Site-directed mutagenesis arabidopsis cryptochrome gene as well as encoding protein and application thereof

Abstract

The invention provides a site-directed mutagenesis arabidopsis cryptochrome gene as well as an encoded protein and application thereof. Compared with the prior art, the site-directed mutagenesis arabidopsis cryptochrome gene is characterized in that a target gene CRY2 sequence is obtained and is connected with a pJL-blue plasmid to construct a recombinant plasmid pJL-blue-CRY2; the method comprises the following steps: designing a mutation primer, and carrying out site-directed mutagenesis by using a recombinant plasmid, so as to construct a mutant plasmid pJL-blue-D392N. The method comprises the following steps: preparing a mutant plasmid and an expression plasmid, then mixing the mutant plasmid and the expression plasmid into a reaction system, recombining a target gene onto an expression plasmid vector PFK272 under the action of an exogenous LR recombinase, and transforming the mutant expression plasmid PFK272-D392N into an arabidopsis thaliana plant to construct a transgenic arabidopsis thaliana plant. Compared with the prior art, the transgenic plant D392N expressed by the invention can accelerate the flowering of arabidopsis thaliana, and the advanced efficiency is about 25%. On the other hand, the number of rosette leaves is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, relates to the field of gene and protein engineering technology, and specifically relates to a site-directed mutated Arabidopsis cryptochrome gene, its encoded protein and application. Background Art

[0002] Cryptochromes, a class of photoreceptors that mediate light responses in plants, are found in algae, mosses, ferns, and seed plants. Cryptochromes primarily function by transducing signals dependent on light conditions and interacting with other light-responsive proteins. The function of cryptochromes in Arabidopsis thaliana is currently best understood. Two cryptochrome genes, cry1 and cry2, have been identified in the plant, with the CRY2 protein, encoded by cry2, primarily regulating flowering. Furthermore, the CO (constans) and FT (flowering locus T) genes in Arabidopsis thaliana are key regulators of flowering in response to photoperiod, integrating light signals and the endogenous circadian clock in their expression. CO is a zinc-finger transcription factor that regulates flowering time primarily by activating the downstream gene FT. FT encodes florigen, a protein central to floral initiation. This long-range signaling molecule migrates from leaves through vascular tissue to the apical meristem, promoting the development of the floral meristem and initiating flowering.

[0003] Light-activated CRY2 interacts with the transcription factor CIB1 (cryptochrome-interacting bHLH 1), directly activating the transcription of the FT gene. FT protein then travels through the phloem to the shoot apical meristem, promoting the transcription of multiple floral tissue signature genes. Simultaneously, as an E3 ubiquitin ligase, CRY2 interacts with constitutive lyphotomorphogenic 1 (COP1) and suppressor of phytochrome A 1 (SPA1), directly participating in the degradation of numerous transcription factors, including CO protein. When oligomerized CRY2 recruits the COP1-SPA1 complex, it inhibits COP1's ubiquitination, thereby preventing CO protein degradation. CO, as the primary transcriptional activator of the FT gene, increases with CO protein stability, leading to increased FT transcription and flowering. Therefore, regardless of which signaling pathway is dominant, cryptochrome protein ultimately influences the transcription of downstream FT genes, regulating flowering time.

[0004] In agriculture, flowering is a particularly relevant trait that determines the timing of fruit and seed production. Determining the correct flowering time is extremely important for plants, ensuring the inheritance of their genes in future populations, and is crucial for crop yield, quality, and adaptability. Currently, most of the knowledge about the mechanism of action and signaling pathways of the photoperiod pathway comes from the model plant Arabidopsis thaliana. In particular, in-depth research on cryptochrome-mediated photoperiod regulation of flowering is of great reference significance for understanding the effects of flowering time on other plants, which suggests that cryptochrome can be used as a molecular target for crop improvement. Therefore, designing and modifying plant cryptochrome proteins can provide a theoretical basis for resolving the contradiction between early maturity and high yield, and provide an effective approach for the molecular design and breeding of high-yield and high-quality crop varieties. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a site-directed mutant Arabidopsis cryptochrome gene.

[0006] The second purpose of the present invention is to provide a site-directed mutation of the Arabidopsis cryptochrome gene encoding protein.

[0007] The third object of the present invention is to provide an expression vector containing a site-directed mutation of the Arabidopsis cryptochrome gene.

[0008] A fourth object of the present invention is to provide a host containing a site-directed mutant of the Arabidopsis cryptochrome gene.

[0009] A fifth object of the present invention is to provide a plant containing a site-directed mutation of the Arabidopsis cryptochrome gene.

[0010] The sixth object of the present invention is to provide an application of a site-directed mutated Arabidopsis cryptochrome gene for regulating the flowering cycle of Arabidopsis.

[0011] The specific technical solutions of the present invention are as follows:

[0012] The present invention provides a site-directed mutation of the Arabidopsis cryptochrome gene, the gene sequence of which is shown in SEQ ID NO: 1.

[0013] AAGATGGACAAAAAGACTATAGTTTGGTTTAGAAGAGACCTAAGGATTGAGGATAATCCTGCATTAGC

[0014] AGCAGCTGCTCACGAAGGATCTGTTTTTCCTGTCTTCATTTGGTGTCCTGAAGAAGAAGGACAGTTTT

[0015] ATCCTGGAAGAGCTTCAAGATGGTGGATGAAACAATCACTTGCTCACTTATCTCAATCCTTGAAGGCT

[0016] CTTGGATCTGACCTCACTTTAATCAAAACCCACAACACGATTTCAGCGATCTTGGATTGTATCCGCGT

[0017] TACCGGTGCTACAAAAGTCGTCTTTAACCACCTCTATGATCCTGTTTCGTTAGTTCGGGACCATACCG

[0018] TAAAGGAGAAGCTGGTGGAACGTGGGATCTCTGTGCAAAGCTACAATGGAGATCTATTGTATGAACCG

[0019] TGGGAGATATACTGCGAAAAGGGCAAACCTTTTACGAGTTTCAATTCTTACTGGAAGAAATGCTTAGA

[0020] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0021] CGATTTGGGCGTGTTCGATTGAAGAACTAGGGCTGGAGAATGAGGCCGAGAAACCGAGCAATGCGTTG

[0022] TTAACTAGAGCTTGGTCTCCAGGATGGAGCAATGCTGATAAGTTACTAAATGAGTTCATCGAGAAGCA

[0023] GTTGATAGATTATGCAAAGAACAGCAAGAAAGTTGTTGGGAATTCTACTTCACTACTTTCTCCGTATC

[0024] TCCATTTCGGGGAAATAAGCGTCAGACACGTTTTCCAGTGTGCCCGGATGAAACAAATTATATGGGCA

[0025] AGAGATAAGAACAGTGAAGGAGAAGAAAGTGCAGATCTTTTTCTTAGGGGAATCGGTTTAAGAGAGTA

[0026] TTCTCGGTATATATGTTTCAACTTCCCGTTTACTCACGAGCAATCGTTGTTGAGTCATCTTCGGTTTT

[0027] TCCCTTGGGATGCTGATGTTGATAAGTTCAAGGCCTGGAGACAAGGCAGGACCGGTTATCCGTTGGTG

[0028] GATGCCGGAATGAGAGAGCTTTGGGCTACCGGATGGATGCATAACAGAATAAGAGTGATTGTTTCAAG

[0029] CTTTGCTGTGAAGTTTCTTCTCCTTCCATGGAAATGGGGAATGAAGTATTTCTGGGATACACTTTTGG

[0030] ATGCTGATTTGGAATGTAATATCCTTGGCTGGCAGTATATCTCTGGGAGTATCCCCGATGGCCACGAG

[0031] CTTGATCGCTTGGACAATCCCGCGTTACAAGGCGCCAAATATGACCCAGAAGGTGAGTACATAAGGCA

[0032] ATGGCTTCCCGAGCTTGCGAGATTGCCAACTGAATGGATCCATCATCCATGGGACGCTCCTTTAACCG

[0033] TACTCAAAGCTTCTGGTGTGGAACTCGGAACAAACTATGCGAAACCCATTGTAGACATCGACACAGCT

[0034] CGTGAGCTACTAGCTAAAGCTATTTCAAGAACCCGTGAAGCACAGATCATGATCGGAGCAGCACCTGA

[0035] TGAGATTGTAGCAGATAGCTTCGAGGCCTTAGGGGCTAATACATTAAAGAACCTGGTCTTTGCCCAT

[0036] CTGTGTCTTCTAATGACCAACAAGTACCTTCGGCTGTTCGTTACAACGGGTCAAAGAGAGTGAAACCT

[0037] GAGGAAGAAGAAGAGAGAGACATGAAGAAATCTAGGGGATTCGATGAAAGGGAGTTGTTTTCGACTGC

[0038] TGAATCTTCTTCTTCTTCGAGTGTGTTTTTCGTTTCGCAGTCTTGCTCGTTGGCATCAGAAGGGAAGA

[0039] ATCTGGAAGGTATTCAAGATTCATCTGATCAGATTACTACAAGTTTGGGAAAAAATGGTTGCAAATGA.

[0040] The present invention provides a site-directed mutated Arabidopsis cryptochrome gene encoding protein, whose amino acid sequence is shown in SEQ ID NO: 2, specifically:

[0041] KMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKA

[0042] LGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEP

[0043] WEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNAL

[0044] LTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWA

[0045] RDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLV

[0046] DAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECNILGWQYISGSIPDGHE

[0047] LDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTA

[0048] RELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK.

[0049] The present invention provides an expression vector containing a site-directed mutated Arabidopsis cryptochrome gene, wherein the expression vector is a recombinant plasmid containing the site-directed mutated Arabidopsis cryptochrome gene;

[0050] The present invention provides a host containing a site-directed mutated Arabidopsis cryptochrome gene, wherein the host is a recombinant engineered bacterium containing a site-directed mutated Arabidopsis cryptochrome gene, and the recombinant engineered bacterium is an Escherichia coli containing an expression vector for the site-directed mutated Arabidopsis cryptochrome gene;

[0051] The present invention provides a plant containing a site-directed mutant Arabidopsis cryptochrome gene, wherein the plant is Arabidopsis thaliana and contains an expression vector of the Arabidopsis thaliana cryptochrome gene containing a site-directed mutation;

[0052] The present invention provides a method for constructing a plant containing a site-directed mutant of an Arabidopsis cryptochrome gene, specifically comprising:

[0053] The target gene cry2 was cloned in vitro using primers designed from total RNA extracted from Arabidopsis thaliana plants and reverse transcribed into a cDNA template. The target gene cry2 was then ligated with the pJL-blue plasmid to construct the recombinant plasmid pJL-blue-CRY2. Mutation primers were designed and site-directed mutagenesis was performed using the recombinant plasmid to construct the mutant recombinant plasmid pJL-blue-D392N. The mutant recombinant plasmid was then combined with the expression plasmid PFK272 by Gateway homologous recombination to construct the PFK272-D392N recombinant plasmid for expression of cryptochrome protein in Arabidopsis thaliana. The PFK272-D392N recombinant plasmid was transformed into Agrobacterium, which was then used to infect Arabidopsis inflorescences for transgenic purposes to obtain Arabidopsis seeds containing the mutant gene. The transgenic Arabidopsis plants were cultured at 20°C with 16 hours / day of light to obtain transgenic mutant plants D392N.

[0054] The sequence of the mutation primer is:

[0055] D392N F:5'-TTGGAATGT AAT ATCCTTGGCTGGCAGTATAT-3′, the sequence of which is shown in SEQ ID No. 3;

[0056] D392N R:5'–CCAAGGAT ATT ACATTCCAAATCAGCATCC-3', the sequence of which is shown in SEQ ID No. 4;

[0057] The invention provides an application of a site-directed mutant Arabidopsis cryptochrome gene for regulating the flowering cycle of Arabidopsis, accelerating the flowering of Arabidopsis, and achieving an earlier flowering cycle than that of transgenic plants expressing the wild-type protein, with a time difference of 7±2 days.

[0058] The application of the site-directed mutated Arabidopsis cryptochrome gene provided by the present invention can also be used to reduce the number of rosette leaves of Arabidopsis.

[0059] The application of the Arabidopsis cryptochrome gene with a site-directed mutation provided by the present invention can also improve the expression level of the Arabidopsis floral gene FT.

[0060] In this invention, a site-directed mutagenesis method is used to modify proteins: through polymerase chain reaction (PCR), mutagenesis primers are used to introduce desired changes (including base additions, deletions, and point mutations) into a target DNA fragment (plasmid, genome). Specifically, the method involves obtaining a gene sequence from a gene library, obtaining the target gene CRY2 sequence, and ligating it with the pJL-blue plasmid to construct the recombinant plasmid pJL-blue-CRY2. Mutagenesis primers are designed, and site-directed mutagenesis is performed using the recombinant plasmid to construct the mutant plasmid pJL-blue-D392N. The mutant plasmid and the expression plasmid are then mixed in a reaction system. Under the action of exogenous LR recombinase, the target gene is recombined into the expression plasmid vector PFK272. The mutant expression plasmid PFK272-D392N is then transformed into Arabidopsis plants to construct transgenic Arabidopsis plants.

[0061] Compared to existing technologies, transgenic plants expressing D392N can accelerate flowering in Arabidopsis, with the flowering cycle significantly earlier than that of transgenic plants expressing the wild-type protein, with a time difference of approximately 7±2 days and an efficiency of approximately 25%. Furthermore, the number of rosette leaves at the base of the plant can also be used as an indicator of flowering time. Typically, early-flowering Arabidopsis plants also have fewer rosette leaves. The basal rosette leaves of transgenic plants show that the number of rosette leaves in D392N is significantly fewer than that of wild-type controls, reducing the number of rosette leaves to 6±1. Furthermore, qRT-PCR results show that the expression level of the floral gene FT in transgenic D392N plants is significantly upregulated compared to wild-type plants, increasing to 1.38±0.02. Therefore, the D392N mutant enzyme is beneficial for regulating the flowering cycle in Arabidopsis and may provide guidance for improving flowering time traits in other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the electrophoresis diagram of the D392N mutant gene in Example 1;

[0063] M is a low molecular weight standard DNA, lanes 1-2 are all mutant genes of D392N; D392N1 and D392N2 refer to two mutant genes constructed in parallel;

[0064] Figure 2 This is a comparison of the flowering phenotypes of the wild type and transgenic Arabidopsis thaliana prepared in Example 2;

[0065] The figure shows the growth of Arabidopsis plants at about 30 days old. Arabidopsis growth: cry2 is a mutant plant with the cry2 gene knocked out, serving as a negative control; WT (wild type) represents wild-type Arabidopsis, serving as a wild-type control; CRY2 and D392N are transgenic plants formed by transferring the cry2 gene and mutant gene D392N into cry2, respectively; D392N-1 and D392N-2 are two transgenic plant lines formed by incorporating a plasmid vector containing two parallel genes into Arabidopsis plants.

[0066] Figure 3 This is a comparison of flowering time between wild-type and transgenic Arabidopsis thaliana prepared in Example 2;

[0067] cry2 is a mutant plant with the Arabidopsis cry2 gene knockout, serving as a negative control; WT (wild type) represents wild-type Arabidopsis, serving as a wild-type control; CRY2 and D392N are transgenic plants formed by transferring the cry2 gene and mutant gene D392N into cry2, respectively;

[0068] Figure 4 This is a comparison of the basal rosette leaves of wild-type and transgenic Arabidopsis thaliana prepared in Example 3;

[0069] cry2 is a mutant plant with the Arabidopsis cry2 gene knockout, serving as a negative control; WT (wild type) represents wild-type Arabidopsis, serving as a wild-type control; CRY2 and D392N are transgenic plants formed by transferring the cry2 gene and mutant gene D392N into cry2, respectively;

[0070] Figure 5 This is a comparison chart of the transcription levels of the floral gene FT between the wild type and transgenic Arabidopsis thaliana prepared in Example 4;

[0071] cry2 is a mutant plant with the Arabidopsis cry2 gene knockout, used as a negative control; WT (wile type) represents wild-type Arabidopsis, used as a wild-type control; CRY2 and D392N are transgenic plants formed by transferring the cry2 gene and mutant gene D392N into cry2, respectively. DETAILED DESCRIPTION

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0073] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0074] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0075] The solution concentration percentages involved in the present invention are all volume concentrations.

[0076] Example 1

[0077] Obtaining the mutant D392N gene and constructing the expression vector:

[0078] 1.1 Obtaining total RNA from wild-type Arabidopsis tissue:

[0079] Collect several leaves (no more than 200 mg) from Arabidopsis thaliana Col-0 seedlings and extract total RNA from Arabidopsis thaliana. The specific steps are as follows:

[0080] (1) Preparation: gun box, small steel balls, Novozyme isolater reagent, 1.5 mL centrifuge tubes and 2 mL centrifuge tubes, tin foil, medicine spoon, all consumables are RNase-free;

[0081] (2) Take an appropriate amount of fresh Arabidopsis leaves (no more than 200 mg), collect them in a 2 mL centrifuge tube, add small steel balls, and then quickly freeze them in liquid nitrogen;

[0082] (3) Grind the quick-frozen leaf tissue thoroughly using a metal grinder;

[0083] (4) Add 1 mL of Novozymes' isolater reagent and vortex to mix.

[0084] (5) After standing on ice for 5 min, centrifuge at 10,000 g for 10 min at 4°C;

[0085] (6) Take 700 μL of supernatant, add 200 μL of chloroform, and vortex to mix;

[0086] (7) After standing on ice for 10 min, centrifuge at 10,000 g for 10 min at 4°C;

[0087] (8) Take 600 μL of supernatant and add an equal volume of isopropanol and mix well;

[0088] (9) After standing on ice for 10 min, centrifuge at 10,000 g for 10 min at 4°C;

[0089] (10) Discard the supernatant, add 1 mL of 75% ethanol solution for washing, and centrifuge at 10,000 g for 5 min at 4°C;

[0090] (11) Discard the supernatant completely, dry the precipitate at room temperature, and add 50 μL of RNase-free deionized water to dissolve it to obtain total RNA;

[0091] (12) Use a microplate reader to monitor RNA concentration and the 260 / 280 nm ratio.

[0092] 1.2 Reverse transcription of Arabidopsis total RNA into cDNA

[0093] The total RNA extracted in 1.1 above was used as a template for reverse transcription of RNA. The Arabidopsis cDNA template was synthesized using the HiScript III RT SuperMix for qPCR kit from Novozymes. The specific steps are as follows:

[0094] (1) Remove gDNA and add samples according to the following system:

[0095] 4× gDNA wiper mix 4 μL;

[0096] RNA 2 μg;

[0097] Add deionized water to 16 μL;

[0098] Reaction conditions: incubate at 42°C for 2 min.

[0099] (2) Reverse transcription: After the previous step, add 4 μL of HiScript III qRT SuperMix;

[0100] (3) Incubate at 37°C for 15 min and heat activate at 85°C for 5 sec;

[0101] (4) Add 80 μL of RNase-free deionized water to obtain the Arabidopsis cDNA template.

[0102] 1.3 Obtaining the wild-type Arabidopsis cryptochrome gene cry2 and constructing the recombinant plasmid

[0103] The Arabidopsis thaliana cryptochrome 2 gene sequence (cry2) was downloaded from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov) and primers were designed. Using the synthesized Arabidopsis cDNA template, a PCR reaction (polymerase chain reaction) was performed to obtain the Arabidopsis thaliana cryptochrome gene cry2. The entry plasmid pJL-blue and the cry2 gene were double-digested using the restriction endonucleases NdeI and XhoI. The plasmid and gene fragments were then ligated using T4 ligase. The ligated system was transformed into Escherichia coli DH5α and screened on ampicillin-resistant plates. Positive clones were obtained and sent to Chuzhou General Biological Company for testing and sequencing confirmation. The DH5α strain was expanded and the successfully constructed entry recombinant plasmid pJL-blue-CRY2 was extracted.

[0104] 1.4 Obtaining the Arabidopsis cryptochrome mutant gene D392N and constructing the entry plasmid

[0105] Using the existing entry recombinant plasmid pJL-blue-CRY2 as a template, a pair of mutation primers were designed using the two-primer method to perform rapid site-directed mutagenesis of the target gene. The mutation primers are:

[0106] D392N F: Its sequence is shown in SEQ ID No. 3;

[0107] D392N R: Its sequence is shown as SEQ ID No. 4;

[0108] The nucleotide sequence of the mutation site in the primer is underlined above;

[0109] Principle of rapid site-directed mutagenesis: The PCR reaction system contains the wild-type recombinant plasmid pJL-blue-CRY2. After the reaction is completed, an extension product containing the mutant gene D392N is generated. Then, the DpnⅠ restriction endonuclease is used for the digestion process. Since the original template plasmid is derived from E. coli, it has been modified by methylation of the dam gene in the bacteria and is sensitive to DpnⅠ and is chopped up (DpnⅠ recognition sequence is methylated G m ATC), and the mutant plasmid synthesized by in vitro PCR reaction is not cut because it is not methylated, and the mutant plasmid clone can be obtained.

[0110] The PCR reaction system (60 μl) is as follows:

[0111]

[0112] The PCR reaction conditions are shown in Table 1.

[0113] Table 1 PCR reaction conditions in step 1.4

[0114]

[0115]

[0116] The enzyme digestion reaction system (20 μl) in step 1.4 is as follows:

[0117] Dpn I 2 μl;

[0118] 10× FastDigest Buffer 2 μl;

[0119] Plasmid pJL-blue-D392N 16 μl.

[0120] The digestion reaction conditions in step 1.4 are as follows:

[0121] 37℃ 90min;

[0122] 80℃ 2min.

[0123] Specifically, PCR amplification was performed using the pJL-blue-CRY2 recombinant plasmid as a template. The reaction conditions were 95°C pre-denaturation for 5 minutes, 94°C denaturation for 45 seconds, 58°C annealing for 45 seconds, and 72°C extension for 90 seconds, for 30 cycles, followed by full extension at 72°C for 5 minutes. After Dpn I digestion, the mutant plasmid pJL-blue-D392N was transformed into E. coli DH5α competent cells. Positive clones were screened for ampicillin resistance and sent to Chuzhou General Biotechnology Co. for sequencing and verification. Sequence alignment confirmed the successful construction of the D392N mutant entry plasmid, and the host cells with the successful mutation were preserved.

[0124] Figure 1 This is the electrophoresis diagram of the D392N mutant gene in Example 1; the D393N gene fragment was amplified by in vitro PCR, and the second band is the target band, which is about 1800 bp.

[0125] 1.5 Construction of expression plasmids for Arabidopsis cryptochrome wild-type CRY2 and mutant gene D392N

[0126] This embodiment adopts traditional Gateway technology to construct transgenic vector, including entry vector (Entry Vector) and expression vector (Destination Vector). Entry plasmid pJL-blue contains two recombination sites of attL1 and attL2. Expression plasmid PFK272, containing Ubiquitin-10 promoter, has attR1 and attR2 recombination sites and ccdB lethal gene at the same time. The successfully verified pJL-blue-CRY2 and pJL-blue-D392N entry plasmids and empty expression plasmid PFK272 are mixed into a reaction system. Using the GatewayLR cloning kit of amb company (Aiming Biotechnology Co., Ltd.), under the action of exogenous LR recombinase, the target gene CRY2 will be recombined into the expression vector, and the ccdB gene therein will be recombined into the entry vector. At this time, the system contains four plasmids: unrecombined entry plasmid, unrecombined expression plasmid, recombined entry plasmid, and recombined expression plasmid. Spectinomycin (100 μg / mL) was used for resistance screening. Positive clones were extracted and transformed into E. coli to obtain expression recombinant plasmids PFK272-CRY2 and PFK272-D392N, which were then sent to General Biotechnology (Chuzhou) for sequencing verification.

[0127] Recombination reaction system (5 μL):

[0128] 2×LR enzyme 2.5 μL;

[0129] Entry vector 1.5 μL;

[0130] Expression vector 1.0 μL;

[0131] Reaction conditions: incubate at 25°C for 5-16 hours.

[0132] Example 2

[0133] Cultivation of transgenic Arabidopsis and statistics of flowering time traits:

[0134] 2.1 Arabidopsis seed disinfection and cultivation

[0135] First, place seeds from the wild-type Arabidopsis strain col-0 and the cry2 knockout mutant cry2 (cry2 is a knockout mutant of the Arabidopsis cry2 gene, obtained through conventional chemical mutagenesis) in a drying oven for approximately 4-5 days. Then, add disinfectant (70% ethanol solution, 0.5% Tween20) to the tube containing the seeds and wash for 10 minutes. Be careful not to exceed 1 / 5 of the volume of the disinfectant to ensure adequate disinfection. Discard the supernatant and then wash twice with 95% ethanol, each for approximately 5 minutes. Finally, spread the seeds flat on filter paper in a clean bench, dry them, and return them to the tube.

[0136] Then prepare the MS medium (1 / 2MS2.2g / L, agar 8‰) specially for Arabidopsis seeds. After the MS medium plate is poured, culture the seeds. Use a small piece of sterile gauze to wrap the mouth of the centrifuge tube, so that the seeds are gently sprinkled on the surface of the MS plate and evenly distributed. Wrap the gap of the plate with sealing film, make detailed marks, and place it on a culture rack with suitable light, long day (16h LD), and culture at a constant temperature of 20℃. Observe the germination of Arabidopsis seeds every day. After about 4-5 days, start preparing for transplanting.

[0137] Before transplanting the seedlings, first prepare the nutrient soil: Purchase high-quality imported nutrient soil, place an appropriate amount in a large basin, and begin manually kneading the soil. Rub the soil until it is fine and soft, removing any hard impurities. Then, add an appropriate amount of water and stir, ensuring the newly prepared soil is sufficiently moist. Next, divide the soil from the large basin into several smaller pots, ensuring it is sufficiently soft. Now, begin transferring the seedlings from the culture medium to the smaller pots, carefully covering the seedling's roots with soil, leaving only the open cotyledons exposed. After transplanting, water the soil with an adequate amount of nutrient solution. Check the seedlings' progress regularly daily. Once the seedlings have bolted and developed sufficient inflorescences, begin preparing for the transgenic stage.

[0138] 2.2 Construction of transgenic Arabidopsis

[0139] First, the Agrobacterium transformation process was carried out. The competent Agrobacterium was thawed on ice, and 2 μl of PFK272-CRY2 and PFK272-D392N plasmids were added respectively, mixed gently, and placed on ice for 20 minutes; placed in a 37°C water bath for 4 minutes; quick-frozen in liquid nitrogen for 1 minute; added 100 μl of LB liquid medium, and cultured on a shaking table for 2 hours at 28°C and 200 rpm; screened according to conventional methods using LB solid medium with the corresponding antibiotics Cam (chloramphenicol), Tet (tetracyclines, tetracycline antibiotics), Kan (kanamycin), and Spe (spectinomycin) and then preserved.

[0140] Streak the Agrobacterium containing the recombinant expression plasmid onto four types of MS medium (Cam, Tet, Kan, Spe) and incubate at 28°C for about 2 days. Pick a single colony into a 5ml test tube and incubate at 28°C for about one day. Then, take 500μl of the bacterial solution and transfer it to 100ml of LB medium and incubate until the OD 600 About 0.8. Then collect the bacteria by centrifugation at 5000rpm at room temperature. At this time, suspend the bacteria in the transformation solution prepared in advance (the transformation solution needs to be stirred for 2 hours in advance; the formula is 1 / 2MS 0.44g; sucrose 20g; Silwet-77 100μl; MES 0.1g; pH 5.7). Carefully pour the Agrobacterium transformation solution into a large culture dish. At this time, remove the excess fresh fruit clips on the bolting seedlings to avoid interference in the transgenic experiment. After that, immerse the inflorescence of the seedling in the culture dish and allow the bacterial solution to fully contact the inflorescence for about 30 seconds each time. Then place several pots of Arabidopsis horizontally in a low light place, let it stand for 24 hours, and then put it back under long-day conditions.

[0141] After Agrobacterium infection of the inflorescence, wait for the Arabidopsis to mature and bear fruit. Seeds from different T0-generation transgenic seedlings are collected. The above process is repeated, and seeds from the T0-generation that have successfully undergone transgenic transformation are replanted and screened. Due to the low efficiency of Agrobacterium transgenesis at this stage, it is necessary to screen positive seedlings on resistant plates and then wait for the seedlings to grow to a sufficient size. After obtaining a portion of the tissue, the genome is extracted using the CTAB method. Gene cloning and PCR are performed to determine if the transgene was successful, and seedlings that have been successfully sequenced are retained. This design results in the cultivation of transgenic plants expressing Arabidopsis CRY2, D392N. Various phenotypes, including the flowering cycle and basal rosette leaves, are observed and analyzed to analyze the apparent differences in the transgenic plants.

[0142] The CTAB method (cetyltrimethylammonium bromide method) was used to extract the genome, and fresh Arabidopsis leaves were selected for genotyping analysis:

[0143] (1) Take an appropriate amount of Arabidopsis leaves and grind them using a tissue grinder;

[0144] (2) Add 500 μL of CTAB solution and mix well. Place at 65°C for 1 hour. Preparation method of CTAB solution (500 mL):

[0145]

[0146] (3) Add 500 μL of chloroform / isoamyl alcohol (volume ratio of 24:1), mix thoroughly, and centrifuge at 10,000 rpm for 10 min.

[0147] (4) Take the supernatant, add an equal volume of isopropanol, mix well, and let stand for 5 minutes;

[0148] (5) Discard the supernatant, add 600 μL of 75% ethanol for washing, and centrifuge at 10,000 rpm for 10 min;

[0149] Completely remove the supernatant and add 100 μL of deionized water to dissolve.

[0150] 2.3 Flowering time phenotype statistics of transgenic Arabidopsis

[0151] It is known that the classic physiological function of AtCRY2 is to control flower development and flowering cycle. Therefore, the present invention analyzes the physiological role of plant cryptochrome 392 site based on the flowering cycle phenotype of Arabidopsis thaliana. Under the conditions of constant temperature of 20℃ and long photoperiod of 16h, the wild-type Arabidopsis thaliana Col-0 line usually flowers in about 30 days. Figure 2 、 Figure 3 , cry2 is a knockout mutant, serving as a negative control; WT represents wild-type Col-0, serving as a blank control; CRY2 represents a transgenic line derived from cry2 and harboring wild-type AtCRY2, serving as a positive control. As can be seen, the D392N mutant flowers significantly earlier than the transgenic CRY2 plant, with a time difference of approximately 7 ± 2 days. This suggests that under natural conditions, the D392N mutation significantly regulates the flowering cycle of cryptochromes, promoting earlier flowering.

[0152] AtCRY2 and CRY2 are synonymous, referring to transgenic plants containing the cry2 gene. cry2 stands for the cryptochrome 2 gene, and CRY2 refers to the plasmid vector containing this gene, or a transgenic Arabidopsis plant.

[0153] In the present invention, D392N refers to the mutation site; D392N1 and D392N2 refer to the two parallel mutant genes constructed, and the recombinant plasmids containing these two parallel mutant genes; D392N-1 and D392N-2 are two transgenic plant lines formed after the plasmid vectors containing the two parallel genes were genetically modified into Arabidopsis plants.

[0154] Example 3

[0155] Statistics of basal rosette leaf traits of transgenic Arabidopsis thaliana:

[0156] During the flowering process, the number of rosette leaves at the base of Arabidopsis is also related to the flowering cycle. Generally, plants that flower earlier will have fewer rosette leaves. Conversely, the later the flowering, the more rosette leaves there are. Therefore, the number of rosette leaves is also used as an indicator to compare the phenotypic differences in the flowering cycle of wild-type Arabidopsis and its transgenic plants. Figure 4The number of rosette leaves in wild-type Arabidopsis thaliana plants (WT) was 8.5±0.5, while the number of rosette leaves in cry2 knockout mutant plants was 12.5±0.5. The number of rosette leaves in wild-type cry2 transgenic plants (CRY2) was 11±1, and the number of rosette leaves in D393N transgenic plants (D393N) carrying the D392N mutant gene was 6±1. By counting the basal rosette leaves of the control plants, it can be seen that the number of rosette leaves in the transgenic D392N plants is far less than that in the CRY2 control. In summary, the D392N mutation significantly affects the function of the cryptochrome protein, promoting the continued regulation of the flowering process by cryptochrome and causing the plants to flower earlier.

[0157] Example 4

[0158] Detection of the transcription level of the FT flowering gene in transgenic Arabidopsis thaliana:

[0159] 4.1 Total RNA extraction from transgenic Arabidopsis plants

[0160] The specific method was the same as that in Example 1, and total RNA was obtained from the control group and the transgenic plants.

[0161] 4.2qRT-PCR experiments

[0162] (1) Using ChamQ Universal SYBR qPCR Master Mix reagent from Novozymes, the sample loading system is as follows:

[0163]

[0164] Primer 1: 5′-AGAAGACTTTAGATGGCTTCTT-3′, the sequence of which is shown in SEQ ID No. 5;

[0165] Primer 2: 5′-TTATCGCATCACACACTATATAAG-3′, the sequence of which is shown in SEQ ID No. 6;

[0166] (2) The program was run using a Roche PCR instrument. The program used a two-step method: pre-denaturation at 95°C for 2 min → denaturation at 95°C for 10 s → annealing and extension at 60°C for 30 s, for a total of 45 cycles. The melting curve stage used the program provided by the Roche PCR instrument.

[0167] (3) Data processing and analysis: Data analysis and processing were performed using Excel, and statistical graphs were drawn using Origin 8.5.

[0168] Cryptochromes are known to act on signaling pathways during flowering, influencing the transcriptional levels of the downstream floral genes, FT, ultimately regulating differences in flowering cycles. The signaling pathways through which AtCRY2 photoperiod regulates flowering typically involve two main mechanisms: 1. Blue light stimulates AtCRY2 to interact with CIB1, directly activating the transcription of the FLOWERING LOCUST T (FT) gene, which encodes the core regulator of floral initiation, or florigen. This gene then migrates from leaves to the apical meristem to promote floral meristem development. 2. COP1, an E3 ubiquitin ligase, directly participates in the degradation of numerous transcription factors, including the CO protein. When blue light activates AtCRY2, the photoactivated CRY2 interacts with SPA1 and COP1 to form a complex that deubiquitinates COP1, thereby inhibiting CO degradation. CO, as the primary transcriptional activator of the FT gene, increases with CO abundance, leading to increased FT transcription levels and ultimately promoting flowering. Therefore, regardless of which signaling pathway is dominant, cryptochromes ultimately influence the transcriptional levels of the downstream FT gene, regulating the flowering cycle. In this example, in order to explore whether the phenotypic differences among different transgenic plants are associated with the altered cryptochrome function caused by the D392N mutation, the transcription level of the flowering-related gene FT was detected.

[0169] The experimental subjects were 14-day-old seedlings that had not yet flowered. Several leaf samples were collected and RNA was extracted and converted into cDNA for qRT-PCR to detect the expression level of the floral gene FT in transgenic plants. The col-0 material was used as the natural control group, cry2 as the negative control group, and various transgenic mutant materials as experimental groups. The qRT-PCR results showed that ( Figure 5 ), the expression levels of the FT gene in the experimental group D392N were upregulated relative to CRY, with T-test results showing a significant difference in FT gene expression in D932N. In wild-type Arabidopsis thaliana plants (WT), the cry2 knockout mutant, CRY2, a transgenic plant expressing the wild-type cry2 gene, and D393N, a transgenic plant expressing the D392N mutant, the FT gene expression levels were 1.04±0.41, 0.47±0.06, 1.15±0.22, and 1.38±0.02, respectively. D393N increased FT gene expression by 20% compared to CRY2.

[0170] This shows that, as previously discovered, the amino acid preference at the D392N site does affect the function of plant cryptochromes. Under natural conditions, the selection of polar amino acids is more conducive to plant growth and development, and thus adaptation to the environment.

[0171] The above embodiments are described to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A site-directed mutagenesis of an Arabidopsis cryptochrome gene, characterized in that: The sequence of the site-directed mutated Arabidopsis cryptochrome gene is shown in SEQ ID NO:

1.

2. A site-directed mutated Arabidopsis cryptochrome gene encoding protein according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

3. An expression vector containing a site-directed mutant of an Arabidopsis cryptochrome gene, characterized in that: The expression vector is a recombinant plasmid, which is a recombinant plasmid containing the above-mentioned site-directed mutated Arabidopsis cryptochrome gene.

4. A host containing a site-directed mutant of an Arabidopsis cryptochrome gene, characterized in that: The host is a recombinant engineered bacterium of Arabidopsis thaliana cryptochrome with a site-directed mutation, and the recombinant engineered bacterium is an Escherichia coli containing an expression vector of the Arabidopsis thaliana cryptochrome gene with a site-directed mutation.

5. A plant containing a site-directed mutation of an Arabidopsis cryptochrome gene, characterized in that: The plant is Arabidopsis thaliana and contains the expression vector of the Arabidopsis thaliana cryptochrome gene containing a site-directed mutation according to claim 3.

6. A method for constructing a plant containing a site-directed mutation of the Arabidopsis cryptochrome gene according to claim 5, specifically comprising: extracting total RNA from Arabidopsis plants, reverse transcribing a cDNA template, designing primers, cloning the target gene cry2 in vitro, and ligating the cDNA template with the pJL-blue plasmid to construct a recombinant plasmid pJL-blue-CRY2; designing mutation primers, performing site-directed mutagenesis using the recombinant plasmid to construct a mutant recombinant plasmid pJL-blue-D392N, and performing Gateway homologous recombination between the mutant recombinant plasmid and the expression plasmid PFK272 to construct a PFK272-D392N recombinant plasmid for expressing cryptochrome protein in Arabidopsis; transforming the PFK272-D392N recombinant plasmid into Agrobacterium, and then infecting Arabidopsis inflorescences with Agrobacterium to perform transgenic transgenesis to obtain Arabidopsis seeds containing the mutant gene; and culturing the transgenic Arabidopsis at 20°C and 16 hours / day of light to obtain a transgenic mutant plant D392N.

7. The construction method according to claim 6, characterized in that: The sequence of the mutation primer is: D392N F: Its sequence is shown in SEQ ID No. 3; D392N R: Its sequence is shown in SEQ ID No.

4.

8. A use of the Arabidopsis cryptochrome gene with site-directed mutation according to claim 1, characterized in that: Used to regulate the flowering cycle of Arabidopsis thaliana, accelerate the flowering of Arabidopsis thaliana, and the flowering cycle of transgenic plants is earlier than that of wild-type proteins, with a time difference of 7±2 days.

9. A use of the Arabidopsis cryptochrome gene with site-directed mutation according to claim 1, characterized in that: Used to reduce the number of rosette leaves in Arabidopsis thaliana, reducing the number of rosette leaves in Arabidopsis thaliana to 6±1.

10. A use of the Arabidopsis cryptochrome gene with site-directed mutation according to claim 1, characterized in that: Used to increase the expression level of the Arabidopsis floral gene FT to 1.38±0.02.

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