Arabidopsis thaliana cryptochrome gene for site-directed mutagenesis, and its encoded protein and application

Abstract

The application provides a site-directed mutant Arabidopsis cryptochrome gene and its coded protein and application, compared with the prior art, the application obtains the sequence of the target gene CRY2 and connects with a pJL-blue plasmid, constructs a recombinant plasmid pJL-blue-CRY2; designs a mutant primer, and uses the recombinant plasmid to carry out site-directed mutation, constructs a mutant plasmid pJL-blue-D392N. Then the mutant plasmid is mixed with an expression plasmid in a reaction system, under the action of an exogenous LR recombinase, the target gene is recombined to the expression plasmid vector PFK272, and the mutant expression plasmid PFK272-D392N is transformed into Arabidopsis plants, and a transgenic Arabidopsis plant is constructed. Compared with the prior art, the transgenic plant D392N expressed by the application can accelerate the flowering of Arabidopsis, and the efficiency is about 25%. On the other hand, the number of rosette leaves will also be less.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, relates to the field of gene and protein engineering, and particularly relates to a site-directed mutant Arabidopsis cryptochrome gene, a protein coded by the gene and application of the gene and the protein. BACKGROUND

[0002] Plant cryptochromes, as a class of light receptors mediating the light response process in plants, are distributed in algae, mosses, ferns, and seed plants. The main way of plant cryptochromes to perform functions is to transduce signals depending on light conditions and interact with other light-responsive proteins. The function of Arabidopsis cryptochromes is the most clear, and two cryptochrome genes cry1 and cry2 have been identified in Arabidopsis, in which the CRY2 protein encoded by cry2 mainly regulates the flowering process. In addition, the CO (constans) and FT (flowering locus T) genes in Arabidopsis are important regulatory factors for regulating flowering in response to photoperiod, and light signals and endogenous biological clocks are integrated in the expression changes of these genes. CO is a zinc finger transcription factor, and it mainly regulates flowering time by activating the expression of downstream gene FT. The latter can encode the core regulatory protein of floral initiation, florigen, as a long-distance signal molecule, migrates from plant leaves to the apical meristem through the vascular tissue, promotes the development of floral meristem, and initiates flowering.

[0003] Light-activated CRY2 interacts with the transcription factor CIB1 (cryptochrome-interacting bHLH 1, CIB1), directly activates the transcription of FT gene, and FT protein reaches the stem tip meristem through phloem, promotes the transcription of multiple flower tissue characteristic genes. At the same time, as an E3 ubiquitin ligase, constitutive lyphotomorphogenic 1 (COP1) and suppressor of phytochrome A (SPA1) interact and directly participate in the protein degradation process of many transcription factors such as CO protein. When oligomerized CRY2 recruits COP1-SPA1 to form a complex, the ubiquitination function of COP1 is inhibited, thereby relieving the degradation of CO protein. As the main transcription activator of FT gene, with the increase of the stability of CO protein, the transcription level of FT is also improved, and flowering is promoted. Therefore, regardless of which signal pathway is dominant, ultimately, the transcription level of downstream FT gene is affected by cryptochrome protein, and flowering time is regulated.

[0004] In agriculture, flowering is a particularly relevant trait that determines the timing of fruit and seed production. Determining the right time to flower is extremely important for plants to ensure that their genes are passed on in future generations, and is crucial for crop yield, quality and adaptability. Most of the current understanding of the mechanisms and signaling pathways of the photoperiod pathway comes from the model plant Arabidopsis thaliana, and in-depth studies of cryptochrome-mediated photoperiod regulation of flowering are of great reference significance for understanding the influence of flowering time in other plants, which implies 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 solving the contradiction between early maturity and high yield, and provide an effective way for molecular design and selection of high-yield and high-quality crop varieties. SUMMARY

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

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

[0007] The third purpose of the present application is to provide an expression vector containing a site-directed mutant Arabidopsis thaliana cryptochrome gene.

[0008] The fourth purpose of the present application is to provide a host containing a site-directed mutant Arabidopsis thaliana cryptochrome gene.

[0009] The fifth purpose of the present application is to provide a plant containing a site-directed mutant Arabidopsis thaliana cryptochrome gene.

[0010] The sixth purpose of the present application is to provide a site-directed mutant Arabidopsis thaliana cryptochrome gene for regulating the flowering period of Arabidopsis thaliana.

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

[0012] The site-directed mutant Arabidopsis thaliana cryptochrome gene provided by the present application has a gene sequence as shown in SEQ ID NO: 1, which is

[0013] AAGATGGACAAAAAGACTATAGTTTGGTTTAGAAGAGACCTAAGGATTGAGGATAATCCTGCATTAGC

[0014] AGCAGCTGCTCACGAAGGATCTGTTTTTCCTGTCTTCATTTGGTGTCCTGAAGAAGAAGGACAGTTTT

[0015] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0016] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0017] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0018] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0019] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0020] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0021] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0022] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0023] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0024] TATGTCGATTGAATCCGTTATGCTTCCTCCTCCTTGGCGGTTGATGCCAATAACTGCAGCGGCTGAAG

[0025] AGAGATAAGAACAGTGAAGGAGAAGAAAGTGCAGATCTTTTTCTTAGGGGAATCGGTTTAAGAGAGTA

[0026] TTCTCGGTATATATGTTTCAACTTCCCGTTTACTCACGAGCAATCGTTGTTGAGTCATCTTCGGTTTT

[0027] TCCCTTGGGATGCTGATGTTGATAAGTTCAAGGCCTGGAGACAAGGCAGGACCGGTTATCCGTTGGTG

[0028] GATGCCGGAATGAGAGAGCTTTGGGCTACCGGATGGATGCATAACAGAATAAGAGTGATTGTTTCAAG

[0029] CTTTGCTGTGAAGTTTCTTCTCCTTCCATGGAAATGGGGAATGAAGTATTTCTGGGATACACTTTTGG

[0030] ATGCTGATTTGGAATGTAATATCCTTGGCTGGCAGTATATCTCTGGGAGTATCCCCGATGGCCACGAG

[0031] CTTGATCGCTTGGACAATCCCGCGTTACAAGGCGCCAAATATGACCCAGAAGGTGAGTACATAAGGCA

[0032] ATGGCTTCCCGAGCTTGCGAGATTGCCAACTGAATGGATCCATCATCCATGGGACGCTCCTTTAACCG

[0033] TACTCAAAGCTTCTGGTGTGGAACTCGGAACAAACTATGCGAAACCCATTGTAGACATCGACACAGCT

[0034] CGTGAGCTACTAGCTAAAGCTATTTCAAGAACCCGTGAAGCACAGATCATGATCGGAGCAGCACCTGA

[0035] TGAGATTGTAGCAGATAGCTTCGAGGCCTTAGGGGCTAATACCATTAAAGAACCTGGTCTTTGCCCAT

[0036] CTGTGTCTTCTAATGACCAACAAGTACCTTCGGCTGTTCGTTACAACGGGTCAAAGAGAGTGAAACCT

[0037] GAGGAAGAAGAAGAGAGAGACATGAAGAAATCTAGGGGATTCGATGAAAGGGAGTTGTTTTCGACTGC

[0038] TGAATCTTCTTCTTCTTCGAGTGTGTTTTTCGTTTCGCAGTCTTGCTCGTTGGCATCAGAAGGGAAGA

[0039] ATCTGGAAGGTATTCAAGATTCATCTGATCAGATTACTACAAGTTTGGGAAAAAATGGTTGCAAATGA。

[0040] The application provides a above-mentioned site-directed mutant Arabidopsis cryptochrome gene encoding protein, and the amino acid sequence is shown as SEQ ID NO: 2, and the protein is specifically as follows:

[0041] KMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKA

[0042] LGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEP

[0043] WEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNAL

[0044] LTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWA

[0045] RDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLV

[0046] DAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECNILGWQYISGSIPDGHE

[0047] LDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTA

[0048] RELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK。

[0049] The application provides an expression vector of Arabidopsis thaliana cryptochrome gene containing a site-directed mutation, and the expression vector is a recombinant plasmid.

[0050] The application provides a host of Arabidopsis thaliana cryptochrome gene containing a site-directed mutation, and the host is a recombinant engineering bacterium of the site-directed mutation Arabidopsis thaliana cryptochrome.

[0051] The application provides a plant of Arabidopsis thaliana cryptochrome gene containing a site-directed mutation, and the plant is Arabidopsis thaliana and contains the expression vector of the Arabidopsis thaliana cryptochrome gene containing a site-directed mutation.

[0052] The application provides a construction method of a plant of Arabidopsis thaliana cryptochrome gene containing a site-directed mutation.

[0053] The cDNA template is reversely transcribed from total RNA extracted from Arabidopsis thaliana plants, primers are designed, and the target gene cry2 is cloned in vitro to obtain a recombinant plasmid pJL-blue-CRY2 by being connected with a pJL-blue plasmid; mutant primers are designed, site-directed mutagenesis is carried out by using the recombinant plasmid, a mutant recombinant plasmid pJL-blue-D392N is constructed, and the mutant recombinant plasmid is subjected to Gateway homologous recombination with an expression plasmid PFK272 to construct a PFK272-D392N recombinant plasmid for in vivo expression of cryptochrome protein of Arabidopsis thaliana; after the PFK272-D392N recombinant plasmid is transformed into Agrobacterium, the Arabidopsis thaliana inflorescences are infected by Agrobacterium, transgenic plants are obtained, and the mutant gene-containing Arabidopsis thaliana seeds are obtained; the transgenic Arabidopsis thaliana is cultured at 20 DEG C, 16 hours / day light, and the transgenic mutant plant D392N is obtained.

[0054] The sequence of the mutant primer is as follows:

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

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

[0057] The application of the site-directed mutant Arabidopsis thaliana cryptochrome gene provided by the application is used for regulating the flowering period of Arabidopsis thaliana, accelerating the flowering of Arabidopsis thaliana, and the flowering period of the transgenic plant is earlier than that of the wild type protein, and the time difference is 7±2 days.

[0058] The application of the site-directed mutant Arabidopsis thaliana cryptochrome gene provided by the application can also be used for reducing the rosette leaf number of Arabidopsis thaliana.

[0059] The application of the site-directed mutant Arabidopsis thaliana cryptochrome gene provided by the application can also improve the expression level of Arabidopsis thaliana flowering gene FT.

[0060] In the present application, the protein is reformed by the method of site-directed mutation: through polymerase chain reaction (PCR), the mutation primer is used to introduce the required change (including the addition, deletion, point mutation of base) in the DNA fragment (plasmid, genome) of the purpose. The present application is as follows: the gene sequence is obtained from the gene library, the sequence of the target gene CRY2 is connected with pJL-blue plasmid, and the recombinant plasmid pJL-blue-CRY2 is constructed. The mutation primer is designed, and the site-directed mutation is carried out by using the recombinant plasmid, and the mutant plasmid pJL-blue-D392N is constructed. Then the mutant plasmid and the expression plasmid are mixed into a reaction system, under the action of exogenous LR recombinase, the target gene is recombined into the expression plasmid vector PFK272, and the mutant expression plasmid PFK272-D392N is transformed into Arabidopsis thaliana plants, and the transgenic Arabidopsis thaliana plant is constructed.

[0061] Compared with the prior art, the transgenic plant D392N expressed in the present application can accelerate the flowering of Arabidopsis thaliana, and the flowering period is significantly earlier than that of the wild type protein transgenic plant, and the time difference is about 7±2 days, and the advance efficiency is about 25%. On the other hand, the number of rosette leaves at the base of the plant can also be used as an indicator reflecting the flowering time. Generally, the Arabidopsis thaliana that flowers early will also have fewer rosette leaves. As can be seen from the rosette leaves at the base of the transgenic plant, the number of D392N rosette leaves is much less than that of the wild type control, and the number of rosette leaves of Arabidopsis thaliana is reduced to 6±1. In addition, the qRT-PCR results show that compared with the wild type, the expression level of the flowering gene FT of the transgenic plant D392N is significantly up-regulated, and is increased to 1.38±0.02. Therefore, the D392N mutant enzyme is beneficial to the regulation of the flowering period of Arabidopsis thaliana, and can provide a direction for the improvement of the flowering time trait of other plants. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 It is the electrophoresis map of the D392N mutant gene in Example 1;

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

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

[0065] Figure 30 days or so of Arabidopsis plant growth; Arabidopsis growth cry2 is Arabidopsis cry2 gene knockout mutant plant, as a negative control; WT (wile type) represents wild type Arabidopsis, as a wild type control; CRY2 and D392N are cry2 gene and mutant gene D392N into cry2 to form a transgenic plant; D392N-1, D392N-2, respectively, are two parallel gene-containing plasmid vector transgenic to Arabidopsis plants, forming two transgenic plant lines;

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

[0067] cry2 is Arabidopsis cry2 gene knockout mutant plant, as a negative control; WT (wile type) represents wild type Arabidopsis, as a wild type control; CRY2 and D392N are cry2 gene and mutant gene D392N into cry2 to form a transgenic plant;

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

[0069] cry2 is Arabidopsis cry2 gene knockout mutant plant, as a negative control; WT (wile type) represents wild type Arabidopsis, as a wild type control; CRY2 and D392N are cry2 gene and mutant gene D392N into cry2 to form a transgenic plant;

[0070] Figure 5 Figure 4 is a comparison chart of FT transcription level of wild type and transgenic Arabidopsis prepared in Example 4;

[0071] cry2 is Arabidopsis cry2 gene knockout mutant plant, as a negative control; WT (wile type) represents wild type Arabidopsis, as a wild type control; CRY2 and D392N are cry2 gene and mutant gene D392N into cry2 to form a transgenic plant. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0073] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels unless otherwise specified.

[0074] The specific techniques or conditions not mentioned in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

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

[0076] Example 1

[0077] Obtaining of mutant D392N gene and construction of expression vector:

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

[0079] A number of Arabidopsis thaliana seedling leaves of Arabidopsis thaliana Col-0 variety (not more than 200 mg) were collected, and the total RNA of Arabidopsis thaliana was extracted according to the following specific operations:

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

[0081] (2) Take an appropriate amount of fresh Arabidopsis thaliana leaves (not more than 200 mg), recover into a 2 mL centrifuge tube, add small steel balls and freeze in liquid nitrogen;

[0082] (3) Use a metal grinder to grind the frozen leaf tissue thoroughly;

[0083] (4) Add 1 mL of isolater reagent of Novozyme Company, and mix well by vortex oscillation;

[0084] (5) After standing on ice for 5 min, centrifuge at 10000g at 4°C for 10 min;

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

[0086] (7) After standing on ice for 10 min, centrifuge at 10000g at 4°C for 10 min;

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

[0088] (9) After standing on ice for 10 min, centrifuge at 10000g at 4°C for 10 min;

[0089] (10) Discard the supernatant, wash with 1 mL of 75% ethanol solution, and centrifuge at 10000g at 4°C for 5 min;

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

[0091] (12) use an enzyme marker to monitor the RNA concentration and the ratio of 260 / 280 nm.

[0092] 1.2 Arabidopsis thaliana total RNA reverse transcription cDNA

[0093] The total RNA extracted in 1.1 above is used as a template to perform an RNA reverse transcription reaction, and a HiScript III RT SuperMix for qPCR kit from Novozyme is used to synthesize an Arabidopsis thaliana cDNA template, and the specific steps are as follows:

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

[0095] 4x gDNA wiper Mix 4 μL;

[0096] RNA 2 μg;

[0097] deionized water, supplement to 16 μL;

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

[0099] (2) reverse transcription: add 4 μL HiScript III qRT SuperMix after the reaction in the previous step is completed;

[0100] (3) 37°C incubation for 15 min, and 85°C heat activation for 5 sec;

[0101] (4) add 80 μL RNase-free deionized water, and obtain an Arabidopsis thaliana cDNA template.

[0102] 1.3 Obtain wild-type Arabidopsis thaliana cryptochrome gene cry2 and construct a recombinant plasmid

[0103] The sequence of the cryptochrome 2 gene (cry2) of Arabidopsis thaliana was searched and downloaded from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov), and the related primers were designed. The Arabidopsis thaliana cryptochrome gene cry2 was obtained by PCR (polymerase chain reaction) using the synthesized Arabidopsis thaliana cDNA template. The double digestion of the entry plasmid pJL-blue and the cry2 gene was performed using restriction endonucleases Nde I and Xho I, and the plasmid and the gene fragment were then connected by T4 ligase. The connected system was transformed into Escherichia coli DH5α, and positive clones were obtained by screening of plates with ampicillin resistance. The sample was sent to Chuzhou General Biotechnology Co., Ltd. for detection, and after confirming the correct sequencing, the DH5α strain was expanded and cultured, and the successfully constructed entry recombinant plasmid pJL-blue-CRY2 was extracted.

[0104] 1.4 Obtaining the Arabidopsis thaliana cryptochrome mutant gene D392N and the construction of the entry plasmid

[0105] Using the existing entry recombinant plasmid pJL-blue-CRY2 as a template, a pair of mutant primers was designed by the two-primer method for rapid site-directed mutagenesis of the target gene. The mutant primers were as follows:

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

[0107] D392N R: The sequence is shown in SEQ ID No. 4;

[0108] The underlined nucleotide sequences in the primers are the mutation sites;

[0109] The principle of rapid site-directed mutagenesis is that the entry recombinant plasmid wild-type pJL-blue-CRY2 is contained in the PCR reaction system, and after the reaction is completed, the extension product containing the mutant gene D392N is produced. Then, the Dpn I restriction endonuclease is used for the enzyme digestion process. Since the original template plasmid is derived from Escherichia coli, it is methylated by the dam gene in the bacterial body and is sensitive to Dpn I, so it is cut into small pieces (the Dpn I recognition sequence is methylated G m ATC), while the mutant plasmid synthesized in vitro is not cut because it is not methylated, and the mutant plasmid clone is obtained.

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

[0111]

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

[0113] PCR reaction conditions in step 1.4 of Table 1

[0114]

[0115]

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

[0117] Dpn I 2 μl;

[0118] 10 x FastDigest Buffer 2 μl;

[0119] Plasmid pJL-blue-D392N 16 μl.

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

[0121] 37°C 90 min;

[0122] 80°C 2 min.

[0123] Specifically, PCR amplification is carried out with the pJL-blue-CRY2 recombinant plasmid as a template, and the reaction conditions are as follows: 95°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 58°C annealing and recombination for 45 s, 72°C extension for 90 s, and 30 cycles; and 72°C sufficient extension for 5 min. After Dpn I enzyme digestion reaction, the mutant plasmid pJL-blue-D392N is transformed into E. coli DH5α competent cells, and positive clones are screened by ampicillin resistance, and are sent to Chuzhou General Biotechnology Co., Ltd. for sequencing identification. Sequence alignment results show that the D392N mutant entry plasmid is successfully constructed, and the mutant host bacteria are preserved.

[0124] Figure 1 It is the electropherogram of the D392N mutant gene in Example 1; the D393N gene fragment is obtained by in vitro PCR amplification, and the second band is the target band, about 1800 bp.

[0125] 1.5 Construction of Arabidopsis thaliana Cryptochrome Wild Type CRY2 and Mutant Gene D392N Expression Plasmid

[0126] This embodiment adopts traditional Gateway technology to construct transgenic vectors, including entry vector and destination vector. The entry plasmid pJL-blue contains two recombination sites attLl and attL2. The expression plasmid PFK272 contains Ubiquitin-10 promoter, and has attRl and attR2 recombination sites and ccdB lethal gene. The verified pJL-blue-CRY2 and pJL-blue-D392N entry plasmids and empty expression plasmid PFK272 are mixed in a reaction system. Using the Gateway LR Cloning Kit of amb company (Aibimeng Biotechnology Co., Ltd.), under the action of exogenous LR recombinase, the target gene CRY2 is recombined into the expression vector, and the ccdB gene is recombined into the entry vector. At this time, the system contains four kinds of plasmids: un-recombined entry plasmid, un-recombined expression plasmid, recombined entry plasmid, and recombined expression plasmid. Spe Spectinomycin (100 μg / mL) antibiotic is used for resistance screening. The positive clones are extracted for E. coli transformation to obtain the expression recombinant plasmids PFK272-CRY2 and PFK272-D392N, which are then sent to General Biotechnology Co., Ltd. (Chuzhou) for sequencing verification.

[0127] Recombination reaction system (5 μL):

[0128] 2x 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 h.

[0132] Example 2

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

[0134] 2.1 Seed disinfection and culture of Arabidopsis

[0135] Firstly, the wild type Arabidopsis thaliana line col-0 and cry2 gene knockout mutant cry2 plant (cry2 is an Arabidopsis thaliana cry2 gene knockout mutant plant obtained by chemical mutagenesis by conventional techniques) seeds are placed in a drying box for about 4-5 days. Then, sterilizing solution (70% ethanol solution by volume, 0.5% Tween 20) is added to the seed-containing tube for 10 min, and the volume of the seed is not more than 1 / 5 of the volume of the sterilizing solution to ensure sufficient sterilization. Discard the supernatant, then wash twice with 95% ethanol for about 5 min each time. Finally, the seeds are spread on filter paper in a clean bench, dried, and then recovered into the tube.

[0136] Then, prepare the MS medium (1 / 2MS 2.2g / L, agar 8‰) specially for Arabidopsis thaliana seeds. After the MS medium plate is poured, the seed culture is carried out. A small piece of sterile gauze is used to wrap the mouth of the centrifuge tube, and the seeds are gently scattered on the surface of the MS plate and evenly distributed. The gap of the plate is wrapped with a sealing film, and after detailed labeling, it is placed in a culture shelf with suitable light, long day (16h LD), and constant temperature culture at 20°C. The germination of Arabidopsis thaliana seeds is observed daily, and after about 4-5 days, the seedlings are prepared for transplanting.

[0137] Before transplanting, first prepare the nutrient soil: purchase high-quality imported nutrient soil, take an appropriate amount into a large pot, and start to rub the soil by hand. Rub the soil fine and soft, and remove the hard impurities, then add an appropriate amount of water and stir to ensure that the newly prepared soil is moist enough. Then, the soil in the large pot is divided into several small pots to ensure sufficient softness. At this time, the seedlings on the culture medium are transferred to the small pots, and the roots of the seedlings are carefully covered with soil, only the opened cotyledons are exposed. After transplanting, irrigate with enough nutrient solution. Check the growth of the seedlings regularly every day. After the seedlings have emerged and grown enough inflorescences, the transgenic stage is prepared.

[0138] 2.2 Construction of transgenic Arabidopsis thaliana

[0139] First, the Agrobacterium transformation process is carried out. The Agrobacterium competent cells are thawed on ice, 2μl PFK272-CRY2 and PFK272-D392N plasmids are added respectively, and the mixture is gently shaken and mixed, placed on ice for 20 min; placed in a 37°C water bath for 4 min; frozen in liquid nitrogen for 1 min; added with 100μl LB liquid medium, and cultured on a shaker for 2h at 28°C and 200rpm; after screening by the conventional method using LB solid medium with corresponding antibiotics Cam (Chloramphenicol), Tet (Tetracyclines), Kan (Kanamycin), and Spe (Spectinomycin), the seeds are preserved.

[0140] Agrobacterium containing recombinant expression plasmid is streaked on four resistant MS medium (Cam, Tet, Kan, Spe), 28°C for about 2 days. Single colony is picked in 5ml test tube, 28°C for about a day, 500ul bacteria liquid is taken to 100ml LB medium, and cultured to OD 600 about 0.8. Then centrifugal collection of bacteria at room temperature, 5000rpm. At this time, the bacteria are suspended with prepared transformation fluid (the transformation fluid needs to be stirred for 2h in advance; formula 1 / 2MS 0.44g; sucrose 20g; Silwet-77 100ul; MES 0.1g; pH 5.7). Carefully pour the Agrobacterium transformation fluid into a large culture dish. At this time, the excess fresh fruit of the bolting seedlings is removed to avoid interference in the transgenic experiment. Then immerse the inflorescences of the seedlings in the culture dish, allowing the bacteria to fully contact the inflorescences, about 30s each time. Then place several pots of Arabidopsis in a weak light, and after 24h, place them in long day conditions again.

[0141] After Agrobacterium infection of the inflorescences, wait for the Arabidopsis to mature. Collect the seeds of different T0 generation transgenic seedlings. Again, according to the above process, replant and screen the T0 generation seeds that successfully transgenated. At this time, due to the low efficiency of Agrobacterium transgenation, positive seedlings need to be screened on resistant plates, and then wait for the seedlings to grow to a sufficient size. After obtaining a portion of the tissue, use the CTAB method to extract the genome, identify whether the transgenation is successful by gene cloning PCR, and reserve the seedlings that have successfully sequenced. Thus, Arabidopsis CRY2, D392N transgenic plants are designed and cultured. Observe and count the flowering period, basal rosette leaves and other phenotypes, and analyze the apparent differences of transgenation.

[0142] Among them, the CTAB method (cetyltrimethylammonium bromide method) for extracting the genome is as follows: fresh Arabidopsis leaves are selected for genotyping analysis:

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

[0144] (2) Add 500ul of CTAB solution and mix well, and place at 65°C for 1h. The CTAB solution is prepared as follows (500ml):

[0145]

[0146] (3) Add 500ul of chloroform / isopentanol (volume ratio 24:1) and mix well, then centrifuge at 10000rpm for 10min;

[0147] (4) Take the supernatant, add an equal volume of isopropyl alcohol, mix well and stand for 5min;

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

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

[0150] 2.3 Statistics of flowering time phenotype of transgenic Arabidopsis

[0151] It is known that the classic physiological function of AtCRY2 is to control the development of flowers and the flowering cycle. Therefore, the present application analyzes the physiological role of the plant cryptochrome 392 site based on the flowering cycle phenotype of Arabidopsis. Under the condition of constant temperature at 20°C and 16h long light, the wild type Arabidopsis Col-0 strain usually flowers at about 30 days. As shown in Figure 2 、 Figure 3 cry2 is a knockout mutant, as a negative control; WT represents the wild type Col-0, as a blank control; CRY2 represents a transgenic strain of wild type AtCRY2 based on cry2, as a positive control. It can be seen that the flowering cycle of the D392N mutant plant is significantly earlier than that of the transgenic plant CRY2, with a difference of about 7±2 days. It shows that under natural conditions, the D392N mutation has a significant regulatory effect on the physiological function of the flowering cycle of the plant cryptochrome, promoting early flowering.

[0152] AtCRY2 and CRY2 refer to the same, both of which are transgenic plants containing the cry2 gene. cry2 represents the cryptochrome 2 gene, CRY2 represents the plasmid vector containing this gene, or the transgenic Arabidopsis plant.

[0153] In the present application, D392N refers to the mutation site; D392N1, D392N2 refer to the construction of two parallel mutant genes, and the construction of recombinant plasmids containing the two parallel mutant genes; D392N-1, D392N-2 are two transgenic plant strains formed after the above-mentioned plasmid vector containing two parallel genes is transgenically introduced into Arabidopsis plants.

[0154] Example 3

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

[0156] During flowering, the number of basal rosette leaves of Arabidopsis is also related to the flowering cycle. Generally, the earlier the flowering, the fewer the number of rosette leaves. Conversely, the later the flowering, the more the number of rosette leaves. Therefore, the number of rosette leaves is also used as an index to compare the differences in flowering cycle phenotype of wild type and transgenic Arabidopsis. As shown in Figure 4The number of rosette leaves of the natural wild type Arabidopsis plant WT was 8.5±0.5, the number of rosette leaves of the knock-out mutant plant cry2 was 12.5±0.5, the number of rosette leaves of the wild type cry2 gene transgenic plant CRY2 was 11±1, and the number of rosette leaves of the mutant gene D392N transgenic plant D393N was 6±1. By counting the above control plants, it can be seen from the basal rosette leaves of the transgenic plants that the number of rosette leaves of the transgenic plant D392N is much less than that of the CRY2 control. Based on the above analysis, the D392N mutation greatly affects the function of the cryptochrome protein, and can promote the sustained regulation of the flowering process by cryptochrome, so that the plant blooms earlier.

[0157] Example 4

[0158] Transcription level detection of FT flowering gene of transgenic Arabidopsis thaliana:

[0159] 4.1 Total RNA extraction of transgenic Arabidopsis thaliana plant

[0160] The specific method is the same as that in Example 1, and the total RNA of the control group and the transgenic plant is obtained, respectively.

[0161] 4.2 qRT-PCR experiment

[0162] (1) The ChamQ Universal SYBR qPCR Master Mix reagent of Novaligen was used, and the sample addition system was as follows:

[0163]

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

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

[0166] (2) The Roche PCR instrument was used to run the program, and the program adopted 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, a total of 45 cycles; the dissolution curve stage used the program of the Roche PCR instrument.

[0167] (3) Process and analyze data: use Excel to analyze and process data, and use Origin 8.5 to draw statistical graphs.

[0168] Cryptochromes are known to act on signaling pathways during flowering, thereby affecting the transcriptional level of downstream flowering genes (FTs) and ultimately regulating differences in the flowering cycle of plants. The AtCRY2 photoperiod regulation of flowering signaling pathways typically involves two main mechanisms: 1. Blue light excites the interaction between AtCRY2 and CIB1, directly activating the transcription of the FLOWERING LOCUST T (FT) gene, which encodes the core vacuolation factor or flowering pigment of flower initiation. This gene then migrates from the plant leaves to the apical meristem to promote the development of the floral meristem; 2. COP1, as an E3 ubiquitination ligase, directly participates in the protein degradation of numerous transcription factors, including CO protein. When blue light activates AtCRY2, the photoactivated CRY2 interacts with SPA1 and COP1 to form a complex protein, de-ubiquitinizing COP1 and inhibiting the degradation of CO protein. Since CO is a major transcriptional activator of FT genes, the transcriptional level of FT increases with CO abundance, ultimately promoting flowering. Therefore, regardless of which signaling pathway is dominant, cryptochromes ultimately influence the transcriptional level of downstream FT genes, regulating the flowering cycle. In this embodiment, in order to explore whether the phenotypic differences among different transgenic plants are associated with the alteration of cryptochrome function caused by the D392N mutation, the transcriptional level of the flowering-related gene FT was detected.

[0169] Using seedlings that were approximately 14 days old and had not yet flowered as experimental subjects, several leaf samples were collected. RNA was extracted, reverse-engineered into cDNA, and then qRT-PCR was performed to detect the expression level of the flowering gene FT in the transgenic plants. The col-0 material served as the natural control group, cry2 as the negative control group, and various transgenic mutant materials as the experimental groups. The qRT-PCR results showed ( Figure 5 In the experimental group, the FT gene expression level of D392N was upregulated compared to CRY, with the T-test showing a significant difference in FT gene expression between D392N and CRY2N. In wild-type Arabidopsis thaliana (WT), the FT gene expression levels of the knockout mutant cry2, the transgenic plant CRY2 (wild-type cry2), and the transgenic plant D393N (D392N) were 1.04±0.41, 0.47±0.06, 1.15±0.22, and 1.38±0.02, respectively. Among these, D393N showed a 20% increase in FT gene expression compared to CRY2.

[0170] This indicates that, consistent with previous findings, the amino acid preference at the D392N site does indeed affect the function of plant cryptochromes. Under natural conditions, the selection of polar amino acids is more conducive to plant growth and development, thereby enabling them to adapt to the environment.

[0171] The above description of the embodiments is made for the purpose of enabling a person of ordinary skill in the art to understand and use the application. It is obvious that those skilled in the art can easily make various modifications to the embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present application without departing from the scope of the present application should be within the scope of the present application.

Claims

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

1.

2. The site-directed mutated Arabidopsis caulinoid gene-encoded protein of claim 1, wherein, The amino acid sequence of the encoded protein is shown as SEQ ID NO:

2.

3. An expression vector for Arabidopsis cryptochrome gene containing a site-directed mutation, characterized in that, The expression vector is a recombinant plasmid, and the recombinant plasmid is a recombinant plasmid containing the site-directed mutant Arabidopsis cryptochrome gene of claim 1.

4. A host containing an Arabidopsis cryptochrome gene with a site-directed mutation, characterized in that, The host is a recombinant engineering bacterium containing the site-directed mutant Arabidopsis cryptochrome gene of claim 1, and the recombinant engineering bacterium is Escherichia coli containing the expression vector containing the site-directed mutant Arabidopsis cryptochrome gene of claim 3.

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

6. The method for constructing the Arabidopsis thaliana acaulescent gene plant containing site-directed mutation according to claim 5, specifically: extracting the total RNA of the Arabidopsis thaliana plant to reversely transcribe a cDNA template, designing a primer, cloning the target gene in vitro to obtain a recombinant plasmid pJL-blue-CRY2; designing a mutation primer, using the recombinant plasmid to perform site-directed mutation, constructing a mutant recombinant plasmid pJL-blue-D392N, and performing Gateway homologous recombination of the mutant recombinant plasmid and an expression plasmid PFK272 to construct a PFK272-D392N recombinant plasmid for expressing the acaulescent protein in vivo of the Arabidopsis thaliana; transforming the PFK272-D392N recombinant plasmid into Agrobacterium, then infecting the Arabidopsis thaliana inflorescence by the Agrobacterium to perform transgenesis, and obtaining the Arabidopsis thaliana seed containing the mutant gene; culturing the transgenic Arabidopsis thaliana at 20℃ with 16 hours / day light to obtain the transgenic mutant plant D392N. cry2 , specifically: extracting the total RNA of the Arabidopsis thaliana plant to reversely transcribe a cDNA template, designing a primer, cloning the target gene in vitro to obtain a recombinant plasmid pJL-blue-CRY2; designing a mutation primer, using the recombinant plasmid to perform site-directed mutation, constructing a mutant recombinant plasmid pJL-blue-D392N, and performing Gateway homologous recombination of the mutant recombinant plasmid and an expression plasmid PFK272 to construct a PFK272-D392N recombinant plasmid for expressing the acaulescent protein in vivo of the Arabidopsis thaliana; transforming the PFK272-D392N recombinant plasmid into Agrobacterium, then infecting the Arabidopsis thaliana inflorescence by the Agrobacterium to perform transgenesis, and obtaining the Arabidopsis thaliana seed containing the mutant gene; culturing the transgenic Arabidopsis thaliana at 20℃ with 16 hours / day light to obtain the transgenic mutant plant D392N. The sequence of the mutant primer is: D392N F: the sequence is shown as SEQ ID No. 3; D392N R: the sequence is shown as SEQ ID No.

4.

7. Use of a site-directed mutated Arabidopsis caulinoid gene according to claim 1, characterized in that, The transgenic plant is used for regulating the flowering period of Arabidopsis, accelerating the flowering of Arabidopsis, and the flowering period is earlier than that of the wild type protein, and the time difference is 7±2 days.

8. Use of the site-directed mutated Arabidopsis caulinoid gene according to claim 1, characterized in that, The transgenic plant is used for reducing the rosette leaf number of Arabidopsis, and reducing the rosette leaf number of Arabidopsis to 6±1.

9. Use of a site-directed mutated Arabidopsis caulinoid gene according to claim 1, characterized in that, Methods for increasing flowering in arabidopsis thaliana FT The expression level was increased to 1.38 ± 0.02.

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