CN111139253B - Cryptomeria fortunei CfCCR gene and application thereof - Google Patents
Cryptomeria fortunei CfCCR gene and application thereof Download PDFInfo
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- CN111139253B CN111139253B CN202010069453.5A CN202010069453A CN111139253B CN 111139253 B CN111139253 B CN 111139253B CN 202010069453 A CN202010069453 A CN 202010069453A CN 111139253 B CN111139253 B CN 111139253B
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Abstract
The invention discloses a cryptomeria fortunei CfCCR gene and application thereof, belonging to the technical field of plant molecular biology and genetic engineering. The cryptomeria xylocarpa lignin synthesis regulation CfCCR gene has a nucleotide sequence shown as SEQ ID No.1, and an amino acid sequence of an expression protein of the cryptomeria xylocarpa lignin synthesis regulation CfCCR gene is shown as SEQ ID No. 2. The CfCCR gene is a key rate-limiting enzyme of a specific lignin synthesis way, regulates the expression of related genes for synthesizing lignin in plants through coding protein, and has important effects on the biosynthesis and stress resistance of the lignin. The CfCCR transgenic tobacco has the advantages of increased lignin expression and developed cell wall. Therefore, the thickness of the xylem cell wall in the stem of the cedar or other plants can be changed by expressing the CfCCR gene, so that the wood density is increased, and the quality of the wood and the stress resistance of the plants are improved.
Description
Technical Field
The invention belongs to the technical field of plant molecular biology and genetic engineering, and particularly relates to a cryptomeria fortunei CfCCR gene and application thereof
Background
Cryptomeria fortunei (Cryptomeria fortunei) belongs to the family Cunningaceae (Taxodiaceae) genus Cryptomeria (Cryptomeria D.Don) plant, also known as Pinus longifola, arbor, crown-shaped cone; the tree bark is reddish brown, fibrous, the big branch is near-recurrent, the small branch is slender and often sagged, the leaf drill shape is slightly bent inwards, the tip is bent inwards, the four sides are provided with air hole lines, the tree is a special tree species in China, the tree species is an important tree species, the wood has wide utilization value, and the tree species can also be used as ornamental plants and medicinal plants. The lignification degree of the cedar can influence the density of the cedar wood, thereby influencing the quality of the wood.
CCR plays a key role in the specific pathway of lignin synthesis, is the first key enzyme of the pathway, catalyzes cinnamoyl-CoA ester to form corresponding cinnamaldehyde, and researches suggest that CCR influences the synthesis of lignin monomers. The previous research finds that: the faster the inhibition of CCR activity decreases, the more the corresponding decrease in lignin content, which has an effect on the growth and development of the plant. Inhibition of CCR activity results in decreased lignin content, decreased secondary wall deposition, relaxed secondary wall structure, and in severe cases abnormal plant development.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a cryptomeria fortunei CfCCR gene which has a relevant bond function in a specific pathway of lignin synthesis and can promote the expression of a plant lignin synthesis gene so as to enhance the biosynthesis of lignin, increase the lignin content and the cell wall thickness, increase the density of cryptomeria fortunei wood and improve the quality of the wood. The invention also aims to provide application of the cedar CfCCR gene.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a Cryptomeria fortunei CfCCR gene has a nucleotide sequence shown in SEQ ID NO. 1.
The amino acid sequence of the expressed protein of the cedar CfCCR gene is shown in SEQ ID NO. 2.
The vector, the recombinant strain or the host cell containing the cedar CfCCR gene.
Furthermore, the vector is a plant recombinant expression vector.
Further, the plant recombinant expression vector is pBI121+ CfCCR.
Further, the host cell is agrobacterium EHA 105.
The cedar CfCCR gene is applied to promotion of plant lignin synthesis, cell wall development or stress resistance.
Further, the application comprises the following steps:
1) constructing a vector of the cedar CfCCR gene;
2) transforming the constructed vector of the cedar CfCCR gene into plants or plant cells;
3) and cultivating and screening to obtain plants with increased lignin, enhanced cell wall development or improved stress resistance.
Further, in the application, the plant is cedar or tobacco.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the cedar CfCCR gene provided by the invention is a key rate-limiting enzyme in a specific path for synthesizing lignin, regulates the expression of a relevant gene for synthesizing the lignin in a plant through a coded protein, and has important effects on the biosynthesis of the lignin and the improvement of the stress resistance of the plant. The CfCCR transgenic tobacco has the advantages of increased lignin expression and developed cell wall. The wooden cell wall thickness in the cryptomeria fortunei or other plant stems can be changed by over-expressing the CfCCR gene, so that the wood density is increased, the quality and quality of wood are improved, and the stress resistance of plants is improved, so that the gene has important application value and significance.
Drawings
FIG. 1 is a pBI121 map of the CfCCR gene expression vector of the invention;
FIG. 2 is a diagram showing the relative expression levels of the CR gene at Cf ℃ at different stages of development according to the present invention;
FIG. 3 is a graph showing the results of the growth process of tobacco expressing CfCCR gene of the present invention;
FIG. 4 is a graph showing the comparison of the phenotype of a tobacco expressing the CfCCR gene of the present invention after maturation, wherein WT is shown on the left side and CfCCR tobacco is shown on the right side;
FIG. 5 is a cross-sectional view of tobacco stems expressing the CfCCR gene of the present invention.
Detailed Description
The invention is further described with reference to specific examples.
The methods used in the following examples are conventional methods unless otherwise specified, all primers in this experiment were synthesized by Shanghai Czeri bioengineering, Inc., and sequencing was performed by Nanjing Kingsri Biotech.
Example 1:
cloning and expression analysis of Cryptomeria fortunei CfCCR gene
1. Cloning of Cryptomeria fortunei CfCCR gene
Selecting good-growing cryptomeria fortunei in a tree garden of Nanjing forestry university, removing the epidermis, scraping vascular tissues by using a blade, extracting total RNA (ribonucleic acid) and performing reverse transcription to obtain cDNA (complementary deoxyribonucleic acid). According to the analysis data of the conserved sequence of the CCR gene of the cedar and the differential expression gene analyzed by the high-throughput sequencing data of the cryptomeria fortunei transcriptome in the laboratory, a CCR homologous sequence is screened out by using the Blast comparison of an NCBI online tool, and the primer design is carried out.
Designing specific amplification primers CCR-F and CCR-R, carrying out PCR amplification by taking cDNA as a template, recovering PCR products, and connecting pBI121 vectors (figure 1).
The specific amplification primers CCR-F and CCR-R are as follows:
CCR-F：5′-TAATTGTTGTAAATACGCTTG-3′
CCR-R：5′-AATTACAGTTCTTATTGGCAT-3′
cDNA synthesized by reverse transcription is taken as a template, and primers CCR-F and CCR-R are used for amplification. The reaction system (50. mu.L) was: 25.0 μ L
Max DNA Polymerase，1.0μl cDNA，22.0μL ddH2O, 1.0. mu.L Forward Primer (10. mu.M), 1.0. mu.L Reverse Primer (10. mu.M). The amplification reaction procedure was: 1min at 98 ℃; 35 cycles of 98 ℃ for 10s, 55 ℃ for 10s, 72 ℃ for 25 s; 5min at 72 ℃; infinity at 4 ℃.
After the PCR product is detected by 1.5% agarose gel electrophoresis, the target fragment is recovered and connected to a vector pBI121, then transferred to escherichia coli competent cells, and the positive clone is detected and sent to Nanjing Kinshire biology company for sequencing.
After sequencing is completed, the sequences of the 3 ', 5' ends and the middle fragment obtained by sequencing are spliced to obtain the CfCCR gene, and then DNAMAN software is used for comparison to ensure that the obtained sequence is a target sequence. The result shows that the obtained CfCCR gene has the nucleotide sequence shown as SEQ ID NO.1 and the size of 975bp, and the amino acid sequence of the coded protein is shown as SEQ ID NO.2 and the size of 324 aa.
2. Real-time fluorescence quantitative expression analysis of Cryptomeria fortunei CfCCR gene
Selecting Actin as an internal reference gene, and designing a real-time quantitative primer of the CfCCR by utilizing Oligo7 according to the nucleotide sequence SEQ ID NO.1 information of the CfCCR gene.
RT-CCR-F：5′-GCTTGCCCTGTTGTAGTACCAA-3′
RT-CCR-R：5′-TCTGCATACTTCCACGCCTC-3′
Real-time fluorescent quantitative PCR reaction system (20 μ Ι _): 10.0. mu.L TB Green Premix Ex Taq II (Tli RnaseH Plus) (2X), 0.8. mu.L Primer-F (10. mu.M), 0.8. mu.L Primer-R (10. mu.M), 0.4. mu.L ROX Reference Dye (50X), 2.0. mu.L DNA template, 6.0. mu.L ddH2And O. The PCR amplification procedure was: 30s at 95 ℃; 5s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 45 cycles; 15s at 95 ℃; lmin at 60 ℃; 15s at 95 ℃; infinity at 4 ℃.
The results of fluorescence quantification (FIG. 2) show: the cedar CfCCR gene shows a trend of descending first and then ascending in 5 growth and development stages, and reaches a maximum value in 11 months, presumably because the CCR gene plays a role in limiting speed in a specific pathway for synthesizing lignin and directly influences the condition of lignification.
Second, construction of pBI121 expression vector and host cell
According to the sequence of the target gene CfCCR which is successfully cloned, a pBI121 vector is linearized by using a double-enzyme cutting method, the transformation background is reduced, and false positive clones are reduced.
According to the obtained amplified CfCCR gene coding region, an amplification primer of the insert fragment is automatically generated by using CE Design, and then PCR amplification, recombination, transformation and identification of the target gene insert fragment are carried out.
And (3) transforming the constructed expression vector pBI121+ CfCCR into an agrobacterium EHA105 competent cell, and detecting a positive colony by using PCR to obtain a transformed host cell.
Genetic transformation, screening and identification of tobacco
The wild tobacco leaf discs without resistance are placed into the diluted bacterial liquid of the transformed agrobacterium EHA105 for infection, and then are subjected to screening culture, seedling hardening and soil culture (as shown in figure 3). And carrying out DNA extraction and PCR detection on the transgenic tobacco to finally obtain three strains of tobacco successfully transformed with the CfCCR gene, which are named as CfCCR-1, CfCCR-2 and CfCCR-3 respectively. Observing the transgenic tobacco:
1. phenotypic observations
The tobacco leaf discs infected by agrobacterium are placed into corresponding culture media for culture, as shown in figure 4, partial leaves of transgenic CfCCR tobacco are observed to have malformation and asymmetric development, and a few transgenic CfCCR tobacco have dwarfing phenomena, the average stem thickness of the transgenic CfCCR tobacco is 7.11mm, and is increased by 1.45 times compared with the average stem thickness of wild tobacco.
2. Observation by scanning electron microscope
As shown in FIG. 5, the increase of the number of xylem cells and the number of cell layers of vascular tissues in the cross section of the transgenic tobacco is obviously greater than that of wild-type tobacco and the ligneous cell walls are arranged closely, and the thickness of the xylem cell walls is also obviously greater than that of the wild-type tobacco. The average thickness of the xylem cell walls of the transgenic CfCCR-1 tobacco is 2.492 mu m, the average thickness of the xylem cell walls of the transgenic CfCCR-2 tobacco is 2.435 mu m, the average thickness of the xylem cell walls of the transgenic CfCCR-3 tobacco is 2.463 mu m, the xylem cell walls of the transgenic CfCCR tobacco plants are obviously thickened, the thickness of the xylem cell walls of the transgenic CfCCR tobacco plants is increased by 1.83, 1.78 and 1.80 times compared with the thickness of the xylem cell walls of wild tobacco respectively, and the xylem cell walls of the transgenic CfCCR tobacco plants reach an extremely obvious level through a variance.
3. Transgenic CfCCR tobacco lignin content determination
And (4) measuring the content of the tobacco lignin by using an ultraviolet spectrophotometry. The sample was placed in a 50 ℃ oven to dry completely and then a small amount of liquid nitrogen was added and ground to a powder. Weighing about 0.006g of sample, placing in a10 mL centrifuge tube, adding 2.5mL of 25% bromoacetyl-acetic acid solution and 0.1mL perchloric acid solution, placing in a water bath at 70 deg.C for 30min, and shaking the tube every 10min until the powder is completely dissolved. Transferring the sample reaction solution into a 50mL volumetric flask, adding 5mL of 2mol/LNaOH and 12.5mL of glacial acetic acid, fixing the volume to 50mL by using the glacial acetic acid, measuring the light absorption value at 260nm by using an ultraviolet spectrophotometer, and calculating according to a standard curve equation to obtain the tobacco lignin content.
The average content of the transgenic CfCCR-1 tobacco lignin is 7.24 percent, the average content of the transgenic CfCCR-2 tobacco lignin is 11.99 percent, the average content of the transgenic CfCCR-3 tobacco lignin is 9.65 percent, and the content of the transgenic CfCCR tobacco lignin is 1.49 to 2.47 percent higher than that of a wild type. The variance test showed that significant levels of CfCCR-1 and very significant levels of CfCCR-2 and CfCCR-3 were achieved (Table 1). This result demonstrates that overexpression of the cedar CfCCR gene increases the lignin content in tobacco in transgenic tobacco with thickened xylem cell walls.
TABLE 1 conversion of CfCCR Gene tobacco Lignin content
The present invention is not limited to the above description of the embodiments, and those skilled in the art who have the benefit of this disclosure will appreciate that many modifications and variations of the present invention are possible within the scope of the present invention without departing from the inventive concepts herein.
Sequence listing
<110> Nanjing university of forestry
<120> Cryptomeria fortunei CfCCR gene and application thereof
<130> 100
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 975
<212> DNA
<213> Cryptomeria fortunei
<400> 1
atgacagaaa atggtaagga gatgtgtgtg acaggaggca caggattcat tgctggtttt 60
ctcattcgtg ctctgctcaa caagggttac tctgttcgca caactgttcg caatccagat 120
aatgtggaga aggtagggta tctatgggat ctgcctggtg caaagcagag gcttaaaata 180
gtgaaagcag atctggtaga tgaaggcagc tttgatgaag cagtaaatgg tgtggatggg 240
gtgtttcata ctgcttgccc tgttgtagta ccaaatgatc acaacatcaa gaccttcctt 300
gatcctgctg tgaatggcac tctcaatgtc ttcaagtctt gttcaagatc accctctgta 360
aagagggtgg tgcttacatc ctcctgctca gctatcagat ataactacaa tactcaagag 420
ctttctcctt tagatgagtc ccattggagc aatccagaat actgcaaaca gtataatctt 480
tggtatgcat atgcaaaaac tacttctgag aaagaggcgt ggaagtatgc agaggagcag 540
aggctcgatt tggttgtggt aaacccatca tttgttgttg gaccacttat tgcacctgaa 600
cctaccagca ctctcgactt tatcctgaac ataataaaag ggggaaataa cagaacatac 660
ccaaatttca gggtgggctt tgttgacatt ggtgatgtaa tatcagcaca tatactggct 720
atggaggcac catcagcatc agggagaatt atatgctctg gtgatgttgc tcactgggaa 780
gaaattgtga agatgctcaa ggaaaaatat cccatgtacc caattgcaga ccagtgtgga 840
acagagcaag gcaatgcgct acctcataca atgaatactg ctaagatcag aagccttgga 900
tttggcaact ttaaaagcat agaccaaatg tttgaagact gcattagaaa tttccaagag 960
aaggggctac tctag 975
<210> 2
<211> 324
<212> PRT
<213> Cryptomeria fortunei
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Met Thr Glu Asn Gly Lys Glu Met Cys Val Thr Gly Gly Thr Gly Phe
1 5 10 15
Ile Ala Gly Phe Leu Ile Arg Ala Leu Leu Asn Lys Gly Tyr Ser Val
20 25 30
Arg Thr Thr Val Arg Asn Pro Asp Asn Val Glu Lys Val Gly Tyr Leu
35 40 45
Trp Asp Leu Pro Gly Ala Lys Gln Arg Leu Lys Ile Val Lys Ala Asp
50 55 60
Leu Val Asp Glu Gly Ser Phe Asp Glu Ala Val Asn Gly Val Asp Gly
65 70 75 80
Val Phe His Thr Ala Cys Pro Val Val Val Pro Asn Asp His Asn Ile
85 90 95
Lys Thr Phe Leu Asp Pro Ala Val Asn Gly Thr Leu Asn Val Phe Lys
100 105 110
Ser Cys Ser Arg Ser Pro Ser Val Lys Arg Val Val Leu Thr Ser Ser
115 120 125
Cys Ser Ala Ile Arg Tyr Asn Tyr Asn Thr Gln Glu Leu Ser Pro Leu
130 135 140
Asp Glu Ser His Trp Ser Asn Pro Glu Tyr Cys Lys Gln Tyr Asn Leu
145 150 155 160
Trp Tyr Ala Tyr Ala Lys Thr Thr Ser Glu Lys Glu Ala Trp Lys Tyr
165 170 175
Ala Glu Glu Gln Arg Leu Asp Leu Val Val Val Asn Pro Ser Phe Val
180 185 190
Val Gly Pro Leu Ile Ala Pro Glu Pro Thr Ser Thr Leu Asp Phe Ile
195 200 205
Leu Asn Ile Ile Lys Gly Gly Asn Asn Arg Thr Tyr Pro Asn Phe Arg
210 215 220
Val Gly Phe Val Asp Ile Gly Asp Val Ile Ser Ala His Ile Leu Ala
225 230 235 240
Met Glu Ala Pro Ser Ala Ser Gly Arg Ile Ile Cys Ser Gly Asp Val
245 250 255
Ala His Trp Glu Glu Ile Val Lys Met Leu Lys Glu Lys Tyr Pro Met
260 265 270
Tyr Pro Ile Ala Asp Gln Cys Gly Thr Glu Gln Gly Asn Ala Leu Pro
275 280 285
His Thr Met Asn Thr Ala Lys Ile Arg Ser Leu Gly Phe Gly Asn Phe
290 295 300
Lys Ser Ile Asp Gln Met Phe Glu Asp Cys Ile Arg Asn Phe Gln Glu
305 310 315 320
Lys Gly Leu Leu
<210> 3
<211> 21
<212> DNA
<213> CCR-F(Artificial)
<400> 3
taattgttgt aaatacgctt g 21
<210> 4
<211> 21
<212> DNA
<213> CCR-R(Artificial)
<400> 4
aattacagtt cttattggca t 21
<210> 5
<211> 22
<212> DNA
<213> RT-CCR-F(Artificial)
<400> 5
gcttgccctg ttgtagtacc aa 22
<210> 6
<211> 20
<212> DNA
<213> RT-CCR-R(Artificial)
<400> 6
tctgcatact tccacgcctc 20
Claims (9)
1. Cryptomeria fortuneiCfCCRThe gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. The Cryptomeria fortunei of claim 1CfCCRThe amino acid sequence of the gene expression protein is shown in SEQ ID NO. 2.
3. Comprising the Cryptomeria fortunei of claim 1CfCCRA vector, a recombinant bacterium or a host cell of the gene.
4. The Cryptomeria fortunei of claim 3CfCCRThe gene vector is characterized in that the vector is a plant recombinant expression vector.
5. The Cryptomeria fortunei of claim 4CfCCRThe gene carrier is characterized in that the plant recombinant expression carrier is pBI121+CfCCR。
6. The Cryptomeria fortunei-containing material of claim 3CfCCRA host cell for a gene, said host cell being Agrobacterium EHA 105.
7. The Cryptomeria fortunei of claim 1CfCCRThe gene is applied to promoting plant lignin synthesis, cell wall development or stress resistance.
8. The use of claim 7, comprising the steps of:
1) construction of Cryptomeria fortuneiCfCCRA vector for the gene;
2) the constructed cryptomeria fortunei is processedCfCCRTransforming a vector of the gene into a plant or plant cell;
3) and cultivating and screening to obtain plants with increased lignin, enhanced cell wall development or improved stress resistance.
9. The use of claim 7 or 8, wherein said plant is cedar or tobacco.
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