Application of BoaVRN1 and BjuVRN1 genes in regulating the content of glucosinolates and the time of bolting and flowering in plants

Abstract

The present invention relates to the field of gene editing and breeding, and in particular to the application of BoaVRN1 gene and BjuVRN1 gene in regulating the glucosinolate content and bolting and flowering time of plants. The BoaVRN1 gene and BjuVRN1 gene provided by the present invention are conducive to in-depth understanding of the regulatory mechanism of glucosinolate biosynthesis and bolting and flowering time of kale and mustard. In the specific embodiment of the present invention, the glucosinolate content of the gene-edited plant obtained by genetic engineering is significantly increased and the bolting and flowering time is significantly delayed. It can be seen that the BoaVRN1 gene and the BjuVRN1 gene can effectively increase the content of glucosinolate in plants and delay the bolting and flowering time. They have application prospects in cultivating and improving high-quality kale and mustard varieties, especially cultivating varieties with high glucosinolate content and resistance to bolting.

Description

Technical Field

[0001] The present invention relates to the field of gene editing and breeding, and in particular to the application of BoaVRN1 gene and BjuVRN1 gene in regulating the glucosinolate content and bolting and flowering time of plants. Background Art

[0002] Glucosinolates (GS) are a class of secondary metabolites rich in sulfur and nitrogen, which are widely found in cruciferous plants such as Brassica vegetables. Glucosinolates play an important role in anti-cancer, plant defense response and flavor formation. Bolting and flowering are important growth and development processes of plants. In cruciferous plants, the process from bolting to flowering determines the transition from the vegetative period to the reproductive period. Premature flowering will cause premature aging of the plant. The timing of bolting and flowering affects the yield, flavor quality and economic benefits of vegetables.

[0003] Chinese kale is a variety of Brassica oleracea in the Brassica family. It is native to southern my country and is now cultivated throughout the country. Chinese kale is mainly edible with stalks and young leaves, and has a unique flavor that is deeply loved by consumers; mustard is an important Brassica vegetable with leaves as the main edible organ. It is widely cultivated in my country, especially in the southwest, and is often used for fresh eating or pickling. Both Chinese kale and mustard are rich in nutrients such as carotenoids and ascorbic acid, especially glucosinolates that are anti-cancer, involved in plant defense reactions and flavor formation. Bolting and flowering directly affect the yield and quality of Chinese kale and mustard. It can be seen that there is a broad development prospect for improving the content of glucosinolates, solving the problem of early bolting or early flowering, and cultivating high-quality Chinese kale and mustard varieties. Therefore, it is very necessary to find a way to increase the content of glucosinolates and delay bolting and flowering. Summary of the invention

[0004] The purpose of the present invention is to provide the application of BoaVRN1 gene and BjuVRN1 gene in regulating the glucosinolate content and bolting and flowering time of plants, so as to solve the problems existing in the above-mentioned prior art. In order to solve the problem of how to increase the glucosinolate content in kale and mustard and delay bolting and flowering, the present invention cloned the BoaVRN1 gene from kale and the BjuVRN1 gene from mustard, and elaborated the function and application prospect of VRN1 gene in regulating the biosynthesis of glucosinolates in kale and mustard and the bolting and flowering time.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of a BoaVRN1 gene or a BjuVRN1 gene in regulating the glucosinolate content and / or bolting and flowering time in a plant. The nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2.

[0007] Preferably, the plant comprises a plant of the family Cruciferae.

[0008] More preferably, the cruciferous plant includes a Brassica plant.

[0009] More preferably, the Brassica plants include mustard, cabbage and kale.

[0010] The present invention provides an application of a protein encoded by the BoaVRN1 gene or a protein encoded by the BjuVRN1 gene in regulating the glucosinolate content and / or bolting and flowering time in a plant. The amino acid sequence of the protein encoded by the BoaVRN1 gene is shown in SEQ ID NO.3; the amino acid sequence of the protein encoded by the BjuVRN1 gene is shown in SEQ ID NO.4.

[0011] Preferably, the plant comprises a plant of the family Cruciferae.

[0012] More preferably, the cruciferous plant includes a Brassica plant.

[0013] More preferably, the Brassica plants include mustard, cabbage and kale.

[0014] The present invention provides the use of a biological material edited by the BoaVRN1 gene or a biological material edited by the BjuVRN1 gene in regulating the glucosinolate content and / or the bolting and flowering time in a plant. The nucleotide sequence of the BoaVRN1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown as SEQ ID NO.2.

[0015] Preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0016] Preferably, the plant comprises a plant of the family Cruciferae.

[0017] More preferably, the cruciferous plant includes a Brassica plant.

[0018] More preferably, the Brassica plants include mustard, cabbage and kale.

[0019] The present invention provides a method for increasing the glucosinolate content in a plant, comprising the steps of editing a BoaVRN1 gene or a BjuVRN1 gene in a plant to increase the glucosinolate content in the plant; the nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2.

[0020] Preferably, the plant comprises a plant of the family Cruciferae.

[0021] More preferably, the cruciferous plant includes a Brassica plant.

[0022] More preferably, the Brassica plants include mustard, cabbage and kale.

[0023] The present invention provides a method for delaying the bolting and flowering time of a plant, comprising the steps of editing a BoaVRN1 gene or a BjuVRN1 gene in a plant to delay the bolting and flowering time of the plant; the nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2.

[0024] Preferably, the plant comprises a plant of the family Cruciferae.

[0025] More preferably, the cruciferous plant includes a Brassica plant.

[0026] More preferably, the Brassica plants include mustard, cabbage and kale.

[0027] The present invention provides a method for cultivating plants with high glucosinolate content and resistance to bolting, comprising the steps of editing the BoaVRN1 gene or the BjuVRN1 gene in the plant to obtain the plants with high glucosinolate content and resistance to bolting; the nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2.

[0028] Preferably, the plant comprises a plant of the family Cruciferae.

[0029] More preferably, the cruciferous plant includes a Brassica plant.

[0030] More preferably, the Brassica plants include mustard, cabbage and kale.

[0031] The present invention discloses the following technical effects:

[0032] The present invention provides the use of BoaVRN1 gene and BjuVRN1 gene in regulating the glucosinolate content and bolting and flowering time of plants, wherein the BoaVRN1 gene is cloned from Chinese kale 'Four Seasons Thick Strips' using PCR amplification technology, and the BjuVRN1 gene is cloned from Brassica juncea 'Er Yue Qing' using PCR amplification technology. The BoaVRN1 gene can increase the glucosinolate content in Chinese kale and delay the bolting and flowering time; the BjuVRN1 gene can increase the glucosinolate content in Brassica juncea and delay the bolting and flowering time. It can be seen that the BoaVRN1 gene and BjuVRN1 gene provided by the present invention are conducive to a deep understanding of the regulatory mechanism of glucosinolate biosynthesis and bolting and flowering time of Chinese kale and Brassica juncea. In the specific embodiment of the present invention, the transgenic plant edited by the gene obtained by genetic engineering has a significantly increased glucosinolate content, and the bolting and flowering time is significantly delayed. It can be seen from this that the BoaVRN1 gene and the BjuVRN1 gene can effectively increase the content of glucosinolates in plants and delay the time of bolting and flowering. They have application prospects in breeding and improving high-quality kale and mustard varieties, especially in breeding varieties with high glucosinolate content and resistance to bolting. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The results of the glucosinolate content of WT kale plants and boavrn1-M kale plants are shown in FIG. A is a bar graph of the total aliphatic mustard oil content, and B is a bar graph of the GNA (3-butenyl glucosinolate) content; WT is an unedited plant (wild type), and M1-M3 are three boavrn1-M kale plants;

[0035] Figure 2 The results of fluorescence quantitative PCR of WT kale plants and boavrn1-M kale plants; AI is the relative expression level of VRN1, AOP2.1, MYB28.2, MYB28.3, MYB29.1, MYB29.2, CYP83A1.1, CYP83A1.2 and CYP79F1; WT is the unedited plant (wild type), M1-M3 are three boavrn1-M kale plants;

[0036] Figure 3The result diagram of bolting and flowering time of WT kale plants and boavrn1-M kale plants; A is a bar graph of bud appearance time, and B is a bar graph of initial flowering time; WT is an unedited plant (wild type), and M1-M3 are three boavrn1-M kale plants;

[0037] Figure 4 The results of glucosinolate content in WT kale plants and boavrn1-N kale plants are shown in Figure 1. A is a bar graph of total aliphatic mustard oil content, and B is a bar graph of GNA content. WT is an unedited plant (wild type), and N1-N3 are three boavrn1-N kale plants.

[0038] Figure 5 The results of fluorescence quantitative PCR of WT kale plants and boavrn1-N kale plants; AI is the relative expression level of VRN1, AOP2.1, MYB28.2, MYB28.3, MYB29.1, MYB29.2, CYP83A1.1, CYP83A1.2 and CYP79F1; WT is the unedited plant (wild type), and N1-N3 are three boavrn1-N kale plants;

[0039] Figure 6 The result diagram of bolting and flowering time of WT kale plants and boavrn1-N kale plants; A is a bar graph of bud appearance time, and B is a bar graph of initial flowering time; WT is an unedited plant (wild type), and N1-N3 are three boavrn1-N kale plants;

[0040] Figure 7 The results of the glucosinolate content of WT mustard plants and bjuvrn1-A mustard plants are shown in the figure; A is a bar graph of the total aliphatic mustard oil content, and B is a bar graph of the SIN (2-propenyl glucosinolate) content; WT is an unedited plant (wild type), and A1 and A2 are two bjuvrn1-A mustard plants;

[0041] Figure 8 The results of fluorescence quantitative PCR of WT mustard plants and bjuvrn1-A mustard plants; AI is the relative expression level of VRN1, MYB28, MYB29, BCAT4, MAM1, CYP79F1, UGT74B1, CYP83A1 and SUR1; WT is the unedited plant (wild type), A1 and A2 are two bjuvrn1-A mustard plants;

[0042] Fig. 9The result diagram of bolting and flowering time of WT mustard plants and bjuvrn1-A mustard plants; A is a bar graph of bud appearance time, and B is a bar graph of initial flowering time; WT is an unedited plant (wild type), and A1 and A2 are two bjuvrn1-A mustard plants;

[0043] Fig.10 The results of glucosinolate content in WT mustard plants and bjuvrn1-B mustard plants are shown in Figure 1. A is a bar graph of total aliphatic mustard oil content, and B is a bar graph of SIN content. WT is an unedited plant (wild type), and B1 and B2 are two bjuvrn1-B mustard plants.

[0044] Fig.11 The results of fluorescence quantitative PCR of WT mustard plants and bjuvrn1-B mustard plants; AI is the relative expression level of VRN1, MYB28, MYB29, BCAT4, MAM1, CYP79F1, UGT74B1, CYP83A1 and SUR1; WT is the unedited plant (wild type), B1 and B2 are two bjuvrn1-B mustard plants;

[0045] Fig.12 The graphs show the bolting and flowering time of WT and bjuvrn1-B mustard plants; A is a bar graph of bud appearance time, and B is a bar graph of initial flowering time; WT is an unedited plant (wild type), and B1 and B2 are two bjuvrn1-B mustard plants. DETAILED DESCRIPTION

[0046] Example 1

[0047] 1. Extraction of total RNA from Chinese kale and synthesis of cDNA

[0048] Total RNA was extracted from mature leaves of Chinese kale 'Si Ji Cu Tiao' using an RNA extraction kit, and the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0049] 2. Cloning of the BoaVRN1 gene from Kale:

[0050]

[0051] The amino acid sequence of the protein encoded by the BoaVRN1 gene is shown in SEQ ID NO.3, specifically: MPRPFFHKLIFSSTIQEKRLRVPDKFVSKFKDELSVAVALTVPDGHVWRVGLRKADNNKIWFQDGWQEFVDRYSIRIGYLLIFRYEGNSAFSVYIYNLSHSEINYHSTGLMDSAHNHFKRARLFEDLEDEDVEVVHPSPLYSSQHPETTAHANKGHTSSAIQSFFAEPPVKAEETTPTPKVPKKRGRKKKNADPEEINSSAPRDDDPEN RSKFYESASARKRTVTAEERERAINAAKTFEPTNPFFRVVLRPSYLYRGCIMYLPSGFAEKYLSGISGFIKVQLGEKQWPVRCLYKAGRAKFSQGWYEFTLENNLGEGDVCVFELLRTRDFVLKVTAFRVNEYV*; wherein, “*” is a stop codon; specific primers for BoaVRN1 gene cloning were designed according to the nucleotide sequence of the BoaVRN1 gene: forward primer: 5′-ATGCCACGCCCTTTCTTCC-3′ (SEQ ID NO.5), reverse primer: 5′-TCAGACGTACTCGTTGACACGAAAG-3′ (SEQ ID NO.6);

[0052] The cDNA of 'Four Seasons Thick Strip' was used as template for amplification. The PCR reaction conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 5s, 72℃ extension for 5s, 34 cycles in total; 72℃ extension for 1min. The PCR product was subjected to 1.0% mass volume fraction agarose gel electrophoresis to recover the target band, connected to the pClone007 vector (Beijing Qingke Biotechnology Co., Ltd.) and transformed into Escherichia coli DH5α. The extracted plasmid was identified by PCR and then commissioned to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0053] 3. Construction of BoaVRN1 gene editing vector

[0054] (1) The target site analysis of the BoaVRN1 sequence obtained by cloning in the early stage was performed, and a sequence located in the exon was selected as the target site. The target site sequence was: GTTGCCCTCACAGTACCTGATGG (SEQ ID NO.7). Target primers were designed according to the target site sequence. The forward primer was: 5′-ATTGTTGCCCTCACAGTACCTGA-3′ (SEQ ID NO.11), and the reverse primer was: 5′-AAACTCAGGTACTGTGAGGGCAA-3′ (SEQ ID NO.12). The target primers were annealed at 98°C for 3 min, naturally cooled to room temperature, and used for ligation;

[0055] (2) The AtU6-26-sgRNA-SK plasmid was digested with Bsa I at 37°C for 60 min, purified, and stored frozen;

[0056] (3) Use T4 DNA ligase to connect the digested AtU6-26-sgRNA-SK vector and the annealed primer at room temperature (20-25°C) for 20-30 minutes;

[0057] (3) Transform the ligated product into E. coli DH5α and coat with Amp + LB plate, after 10-12 hours, single clones were selected for colony PCR identification and sequencing to confirm whether the target was connected to the vector, forward primer: 5′-CTCACTATAGGGCGAATTGG-3′ (SEQ ID NO.19), reverse primer: reverse primer of the target (SEQ ID NO.12), the correct clone was expanded and cultured, the plasmid was extracted, double-digested with Nhe I and Spe I, and the recovered fragment was the intermediate vector of the constructed single guide RNA (sgRNA) cassette;

[0058] (4) pCAMBIA1300-pYAO:Cas9 plasmid was digested with Spe I at 37°C for 10-30 min, inactivated at 80°C for 20 min, and stored frozen after gel electrophoresis.

[0059] (5) Use T4 DNA ligase to connect the digested pCAMBIA1300-pYAO:Cas9 plasmid and sgRNAcassette vector at room temperature (20-25°C) for 20-30 minutes;

[0060] (6) Transform 10 μL of the ligation product into DH5α and coat +LB plate, single clones were selected after 14-16 hours, and the primer sequences on the binary vector were used for colony PCR identification. The correct single clones were selected for expansion culture, plasmids were extracted, and restriction enzymes were used for verification by SalI and Kpn I. The length of the restriction enzyme fragment was about 670bp. The primers for colony PCR identification were located at both ends of the Spe I restriction site, forward primer: 5′-CCAGTCACGACGTTGTAAAAC-3′ (SEQ ID NO.20); reverse primer: 5′-CAATGAATTTCCCATCGTCGAG-3′ (SEQ ID NO.21), the PCR fragment length was about 750bp, and the unconnected clone PCR fragment was only about 100bp. The correct plasmid was the final vector pCAMBIA1300-pYAO:Cas9 used for plant transformation, named pCase-BoaVRN1-M, and the correctly identified plasmid was transformed into Agrobacterium to infect plants.

[0061] 4. Transform the recombinant vector into Agrobacterium GV3101:

[0062] (1) Add 0.5 μg of the recombinant vector pCas9-BoaVRN1-M to every 50 μL of GV3101 Agrobacterium competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 h;

[0063] (2) Centrifuge at 6000 rpm for 1 min to collect the cells. Take about 100 μL of the supernatant, gently blow to resuspend the cells, and evenly spread it on a plate containing 50 μg mL -1 Kanamycin (kan), 20 μg mL -1 The cells were placed on LB solid medium containing rifampin (rif), inverted and placed in a 28°C incubator for 2 days, and several positive clones were picked out to verify the results using colony PCR.

[0064] 5. Explant culture of Chinese kale

[0065] Sterile kale seeds were sown on 1 / 2MS solid culture medium and placed in a tissue culture room for culture. The conditions in the tissue culture room were: temperature 25 / 20°C (day / night), photoperiod 16 / 8h (day / night), and light intensity 36μmol·m -2 ·s -1 After 7 days of culture, kale cotyledons with 1-2 mm petioles were cut and used as explants for Agrobacterium-mediated transformation.

[0066] 6. Genetic transformation of Chinese kale

[0067] (1) Explants were pre-cultured in MS medium for 3 days with a photoperiod of 16 h light / 8 h dark and a light intensity of 80 μmol·m -2 ·s -1 ;

[0068] (2) Infect with Agrobacterium strain GV3101 into which the recombinant vector has been transferred for 1-2 minutes;

[0069] (3) The explants were co-cultured with Agrobacterium in MS medium for 3 days under dark conditions;

[0070] (4) Transfer the explants to delayed screening medium (0.03 mg·L -1 NAA+0.75mg·L -1 6-BA+0.8% agar+325mg·L -1 Carb+325mg·L -1 Timentin) and grown for 7 days;

[0071] (5) The explants were transferred to a resistance screening medium containing selective antibiotics (0.03 mg·L -1 NAA+0.75mg·L -1 6-BA+0.8% agar+325mg·L -1 Carb+325mg·L -1 Timentin+12mg·L -1 Hyg) for resistance screening, subculture once every 20 days or so, and the subculture medium used is the same as the resistance screening medium;

[0072] (6) After the explants grew for 3 months, hygromycin-resistant plants were obtained and then transplanted into a matrix composed of nutrient soil, perlite, and vermiculite in a volume ratio of 3:1:1. The explants were placed under a light intensity of 80 μmol·m -2 ·s -1 , cultured in an artificial climate box with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%.

[0073] 7. Positive plant screening

[0074] Specific primers were designed according to the hygromycin sequence contained in the vector, including forward primer: 5′-CGATTGCGTCGCATCGACC-3′ (SEQ ID NO.22) and reverse primer: 5′-TTCTACAACCGGTCGCGGAG-3′ (SEQID NO.23). PCR amplification was performed using the genomic DNA of each gene-edited plant as a template. After detection by agarose gel electrophoresis, gene-edited positive plants were screened and seeds were retained to obtain the BoaVRN1 gene-edited kale T1 generation, named boavrn1-M, or M for short.

[0075] 8. Phenotypic observation

[0076] (1) K. oleracea (WT) and T1 generation seeds of BoaVRN1 gene-edited K. oleracea were sown simultaneously in a matrix with a volume ratio of 3:1:1 of nutrient soil, perlite, and vermiculite. The seeds were grown under a light intensity of 80 μmol·m -2 ·s -1 , cultured in an artificial climate chamber with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%;

[0077] (2) Take fresh kale leaves (0.5 g) and boil them in 5 mL of water for 10 min. Collect the supernatant after centrifugation (5 min, 7000×g). Load the extract onto a DEAE-Sephadex A-25 column, wash three times with 1 mL of 0.02% pyridine acetate, and wash twice with 1 mL of ddH2O. Treat with 100 μL of 0.1% sulfatase overnight to convert glucosinolates into their desulfurized products, and finally wash with 1 mL of water to remove the desulfurized glucosinolates. HPLC analysis of glucosinolates was performed using a liquid chromatograph at 30°C using acetonitrile and water at a flow rate of 1 mL / min on a Waters Spherisorb C 18 The sample was separated on a chromatographic column. The program was to use 1.5% acetonitrile for the first 5 min, a linear gradient to 20% acetonitrile for the next 15 min, and then 20% acetonitrile for the last 10 min.

[0078] HPLC analysis results showed that the boavrn1-M glucosinolate content in the T1 generation of BoaVRN1 gene-edited kale was significantly higher than that in the WT plants. Figure 1 shown.

[0079] 9. Quantitative analysis of glucosinolate-related genes in gene-edited kale plants

[0080] (1) The middle leaves of WT and boavrn1-M plants grown for 60 days were used as materials, immediately frozen with liquid nitrogen, and stored in a -80°C refrigerator;

[0081] (2) RNA and cDNA were obtained according to the method in “1. Extraction of total RNA from kale and synthesis of cDNA”;

[0082] (3) Specific primers for detecting the expression of kale mustard oil glycoside-related genes (VRN1, AOP2.1, MYB28.2, MYB28.3, MYB29.1, MYB29.2, CYP83A1.1, CYP83A1.2 and CYP79F1) and the internal reference gene β-actin were designed for fluorescence quantitative detection. The primers used are shown in Table 1.

[0083] Table 1 Detailed information of primers

[0084]

[0085] (4) Real-time quantitative PCR using TB Green Premix Ex Taq TM The expression levels of kale glucosinolate biosynthesis genes were detected according to the instructions of the II qPCR Kit (Tli RNaseH Plus, Takara).

[0086] (5) The formula for calculating the relative transcriptional expression level of the target gene is 2 -ΔΔCt , ΔΔCt=(Ct target gene-CtActin) treatment group-(Ct target gene-Ct Actin) control group.

[0087] The results of fluorescence quantitative PCR showed that in the BoaVRN1 gene-edited kale T1 plant boavrn1-M, the expression level of VRN1 was lower than that of WT, and the expression levels of eight glucosinolate biosynthesis-related genes (AOP2.1, MYB28.2, MYB28.3, MYB29.1, MYB29.2, CYP83A1.1, CYP83A1.2 and CYP79F1) were higher than those of WT. Figure 2 shown.

[0088] 10. Analysis of bolting and flowering time of gene-edited kale plants

[0089] The phenotypic traits of the T1 generation BoaVRN1 gene-edited plants were observed and measured, and the bolting and flowering times of the boavrn1-M plants were recorded. Bolting was marked by the appearance of budding traits, and flowering was marked by the appearance of initial flowering traits. Figure 3 It can be seen that compared with WT plants, the bolting and flowering time of the T1 generation boavrn1-M plants edited with the BoaVRN1 gene was significantly delayed, and the bolting and flowering time was significantly later than that of the WT plants.

[0090] The above-mentioned physiological index analysis and bolting and flowering phenotype results prove that editing the BoaVRN1 gene of kale can significantly increase the content of glucosinolates in kale and delay the time of bolting and flowering.

[0091] Example 2

[0092] 1. Extraction of total RNA from Chinese kale and synthesis of cDNA

[0093] At the same time, Example 1.

[0094] 2. Cloning of the BoaVRN1 gene from Kale:

[0095] Same as Example 1.

[0096] 3. Construction of BoaVRN1 gene editing vector

[0097] The same as Example 1, except that the target site sequence is different, specifically: CTACCACTCCACCGGTCTCATGG (SEQID NO.8), target primer, forward primer: 5′-ATTGCTACCACTCCACCGGTCTCATGG-3′ (SEQ ID NO.13), reverse primer: 5′-AAACTGAGACCGGTGGAGTGGTAG-3′ (SEQ ID NO.14), the constructed vector is named pCase-BoaVRN1-N, and is used for Agrobacterium transformation; to confirm whether the target site is connected to the vector, the forward primer is shown in SEQ ID NO.19, and the reverse primer is shown in SEQ ID NO.14.

[0098] 4. Transform the recombinant vector into Agrobacterium GV3101:

[0099] The same as Example 1, except that 0.5 μg of the recombinant vector pCas9-BoaVRN1-N was added to every 50 μL of GV3101 Agrobacterium competent cells.

[0100] 5. Explant culture of Chinese kale

[0101] Same as Example 1.

[0102] 6. Genetic transformation of Chinese kale

[0103] Same as Example 1.

[0104] 7. Positive plant screening

[0105] As in Example 1, the BoaVRN1 gene-edited kale T1 generation was obtained and named boavrn1-N, abbreviated as N.

[0106] 8. Phenotypic observation

[0107] Same as Example 1. The HPLC analysis results showed that the boavrn1-N glucosinolate content of the BoaVRN1 gene-edited kale T1 plants was significantly higher than that of the WT plants. Figure 4 shown.

[0108] 9. Quantitative analysis of glucosinolate-related genes in gene-edited kale plants

[0109] The same as Example 1, except that the samples were the middle leaves of WT and boavrn1-N plants grown for 60 days. The results of quantitative fluorescence PCR showed that in boavrn1-N plants, the expression level of VRN1 was lower than that of WT, and the expression levels of 8 glucosinolate biosynthesis-related genes (AOP2.1, MYB28.2, MYB28.3, MYB29.1, MYB29.2, CYP83A1.1, CYP83A1.2 and CYP79F1) were all higher than those of WT. The results are as follows Figure 5 shown.

[0110] 10. Analysis of bolting and flowering time of gene-edited kale plants

[0111] The phenotypic traits of the T1 generation BoaVRN1 gene-edited plants were observed and measured, and the bolting and flowering times of the boavrn1-N plants were recorded. Bolting was marked by the appearance of budding traits, and flowering was marked by the appearance of initial flowering traits. Figure 6 It can be seen that compared with WT plants, the bolting and flowering time of the T1 generation plant boavrn1-N edited by the BoaVRN1 gene was significantly delayed, and the bolting and flowering time was significantly later than that of the WT plants.

[0112] The above-mentioned physiological index analysis and bolting and flowering phenotype results prove that editing the BoaVRN1 gene of kale can significantly increase the content of glucosinolates in kale and delay the time of bolting and flowering.

[0113] Example 3

[0114] 1. Extraction of total RNA from Brassica juncea and synthesis of cDNA

[0115] The total RNA was extracted from mature leaves of Brassica juncea 'Eryueqing' using the CTAB method.

[0116] (1) Add 15 mL of CTAB extraction buffer (300 μL of β-mercaptoethanol is required) to a 50 mL centrifuge tube and preheat to 65°C.

[0117] (2) Weigh 2-3 g of fresh plant material and grind it into powder in liquid nitrogen;

[0118] (3) The sample was transferred to a 50 mL centrifuge tube and rapidly shaken to mix until no lumps formed. The sample was incubated at 65°C for 10 min, and then 15 mL of chloroform was added and mixed.

[0119] (4) Centrifuge at 10,000 rpm for 15 min at room temperature, transfer the upper aqueous phase into a new centrifuge tube, add an equal volume of chloroform, mix well, and extract again;

[0120] (5) Transfer the upper aqueous phase to another centrifuge tube, add 8 M LiCl to a final concentration of 2 M (approximately 1 / 3 of the aqueous phase), and incubate at 4 °C overnight;

[0121] (6) Centrifuge at 12,000 rpm for 20 min at 4°C to precipitate RNA, discard the supernatant, wash the precipitate with 70% (volume percentage) ethanol, and air dry;

[0122] (7) Add 500 μL SSTE buffer to dissolve the precipitate, transfer to a 1.5 mL centrifuge tube, add an equal volume of acid phenol / chloroform (the volume ratio of acid phenol to chloroform is 5:1) and mix well;

[0123] (8) Centrifuge at 13,000 rpm for 10 min at room temperature, transfer the upper aqueous phase to another 1.5 mL centrifuge tube, add an equal volume of chloroform and mix;

[0124] (9) Centrifuge at 13,000 rpm for 10 min at room temperature, transfer the upper aqueous phase to a new tube, add 1 / 10 volume of 3 M NaAC and 2 volumes of anhydrous ethanol, and incubate at -80 °C for 30 min;

[0125] (10) Centrifuge at 13,000 rpm for 20 min at 4°C, wash the precipitate with 70% (volume percentage) ethanol, dry it, add 50 μL of DEPC-treated H2O to dissolve the RNA, take 1 μL for electrophoresis, and store it at -80°C for later use.

[0126] The extracted total RNA was reverse transcribed into cDNA using PrimeScript 1st Strand cDNA Synthesis Kit (Baoriyi Biotechnology (Beijing) Co., Ltd.).

[0127] 2. Cloning of B. juncea BjuVRN1 gene

[0128]

[0129] The amino acid sequence of the protein encoded by the BjuVRN1 gene is shown in SEQ ID NO.4, specifically: MPRPFFHKLIFSSTIQEKRLRVPDKFVSKFKDELSVAVALTVPDGHVWRVGLRKADNNKIWFQDGWQEFVDRYSIRIGYLLIFRYEGNSAFSVYIYNLSHSEINYHSTGLMDSAHNHFKRARLFEELEDEDVEVVHPSSLYPSHHPGTTVAASKGHTSSAIQSFFTGPVKAEETTPTPTPKVPKKRGRKKKNAVAGPEEINSSAPPDDDPENRSKFYESASARKRTVTAEERERAINAAKTFEPTNPFFRVVLRPSYLYRGCIMYLPSGFAEKYLSGISGFIKVQLGEKQWPVRCLYKAGRAKFSQGWYEFTLENNLGEGDVCVFELLRTRDFVLKVTAFRVNEYV*; wherein, “*” is a stop codon.

[0130] Specific primers for BjuVRN1 gene cloning were designed according to the nucleotide sequence of the BjuVRN1 gene: the forward primer was shown in SEQ ID NO.5, and the reverse primer was shown in SEQ ID NO.6.

[0131] The cDNA of Eryueqing was used as template for amplification. The PCR reaction conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 5s, 72℃ extension for 5s, 34 cycles in total; 72℃ extension for 1min. The PCR product was subjected to 1.0% agarose gel electrophoresis to recover the target band, connected to the pClone007 vector (Beijing Qingke Biotechnology Co., Ltd.) and transformed into Escherichia coli DH5α. The extracted plasmid was identified by PCR and then commissioned to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0132] 3. Construction of BjuVRN1 gene editing vector

[0133] The target site analysis of the BjuVRN1 sequence obtained by cloning in the early stage was performed, and a sequence located in the exon was selected as the target site. The target site sequence was: CTACTGTAGCTGCTAGTAAAGGG (SEQ ID NO.9). According to the target site sequence, the target primers were designed, the forward primer was: 5′-ATTGCTACTGTAGCTGCTAGTAAAGGG-3′ (SEQ ID NO.15), the reverse primer was: 5′-AAACCCCTTTACTAGCAGCTACAGTAG-3′ (SEQ ID NO.16), the target primers were annealed at 98°C for 3 minutes, cooled naturally to room temperature, and used for ligation;

[0134] (2) The AtU6-26-sgRNA-SK plasmid was digested with Bsa I at 37°C for 60 min, purified, and stored frozen.

[0135] (3) Use T4 DNA ligase to connect the digested AtU6-26-sgRNA-SK vector and the annealed primer at room temperature (20-25°C) for 20-30 minutes;

[0136] (3) Transform the ligated product into E. coli DH5α and coat with Amp + LB plate, after 10-12 hours, single clones were selected for colony PCR identification and sequencing to confirm whether the target was connected to the vector. The forward primer was shown in SEQ ID NO.19, and the reverse primer was shown in SEQ ID NO.16. The correct clone was expanded and cultured, and the plasmid was extracted and double-digested with Nhe I and Spe I. The recovered fragment was the intermediate vector of the constructed single guide RNA (sgRNA) cassette;

[0137] (4) pCAMBIA1300-pYAO:Cas9 plasmid was digested with Spe I at 37°C for 10-30 min, inactivated at 80°C for 20 min, and stored frozen after gel electrophoresis.

[0138] (5) Use T4 DNA ligase to connect the digested pCAMBIA1300-pYAO:Cas9 plasmid and sgRNAcassette vector at room temperature (20-25°C) for 20-30 minutes;

[0139] (6) Transform 10 μL of the ligation product into DH5α and coat +LB plate, single clones were selected after 14-16 hours, and the primer sequences on the binary vector were used for colony PCR identification. The single clones with correct identification were selected for expansion culture, plasmids were extracted, and restriction enzyme verification was performed using Sal I and Kpn I. The length of the restriction enzyme fragment was about 670bp. The primers for colony PCR identification were located at both ends of the Spe I restriction site, the forward primer was shown in SEQ ID NO.20; the reverse primer was shown in SEQ ID NO.21, the PCR fragment length was about 750bp, and the PCR fragment of the clone that was not successfully connected was only about 100bp. The correct plasmid was the final vector pCAMBIA1300-pYAO:Cas9 for plant transformation, named pCase-BjuVRN1-A, and the correctly identified plasmid was transformed into Agrobacterium to infect plants.

[0140] 4. Transform the recombinant vector into Agrobacterium EHA105

[0141] (1) Add 0.5 μg of the recombinant vector pCas9-BjuVRN1-A to every 50 μL of EHA105 Agrobacterium competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 h;

[0142] (2) Centrifuge at 6000 rpm for 1 min to collect the cells. Take about 100 μL of the supernatant, gently blow to resuspend the cells, and evenly spread it on a plate containing 50 μg mL -1 Kanamycin (kan), 20 μg mL -1 The cells were placed on LB solid medium containing rifampin (rif), inverted and placed in a 28°C incubator for 2 days, and several positive clones were picked out to verify the results using colony PCR.

[0143] 5. Explant culture of Brassica juncea

[0144] Sterile seeds were sown on 1 / 2MS solid medium and cultured in a tissue culture room. The conditions in the tissue culture room were: temperature 25 / 20°C (day / night), photoperiod 16 / 8h (day / night), and light intensity 36μmol·m -2 ·s -1 After 5 days of culture, cotyledons of Brassica juncea with 1-2 mm petioles were cut and used as explants for Agrobacterium-mediated transformation.

[0145] 6. Genetic transformation of Brassica juncea

[0146] (1) Explants were pre-cultured in MS medium for 2 days with a photoperiod of 16 h light / 8 h dark and a light intensity of 80 μmol·m-2 ·s -1 ;

[0147] (2) Infect with Agrobacterium strain EHA105 into which the recombinant vector has been transferred for 1-2 minutes;

[0148] (3) The explants were co-cultured with Agrobacterium in MS medium for 3 days under dark conditions;

[0149] (4) Transfer the explants to delayed screening medium (0.03 mg·L -1 NAA+0.75mg·L -1 6-BA+0.8% agar+325mg·L -1 Carb+325mg·L -1 Timentin) and grown for 7 days;

[0150] (5) The explants were transferred to a resistance screening medium containing selective antibiotics (0.03 mg·L -1 NAA+0.75mg·L -1 6-BA+0.8% agar+325mg·L -1 Carb+325mg·L -1 Timentin+12mg·L -1 Hyg) for resistance screening, subculture once every 20 days or so, and the subculture medium used is the same as the resistance screening medium;

[0151] (6) After the explants grew for 3 months, hygromycin-resistant plants were obtained and then transplanted into a matrix composed of nutrient soil, perlite, and vermiculite in a volume ratio of 3:1:1. The explants were placed under a light intensity of 80 μmol·m -2 ·s -1 , cultured in an artificial climate box with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%.

[0152] 7. Positive plant screening

[0153] Specific primers were designed according to the hygromycin sequence contained in the vector, the forward primer was shown in SEQ ID NO.22, and the reverse primer was shown in SEQ ID NO.23. PCR amplification was performed using the genomic DNA of each gene-edited plant as a template. After detection by agarose gel electrophoresis, gene-edited positive plants were screened and seeds were retained to obtain the BjuVRN1 gene-edited T1 generation of Brassica juncea, which was named bjuvrn1-A (abbreviated as A).

[0154] 8. Analysis of glucosinolate components and contents in gene-edited mustard plants

[0155] (1) Seeds of mustard (WT) and BjuVRN1 gene-edited mustard T1 generation bjuvrn1-A were sown simultaneously in a matrix with a volume ratio of 3:1:1 of nutrient soil, perlite, and vermiculite. The seeds were grown under a light intensity of 80 μmol·m -2 ·s -1 , cultured in an artificial climate chamber with a temperature of 25 / 20°C (day / night), a photoperiod of 12 / 12h (day / night), and a relative humidity of 75%;

[0156] (2) Take fresh mustard leaves (0.5 g) and boil them in 5 mL of water for 10 min. Collect the supernatant after centrifugation (5 min, 7000×g). Load the extract onto a DEAE-Sephadex A-25 column, wash three times with 1 mL of 0.02% pyridine acetate, and wash twice with 1 mL of ddH2O. Treat with 100 μL of 0.1% sulfatase overnight to convert glucosinolates into their desulfurized products, and finally wash with 1 mL of water to remove the desulfurized glucosinolates. HPLC analysis of glucosinolates was performed using a liquid chromatograph at 30°C using acetonitrile and water at a flow rate of 1 mL / min on a Waters Spherisorb C 18 The sample was separated on a chromatographic column. The program was to use 1.5% acetonitrile for the first 5 min, a linear gradient to 20% acetonitrile for the next 15 min, and then 20% acetonitrile for the last 10 min.

[0157] HPLC analysis results showed that the bjuvrn1-A glucosinolate content in BjuVRN1 gene-edited B. juncea T1 plants was significantly higher than that in WT plants. Figure 7 shown.

[0158] 9. Quantitative analysis of glucosinolate-related genes in gene-edited mustard plants

[0159] (1) The middle leaves of 90-day-old Brassica juncea WT and bjuvrn1-A plants were used as materials, immediately frozen with liquid nitrogen, and stored in a -80°C freezer;

[0160] (2) obtaining RNA and cDNA according to the method in “1. Extraction of total RNA from Brassica juncea and synthesis of cDNA”;

[0161] (3) Design specific primers for gene expression detection based on the sequences of mustard glucosinolate-related genes (VRN1, MYB28, MYB29, BCAT4, MAM1, CYP79F1, UGT74B1, CYP83A1 and SUR1) and the internal reference gene β-actin for fluorescence quantitative detection. The primers are shown in Table 2.

[0162] Table 2 Primer information

[0163]

[0164] (4) Real-time quantitative PCR using TB Green Premix Ex Taq TM II qPCR Kit (Tli RNaseH Plus, Takara) fluorescence quantitative kit instructions were used to detect the expression of mustard glucosinolate biosynthesis genes;

[0165] (5) The formula for calculating the relative transcriptional expression level of the target gene is 2 -ΔΔCt , ΔΔCt=(Ct target gene-CtActin) treatment group-(Ct target gene-Ct Actin) control group.

[0166] The results of fluorescence quantitative PCR showed that in BjuVRN1-A plants, the expression level of VRN1 was lower than that of WT, and the expression levels of eight glucosinolate biosynthesis-related genes (MYB28, MYB29, BCAT4, MAM1, CYP79F1, UGT74B1, CYP83A1 and SUR1) were higher than those of WT. Figure 8 As shown.

[0167] 10. Analysis of bolting and flowering time of gene-edited mustard plants

[0168] The phenotypic traits of the T1 edited plants were observed and measured, and the bolting and flowering times of the bjuvrn1-A plants were recorded. Bolting was marked by the appearance of budding, and flowering was marked by the appearance of the initial flower shape. Fig. 9 It can be seen that compared with WT plants, the bolting and flowering time of the T1 generation plants bjuvrn1-A edited with BjuVRN1 gene was significantly delayed, and the bolting and flowering time was significantly later than that of WT plants.

[0169] The above-mentioned physiological index analysis and bolting and flowering phenotype results prove that editing the BjuVRN1 gene of mustard can significantly increase the glucosinolate content of mustard and delay the bolting and flowering time.

[0170] Example 4

[0171] 1. Extraction of total RNA from Brassica juncea and synthesis of cDNA

[0172] Same as Example 3.

[0173] 2. Cloning of B. juncea BjuVRN1 gene

[0174] Same as Example 3.

[0175] 3. Construction of editing vector for BjuVRN1 gene

[0176] The same as Example 3, except that the target site sequence is different, specifically: CGACCATCCTATCTTTACAGAGG (SEQ ID NO.10), the target primers are designed according to the target site sequence, the forward primer is: 5′-ATTGCGACCATCCTATCTTTACAGAGG-3′ (SEQ ID NO.17), the reverse primer is: 5′-AAACCTCTGTAAAGATAGGATGGTC-3′ (SEQ ID NO.18), the constructed vector is named pCase-BjuVRN1-B, and whether the target site is connected to the vector is confirmed. The forward primer is shown in SEQ ID NO.19, and the reverse primer is shown in SEQ ID NO.18.

[0177] 4. Transform the recombinant vector into Agrobacterium EHA105

[0178] Same as Example 3, except that 0.5 μg of the recombinant vector pCase-BjuVRN1-B was used instead of 0.5 μg of the recombinant vector pCase-BjuVRN1-A.

[0179] 5. Explant culture of Brassica juncea

[0180] Same as Example 3.

[0181] 6. Genetic transformation of Brassica juncea

[0182] Same as Example 3.

[0183] 7. Positive plant screening

[0184] As in Example 3, the BjuVRN1 gene-edited T1 generation of Brassica juncea was obtained and named bjuvrn1-B (abbreviated as B).

[0185] 8. Analysis of glucosinolate components and contents in gene-edited mustard plants

[0186] Same as Example 3. HPLC analysis results showed that the bjuvrn1-B glucosinolate content in the BjuVRN1 gene-edited mustard T1 plants was significantly higher than that in the WT plants. Fig.10 shown.

[0187] 9. Quantitative analysis of glucosinolate-related genes in gene-edited mustard plants

[0188] The same as Example 3, except that the samples were the middle leaves of WT and bjuvrn1-B plants grown for 60 days. The results of quantitative fluorescence PCR showed that the expression level of VRN1 in BjuVRN1-B plants was lower than that in WT, and the expression levels of 8 glucosinolate biosynthesis-related genes (MYB28, MYB29, BCAT4, MAM1, CYP79F1, UGT74B1, CYP83A1 and SUR1) were all higher than those in WT. Fig.11 shown.

[0189] 10. Analysis of bolting and flowering time of gene-edited mustard plants

[0190] The phenotypic traits of the T1 generation edited plants were observed and measured, and the bolting and flowering times of bjuvrn1-B plants were recorded. Bolting was marked by the appearance of budding traits, and flowering was marked by the appearance of initial flowering traits.

[0191] Depend on Fig.12 It can be seen that compared with WT plants, the bolting and flowering time of BjuVRN1 gene-edited mustard T1 generation bjuvrn1-B plants was significantly delayed, and the bolting and flowering time was significantly later than that of WT plants.

[0192] The above-mentioned physiological index analysis and bolting and flowering phenotype results prove that editing the BjuVRN1 gene of mustard can significantly increase the glucosinolate content of mustard and delay the bolting and flowering time.

[0193] In summary, it can be seen that the gene-edited plant obtained by genetic engineering in the specific embodiment of the present invention has a significantly increased glucosinolate content, and the bolting and flowering time is significantly delayed. It can be seen that the kale BoaVRN1 gene and the mustard BjuVRN1 gene can effectively increase the content of glucosinolates in plants, and have application prospects in cultivating and improving high-quality cruciferous plant varieties, especially cultivating kale and mustard varieties with high glucosinolate content and resistance to bolting.

[0194] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of BoaVRN1 gene or BjuVRN1 gene in regulating the glucosinolate content and bolting and flowering time in plants, characterized in that: The nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2; and the plant is kale or mustard.

2. Use of a protein encoded by the BoaVRN1 gene or a protein encoded by the BjuVRN1 gene in regulating the glucosinolate content and the bolting and flowering time in a plant, characterized in that: The amino acid sequence of the protein encoded by the BoaVRN1 gene is shown in SEQ ID NO.3; the amino acid sequence of the protein encoded by the BjuVRN1 gene is shown in SEQ ID NO.4; and the plant is kale or mustard.

3. Application of a biomaterial edited by the BoaVRN1 gene or a biomaterial edited by the BjuVRN1 gene in regulating the glucosinolate content and the bolting and flowering time in a plant, characterized in that: The nucleotide sequence of the BoaVRN1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown as SEQ ID NO.2; and the plant is kale or mustard.

4. The use according to claim 3, characterized in that: The biological material includes a recombinant vector or a recombinant bacterium.

5. A method for increasing the glucosinolate content in a plant, characterized in that: The method comprises the steps of reducing the BoaVRN1 gene or BjuVRN1 gene in the plant and increasing the content of glucosinolate in the plant; the nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2; and the plant is kale or mustard.

6. A method for cultivating plants with high glucosinolate content and resistance to bolting, characterized in that: The method comprises the steps of reducing the BoaVRN1 gene or BjuVRN1 gene in the plant to obtain a plant with high glucosinolate content and resistance to bolting; the nucleotide sequence of the BoaVRN1 gene is shown in SEQ ID NO.1; the nucleotide sequence of the BjuVRN1 gene is shown in SEQ ID NO.2; and the plant is kale or mustard.

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