CN111826386A

CN111826386A - Fusion gene for regulating and controlling color development of cotton fibers, expression vector and application thereof

Info

Publication number: CN111826386A
Application number: CN202010754544.2A
Authority: CN
Inventors: 冉玲芳; 肖月华; 李耀华; 曾健晏; 王毅; 莫童; 梁爱敏; 罗明
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2020-10-27
Anticipated expiration: 2040-07-30
Also published as: CN111826386B

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

Fusion gene for regulating and controlling color development of cotton fibers, expression vector and application thereof

Technical Field

The invention belongs to the technical field of plant transgenic breeding, and particularly relates to a fusion gene for regulating and controlling color development of cotton fibers, and an expression vector and application thereof.

Background

Natural colored cotton is a cotton material that can synthesize and accumulate natural pigments during the fiber development process, thereby giving the mature fibers a natural color. Compared with the conventional white cotton and chemical fiber, the colored cotton has natural color, so that bleaching and printing and dyeing treatment are not needed in the textile processing process, the pollution of toxic and harmful residues such as dye, organic solvent, heavy metal and the like to the textile and the harm to human bodies are avoided, and the green production level of the textile can be obviously improved. The color cotton material applied to the textile at present only has two colors of green and brown, and can not meet the requirements of consumers and textile industry on color diversity. Therefore, the color range of the mature fiber is expanded, which is a necessary condition for further development of the color cotton industry.

Disclosure of Invention

In view of the above, the invention aims to provide a fusion gene for regulating and controlling color development of cotton fibers, an expression vector and application thereof, which can significantly improve the anthocyanidin content of the cotton fibers, thereby obtaining natural purplish red cotton fibers.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a fusion gene for regulating and controlling color generation of cotton fibers, which structurally comprises a nucleotide sequence of a 5 '-corn leaf color gene Lc-peptide segment 2A-a cotton anthocyanidin synthesis regulation gene GhPAP 1D-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 as SEQ ID NO. 2; the nucleotide sequence of the cotton anthocyanidin synthesis regulatory gene GhPAP1D is shown in SEQ ID No. 3.

Preferably, the coding 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 as SEQ ID NO. 5; the coding amino acid sequence of the cotton anthocyanidin synthesis regulatory gene GhPAP1D is shown in SEQ ID NO. 6.

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

Preferably, the primers for amplifying the cotton anthocyanidin synthesis regulatory gene GhPAP1D comprise GhPAP1D-U and GhPAP 1D-D; the nucleotide sequence of the GhPAP1D-U is shown as SEQ ID NO.9, and the nucleotide sequence of the GhPAP1D-D is shown as SEQ ID NO. 10.

It is another object of the present invention to provide an expression vector comprising the above fusion gene, the gene structure of the T-DNA region of the expression vector comprising: right border of T-DNA-recombinase recognition site loxpfqrt-fusion gene expression cassette of cauliflower mosaic virus 35S promoter-reporter gene GUS and marker gene NPTII of tandem two enhancers-agrobacterium opine synthase gene terminator nos-recombinase recognition site loxpfqrt-cotton fiber secondary wall synthesis period specific promoter pFbl 2A-fusion gene-agrobacterium opine synthase gene terminator nos-T-DNA left border;

the nucleotide sequence of the specific promoter pFbl2A in the synthesis period of the secondary wall of the cotton fiber is shown as SEQID NO. 11.

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

Preferably, primers for amplifying the cotton fiber secondary wall synthesis period specific promoter pFBl2A comprise pFBl2A-F and pFBl 2A-R; the nucleotide sequence of the pFBl2A-F is shown as SEQ ID NO.12, and the nucleotide sequence of the pFBl2A-R is shown as SEQ ID NO. 13.

The invention also aims to provide an application of the fusion gene or the expression vector in constructing transgenic cotton, wherein mature fibers of the transgenic cotton are purple red.

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

Preferably, the overexpression method comprises transforming the expression vector into agrobacterium LBA4404, and performing genetic transformation of cotton by agrobacterium tumefaciens mediated method.

The invention provides a fusion gene for controlling color development of cotton fibers, which is characterized in that 2A peptide capable of being automatically cracked into small fragment peptide is utilized, Lc and GhPAP1D genomes are synthesized into the fusion gene, and a fiber secondary wall specific promoter pFBl2A is adopted to start the over-expression of the fusion gene in the cotton fibers, so that transgenic cotton generates purple red fibers. In the embodiment of the invention, the result of measuring the anthocyanin content in the transgenic cotton shows that the relative content of the anthocyanin in the transgenic fiber is increased by 4093 times compared with that in white cotton.

Drawings

FIG. 1 is a gene structure diagram of the T-DNA region of an expression vector of pLGN-pFBLA2-Lc-2A-GhPAP 1D;

FIG. 2 is a process for constructing a fiber-specific expression vector pLGN-pFBl 2A-Lc-2A-GhCAP 1D;

FIG. 3 is a graph comparing fiber color at 25 and 50 days after cotton flowering, where WF is white fiber, RF is transgenic red fiber, and DPA is days after flowering.

Detailed Description

The coding amino acid sequence of the corn leaf color gene Lc is shown in SEQ ID NO. 4: MALSASRVQQAEELLQRPAERQLMRSQLAAAARSINWSYALFWSISDTQPGVLTWTDGFYNGEVKTRKISNSVELTSDQLVMQRSDQLRELYEALLSGEGDRRAAPARPAGSLSPEDLGDTEWYYVVSMTYAFRPGQGLPGRSFASDEHVWLCNAHLAGSKAFPRALLAKSASIQSILCIPVMGGVLELGTTDTVPEAPDLVSRATAAFWEPQCPSSSPSGRANETGEAAADDGTFAFEELDHNNGMDDIEAMTAAGGHGQEEELRLREAEALSDDASLEHITKEIEEFYSLCDEMDLQALPLPLEDGWTVDASNFEVPCSSPQPAPPPVDRATANVAADASRAPVYGSRATSFMAWTRSSQQSSCSDDAAPAAVVPAIEEPQRLLKKVVAGGGAWESCGGATGAAQEMSGTGTKNHVMSERKRREKLNEMFLVLKSLLPSIHRVNKASILAETIAYLKELQRRVQELESSREPASRPSETTTRLITRPSRGNNESVRKEVCAGSKRKSPELGRDDVERPPVLTMDAGTSNVTVTVSDKDVLLEVQCRWEELLMTRVFDAIKSLHLDVLSVQASAPDGFMGLKIRAQFAGSGAVVPWMISEALRKAIGKR are provided. The amino acid sequence of the peptide segment 2A is shown as SEQ ID NO. 5: QLLNFDLLKLAGDVESNPGP, the peptide fragment 2A can be self-cleaved into small fragments. The gene of the small fragment peptide 2A peptide and the leaf color gene Lc are preferably synthesized by Huada optimized fusion, primers Lc-2A-U and Lc-2A-D are designed based on the synthesized fragment, and the nucleotide sequence of Lc-2A-U is preferably shown as SEQ ID NO. 7: ggatccATGGCTCTTTCTGCTTCT, the nucleotide sequence of Lc-2A-D is preferably as shown in SEQ ID NO. 8: gagccttccaTAGGACCAGGGTTAGATTCA are provided. The 5 'end of the Lc-2A fragment obtained by amplification is preferably added with a BamHI enzyme cutting site, and the 3' end is added with a homologous arm of a 2A peptide. The system for amplification according to the invention is preferably a 10. mu.l system comprising: 2 XPrimeSTAR MAX Premix 5. mu.l, 1. mu.l each of primers (5. mu. mol/L), about 60ng of template DNA, ddH was added₂O to 10. mu.l. The procedure for amplification according to the invention is preferably: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 60s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.

The coding amino acid sequence of cotton anthocyanidin synthesis regulation gene GhPAP1D in the fusion gene is shown in SEQ ID NO. 6: MEGSSLRVRK GAWTEEEDLL LKKCIEKYGEGKWHQVPARAGLNRCRKSCRLRWLNYLKPNIKRGYFAADEVDLIIRLHNLLGNRWSLIAGRLPGRTANDVKNYWNTHLLKKNIDTSGKNSKPKSYQPNPNTKIIKPRPHILSKHSFLISLDEYNNNNNNNHAEASNNVALANDGNNDYGYCFPNDHDEMMWWENMMINEKEVDGYQLQCSANDFDQSMLDQPMNEENYGSTIDEVFLDEELWNVFNP are provided. The invention preferably utilizes an amplification method, takes the cDNA of the leaf of the cotton red strain T586 as a template, and obtains a target fragment by amplification, wherein an amplification primer comprises GhPAP1D-U and GhPAP 1D-D; the nucleotide sequence of the GhPAP1D-U is shown in SEQ ID NO. 9: cc (cc)ctggtcctATGGAAGGCTCATCTTTAAG, the nucleotide sequence of the GhPAP1D-D is shown in SEQ ID NO. 10: cgggcccTATGGGTTGAACACAT are provided. In the present invention, the 5 'end of the GhPAP1D fragment obtained by the above amplification is preferably added with a homology arm of 2A peptide, and the 3' end is preferably added with an ApaI enzyme cutting site. The system for amplification according to the invention is preferably a 10. mu.l system comprising: 2 XPrimeSTAR MAX Premix 5. mu.l, primers (5. mu. mol/L) each 1. mu.l, about 60ng of template DNA, ddH was added₂O to 10. mu.l. The amplification procedure of the invention is preferably pre-denaturation at 98 ℃ for 3 min; then denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 60s for 30 cycles; finally, extension is carried out for 10min at 72 ℃. .

The method for fusing the two genes preferably comprises the step of performing overlapping PCR on the two Lc-2A, GhPAP1D fragments, and the system for amplifying is preferably a 10-microliter system, and comprises the following steps: 2.mu.l of 2 XPrimeSTAR MAXPREmix, 1. mu.l each of primers Lc-2A-U and GhPAP1D-D (5. mu. mol/L), about 60ng of template DNA, ddH added₂O to 10. mu.l. The amplification procedure was: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 15s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 90s for 10 cycles; finally, extension is carried out for 10min at 72 ℃, an amplification product is taken out, primers Lc-2A-U and GhPAP1D-D (5 mu mol/L) are added into each 1 mu L, and amplification is carried out again, wherein the amplification procedure is as follows: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 15s, extension at 68 ℃ for 90s, 30 cycles; finally, extension is carried out for 10min at 72 ℃. After the amplified product was taken out, 10. mu.l of 2 XTaq Mix was added, and extension was carried out at 72 ℃ for 10min, and A tails were added to both ends of the fragment. Finally, the gel of the amplification product after electrophoresis is recovered. After recovery, the DNA fragment was ligated to pGEM-T-easy vector and sent to Huada Gene Co for sequencing. After the sequencing is correct, enzyme digestion is carried out by Hind III and Apa I, and the gel is recovered after enzyme 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 comprising: right border of T-DNA-recombinase recognition site loxpfqrt-fusion gene expression cassette of cauliflower mosaic virus 35S promoter-reporter gene GUS and marker gene NPTII of tandem two enhancers-agrobacterium opine synthase gene terminator nos-recombinase recognition site loxpfqrt-cotton fiber secondary wall synthesis period specific promoter pFbl 2A-fusion gene-agrobacterium opine synthase gene terminator nos-T-DNA left border; the nucleotide sequence of the specific promoter pFBl2A in the synthesis period of the secondary wall of the cotton fiber is shown as 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 a plant expression vector pLGN. The pLGN of the present invention is preferably a binary plant expression vector modified from the conventional plant expression vector pBI121, in which the T-DNA segment (region between RB and LB, FIG. 2) replaces the fusion gene expression cassette of the reporter gene GUS and the marker gene NPTII, which is controlled by the constitutive 2X 35S promoter (2X 35S-P).

The specific promoter pFBl2A in the secondary wall synthesis period of the cotton fiber is preferably obtained by amplification, the promoter pFBl2A is amplified and cloned from a genome of No. 14 Ji cotton of a cotton strain, and the amplified primers preferably comprise pFBl2A-F and pFBl 2A-R; the nucleotide sequence of the pFabl 2A-F is preferably shown in SEQ ID NO. 12: aagcttGATATCGAATTCCTGCAG, the nucleotide sequence of the pFBl2A-R is preferably as shown in SEQ ID NO. 13: ggatccTGTAATTGTAAATAGTAATTGTAA, for convenient subsequent ligation of vectors, HindIII site was added to the 5 'end of the promoter and BamHI site was added to the 3' end of the promoter. The system for amplification according to the invention is preferably a 10. mu.l system comprising: 2 XPrimeSTAR MAX Premix 5. mu.l, 1. mu.l each of primers (5. mu. mol/L), about 60ng of template DNA, ddH was added₂O to 10. mu.l. The procedure for amplification according to the invention is preferably: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 60s, and 35 cycles; finally, extension is carried out for 10min at 72 ℃. After the amplified product was taken out, 10. mu.l of 2 XTaq Mix was added, and extension was carried out at 72 ℃ for 10min, and A tails were added to both ends of the fragment. Finally, the gel of the amplification product after electrophoresis is recovered. After recovery, the DNA fragment was ligated to pGEM-T-easy vector and sent to Huada Gene Co for sequencing. After the sequencing is correct, HindIII and BamHI are used for enzyme digestion, and after the enzyme digestion, glue is recovered to obtain a DNA fragment of the promoter pFBl 2A.

The invention also provides application of the fusion gene or the expression vector in constructing transgenic cotton, wherein mature fibers of the transgenic cotton are purple red. The fiber of the transgenic cotton of the invention over-expresses the fusion gene, and the content of the pigment of the transgenic cotton fiber can reach 4093 times of that of white cotton.

The invention also provides a method for constructing transgenic cotton with purple-red mature fibers, which comprises the following steps: the fusion gene is overexpressed in cotton fibers of transgenic cotton. The overexpression method preferably comprises transforming the expression vector into agrobacterium LBA4404, and performing genetic transformation of cotton by an agrobacterium tumefaciens mediated method. The method of genetic transformation is not particularly limited in the present invention, and a conventional genetic transformation method in the art may be used.

The fusion gene and expression vector for regulating color development of cotton fiber and the application thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Obtaining of corn leaf color gene Lc gene, small fragment peptide 2A peptide gene and cotton anthocyanidin synthesis regulation gene GhPAP1D

The small fragment peptide 2A peptide gene and the Lc gene are synthesized by Huada optimized fusion, a synthetic fragment is used as a template, primers (table 1, Lc-2A-U and Lc-2A-D) are designed, the 5 'end of the primer Lc-2A-U is added with a BamHI enzyme cutting site, and the 5' end of the Lc-2A-D is added with a GhPAP1D gene 10bp homologous arm. The system for amplification is preferably a 10. mu.l system comprising: 2 XPrimeSTAR MAX Premix 5. mu.l, primers Lc-2A-U and Lc-2A-D (5. mu. mol/L) each 1. mu.l, template DNA about 60ng, ddH added₂O to 10. mu.l. The amplification procedure was: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 60s for 35 cycles; finally, extension is carried out for 10min at 72 ℃.

The CDS sequence of Gohir.D7G082100 is used as a reference design primer (table 1, GhPAP1D-U and GhPAP1D-D), the 5 'end of the primer GhPAP1D-U is added with a 10bp homologous arm of a 2A peptide gene, and the 5' end of the primer GhPAP1D-D is added with ApaI enzyme cutting site. Leaf cDNA of cotton red strain T586 (Li Xin. transgenic functional study of cotton GhPAP 1. published in Master academic paper 2014 at the university of southwest) was used as a template. The system for amplification is preferably a 10. mu.l system comprising: 2.5. mu.l of PrimeStarXPremix, primers GhPAP1D-U and GhPAP1D-D (5. mu. mo)L/L) 1. mu.l each, about 60ng of template DNA, ddH was added₂O to 10. mu.l. The procedure for amplification according to the invention is preferably: pre-denaturation at 98 ℃ for 3 min; then denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 60s for 30 cycles; finally, extension is carried out for 10min at 72 ℃. .

And (3) performing overlapping PCR on the two Lc-2A, GhPAP1D fragments: 2 XPrimeSTAR MAX Premix 5. mu.l, primers Lc-2A-U and GhPAP1D-D (5. mu. mol/L) each 1. mu.l, template DNA about 60ng, ddH was added₂O to 10. mu.l. The amplification procedure was: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 15s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 90s for 10 cycles; finally, extension is carried out for 10min at 72 ℃, an amplification product is taken out, primers Lc-2A-U and GhPAP1D-D (5 mu mol/L) are added into each 1 mu L, and amplification is carried out again, wherein the amplification procedure is as follows: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 15s, extension at 68 ℃ for 90s, 30 cycles; finally, extension is carried out for 10min at 72 ℃. After the amplified product was taken out, 10. mu.l of 2 XTaq Mix was added, and extension was carried out at 72 ℃ for 10min, and A tails were added to both ends of the fragment. Finally, the gel of the amplification product after electrophoresis is recovered. After recovery, the DNA fragment was ligated to pGEM-T-easy vector and sent to Huada Gene Co for sequencing.

TABLE 1 primer information used in the examples

Example 2

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

The procedure for constructing the coding sequence of Lc-2A, GhPAP1D gene into plant expression vector pLGN is shown in FIG. 2. pLGN is a binary plant expression vector engineered from the conventional plant expression vector pBI 121. The T-DNA segment (region between RB and LB, FIG. 2) replaces the fusion gene expression cassette of the reporter gene GUS and the marker gene NPTII controlled by the 2X 35S promoter (2X 35S-P) for constitutive purposes. Cloning of the promoter Fbl2A from the genome of Ji Cotton No. 14 of a cotton plant was carried out by amplifying the primers pFBl2A-F and pFBl2A-R (see Table 1 for sequence), the promoter having a HindIII cleavage site at its 5 'end and a BamHI cleavage site at its 3' end. The amplification system of the present invention is preferably 10μ l system comprising: 2.mu.l of 2 XPPrimeSTAR MAXPREmix, 1. mu.l each of primers pFBl2A-F and pFBl2A-R (5. mu. mol/L), about 60ng of template DNA, ddH added₂O to 10. mu.l. The procedure for amplification according to the invention is preferably: pre-denaturation at 98 ℃ for 5 min; then denaturation at 98 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 60s, and 35 cycles; finally, extension is carried out for 10min at 72 ℃. After the amplified product is taken out, 10 mul of 2 XTaqMix is added, extension is carried out for 10min at 72 ℃, and A tails are added at two ends of the fragment. Finally, the gel of the amplification product after electrophoresis is recovered. After recovery, the DNA fragment was ligated to pGEM-T-easy vector and sent to Huada Gene Co for sequencing. After the sequencing is correct, HindIII and BamHI are used for enzyme digestion, and the gel is recovered after enzyme digestion. A DNA fragment of the promoter pFBl2A was obtained.

The Lc-2A-PAP1D fused DNA fragment connected to the pGEM-T-easy vector is cut by Hind III and Apa I after being sequenced correctly, and the gel is recovered after the cutting. The Lc-2A-PAP1D fusion DNA fragment was obtained.

Cutting pLGN vector with Hind III/Apa I, recovering linear vector fragment by electrophoresis, connecting the fused DNA fragment with promoter pFBl2A and Lc-2A-PAP1D by T4 DNA ligase, wherein the connecting system is 20 μ l and comprises: t4 DNA Ligase 1. mu.l, 10 XT 4 DNALigase Buffer 2. mu.l, pLGN-pFBL2A vector about 50ng, promoter pFBL2A about 50ng, Lc-2A-PAP1D fusion DNA fragment about 50ng, add ddH₂O to 20. mu.l. Reacting for 6 hours at constant temperature of 20 ℃. The ligation product was transformed into E.coli (Escherichia coli) competent DH 5. alpha. and the plasmid was extracted and verified by restriction. Finally, the specific expression vector pLGN-pFBl2A-Lc-2A-GhPAP1D of Lc-2A-GhPAP1D is obtained.

The above vector was introduced into Agrobacterium LBA4404 by electroporation, referred to Bio-RAD MicroPulser user instructions.

Example 3

Genetic transformation of cotton

The cotton genetic transformation of the above expression vector was performed by Agrobacterium tumefaciens mediated method, and the medium formulation used is shown in Table 2. The specific method comprises the following steps: removing hull from plump cotton seeds of wild type upland cotton YZ-1, placing a small amount (about 20-40) of the seeds in a sterilized 100mL triangular flask, pre-washing the seeds with 75% alcohol for 1min, pouring out the alcohol, and adding 0.1% HgCl₂Sterilizing about 12min (continuously shaking the triangular flask for sterilization), pouring out mercuric chloride gently, adding sterile water to rinse the seeds fully, and rinsing for about 10 times. And (3) placing the killed seeds on a germination culture medium, after the radicle grows to about 1cm (about 36-48 h), lightly inserting the radicle into the germination culture medium, and culturing the hypocotyl to about 7cm (about 7 days) at about 30 ℃ in the dark. Inoculating Agrobacterium single colony carrying genetic transformation vector to liquid YEB culture medium containing 50mg/L Km and 125mg/L Sm about 20h before infecting embryo section, placing in 28 deg.C shaking table (200rpm), and measuring bacterial liquid OD (OD) after culturing for about 20h₆₀₀)，OD₆₀₀The transformation is suitable at 0.4-0.6. The activated Agrobacterium solution was collected and centrifuged at 8000rpm for 1min, the supernatant was discarded, the cells were resuspended in MGL broth (containing 100. mu. mo1/L AS, acetosyringone) of the same volume, the resuspended cells were collected in a sterile 100mL Erlenmeyer flask and cultured in a shaker (28 ℃ C., 100rpm) for about 40 min. Cutting hypocotyls into small sections with the length of 0.8-1 cm by using a sterile scalpel, then placing the small sections into a resuspension solution, infecting the small sections for 40min on a shaking table (28 ℃, 100rpm), removing the liquid, taking out the hypocotyls, gently placing the hypocotyls on the surface of a solid co-culture medium, and carrying out dark culture for about 48 h. And (3) transferring the hypocotyl section into a solid one-screen culture medium for culture after dark culture (30 ℃, 16h of light/8 h of dark, the same below), transferring into a solid IBA culture medium after 30 days, subculturing once every 30 days or so until green somatic embryos are formed on the callus, and transferring the green somatic embryos onto a solid differentiation culture medium for culture. After the somatic embryo grows to about 3cm, inserting the somatic embryo into a rooting culture medium until seedlings grow out. The above operations must be performed under strict aseptic conditions.

The vigorous regenerated cotton seedlings are transplanted to a planting pot and are managed in a greenhouse until the cotton fibers and seeds are mature. Transgenic cotton seeds of T0 generation were harvested, and T1 generation continued planting and GUS staining. Homozygous transgenic T1 generation lines (all GUS positive or GUS negative plants) were selected. And detecting the expression levels of the target genes GhPAP1 and Lc and comparing the fiber anthocyanidin content and the change of fiber color.

TABLE 2 Agrobacterium tumefaciens-mediated culture medium for genetic transformation of cotton

Example 4

Cotton fiber color observation, anthocyanidin content detection and gene expression amount detection

Cotton fiber color was photographed 25 days and 50 days after flowering (FIG. 3).

White fibers and red fibers 25 days after blooming are sent to a Meiwei metabolism company for transcriptome and metabolome detection, wherein a metabolome data acquisition instrument system mainly comprises a high performance liquid chromatography and a tandem mass spectrum, and qualitative analysis is carried out on primary spectrum data and secondary spectrum data of mass spectrum detection based on an MWDB (Metal database) and a metabolite information public database built by the Meiwei metabolism company. The quantification of the metabolites is completed by utilizing the multi-reaction detection mode (MRM) analysis of triple quadrupole mass spectrometry, characteristic ions of each substance are screened out through the triple quadrupole, the signal intensity (CPS) of the characteristic ions is obtained in a detector, a sample off-line file is opened by using MultiaQuant software, the integration and the correction of chromatographic peaks are carried out, and the peak Area (Area) of each chromatographic peak represents the relative content of the corresponding substance. The anthocyanidin content and gene expression level are obtained from the feedback data, and the results are shown in table 3, 8 anthocyanins are detected in total, and are remarkably improved compared with white cotton, and the total quantity of the anthocyanidins is 4093 times of that of the white cotton. As shown in table 4, the expression levels of the phenylpropane pathway and part of the structural genes and regulatory genes of the anthocyanidin precursor synthesis pathway are significantly improved compared with white cotton.

TABLE 3 relative content of anthocyanins in white fibers and transgenic red fibers 25 days after flowering

TABLE 4 expression levels of genes involved in anthocyanin synthesis in white fibers and transgenic red fibers 25 days after flowering

RF red cotton; WF is white cotton; fpkm, the number of reads from map to exon per 1K bases in reads per 1 million maps; GhPAL is phenylalanine lyase; GhCHHS: chalcone synthase; GhDFR: a flavanonol reductase; GhANS: an anthocyanidin synthase; GhUFGT: anthocyanidin glucosyltransferase; GhGST: glutathione transferase.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of southwest

<120> fusion gene for regulating cotton fiber color development, expression vector and application thereof

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atggctcttt ctgcttctag agttcaacaa gctgaagagc ttcttcaaag acctgctgaa 60

agacaactta tgaggtcaca acttgctgct gctgctagat ctatcaattg gtcttacgct 120

cttttctggt ctatctctga tactcaacct ggtgttctta cttggactga cggtttctat 180

aacggtgagg tgaagactag aaagatctcc aactctgtgg aacttacttc tgatcagctt 240

gtgatgcaaa gatctgacca gcttagagaa ctttacgagg ctcttctttc tggtgaaggt 300

gatagaagag ctgctcctgc tagacctgct ggttctcttt ctcctgaaga tcttggtgat 360

actgaatggt attacgtggt gtctatgact tatgctttca gacctggtca aggtcttcct 420

ggtagatctt tcgcttctga tgaacatgtt tggctttgca atgctcatct tgctggttct 480

aaggctttcc ctagagctct tcttgctaag tctgcttcta tccagtctat tctctgtatc 540

cctgttatgg gtggtgttct tgaacttggt accactgata ctgttcctga agctcctgat 600

cttgtttcta gagctactgc tgctttctgg gaacctcaat gtccttcttc ttctccttct 660

ggtagagcta atgaaactgg tgaagctgct gctgatgatg gtactttcgc tttcgaagaa 720

cttgatcaca acaatggtat ggatgacatc gaagctatga ctgctgctgg tggtcatggt 780

caagaagaag agcttagact tagagaagct gaagctcttt ctgatgatgc ttctcttgag 840

cacattacta aggaaatcga ggagttctat tctctttgtg acgagatgga tcttcaagct 900

cttcctcttc ctcttgaaga tggttggact gttgatgctt ctaacttcga agttccttgt 960

tcttctcctc aacctgctcc tcctcctgtt gatagagcta ctgctaatgt tgctgctgat 1020

gcttctagag ctcctgttta tggttctaga gccacttctt tcatggcttg gactagatct 1080

tctcaacagt cttcttgctc tgatgatgct gctcctgctg ctgttgttcc tgctattgaa 1140

gaacctcaga gacttcttaa gaaggttgtt gctggtggtg gtgcttggga atcttgtggt 1200

ggtgctactg gtgctgctca agaaatgtct ggtaccggta ctaagaatca cgtgatgtct 1260

gaaagaaaga ggcgcgagaa gcttaatgag atgttcctcg tgcttaagtc tcttcttcct 1320

tctattcaca gggttaacaa ggcttctatc cttgctgaaa ctatcgctta tctcaaggag 1380

cttcaaagaa gggttcagga acttgaatct tctagagaac ctgcttctag accttctgaa 1440

actactacta ggcttatcac taggccttct agaggtaata acgagtctgt tcgcaaggaa 1500

gtttgtgctg gttctaagag aaagtctcct gaacttggta gagatgatgt tgaaagacct 1560

cctgttctta ctatggatgc tggtacttct aacgttactg tgactgtttc tgataaggac 1620

gtgcttcttg aagttcaatg tagatgggag gaacttctta tgactagagt gttcgacgct 1680

attaagtctc ttcaccttga tgtgctttct gttcaagctt ctgctcctga tggtttcatg 1740

ggtcttaaga tcagagctca attcgctggt tctggtgctg ttgttccttg gatgatttct 1800

gaagctctca ggaaggctat tggtaagagg 1830

<210>2

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

caattgctta acttcgattt gcttaagttg gctggtgatg ttgaatctaa ccctggtcct 60

<210>3

<211>750

<212>DNA

<213>Gossypium spp

<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 ThrTrp 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 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 (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 AsnAsn

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 (Artificial Sequence)

<400>7

ggatccatgg ctctttctgc ttct 24

<210>8

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gagccttcca taggaccagg gttagattca 30

<210>9

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ccctggtcct atggaaggct catctttaag 30

<210>10

<211>23

<212>DNA

<213> Artificial Sequence (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 gagtttttta gataattatt 180

ttaaatattc tgcataattt ttctgttatt tgaaaaggat gttcgaattt tttttcaaaa 240

ttgaaacgtt taagaatttt tactactgca aattcagaat aagtgaattt gttttttaga 300

aagattaaat aagttagtat tacgattttt agtttgattt ggtggaaagt aatgtatgtt 360

tttgaacata attatttgac aataattaag ttttctagga aataaacgga aatatcttct 420

tttttttttg taaaattact aatgcaagaa caaacaacgt tttgggaagc aaataatcta 480

gctttaagta gtcagtgtaa ctctcaaaat ctggtcataa cttctaggct gagtttgctg 540

tgctacagta gtaagtctat agaaacttac ctgacaaaac gacatgacgt cagggtcgaa 600

tctacaactt ttcctttttc 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 (Artificial Sequence)

<400>12

aagcttgata tcgaattcct gcag 24

<210>13

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

ggatcctgta attgtaaata gtaattgtaa 30

Claims

1. A fusion gene for regulating and controlling the color of cotton fibers is characterized in that the structure of the fusion gene comprises a nucleotide sequence of a 5 '-corn leaf color gene Lc-peptide segment 2A-a cotton anthocyanidin synthesis regulation gene GhPAP 1D-3';

2. The fusion gene of claim 1, wherein the amino acid sequence of the maize leaf color gene Lc is shown as SEQ ID No. 4; the amino acid sequence of the peptide segment 2A is shown as SEQ ID NO. 5; the amino acid sequence of the coding protein of the cotton anthocyanidin synthesis regulatory gene GhPAP1D is shown in SEQ ID NO. 6.

3. The fusion gene of claim 1 or 2, wherein the primers for amplifying the maize leaf color gene Lc comprise Lc-2A-U and Lc-2A-D; the nucleotide sequence of the Lc-2A-U is shown in SEQ ID NO.7, and the nucleotide sequence of the Lc-2A-D is shown in SEQ ID NO. 8.

4. The fusion gene of claim 1 or 2, wherein the primers for amplifying the cotton anthocyanin synthesis regulatory gene GhPAP1D comprise GhPAP1D-U and GhPAP 1D-D; the nucleotide sequence of the GhPAP1D-U is shown as SEQ ID NO.9, and the nucleotide sequence of the GhPAP1D-D is shown as SEQ ID NO. 10.

5. An expression vector comprising the fusion gene of any one of claims 1 to 4, wherein the T-DNA region of the expression vector has a gene structure comprising: right border of T-DNA-recombinase recognition site loxpfqrt-fusion gene expression cassette of cauliflower mosaic virus 35S promoter-reporter gene GUS and marker gene NPTII of tandem two enhancers-agrobacterium opine synthase gene terminator nos-recombinase recognition site loxpfqrt-cotton fiber secondary wall synthesis period specific promoter pFbl 2A-fusion gene-agrobacterium opine synthase gene terminator nos-T-DNA left border;

the nucleotide sequence of the specific promoter pFBl2A in the synthesis period of the secondary wall of the cotton fiber is shown as SEQ ID NO. 11.

6. The expression vector of claim 5, wherein the base vector of the expression vector comprises the plant expression vector pLGN.

7. The expression vector of claim 5, wherein the primers for amplifying the cotton fiber secondary wall synthesis phase specific promoter pFBl2A comprise pFBl2A-F and pFBl 2A-R; the nucleotide sequence of the pFBl2A-F is shown as SEQ ID NO.12, and the nucleotide sequence of the pFBl2A-R is shown as SEQ ID NO. 13.

8. Use of the fusion gene of any one of claims 1 to 4 or the expression vector of any one of claims 5 to 7 for constructing transgenic cotton, wherein mature fibers of the transgenic cotton exhibit a purple-red color.

9. A method for constructing transgenic cotton with purple red mature fibers is characterized by comprising the following steps: overexpressing the fusion gene of any one of claims 1 to 4 in cotton fibers of transgenic cotton.

10. The method of claim 9, wherein the overexpression method comprises transforming the expression vector of any one of claims 5 to 7 into agrobacterium LBA4404, and performing genetic transformation of cotton by agrobacterium tumefaciens mediated method.