, including 5 μL of quantitative reaction mix, and 10 μmol·L of upstream and downstream primers. -1) 1 μL each, 2 μL cDNA template, add water to 10 μL. The PCR reaction procedure is a three-step method, and the specific process is carried out according to the Bimake SYBR Green qPCRMasterMix manual. The relative expression level is calculated using the 2-ΔΔCT method.

[0048] Figure 1 This is the expression level of the FaCOL1 gene in various tissues of strawberry in normally growing strawberry plants.

[0049] Figure 2 is the gene expression level of FaCOL1 in the leaves of strawberry plants after low temperature treatment, where CK refers to 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 in the figure, with the increase of treatment time, the expression level of FaCOL1 continued to increase in the first 12 hours. Therefore, it can be determined that FaCOL1 has an anti-stress effect.

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

[0051] Ⅰ. Construction of FaCOL1 transient silencing vector

[0052] The plasmid was extracted from the bacterial solution prepared above (the bacterial solution prepared in the E. coli transformation step in Example 1) and connected to the TRV vector. -1 MES, 10mmol·L -1 MgCl2, 250 μmol·L -1 The TRV1 and TRV2 cells were resuspended in acetosyringone to an OD600 value of 2, and mixed at a ratio of 1:1 to form an empty vector. The plasmid (containing FaCOL1) was transformed into Agrobacterium competent GV3101 for culture and propagation. After the cells were collected, the infection solution was resuspended to an OD600 value of 1. The resuspended solution (10 mmol·L -1 MES, 10mmol·L -1 MgCl2, 250 μmol·L -1 Acetosyringone) was added to adjust the bacterial suspension to OD600 = 2, and it was mixed with TRV1 Agrobacterium at a ratio of 1:1 to obtain a resuspended Agrobacterium containing FaCOL1. The bacterial suspension was drawn up with a 1 ml syringe and injected into the strawberries. The strawberries were placed in an incubator (20°C, 16 h of light) and cultured for 7 days. Photos were taken and the results were as follows: Figure 3 shown.

[0053] Ⅱ. Analysis of tissue expression characteristics of FaCOL1 gene

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

[0055] The quantitative primer qFaCOL1 was designed based on the cloned gene sequence. Fluorescence quantitative primer:

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

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

[0058] The expression level was detected by SYBR Green fluorescent dye method. The fluorescence quantitative PCR instrument was CFX96 from Bio-Rad. The reaction system was 10 μL, including 5 μL of quantitative reaction mix, and 10 μmol·L of upstream and downstream primers. -1) 1 μL each, 2 μL cDNA template, add water to 10 μL. The PCR reaction procedure is a three-step method, and the specific process is carried out according to the Bimake SYBR Green qPCRMasterMix manual. The relative expression level is calculated using the 2-ΔΔCT method.

[0059] III. VIGS fruit anthocyanin, soluble sugar, acid determination;

[0060] Anthocyanins were determined using the Ph differential method; the composition and content of soluble sugars were determined using an Agilent 1290infinity II ultra-high performance liquid chromatograph (HPLC). Figure 3 As shown in the figure, the anthocyanin content and the sucrose, glucose and fructose contents in the strawberry fruits after silencing treatment were reduced. It can be seen that FaCOL1 is positively correlated with the anthocyanin and soluble sugar content in strawberries.

[0061] Figure 3 In the figure, CK refers to strawberry fruit injected with a mixed suspension of TRV1 and TRV2, and VIGS refers to strawberry fruit injected with a suspension of Agrobacterium containing FaCOL1. (a) is a photo of strawberry fruit after infection, (b) is the anthocyanin content measured 7 days after infection, (c) is the sucrose content measured 7 days after infection, (d) is the glucose content measured 7 days after infection, and (e) is the fructose content measured 7 days after infection.

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

Claims

1. A strawberry stress resistance and fruit quality regulation gene FaCOL1, characterized in that: The nucleotide sequence of the gene FaCOL1 is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein of the gene FaCOL1 is shown as SEQ ID NO.

2.

2. A silencing vector containing the strawberry stress resistance and fruit quality regulating gene FaCOL1 according to claim 1.

3. The use of the silencing vector according to claim 2, characterized in that: The silencing vector is used to inhibit the expression of the strawberry gene FaCOL1.

4. The use according to claim 3, characterized in that: The steps of constructing the silencing vector and transiently transforming strawberries are as follows: Step (1), using total RNA from strawberry leaves as a template for reverse transcription, performing PCR reaction on the reverse transcription product, connecting the PCR product with a blunt-zero cloning vector, adding the connection product to Escherichia coli competent cells for Escherichia coli transformation, and obtaining an Escherichia coli bacterial solution containing the FaCOL1 gene after sequencing; In the PCR reaction, the ORF upstream primer sequence of FaCOL1 is shown as SEQ ID NO.3; the ORF upstream primer sequence is shown as SEQ ID NO.4; Step (2), extracting plasmid from the Escherichia coli culture medium containing the FaCOL1 gene, connecting to the TRV vector, mixing TRAV1 and TRAV2 as an empty vector, and transforming the plasmid into the Agrobacterium competent GV3101 for culture and propagation to obtain the FaCOL1 gene silencing vector; Step (3), collecting Agrobacterium and resuspending it in infection solution; Step (4), injecting the infection solution into the strawberry ginkgo nuts.

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