CN113549622A

CN113549622A - Fiber-specific promoter proGhROP6 and application thereof

Info

Publication number: CN113549622A
Application number: CN202110813319.6A
Authority: CN
Inventors: 张觅; 李保霞; 张柳琴; 席敬; 付兴贤; 裴炎; 侯磊
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-07-19
Filing date: 2021-07-19
Publication date: 2021-10-26
Also published as: CN113774062A; CN113774062B

Abstract

The technical field of plant genetic engineering, in particular to a fiber-specific promoter proGhROP6 and application thereof. The nucleotide sequence of the fiber-specific promoter proGhROP6 is shown in SEQ ID No. 1. The invention further provides application of the promoter. The cloned proGhROP6 promoter is specifically expressed in the initial stage of cotton fiber, has mild strength and has great application value in genetic improvement of cotton fiber characters.

Description

Fiber-specific promoter proGhROP6 and application thereof

Technical Field

The technical field of plant genetic engineering, in particular to a fiber-specific promoter proGhROP6 and application thereof.

Background

Cotton is an important economic crop and a natural fiber raw material provided in the global textile industry. In response to the demand of cotton production, genetic engineering has become a rapid and effective means for genetic improvement of cotton. Currently, the use of transgenic cotton has exceeded 70% in global cotton production (Zhang et al, 2015). But such attempts have been very challenging in improving fiber properties. The most important of these is how to express the desired gene of interest at a particular time and in a particular tissue. Since some genes that promote fiber development affect the growth and development of plant tissues. For example, auxin is an important plant hormone that promotes fiber development (Seagull and Giavalis, 2004). Inappropriate manipulation of auxin biosynthesis in plants often leads to abnormalities in transgenic cotton (Zhang et al, 2011). In addition, controlling the precise expression of the gene of interest in the fiber also avoids any systemic adverse effects. Therefore, it is important to isolate and identify promoters related to specific development of cotton fibers.

Cotton fibers were differentiated from ovule expressing cells. The development of cotton fibers is divided into five successive stages: initiation, elongation, transformation, secondary cell wall synthesis and maturation (Haigler et al, 2012). Currently, a series of fiber-associated promoters have been discovered. Most are active in the fiber at the stage of elongation to secondary cell wall synthesis. E6 and FbL2A are two promoters isolated for their extensive transcription in cotton fiber cDNA libraries, and have significant activity at this stage (John and Crow, 1992; Rinehart et al, 1996). E6 and FbL2A showed very good fiber specificity, with activity intensities of about one ninth and one third of that of the constitutive promoter CaMV35S (Rinehart et al, 1996). The promoter of the lipase/hydrolase gene (GhGDSL) is active in the fibers at the secondary cell wall synthesis stage (Yadav et al, 2017). Similar activities were also observed on the promoters of chitinase-like protein (GhCTL) and TCP transcription factor (GbTCP). However, these two promoters also have some activity in anthers, xylem or young cotyledons and roots (Zhang et al, 2004; Hao et al, 2012).

Another part of the cotton fiber-associated promoter is mainly active in the initiation and elongation stages of the fiber. The promoter of expansin GbEXP2de is active mainly in elongated fibers, not in the starting fibers, and also in other enlarged tissues, such as the seed coat, young leaves and hypocotyl (Li et al, 2015). Microtubules and microfilaments are closely related to the development of cotton fibers. The promoter of β -tubulin ghtoub 1 was also active in developing fibers, but expression could also be detected in tissues such as pollen, ovaries, style and cotyledons (Li et al, 2002). The promoter of the ACTIN gene (GhACT1) also features very similar thereto (Li et al, 2005). The initiation and elongation of cotton fibers are similar to other trichome cells, and many transcription factors are involved. Regulatory sequences for some transcription factors have also been identified accordingly. The promoters of RD22-like1(RDL1), R2R3 MYB factors (GhMYB25, GhMYB25-like) and HD-ZIP factors (GhHD-1) show typical trichome-specific expression activity not only in developing fibers but also in other trichome cells (Wang et al, 2004; Machado et al, 2009; Walford et al, 2011, 2012). Furthermore, some activity leakage was observed in tissues such as anthers, pollen grains, xylem vessels, or roots of the promoters of GhMYB25, GhMYB25-Like, and GhHD-1 (Machado et al, 2009; Walford et al, 2011, 2012). In addition, the promoter of another MYB transcription factor (GhMYB109) has very good fiber specificity and is highly active in the fiber initiation phase, especially in fibers 5 days after flowering (Pu et al, 2008). This better specificity also appears in the promoter of a fiber-specific protease (GhSCFP), whose activity continues from the day of flowering to the period of secondary wall synthesis (Hou et al, 2008), and which is also of higher activity intensity (Zhang et al, 2011). GbPDF1 is a PROTODERMAL FACTOR1 that regulates cotton fiber initiation, with a promoter that is active primarily in the ovule epidermis and developing fibers, but is also very active in some reproductive tissues and seedlings (Deng et al, 2012). In addition to the cotton endogenous promoter, some heterologous promoters also exhibit fiber-related activity. Both the petunia-derived FBP7 promoter and the arabidopsis derived BAN promoter showed strong activity in ovule epidermal cells at the fiber initiation stage (Zhang et al, 2011). With the application of high-throughput technology, a large number of genes that are preferentially expressed in fibers have been identified. However, regulatory sequences capable of specifically controlling gene transcription in fibers remain quite limited, especially those promoters with detailed activity identification. This hinders progress in understanding the mechanisms of fiber development and improving the properties of cotton fibers.

Disclosure of Invention

The invention aims to provide a promoter which can be used for genetic improvement of cotton fiber characters.

The technical scheme of the invention is that a fiber-specific promoter proGhROP6, the nucleotide sequence of which is shown in SEQ ID No. 1.

The invention also provides a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the promoter.

The invention also provides the fiber-specific promoter proGhROP6, and application of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the promoter in regulation and control of cotton fiber growth and development, wherein the nucleotide sequence of proGhROP6 is shown in SEQ ID No. 1.

Furthermore, the invention also provides the fiber-specific promoter proGhROP6 and application of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the promoter in regulation of cotton fiber growth and development at a fiber initiation stage.

The invention has the beneficial effects that: the GhROP6 regulatory sequence specifically expressed in the cotton fiber development process is cloned, and the test proves that the GhROP6 regulatory sequence is a promoter which has mild strength, high specificity in fibers and is mainly active in the fiber initiation stage. The invention provides a new element for the fine control of gene expression required by cotton fiber research and fiber character improvement.

Drawings

FIG. 1: proGhROP6 identification of GUS transgenic plants. N stands for negative control, P stands for positive control, M2000 stands for Marker 2000, 1-100 stands for different transgenic lines.

FIG. 2: proGhROP6 results of GUS staining in different tissues of upland cotton.

Numbers #

1, 5, 19 and 21 represent different transgenic lines; ovule stands for Ovule, Leaf for Leaf, Stem for Stem, Petal for petals, Stamen + pistil for Stamen and pistil.

FIG. 3: proGhROP6 shows GUS staining results of GUS in different development stages of upland cotton fibers.

Numbers #

1, 19 and 21 represent different transgenic lines; DPA is an abbreviation for day(s) post anthesis, and represents the number of days after flowering.

FIG. 4: proGhROP6 shows GUS enzyme activity of GUS transgenic plant in different stages of fiber development. The abscissa represents the number of days (cotton) flowering; the ordinate represents GUS enzyme activity expressed as the amount of 4-MU produced per minute per mg protein (pmol). 3 biological replicates were set up for each experiment.

FIG. 5: CaMV35S, GUS and proGhROP6, and GUS enzyme activity in ovules on the day of GUS flowering. The abscissa represents the number of days (cotton) flowering; the ordinate represents GUS enzyme activity expressed as the amount of 4-MU produced per minute per mg protein (pmol). 3 biological replicates were set up for each experiment.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings, but the following description is not intended to limit the present invention, and any modifications and changes may be made thereto without departing from the spirit of the present invention, which is defined in the appended claims.

The reagent drugs in the examples of the present invention are not specifically described and are generally commercially available. Materials methods, not specifically described, are referred to in the molecular cloning protocols (Sambrook and Russell, 2001).

EXAMPLE 1 construction of the vector

1.1 plant RNA extraction

Selecting about 1g of fresh cotton material, adding liquid nitrogen into a mortar, fully grinding into fine powder, extracting RNA by using an EASYspin Plant RNA Kit of Eldely Bio, and performing the extraction steps according to the attached instructions. After extraction, 2. mu.L of the extract was subjected to agarose gel electrophoresis to examine the quality of the extracted RNA.

1.2cDNA Synthesis

After completion of RNA extraction, cDNA synthesis was performed using PrimeScript RT reagent Kit with gDNA Eraser from TaKaRa, and the procedure was performed according to the instructions.

1.3 obtaining of promoter of Cotton Gene GhROP6

Based on research experience, the applicant designs primers (SEQ ID NO.2 and SEQ ID NO.3) according to the upstream sequence of GhROP6(JGI accession number: Gohir. D01G159200), and obtains a nucleotide sequence (SEQ ID NO.1) of 1240bp through amplification by using ovule gDNA of the day of cotton blossoming as a template, wherein the nucleotide sequence is promoter proGhROP 6.

SEQ ID NO. 1: gene from upland cotton

1 cagaactttc ttatttcact gttaatttcc ttttaattgc attttctgtc attcaatcat

61 tcttatcgaa tcattaaaat acattattat taaaaaaaca aataaatact taatttaaaa

121 ttttaaaaat atgtcatact tgaaataact caataagccc ttcaatgaac tttttaggta

181 taataataaa ataaataata aattatttat ggaaaatata ttgttgtaat gtaagcagat

241 agctgtccaa gagctgtctt tcctttccct aatgattctc tctctttctt tcgggcaatg

301 tctttgatgt cttccagttt tacttagtcc ttcatcttct tcttcatttc agtgtttttg

361 ggtttgtttt ttttcctact gttttggaca ataattgaag caagagataa ccttgggggc

421 ggcggcggag ggagaggaac atctctgaaa ttactttcac ttttgccggt gggagtaaaa

481 tgagtgcatc aaggttcatc aaatgtgtca ctgttggtga cggtgccgtc ggcaagactt

541 gcatgctcat ctcctacacc agcaatactt tccctactgt gagttccaaa aagaaagaaa

601 ctaacccatt ttttttgttt ggtctctgag aaaatatggg gaatggaaaa ggaagtttgg

661 ctggccctta acgttttctt tacttggatt atcatggatt ttcttttctt tttctttccc

721 tttttgtttc ccctattttc ttggccacca agcagaccct acactggttt ctggtggtca

781 atgtaacttt ggtaaagttg ctttctttat tgggtttttt tttgtttctt tttcccattt

841 tgttgtactt gtctttggaa tcttgaattt ggggcatagt atctttctga ataatagaat

901 cattcttcgt aaattgtgtt caagtgagca atttcttagg gttttttgct tcaaaaactt

961 gcagaaaatt tattgccttt ttgtggttta taccttctta tttcttctgt ttgtgttttg

1021 atgatgcatc atctaggata aagattcttt gtcttgtagg ttttcatttc aattagtgag

1081 ttaaggtcaa aagttaaaac tggaaacatt atatcctgct actcccctgt tttttgctgg

1141 aggagaaaga aaacaaattg ctaaatctga aactattgct ggtagaaagt ctcatttaga

1201 gatttcataa taggtgcttt cattactgcg aatcttgggt

SEQ ID NO. 2: proGhROP6 amplification upstream primer, 5'-attaagcttcagaactttcttatttcactg-3'

SEQ ID NO. 3: proGhROP6 downstream primer for amplification, 5'-attggatcc acccaagattcgcagtaatgaaagc-3'

1.4 construction of proGhROP6 GUS plant overexpression vector

The preferred framework vector for constructing the plant expression vector is pBI121(Genebank accession number AF485783), is a commonly used plant expression binary vector and has a GUS gene under the regulation of a CaMV35S promoter.

pBI121 plant expression vectors were linearized by digestion with BamHI and HindIII. The restriction enzymes were obtained from Thermo Scientific company and were digested as described in the specification. The sequence of proGhROP6 amplified with primers (SEQ ID NO.2 and SEQ ID NO.3) having cleavage sites and the plant expression vector pBI121 were cleaved with BamHI and HindIII and ligated together. The obtained proGhROP6 is used for genetic transformation of cotton in a GUS plant expression vector, and the used cotton experimental material is upland cotton (Gossypium hirsutum L).

Example 2 transformation of Cotton

2.1 introduction of the constructed plant expression vector plasmid into Agrobacterium GV3101 by Electrical stimulation

The above vector was introduced into Agrobacterium GV3101 by electro-stimulated transformation, referred to Bio-RAD MicroPulser user instructions.

The agrobacterium electric shock transformation step comprises: and taking out the agrobacterium-infected cells, placing the agrobacterium-infected cells on ice to melt, washing an electric shock cup used for electric shock transformation for 5-6 times by using sterile water, and placing the electric shock cup on ice for later use. Sucking 20-50 ng of plasmid and mixing with competent cells, and adding into an electric shock cup. The shock converter of Bio-Rad was turned on and the Agr/Bacteria mode was selected for shock. Immediately after the electric shock, 800. mu.L of YEB liquid medium was added, and shaking culture was performed at 28 ℃ and 200rpm for 3 hours. Centrifuging at 10000rpm for 1min, discarding the supernatant, leaving about 100 μ L of the culture medium to suspend the thallus, spreading the thallus on YEB plate with corresponding antibiotic (such as 50 μ g/mL Kan), and culturing at 28 deg.C for 48 h.

2.2 integration of vectors constitutively expressing GUS binding to proGhROP6 into the Cotton genome

Genetic transformation of cotton was performed by Agrobacterium tumefaciens-mediated transformation (the medium used in the transformation process is shown in Table 1), and the T-DNA segment of the above plant expression vector was introduced into cotton by Agrobacterium-mediated embryogenic callus. The specific method comprises the following steps:

(1) germinating cotton seeds: peeling off shells of koji cotton seeds of upland cotton cultivars, selecting large and full seeds, placing the seeds in a triangular flask, sterilizing the seeds with 75% alcohol for 1-2 min, and rinsing the seeds with sterile water for 2 times; 0.1% mercuric chloride (HgCl) was added again₂) Sterilizing for 10min, and rinsing with sterile water for 6 times. Shaking and culturing with sterile water at normal temperature on a shaking table at 120rpm for 12 h. And (3) downwards inoculating the exposed and white seed kernel radicle into a germination culture medium, and placing the seed kernel radicle under the dark condition of 28 ℃ for germination for 2-3 d to obtain the sterile cotton seedling.

(2) Culturing of transformed Agrobacterium the Agrobacterium strain containing the above plant expression vector was inoculated into YEB solid medium (0.5% sucrose (W/V), 0.1% yeast extract for bacteria (W/V), 1% tryptone for bacteria (W/V), 0.05% MgSO 0/V) containing 50mg/L kanamycin and 125mg/L streptomycin₄·7H₂O (W/V), 1.5% agar powder (W/V) pH 7.0). Inoculating a single colony of newly-scribed activated agrobacterium to 5mL of YEB liquid medium containing the same antibiotic, and performing shaking culture at 28 ℃ and 200rpm overnight. Taking 1mL of bacterial liquid to perform secondary culture in 20-25 mL of YEB liquid culture medium containing the same antibioticsPerforming secondary activation, and performing shaking culture at 28 ℃ and 200rpm until the OD600 is about 0.8-1.0. Centrifuging at 10000rpm at room temperature for 1min, collecting the thallus, and resuspending the thallus with an equal volume of liquid co-culture medium (containing AS) AS a infection solution for later use.

(3) Dip dyeing and co-culturing, cutting cotton sterile hypocotyl into small segments of about 0.5cm, placing in a triangular flask containing the dye-soaking solution, and performing shake cultivation at 28 deg.C and 100rpm for 45 min. Discarding the bacterial liquid, inoculating the infected hypocotyl section onto a solid co-culture medium (containing AS), and culturing in the dark at 28 ℃ for 2 d.

(4) And (3) screening transformants, namely transferring the hypocotyl section to a screening culture medium for degerming and selective culture after the co-culture is finished, and carrying out subculture once every 2 weeks at 28 ℃ for 16h under illumination. Most of the calli are browned and dead after 1-2 months, and a small part of the calli show kanamycin resistance, so that fresh embryogenic calli grow. Subculturing the callus blocks, inoculating each block of tissue to liquid suspension culture medium when the tissue proliferates to 2.0-3.0g, and performing suspension culture on shaking table at 120rpm to obtain a large amount of somatic embryos. After suspending for 2 weeks, the suspension cultured tissue was filtered through a 30-mesh screen, and the undersize pellet was transferred to a somatic embryo maturation medium. The germinated mature embryo is transferred to a somatic embryo elongation inducing culture medium and cultured for 2 weeks under the dark condition at the temperature of 28 ℃ to induce the somatic embryo to elongate and germinate. Transferring the bigger germinated embryo (>0.5cm) into SH culture medium to form seedling, and culturing at 28 deg.C under 16h light. When the seedling grows to about 2cm high, cutting the seedling and grafting the seedling to a cotton seedling with 3-4 true leaves.

TABLE 3 Agrobacterium tumefaciens-mediated culture media for genetic transformation of cotton

Gelrite: sigma, cat No.: g1910; SH: schenk & Hildebrandt, 1972.

2.3 obtaining transgenic Cotton

Transgenic cotton obtained at T1 generation was cultivated in greenhouse and managed routinely. And collecting the mature fiber for character analysis. The obtained transgenic cotton has no obvious difference in phenotype and growth development from the wild type control.

Example 3GhROP6 selection of GUS transgenic Cotton transgenic plants

The plant expression vector backbone pBI121 is a binary vector, and the T-DNA region contains an NPTII expression cassette for resistance selection. Thus, the gene NPTII (SEQ ID NO.4) can be amplified to identify proGhROP6:: GUS transgenic cotton. The gDNA of cotton leaf is used as template to amplify target sequence with NPTII specific primer (SEQ ID No.5 and SEQ ID No.6) to screen proGhROP6, GUS transgenic plant.

SEQ ID NO. 4: NPTII gene sequence

1 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc

61 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca

121 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg

181 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg

241 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag

301 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg

361 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc

421 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa

481 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac

541 ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat

601 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac

661 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc

721 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt

781 gacgagttct tctga

SEQ ID No. 5: NPTII gene amplification primer 1, 5'-atgattgaacaagatggattgcacg-3'

SEQ ID NO. 6: NPTII gene amplification primer 2, 5'-tcagaagaactcgtcaagaaggcga-3'

The specific operation steps are as follows:

the conditions for PCR in vitro amplification verification of transgenic plant DNA are as follows: the total reaction volume was 25. mu.L, including 10. mu.L of LA Taq buffer 2.5. mu.L, 100. mu. mol/L of each dNTP, 1.5mmol/L of MgCl2, 10ng of template DNA, 400nmol/L of each of the upstream and downstream primers, and 1 unit of LA Taq DNA polymerase (TaKaRa Co.).

Amplification conditions: denaturation at 94 ℃ for 4min, followed by denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles, and finally extension at 72 ℃ for 10 min. The amplification products were electrophoresed on a 1% agarose gel containing ethidium bromide at a voltage of 5V/cm and recorded under an ultraviolet lamp. The results of PCR validation of transgenic Arabidopsis are shown in FIG. 1. The presence of the NPTII gene was detected in the transgenic cotton DNA (lanes 1-100) as in the positive plasmid D (lane P), while the wild-type cotton DNA (lane N) was negative. This indicates that all the obtained transgenic cotton had integrated the transgene sequence of interest into the genome.

EXAMPLE 4 histochemical staining and visualization of GUS

Fresh transgenic cotton ovules are taken and placed in a 1.5mL centrifuge tube, and GUS staining solution (10mmol/L EDTA, 100mmol/L phosphate buffer (pH7.0)) and 0.5mol/L K are added₃[Fe(CN)₆]，0.5mol/L K₄[Fe(CN)₆]0.1% (V/V) Triton X-100, 1.0mmol/L X-Gluc). The centrifuge tube containing the plant material and GUS dye was placed in a 37.0 ℃ incubator overnight for staining. Finally, the staining solution was removed, and after decolorizing with 70% ethanol, photography was observed.

Transgenic proGhROP6 GUS gene cotton showed GUS staining as shown in FIGS. 2 and 3. The staining results of different tissues of transgenic cotton show that the promoter proGhROP6 in multiple transgenic lines all show consistent specificity, and only has expression activity in ovule epidermis, but not in tissues such as leaves, petals, stigma, stems and the like (FIG. 2). The results of ovule staining at different stages of fiber development further showed that progherop 6 was expressed in the ovule outer skin and fibers, and that expression in fibers was clearly superior (0DPA ovule in figure 3). The expression phase is mainly focused on the fiber initiation phase and elongation phase. This indicates that the GhROP6 promoter has better fiber expression specificity (fig. 3).

EXAMPLE 5 GUS enzyme Activity assay

The method for measuring GUS enzyme activity was performed (2008) with reference to the method described by Hou et al, and was partially modified.

(1) The protein concentration was determined by the Bradford method (1976) as follows:

50 fresh ovules (0DPA ovules) were each ground to powder with liquid nitrogen, GUS extraction buffer was added at a ratio of 1:2 sample/extract (m/v), and extracted on ice for 1 h. ② 13000rpm is centrifuged for 10min at room temperature, and the supernatant is GUS extracting solution. ③ diluting 3. mu.L of GUS extract with 900. mu.L of Bradford working solution, and measuring absorbance at OD595 with a microplate reader Varioskan LUX (Thermo Fisher Co., Ltd.) to obtain 200. mu.L of the diluted GUS extract. And fourthly, calculating the total protein concentration according to the BSA standard curve.

(2) The GUS enzyme activity determination method specifically comprises the following steps:

adding 195 microliter of GUS reaction solution into a blank enzyme label plate with 96 holes according to the experiment requirement, and preheating at 37 ℃ for standby. ② taking a blank enzyme-linked immunosorbent assay plate with 96 holes again, and adding 180 mu L of GUS reaction termination solution into each hole according to the experimental requirement for standby. ③ adding 5 MU L of GUS extracting solution into each hole of the preheated GUS reaction solution, mixing uniformly, immediately adding 20 MU L of the reaction solution into 180 MU L of GUS reaction termination solution, and measuring the 4-MU fluorescence value at 0min by using a microplate reader Varioskan LUX (Thermo Fisher Co.). The excitation wavelength was measured at 360nm, the emission wavelength at 460nm and the Gain value at 63 (consistent with the values used for the calibration). And fourthly, adding 20 MU L of reaction solution into 180 MU L of GUS reaction termination solution when the time is 30min, and measuring the 4-MU fluorescence value of the solution for 30 min. And fifthly, calculating the GUS enzyme activity according to the 4-MU fluorescence value standard curve. Nmol 4-MU-minute for GUS enzyme activity^-1·mg^-1total protein represents. 3 biological replicates were set up for each experiment.

The results showed that proghrup 6 had the highest activity intensity at the initial stage of fiber and was at a lower level 5 days after flowering (fig. 4). At the same time, the promoter strength was mild, about one twenty-fifth of that of the constitutive promoter 35S (FIG. 5).

The above examples show that the cloned proGhROP6 promoter nucleotide has a length of 1240bp, and when the promoter is fused with a reporter gene, the promoter can guide the GUS reporter gene to be specifically expressed in the fiber at the initial stage in cotton, and has mild strength and great application value in genetic improvement of cotton fiber traits.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> university of southwest

<120> fiber-specific promoter proGhROP6 and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1240

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagaactttc ttatttcact gttaatttcc ttttaattgc attttctgtc attcaatcat 60

tcttatcgaa tcattaaaat acattattat taaaaaaaca aataaatact taatttaaaa 120

ttttaaaaat atgtcatact tgaaataact caataagccc ttcaatgaac tttttaggta 180

taataataaa ataaataata aattatttat ggaaaatata ttgttgtaat gtaagcagat 240

agctgtccaa gagctgtctt tcctttccct aatgattctc tctctttctt tcgggcaatg 300

tctttgatgt cttccagttt tacttagtcc ttcatcttct tcttcatttc agtgtttttg 360

ggtttgtttt ttttcctact gttttggaca ataattgaag caagagataa ccttgggggc 420

ggcggcggag ggagaggaac atctctgaaa ttactttcac ttttgccggt gggagtaaaa 480

tgagtgcatc aaggttcatc aaatgtgtca ctgttggtga cggtgccgtc ggcaagactt 540

gcatgctcat ctcctacacc agcaatactt tccctactgt gagttccaaa aagaaagaaa 600

ctaacccatt ttttttgttt ggtctctgag aaaatatggg gaatggaaaa ggaagtttgg 660

ctggccctta acgttttctt tacttggatt atcatggatt ttcttttctt tttctttccc 720

tttttgtttc ccctattttc ttggccacca agcagaccct acactggttt ctggtggtca 780

atgtaacttt ggtaaagttg ctttctttat tgggtttttt tttgtttctt tttcccattt 840

tgttgtactt gtctttggaa tcttgaattt ggggcatagt atctttctga ataatagaat 900

cattcttcgt aaattgtgtt caagtgagca atttcttagg gttttttgct tcaaaaactt 960

gcagaaaatt tattgccttt ttgtggttta taccttctta tttcttctgt ttgtgttttg 1020

atgatgcatc atctaggata aagattcttt gtcttgtagg ttttcatttc aattagtgag 1080

ttaaggtcaa aagttaaaac tggaaacatt atatcctgct actcccctgt tttttgctgg 1140

aggagaaaga aaacaaattg ctaaatctga aactattgct ggtagaaagt ctcatttaga 1200

gatttcataa taggtgcttt cattactgcg aatcttgggt 1240

<210> 2

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attaagcttc agaactttct tatttcactg 30

<210> 3

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attggatcca cccaagattc gcagtaatga aagc 34

<210> 4

<211> 795

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540

ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgattgaac aagatggatt gcacg 25

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cagaagaact cgtcaagaag gcga 24

Claims

1. The fiber-specific promoter proGhROP6 is characterized in that the nucleotide sequence is shown in SEQ ID No. 1.

2. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the promoter of claim 1.

3. The use of the fiber-specific promoter proGhROP6 and the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the promoter in regulating the growth and development of cotton fibers according to claim 1.

4. The use of the fiber-specific promoter proGhROP6 and the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the promoter in the fiber initiation stage to regulate the growth and development of cotton fibers.