CN111826386A - A fusion gene for regulating cotton fiber color, its expression vector and application - Google Patents

Abstract

The invention provides a fusion gene for regulating and controlling color development of cotton fibers, an expression vector and application thereof, and relates to the technical field of plant transgenic breeding. The invention utilizes 2A peptide which can be automatically cracked into small fragment peptide, Lc and GhPAP1 genomes are synthesized into a fusion gene, a fiber secondary wall specific promoter pFBl2A is adopted to start the over-expression of the fusion gene in cotton fibers, the anthocyanin content of the obtained transgenic cotton fibers is obviously improved compared with white fibers, the total anthocyanin content in the transgenic fibers is increased by 4093 times compared with the white fibers, the transgenic cotton generates purple red fibers, and the color range of mature fibers is obviously expanded.

Description

A fusion gene for regulating cotton fiber color, its expression vector and application

technical field

The invention belongs to the technical field of plant transgenic breeding, and in particular relates to a fusion gene for regulating the coloration of cotton fibers, an expression vector and application thereof.

Background technique

Natural colored cotton is a cotton material that can synthesize and accumulate natural pigments during fiber development, so that mature fibers have a natural color. Compared with conventional white cotton and chemical fibers, colored cotton does not need bleaching and dyeing treatment during textile processing because of its natural color, avoiding the pollution of dyes, organic solvents, heavy metals and other toxic and harmful residues on textiles and harm to human body. , can significantly improve the green production level of textiles. At present, the colored cotton materials used in textiles are only green and brown, which cannot meet the needs of consumers and the textile industry for color diversity. Therefore, expanding the color range of mature fibers is a necessary condition for the further development of the colored cotton industry.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is a fusion gene that regulates the coloration of cotton fibers, an expression vector and application thereof, and significantly increases the anthocyanin content of cotton fibers, thereby obtaining natural purple-red cotton fibers.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a fusion gene for regulating the coloration of cotton fibers. The structure of the fusion gene includes the nucleotide sequence of 5'-maize leaf color gene Lc-peptide segment 2A-cotton anthocyanin synthesis regulation gene GhPAP1D-3' ;

The nucleotide sequence of the corn leaf color gene Lc is shown in SEQ ID NO.1; the nucleotide sequence of the peptide segment 2A is shown in SEQ ID NO.2; the cotton anthocyanin synthesis regulation gene GhPAP1D The nucleotide sequence is shown in SEQ ID NO.3.

Preferably, the encoded amino acid sequence of the corn leaf color gene Lc is shown in SEQ ID NO.4; the amino acid sequence of the peptide segment 2A is shown in SEQ ID NO.5; the cotton anthocyanin synthesis regulation gene GhPAP1D The encoded amino acid sequence is shown in SEQ ID NO.6.

Preferably, the primers for amplifying the maize leaf color gene Lc include Lc-2A-U and Lc-2A-D; the nucleotide sequence of the Lc-2A-U is shown in SEQ ID NO. 7, and the The nucleotide sequence of Lc-2A-D is shown in SEQ ID NO.8.

Preferably, the primers for amplifying the cotton anthocyanin synthesis regulation gene GhPAP1D include GhPAP1D-U and GhPAP1D-D; the nucleotide sequence of the GhPAP1D-U is shown in SEQ ID NO. The nucleotide sequence is shown in SEQ ID NO.10.

Another object of the present invention is to provide an expression vector comprising the above fusion gene. The gene structure of the T-DNA region of the expression vector includes: the right border of the T-DNA-recombinase recognition site LoxpFRT-two in series Cauliflower mosaic virus 35S promoter of enhancer - fusion gene expression cassette of reporter gene GUS and marker gene NPTII - Agrobacterium opine synthase gene terminator nos - recombinase recognition site LoxpFRT - cotton fiber secondary wall synthesis stage Specific promoter pFbl2A-fusion gene-Agrobacterium opine synthase gene terminator nos-T-DNA left border;

The nucleotide sequence of the specific promoter pFbl2A during the synthesis period of the cotton fiber secondary wall is shown in SEQ ID NO.11.

Preferably, the base vector of the expression vector includes the plant expression vector pLGN.

Preferably, the primers for amplifying the specific promoter pFbl2A during the synthesis stage of the cotton fiber secondary wall include pFbl2A-F and pFbl2A-R; the nucleotide sequence of the pFbl2A-F is shown in SEQ ID NO. 12, and the The nucleotide sequence of pFbl2A-R is shown in SEQ ID NO.13.

Another object of the present invention is to provide an application of the above-mentioned fusion gene or the above-mentioned expression vector in constructing transgenic cotton, and the mature fiber of the transgenic cotton is purple-red.

Another object of the present invention is to provide a method for constructing transgenic cotton whose mature fibers are purple-red, comprising the following steps: overexpressing the fusion gene in the cotton fiber of the transgenic cotton.

Preferably, the method for overexpression includes transforming the above-mentioned expression vector into Agrobacterium LBA4404, and genetically transforming cotton by a method mediated by Agrobacterium tumefaciens.

The present invention provides a fusion gene for controlling the coloration of cotton fibers. Lc and GhPAP1D genes are combined into a fusion gene using a 2A peptide that can be self-cleaved into small fragment peptides, and the fiber secondary wall-specific promoter pFbl2A is used to start the fusion gene. The fusion gene was overexpressed in cotton fibers, making transgenic cotton produce purple-red fibers. In the example of the present invention, the anthocyanin content of the transgenic cotton was measured, and the result showed that the relative content of anthocyanin in the transgenic fiber was 4093 times higher than that of the white cotton.

Description of drawings

Fig. 1 is the gene structure diagram of the T-DNA region of the expression vector of pLGN-pFblA2-Lc-2A-GhPAP1D;

Fig. 2 is the construction process of fiber-specific expression vector pLGN-pFbl2A-Lc-2A-GhPAP1D;

Figure 3 is a comparison chart of fiber color and lustre of cotton at 25 and 50 days after flowering, wherein WF is white fiber, RF is transgenic red fiber, and DPA is days after flowering.

Detailed ways

The present invention provides a fusion gene for regulating the coloration of cotton fibers. The structure of the fusion gene includes the nucleotide sequence of 5'-maize leaf color gene Lc-peptide segment 2A-cotton anthocyanin synthesis regulation gene GhPAP1D-3' ;

The nucleotide sequence of the corn leaf color gene Lc is shown in SEQ ID NO.1; the nucleotide sequence of the peptide segment 2A is shown in SEQ ID NO.2; the cotton anthocyanin synthesis regulation gene GhPAP1D The nucleotide sequence is shown in SEQ ID NO.3.

The encoded amino acid sequence of the corn leaf color gene Lc of the present invention is shown in SEQ ID NO.4: . The amino acid sequence of the peptide segment 2A of the present invention is shown in SEQ ID NO. 5: QLLNFDLLKLAGDVESNPGP, and the peptide segment 2A can be self-cleaved into small fragments. The gene of the small fragment peptide 2A peptide and the leaf color gene Lc of the present invention are preferably synthesized by optimized fusion of BGI, and primers Lc-2A-U and Lc-2A-D are designed based on the synthesized fragments, and the primers of Lc-2A-U are designed The nucleotide sequence is preferably shown in SEQ ID NO. 7: ggatccATGGCTCTTTCTGCTTCT, and the nucleotide sequence of Lc-2A-D is preferably shown in SEQ ID NO. 8: gagccttccaTAGGACCAGGGTTAGATTCA. In the present invention, the 5' end of the Lc-2A fragment obtained by the above amplification is preferably added with a BamHI enzyme cleavage site, and the 3' end is added with the homology arm of the 2A peptide. The amplification system of the present invention is preferably a 10 μl system, including: 5 μl of 2×PrimeSTAR MAX Premix, 1 μl of primers (5 μmol/L) each, about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification procedure of the present invention is preferably: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 30s, annealing at 56°C for 30s, extension at 72°C for 60s, 35 cycles; and finally extension at 72°C for 10 min.

本发明所述融合基因中棉花花色素合成调控基因GhPAP1D的编码氨基酸序列如SEQ ID NO.6所示：MEGSSLRVRK GAWTEEEDLL LKKCIEKYGEGKWHQVPARAGLNRCRKSCRLRWLNYLKPNIKRGYFAADEVDLIIRLHNLLGNRWSLIAGRLPGRTANDVKNYWNTHLLKKNIDTSGKNSKPKSYQPNPNTKIIKPRPHILSKHSFLISLDEYNNNNNNNHAEASNNVALANDGNNDYGYCFPNDHDEMMWWENMMINEKEVDGYQLQCSANDFDQSMLDQPMNEENYGSTIDEVFLDEELWNVFNP。 In the present invention, the amplification method is preferably used, and the target fragment is obtained by amplifying the leaf cDNA of cotton red strain T586 as a template, and the amplification primers include GhPAP1D-U and GhPAP1D-D; the nucleotide sequence of the GhPAP1D-U is as shown in SEQ ID NO. 9: ccctggtcctATGGAAGGCTCATCTTTAAG, and the nucleotide sequence of the GhPAP1D-D is shown in SEQ ID NO. 10: cgggcccTATGGGTTGAACACAT. In the present invention, the 5' end of the GhPAP1D fragment obtained by the above amplification is preferably added with the homology arm of the 2A peptide, and the 3' end is added with the ApaI restriction site. The amplification system of the present invention is preferably a 10 μl system, including: 5 μl of 2×PrimeSTAR MAX Premix, 1 μl of each primer (5 μmol/L), about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification procedure of the present invention is preferably pre-denaturation at 98°C for 3 minutes; then denaturation at 98°C for 10s, annealing at 56°C for 30s, extension at 72°C for 60s, 30 cycles; and finally extension at 72°C for 10 minutes. .

The method for fusing the above two genes in the present invention preferably includes overlapping PCR of two fragments of Lc-2A and GhPAP1D, and the amplification system is preferably a 10 μl system, including: 2×PrimeSTAR MAXPremix 5 μl, primers Lc-2A-U and GhPAP1D- D (5 μmol/L) is 1 μl each, template DNA is about 60 ng, and ddH ₂ O is added to 10 μl. The amplification procedure was as follows: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 15s, annealing at 52°C for 30s, extension at 72°C for 90s, 10 cycles; last extension at 72°C for 10 min, take out the amplification product, add primers Lc-2A-U and 1 μl of GhPAP1D-D (5 μmol/L) each was amplified again. The amplification procedure was as follows: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 15s, extension at 68°C for 90s, 30 cycles; and final extension at 72°C for 10 min. After the amplification product was taken out, 10 μl of 2×Taq Mix was added, and the extension was carried out at 72°C for 10 min. A tail was added to both ends of the fragment. Finally, the amplified products were recovered by gel after electrophoresis. After recovery, it was connected to pGEM-T-easy vector and sent to BGI for sequencing. After the sequencing was correct, it was digested with Hind III and Apa I, and the gel was recovered after the digestion. The Lc-2A-PAP1D fusion DNA fragment was obtained.

The present invention provides an expression vector comprising the above fusion gene. The gene structure of the T-DNA region of the expression vector includes: the right border of the T-DNA-recombinase recognition site LoxpFRT-cauliflower flower with two enhancers in series Leaf virus 35S promoter- fusion gene expression cassette of reporter gene GUS and marker gene NPTII- Agrobacterium opine synthase gene terminator nos- recombinase recognition site LoxpFRT- cotton fiber secondary wall synthesis stage specific promoter pFbl2A- Fusion gene-Agrobacterium opine synthase gene terminator nos-T-DNA left border; the nucleotide sequence of the specific promoter pFbl2A during the synthesis stage of cotton fiber secondary wall is shown in SEQ ID NO.11.

The gene structure of the T-DNA region of the expression vector of the present invention is preferably as shown in FIG. 1 , and the base vector of the expression vector preferably includes the plant expression vector pLGN. The pLGN of the present invention is preferably a binary plant expression vector transformed from the traditional plant expression vector pBI121, and its T-DNA segment (region between RB and LB, Figure 2) is replaced with a constitutive 2×35S promoter A fusion gene expression cassette of the reporter gene GUS and the marker gene NPTII controlled by a sub (2×35S-P) gene.

The specific promoter pFbl2A of the cotton fiber secondary wall synthesis stage of the present invention is preferably obtained by amplification, and the promoter pFbl2A is amplified and cloned from the genome of cotton strain Jimian No. 14, and the amplified primers preferably include pFbl2A-F and pFbl2A-R; the nucleotide sequence of the pFbl2A-F is preferably as shown in SEQ ID NO.12: aagcttGATATCGAATTCCTGCAG, and the nucleotide sequence of the pFbl2A-R is preferably as shown in SEQ ID NO.13: ggatccTGTAATTGTAAATAGTAATTGTAA, in order to facilitate the subsequent ligation of the vector, a Hind III restriction site was added to the 5' end of the promoter, and a 'BamHI restriction site was added to the 3 end of the promoter. The amplification system of the present invention is preferably a 10 μl system, including: 5 μl of 2×PrimeSTAR MAX Premix, 1 μl of each primer (5 μmol/L), about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification procedure of the present invention is preferably: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 30s, annealing at 58°C for 30s, extension at 72°C for 60s, 35 cycles; and final extension at 72°C for 10 min. After the amplification product was taken out, add 10 μl of 2×Taq Mix, extend at 72°C for 10 min, and add A tails to both ends of the fragment. Finally, the amplified products were recovered by gel after electrophoresis. After recovery, it was connected to pGEM-T-easy vector and sent to BGI for sequencing. After the sequencing was correct, it was digested with Hind III and BamHI, and the gel was recovered after the digestion to obtain the DNA fragment of the promoter pFbl2A.

The present invention also provides the application of the above fusion gene or the above expression vector in constructing transgenic cotton, and the mature fiber of the transgenic cotton is purple-red. The fusion gene is overexpressed in the fiber of the transgenic cotton of the present invention, and the anthocyanin content of the transgenic cotton fiber can reach 4093 times that of white cotton.

The present invention also provides a method for constructing transgenic cotton whose mature fibers are purple-red, comprising the following steps: overexpressing the above fusion gene in the cotton fibers of the transgenic cotton. The overexpression method of the present invention preferably includes transforming the above-mentioned expression vector into Agrobacterium LBA4404, and genetically transforming cotton by a method mediated by Agrobacterium tumefaciens. The method of genetic transformation is not particularly limited in the present invention, and conventional genetic transformation methods in the field can be used.

The fusion gene and expression vector for regulating the coloration of cotton fibers provided by the present invention and their applications are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

The acquisition of maize leaf color gene Lc gene, small fragment peptide 2A peptide gene, cotton anthocyanin synthesis regulation gene GhPAP1D

The small fragment peptide 2A peptide gene and Lc gene were synthesized by optimized fusion of BGI. Using the synthetic fragment as a template, primers were designed (Table 1, Lc-2A-U and Lc-2A-D), the 5' of primer Lc-2A-U A Bam HI restriction site was added to the end, and a 10bp homology arm of the GhPAP1D gene was added to the 5' end of Lc-2A-D. The amplification system is preferably a 10 μl system, including: 5 μl of 2×PrimeSTAR MAX Premix, 1 μl each of primers Lc-2A-U and Lc-2A-D (5 μmol/L), about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification program was: pre-denaturation at 98 °C for 5 min; then denaturation at 98 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 60 s, 35 cycles; and a final extension at 72 °C for 10 min.

Using the CDS sequence of Gohir.D7G082100 as a reference, primers were designed (Table 1, GhPAP1D-U and GhPAP1D-D), the 5' end of the primer GhPAP1D-U added a 10bp homology arm of the 2A peptide gene, and the 5' end of the primer GhPAP1D-D Add the ApaI restriction site. The leaf cDNA of cotton red line T586 (Li Xin. Research on the transgenic function of cotton GhPAP1. Southwest University Master's thesis. 2014 has been published) was used as the template. The amplification system is preferably a 10 μl system, including: 5 μl of 2×PrimeSTARMAXPremix, 1 μl each of primers GhPAP1D-U and GhPAP1D-D (5 μmol/L), about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification procedure of the present invention is preferably: pre-denaturation at 98°C for 3 min; then denaturation at 98°C for 10s, annealing at 56°C for 30s, extension at 72°C for 60s, 30 cycles; and final extension at 72°C for 10 min. .

The two fragments of Lc-2A and GhPAP1D were subjected to overlapping PCR, and the amplification system: 5μl of 2×PrimeSTAR MAX Premix, 1μl of primers Lc-2A-U and GhPAP1D-D (5μmol/L), about 60ng of template DNA, and ddH ₂ 0 to 10 μl. The amplification procedure was as follows: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 15s, annealing at 52°C for 30s, extension at 72°C for 90s, 10 cycles; last extension at 72°C for 10 min, take out the amplification product, add primers Lc-2A-U and 1 μl of GhPAP1D-D (5 μmol/L) each was amplified again. The amplification procedure was as follows: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 15s, extension at 68°C for 90s, 30 cycles; and final extension at 72°C for 10 min. After the amplification product was taken out, 10 μl of 2×Taq Mix was added, and the extension was carried out at 72°C for 10 min. A tail was added to both ends of the fragment. Finally, the amplified products were recovered by gel after electrophoresis. After recovery, it was connected to pGEM-T-easy vector and sent to BGI for sequencing.

Primer information used in the examples of Table 1

Example 2

Construction of plant expression vector specifically expressing Lc and GhPAP1D fusion gene during fiber secondary wall synthesis

Figure 2 shows the process of constructing the coding sequences of Lc-2A and GhPAP1D genes into the plant expression vector pLGN. pLGN is a binary plant expression vector transformed from the traditional plant expression vector pBI121. Its T-DNA segment (region between RB and LB, Figure 2) was replaced with a fusion gene expression cassette of reporter gene GUS and marker gene NPTII controlled by a constitutive 2x35S promoter (2x35S-P). Using primers pFbl2A-F and pFbl2A-R (see Table 1 for the sequence), the cloned promoter Fbl2A was amplified from the genome of cotton strain Jimian No. 14, and the 5' end of the promoter was added with a Hind III restriction site to initiate A BamHI restriction site was added to the 3' end of the sub. The amplification system of the present invention is preferably a 10 μl system, including: 5 μl of 2× PrimeSTAR MAXPremix, 1 μl of primers pFbl2A-F and pFbl2A-R (5 μmol/L), about 60 ng of template DNA, and ddH ₂ O to 10 μl. The amplification procedure of the present invention is preferably: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 30s, annealing at 58°C for 30s, extension at 72°C for 60s, 35 cycles; and finally extension at 72°C for 10 min. After the amplification product was taken out, add 10 μl of 2×TaqMix, extend at 72°C for 10 min, and add A tails to both ends of the fragment. Finally, the amplified products were recovered by gel after electrophoresis. After recovery, it was connected to pGEM-T-easy vector and sent to BGI for sequencing. After the sequencing was correct, it was digested with Hind III and BamHI, and the gel was recovered after the digestion. A DNA fragment of the promoter pFbl2A was obtained.

The Lc-2A-PAP1D fusion DNA fragment ligated to the pGEM-T-easy vector was sequenced correctly and digested with Hind III and Apa I enzymes, and the gel was recovered after the digestion. The Lc-2A-PAP1D fusion DNA fragment was obtained.

The pLGN vector was digested with Hind III/Apa I and electrophoresed to recover the linear vector fragment, and ligated with the promoter pFbl2A, Lc-2A-PAP1D fusion DNA fragment with T4 DNA ligase, the ligation system was 20 μl, including: T4 DNA Ligase 1 μl , 10×T4 DNALigase Buffer 2μl, pLGN-pFbl2A vector about 50ng, promoter pFbl2A about 50ng, Lc-2A-PAP1D fusion DNA fragment about 50ng, add ddH ₂ O to 20μl. 20 ℃ constant temperature reaction 6h. The ligation product was transformed into Escherichia coli competent DH5α, and the extracted plasmid was digested for verification. Finally, the specific expression vector pLGN-pFbl2A-Lc-2A-GhPAP1D of Lc-2A-GhPAP1D was obtained.

Referring to the user manual of Bio-RAD MicroPulser, the above vector was introduced into Agrobacterium LBA4404 by electroporation method.

Example 3

Genetic transformation of cotton

The cotton genetic transformation of the above expression vector was carried out by the method mediated by Agrobacterium tumefaciens, and the medium formula used is shown in Table 2. The specific method is as follows: Dehulling the plump cotton seeds of wild-type upland cotton YZ-1, placing a small amount (about 20 to 40) of the dehulled seeds in a sterilized 100mL conical flask, first with 75% alcohol Pre-wash the seeds for 1min, pour out the alcohol and then add 0.1% HgCl ₂ for sterilization for about 12min (constantly shake the triangular flask for sterilization), pour out the mercury chloride gently, add sterile water to fully rinse the seeds, and rinse about 10 times. Place the destroyed seeds on the germination medium. After the radicle grows about 1cm (about 36-48h), gently insert the radicle into the germination medium, and cultivate the hypocotyl to about 7cm in the dark at about 30°C. (about 7 days). About 20h before infecting the hypoblast, a single colony of Agrobacterium carrying a genetic transformation vector was inoculated into a liquid YEB medium containing 50 mg/L Km and 125 mg/L Sm, placed on a shaker (200 rpm) at 28°C, and cultured for about 20 hours. After 20 hours, the OD value (OD ₆₀₀ ) of the bacterial solution was measured, and the OD ₆₀₀ of 0.4-0.6 was suitable for transformation. Collect the activated Agrobacterium solution, centrifuge at 8000 rpm for 1 min, discard the supernatant and resuspend the bacteria with the same volume of MGL liquid medium (containing 100 μmol/L AS, acetosyringone), and collect the resuspended bacteria in a sterile 100 mL conical flask The body was placed on a shaker (28 °C, 100 rpm) for about 40 min. Cut the hypocotyl into 0.8-1cm lengths with a sterile scalpel, then place it in the resuspension and infect it on a shaker (28°C, 100rpm) for 40min, discard the liquid and then take out the hypocotyl and place it gently. The surface of the solid co-culture medium was cultured in the dark for about 48h. After dark culture, the hypoblasts were transferred to solid one-sieve medium for culture (30°C, 16h light/8h dark, the same below), and then transferred to solid IBA medium after 30 days, and subcultured every 30 days or so until healing. Green somatic embryos were formed on the wound, and the green somatic embryos were transferred to solid differentiation medium for culture. After the somatic embryos grow to about 3 cm, they are inserted into the rooting medium until the seedlings grow. The above operations must be completed under strict aseptic conditions.

Robust regenerated cotton seedlings were transplanted to planting pots and routinely managed in the greenhouse until cotton fibers and seeds matured. The T0 generation transgenic cotton seeds were harvested, and the T1 generation was continued to be planted and stained with GUS. Homozygous transgenic T1 generation lines (all GUS-positive or GUS-negative plants) were screened out. The expression levels of target genes GhPAP1 and Lc were detected, and the changes of fibroanthocyanidin content and fiber color were compared.

Table 2 Medium for cotton genetic transformation mediated by Agrobacterium tumefaciens

Example 4

Cotton fiber color observation, anthocyanin content detection and gene expression detection

Cotton fiber color was taken 25 days and 50 days after flowering to observe and photograph (Fig. 3).

The white fibers and red fibers 25 days after flowering were sent to Maiwei Metabolism Company for transcriptome and metabolome detection. The metabolome data acquisition instrument system mainly includes high performance liquid chromatography and tandem mass spectrometry, based on the self-built database MWDB of Maiwei Metabolism Company. (metware database) and metabolite information public database, qualitative analysis of primary and secondary spectrum data detected by mass spectrometry. The quantification of metabolites is completed by the multiple reaction monitoring (MRM) analysis of triple quadrupole mass spectrometry. CPS), use the MultiaQuant software to open the sample download file, and perform the integration and calibration of the chromatographic peaks. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance. The anthocyanin content and gene expression were obtained from the feedback data. The results are shown in Table 3. A total of 8 kinds of anthocyanins were detected, which were significantly improved compared with white cotton, and the total amount of anthocyanins was 4093 times that of white cotton. As shown in Table 4, the phenylpropane pathway for synthesizing anthocyanin precursor substances and the expression of some structural genes and regulatory genes of the anthocyanin synthesis pathway were significantly improved compared with those of white cotton.

Table 3 Relative content of anthocyanins in white fibers and transgenic red fibers 25 days after flowering

Table 4 Expression levels of anthocyanin synthesis-related genes in white fibers and transgenic red fibers 25 days after flowering

RF: red cotton; WF: white cotton; fpkm: the number of reads per 1K bases mapped to exons per 1 million reads per map; GhPAL: phenylalanine lyase; GhCHS: Chalcone synthase; GhDFR: dihydroflavonol reductase; GhANS: anthocyanin synthase; GhUFGT: anthocyanin glucosyltransferase; GhGST: glutathione transferase.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

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<400> 3

atggaaggct catctttaag agttagaaaa ggtgcatgga ctgaagaaga agaccttctt 60

cttaagaaat gtattgagaa atatggtgaa gggaaatggc atcaagtgcc tgctagagct 120

ggcttgaatc gttgccggaa aagctgtaga ctgcggtggc tgaattattt gaagcctaat 180

atcaagagag gatattttgc agctgatgaa gttgacctca ttattcgcct ccataacctc 240

ctaggtaata gatggtcact gattgctggt agactgccag gaagaacagc aaacgatgtg 300

aaaaactatt ggaacaccca cttgcttaaa aaaaatatag atacgtccgg taaaaactca 360

aaaccaaaat cttatcaacc aaatcccaat accaaaatta tcaagcctcg gcctcatatc 420

ttatcaaagc acagttttct tataagtttg gatgagtaca ataataacaa caacaacaac 480

catgcagaag caagtaacaa cgtggcatta gctaacgacg gtaataacga ctatgggtat 540

tgtttcccta atgaccacga tgagatgatg tggtgggaaa atatgatgat aaatgaaaag 600

gaggttgatg gctatcagct acagtgttca gccaatgatt ttgatcaaag catgttggac 660

caacctatga atgaagagaa ttatggcagt actatagatg aggtttttct tgatgaggaa 720

ctgtggaatg tgttcaaccc atagggcccg 750

<210> 4

<211> 610

<212> PRT

<213> Zea mays L.

<400> 4

Met Ala Leu Ser Ala Ser Arg Val Gln Gln Ala Glu Glu Leu Leu Gln

1 5 10 15

Arg Pro Ala Glu Arg Gln Leu Met Arg Ser Gln Leu Ala Ala Ala Ala

20 25 30

Arg Ser Ile Asn Trp Ser Tyr Ala Leu Phe Trp Ser Ile Ser Asp Thr

35 40 45

Gln Pro Gly Val Leu Thr Trp Thr Asp Gly Phe Tyr Asn Gly Glu Val

50 55 60

Lys Thr Arg Lys Ile Ser Asn Ser Val Glu Leu Thr Ser Asp Gln Leu

65 70 75 80

Val Met Gln Arg Ser Asp Gln Leu Arg Glu Leu Tyr Glu Ala Leu Leu

85 90 95

Ser Gly Glu Gly Asp Arg Arg Ala Ala Pro Ala Arg Pro Ala Gly Ser

100 105 110

Leu Ser Pro Glu Asp Leu Gly Asp Thr Glu Trp Tyr Tyr Val Val Ser

115 120 125

Met Thr Tyr Ala Phe Arg Pro Gly Gln Gly Leu Pro Gly Arg Ser Phe

130 135 140

Ala Ser Asp Glu His Val Trp Leu Cys Asn Ala His Leu Ala Gly Ser

145 150 155 160

Lys Ala Phe Pro Arg Ala Leu Leu Ala Lys Ser Ala Ser Ile Gln Ser

165 170 175

Ile Leu Cys Ile Pro Val Met Gly Gly Val Leu Glu Leu Gly Thr Thr

180 185 190

Asp Thr Val Pro Glu Ala Pro Asp Leu Val Ser Arg Ala Thr Ala Ala

195 200 205

Phe Trp Glu Pro Gln Cys Pro Ser Ser Ser Ser Pro Ser Gly Arg Ala Asn

210 215 220

Glu Thr Gly Glu Ala Ala Ala Asp Asp Gly Thr Phe Ala Phe Glu Glu

225 230 235 240

Leu Asp His Asn Asn Gly Met Asp Asp Ile Glu Ala Met Thr Ala Ala

245 250 255

Gly Gly His Gly Gln Glu Glu Glu Leu Arg Leu Arg Glu Ala Glu Ala

260 265 270

Leu Ser Asp Asp Ala Ser Leu Glu His Ile Thr Lys Glu Ile Glu Glu

275 280 285

Phe Tyr Ser Leu Cys Asp Glu Met Asp Leu Gln Ala Leu Pro Leu Pro

290 295 300

Leu Glu Asp Gly Trp Thr Val Asp Ala Ser Asn Phe Glu Val Pro Cys

305 310 315 320

Ser Ser Pro Gln Pro Ala Pro Pro Pro Val Asp Arg Ala Thr Ala Asn

325 330 335

Val Ala Ala Asp Ala Ser Arg Ala Pro Val Tyr Gly Ser Arg Ala Thr

340 345 350

Ser Phe Met Ala Trp Thr Arg Ser Ser Gln Gln Ser Ser Cys Ser Asp

355 360 365

Asp Ala Ala Pro Ala Ala Val Val Pro Ala Ile Glu Glu Pro Gln Arg

370 375 380

Leu Leu Lys Lys Val Val Ala Gly Gly Gly Ala Trp Glu Ser Cys Gly

385 390 395 400

Gly Ala Thr Gly Ala Ala Gln Glu Met Ser Gly Thr Gly Thr Lys Asn

405 410 415

His Val Met Ser Glu Arg Lys Arg Arg Glu Lys Leu Asn Glu Met Phe

420 425 430

Leu Val Leu Lys Ser Leu Leu Pro Ser Ile His Arg Val Asn Lys Ala

435 440 445

Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys Glu Leu Gln Arg Arg

450 455 460

Val Gln Glu Leu Glu Ser Ser Arg Glu Pro Ala Ser Arg Pro Ser Glu

465 470 475 480

Thr Thr Thr Arg Leu Ile Thr Arg Pro Ser Arg Gly Asn Asn Glu Ser

485 490 495

Val Arg Lys Glu Val Cys Ala Gly Ser Lys Arg Lys Ser Pro Glu Leu

500 505 510

Gly Arg Asp Asp Val Glu Arg Pro Pro Val Leu Thr Met Asp Ala Gly

515 520 525

Thr Ser Asn Val Thr Val Thr Val Ser Asp Lys Asp Val Leu Leu Glu

530 535 540

Val Gln Cys Arg Trp Glu Glu Leu Leu Met Thr Arg Val Phe Asp Ala

545 550 555 560

Ile Lys Ser Leu His Leu Asp Val Leu Ser Val Gln Ala Ser Ala Pro

565 570 575

Asp Gly Phe Met Gly Leu Lys Ile Arg Ala Gln Phe Ala Gly Ser Gly

580 585 590

Ala Val Val Pro Trp Met Ile Ser Glu Ala Leu Arg Lys Ala Ile Gly

595 600 605

Lys Arg

610

<210> 5

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 5

Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 6

<211> 249

<212> PRT

<213> Gossypium spp

<400> 6

Met Glu Gly Ser Ser Leu Arg Val Arg Lys Gly Ala Trp Thr Glu Glu

1 5 10 15

Glu Asp Leu Leu Leu Lys Lys Cys Ile Glu Lys Tyr Gly Glu Gly Lys

20 25 30

Trp His Gln Val Pro Ala Arg Ala Gly Leu Asn Arg Cys Arg Lys Ser

35 40 45

Cys Arg Leu Arg Trp Leu Asn Tyr Leu Lys Pro Asn Ile Lys Arg Gly

50 55 60

Tyr Phe Ala Ala Asp Glu Val Asp Leu Ile Ile Arg Leu His Asn Leu

65 70 75 80

Leu Gly Asn Arg Trp Ser Leu Ile Ala Gly Arg Leu Pro Gly Arg Thr

85 90 95

Ala Asn Asp Val Lys Asn Tyr Trp Asn Thr His Leu Leu Lys Lys Asn

100 105 110

Ile Asp Thr Ser Gly Lys Asn Ser Lys Pro Lys Ser Tyr Gln Pro Asn

115 120 125

Pro Asn Thr Lys Ile Ile Lys Pro Arg Pro His Ile Leu Ser Lys His

130 135 140

Ser Phe Leu Ile Ser Leu Asp Glu Tyr Asn Asn Asn Asn Asn Asn Asn

145 150 155 160

His Ala Glu Ala Ser Asn Asn Val Ala Leu Ala Asn Asp Gly Asn Asn

165 170 175

Asp Tyr Gly Tyr Cys Phe Pro Asn Asp His Asp Glu Met Met Trp Trp

180 185 190

Glu Asn Met Met Ile Asn Glu Lys Glu Val Asp Gly Tyr Gln Leu Gln

195 200 205

Cys Ser Ala Asn Asp Phe Asp Gln Ser Met Leu Asp Gln Pro Met Asn

210 215 220

Glu Glu Asn Tyr Gly Ser Thr Ile Asp Glu Val Phe Leu Asp Glu Glu

225 230 235 240

Leu Trp Asn Val Phe Asn Pro Gly Pro

245

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

ggatccatgg ctctttctgc ttct 24

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 8

gagccttcca taggaccagg gttagattca 30

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 9

ccctggtcct atggaaggct catctttaag 30

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 10

cgggccctat gggttgaaca cat 23

<210> 11

<211> 2331

<212> DNA

<213> Gossypium spp

<400> 11

cttgatatcg aattcctgca gacttaggat tggatggcgt tcaggagctt ggattggttt 60

tctcacatca tattttatta aataattatt aattaaaatt tatggacttt tggactgtct 120

gactaatttt cagaatttta ttttggtttt gggttttgtt gagttttttta gataattatt 180

ttaaatattc tgcataattt ttctgttatt tgaaaaggat gttcgaattt tttttcaaaa 240

ttgaaacgtt taagaatttt tactactgca aattcagaat aagtgaattt gtttttttaga 300

aagattaaat aagttagtat tacgattttt agtttgattt ggtggaaagt aatgtatgtt 360

tttgaacata attatttgac aataattaag ttttctagga aataaacgga aatatcttct 420

ttttttttttg taaaattact aatgcaagaa caaacaacgt tttgggaagc aaataatcta 480

gctttaagta gtcagtgtaa ctctcaaaat ctggtcataa cttctaggct gagtttgctg 540

tgctacagta gtaagtctat agaaacttac ctgacaaaac gacatgacgt cagggtcgaa 600

tctacaactt ttccttttttc ttcaattaac atatggttga ttcaagttcc gatctataat 660

aatttattac gatttatcaa tttcaattac cttatatcat cctattataa atataagtca 720

gttcaattca gttttcgaaa gttccctaaa attttgaatt ttattaaatt tattccctaa 780

aaccgaaata gtgatatctt tcaaatttaa gtttcatttt tcaatccgat ttcaatttca 840

tccttttata actctctatg atctataatt acataaattt caaactaatt ttgaaatata 900

tacactttag tccctaagtt caaaactata aattttcact ttagaaatta atcatttttc 960

acatctaagc atcaaattta accaaatgac acaaatttca tgattagtta gatcaagctt 1020

ttgagtcttc aaaaacataa aaattacaaa aaaaaaaaaa caaacttaaa atcatttatc 1080

aatttgaaca acaaagcttg gccgaatgct aagagcttaa aaatggcttc ttttgtttct 1140

ttttgttgca aacggtggag agaagaggga aatgaagatt gaccatattt ttttattatg 1200

ttttaacata taatattaat aatttaatca taattatact ttggtgaatg tgacagtggg 1260

gagatacgta aagtatataa cattatactt tttgcaagca gttggctggt ctatccaaga 1320

gtgatcaaag tttgagctgc cttcaatgag ccaatttttg cccataatgg ataaaggcaa 1380

tttgtttagt tcaactgctc acagaataat gttaaaatga aattaaaata aggtggcctg 1440

gtcacacaca cacaaaaaaa aaactaatgt tggttggttg aattttatat tacggaatgt 1500

aatgttatat tttaaaataa aattatgtta tttagattct taatattttg agcattccat 1560

actataatct cgtatacata atattaaaat atagtaatat aaagtgtaat taactttaaa 1620

ttacaagcat aatattaaat tttgaatcaa ttaattttta tttctattat tttaattaat 1680

ttagtctatt ttttcaaaat aaaatttaaa tctaaataaa aataattttt ccttaatatt 1740

attaataaat ttatttcaac atcatatatt tacttattaa tacataaatt ataataattt 1800

atcataattt tatggaaatt gagaccaaga aacattaaga gaacaaattc tataacaaag 1860

acaatttagt aaaaatgtac ttttaggtaa ttttaagtac tcttaaccaa acacaaaaat 1920

tcaaatcaaa tgaaccaaat aagataatat aacatacaga atatcctact tgtattctta 1980

cattcccgta atcatattat gaaaagtaat attatattac ctgagccaaa tgctctcaca 2040

aactattatc caaaaaaaaa atgttgaata taatttttat aacatttttt catatatttg 2100

caagattata ttttgtatat ttacgtaaaa atatttgaca tagattgaac accttcttaa 2160

cataatccca ccataagtca agtatgtaga tgagaaattg gtacaaacaa cgtggggcca 2220

aatcccacca aaccatctct catcctctcc tataaaaggc tagttacaca tacacaacaa 2280

tccacacaca aatacactca aaattctttg ctttgtattt cggttggggg a 2331

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 12

aagcttgata tcgaattcct gcag 24

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 13

ggatcctgta attgtaaata gtaattgtaa 30

Claims (10)

1. a fusion gene that regulates and controls cotton fiber coloring, it is characterized in that, the structure of described fusion gene comprises the nucleotide sequence of 5 '-maize leaf color gene Lc-peptide segment 2A-cotton anthocyanin synthesis regulation gene GhPAP1D- 3'; The nucleotide sequence of the corn leaf color gene Lc is shown in SEQ ID NO.1; the nucleotide sequence of the peptide segment 2A is shown in SEQ ID NO.2; the cotton anthocyanin synthesis regulation gene GhPAP1D The nucleotide sequence is shown in SEQ ID NO.3. 2. The fusion gene according to claim 1, wherein the encoded amino acid sequence of the corn leaf color gene Lc is as shown in SEQ ID NO.4; the amino acid sequence of the peptide segment 2A is as shown in SEQ ID NO.5 The amino acid sequence of the encoded protein of the cotton anthocyanin synthesis regulation gene GhPAP1D is shown in SEQ ID NO.6. 3. according to the described fusion gene of claim 1 or 2, it is characterized in that, the primer that amplifies described maize leaf color gene Lc comprises Lc-2A-U and Lc-2A-D; The nucleus of described Lc-2A-U The nucleotide sequence is shown in SEQ ID NO.7, and the nucleotide sequence of Lc-2A-D is shown in SEQ ID NO.8. 4. fusion gene according to claim 1 or 2 is characterized in that, the primer that amplifies described cotton anthocyanin synthesis regulation gene GhPAP1D comprises GhPAP1D-U and GhPAP1D-D; The nucleotide sequence of described GhPAP1D-U is as follows: As shown in SEQ ID NO.9, the nucleotide sequence of the GhPAP1D-D is shown in SEQ ID NO.10. 5. An expression vector comprising the fusion gene according to any one of claims 1 to 4, wherein the gene structure of the T-DNA region of the expression vector comprises: the right border of T-DNA-recombinase recognition LoxpFRT-Cauliflower mosaic virus 35S promoter with two enhancers in tandem-fusion gene expression cassette of reporter gene GUS and marker gene NPTII-Agrobacterium opine synthase gene terminator nos-recombinase recognition site LoxpFRT- Cotton fiber secondary wall synthesis stage specific promoter pFbl2A-fusion gene-Agrobacterium opine synthase gene terminator nos-T-DNA left border; The nucleotide sequence of the specific promoter pFbl2A during the synthesis stage of the cotton fiber secondary wall is shown in SEQ ID NO.11. 6. The expression vector according to claim 5, wherein the base vector of the expression vector comprises a plant expression vector pLGN. 7. according to the described expression vector of claim 5, it is characterized in that, the primer of the specific promoter pFbl2A of the synthetic stage of described cotton fiber secondary wall synthesis comprises pFbl2A-F and pFbl2A-R; The nucleotide of described pFbl2A-F The sequence is shown in SEQ ID NO.12, and the nucleotide sequence of the pFbl2A-R is shown in SEQ ID NO.13. 8 . The application of the fusion gene of any one of claims 1 to 4 or the expression vector of any one of claims 5 to 7 in constructing transgenic cotton, wherein the mature fibers of the transgenic cotton are purple-red. 9 . A method for constructing transgenic cotton whose mature fibers are purple-red, comprising the following steps: overexpressing the fusion gene according to any one of claims 1 to 4 in cotton fibers of the transgenic cotton. 10 . 10. The method according to claim 9, characterized in that, the method for overexpression comprises transforming the expression vector of any one of claims 5 to 7 into Agrobacterium LBA4404, and through Agrobacterium tumefaciens-mediated Methods The genetic transformation of cotton was carried out.

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