CN112481267B - Cotton promoter PCGP1And uses thereof - Google Patents
Cotton promoter PCGP1And uses thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, relates to a promoter related to cotton pigment gland expression, and particularly relates to a cotton promoter PCGP1And applications thereof. The P isCGP1The nucleotide sequence of the promoter is shown in SEQ ID NO. 1. The invention provides a new method for molecular breeding. The method comprises adding PCGP1The promoter is connected with a target gene and then transferred into cotton, and the synthesis, accumulation and extraction efficiency of secondary metabolites of pigment glands is improved by driving the expression of the gene in gland cells through the promoter. The cloned promoter is connected with a target gene and is transferred into a plant, so that adverse effects caused by the over-expression of the target gene by the plant can be avoided.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a promoter related to cotton pigment gland expression, and particularly relates to a cotton promoter PCGP1And applications thereof.
Background
The promoter is a segment located in the structural gene 5’The DNA sequence of the terminal upstream region can activate RNA polymerase to accurately bind with the template DNA and has the specificity of transcription initiation. The promoter is an important cis-acting element for regulating gene expression, mainly composed of nucleusThe heart promoter region and the upstream promoter element thereof. The TATA box located-20 to-30 bp upstream of the transcription initiation site is a promoter core element, and is an AT-rich conserved sequence region, which functions to accurately start transcription and is necessary for the correct expression of most plant promoters. The conserved sequence at the upstream of the TATA box is called as a promoter upstream element, and comprises a CAAT box at the upstream of-70 to-80 bp, a GC box near the upstream of-80 to-110 bp, and other general upstream promoter elements and other special upstream elements. Upstream promoter elements regulate the expression site, expression period and expression intensity of genes (heroic et al, 2019; Bucsenez et al, 2012).
The pigmented glands are a lysoluminal structure characteristic of cotton plants, which contains a large number of secondary metabolites and appears as black punctate structures (Fryxell P a, 1965). The development of the pigmented glands involves a process of cell lysis and programmed cell death. The pigmented gland originates from a cluster of glandular blasts beneath the epidermis, which are generally characterized by a high density of cytoplasm and large nucleoli. The mature gland consists of a lysogenic cavity, which is degraded by the central primitive cells to form a large lysogenic cavity surrounded by multiple layers of sclerenchyma cells (Zhouyu, 2011; Liuet al, 2010). The chromoglandular lysosomes store a large number of secondary metabolites that protect plants from pathogens, insects, and herbivores, and often have high medicinal value. Among them, gossypol is the most studied secondary metabolite in the pigmented glands. Gossypol is a yellow polyphenol hydroxy-bis-naphthaldehyde compound, mainly exists in roots, stems, leaves, seeds and petals of cotton, and has the highest content in cottonseed kernels (Shidong and Qujiamu, 2006). Gossypol has effects of inhibiting spermatogenesis and sperm motility. Can be used as an effective male contraceptive. Gossypol is also used to treat gynecological disorders including menorrhagia or disorders, uterine fibroids, endometriosis, and the like. Gossypol also has an effect on anti-tumor, and in vitro experiments show that gossypol has obvious proliferation inhibition activity on a plurality of tumor cell lines originating from lymph and granulocytes, adrenal gland, mammary gland, cervix, rectum and central nervous system (Liu et al, 2001; Shailendra Kapoor, 2013). The pigment gland is a main storage place of secondary metabolites of cotton plants and can be used as a biological container for gossypol and other substances. The research on the pigment gland specific promoter has important significance on the accumulation and application of cotton breeding, gossypol and other substances.
Disclosure of Invention
The invention provides a cotton promoter PCGP1And the application thereof, which solves the technical problem of starting the specific expression of the target gene in the cotton pigment gland cells.
The technical scheme of the invention is realized as follows:
cotton promoter PCGP1Said P isCGP1The nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
The P isCGP1The nucleotide sequence of the promoter is a nucleotide sequence with homology of more than 70 percent with the sequence shown in SEQ ID NO.1 or a subfragment with equivalent function with the sequence shown in SEQ ID NO. 1.
The above cotton promoter PCGP1The application of the promoter as a probe to screening a promoter with a nucleotide sequence shown as SEQ ID NO.1 or a homologous promoter thereof from a genome library.
The above cotton promoter PCGP1The application in driving gene transcription.
The above cotton promoter PCGP1The application of the gene in the specific expression of the regulatory gene in cotton pigment gland cells.
The application comprises the following steps: the cotton promoter PCGP1The target gene is transferred into plant cells after being connected to construct transgenic plants.
The invention has the following beneficial effects:
the invention provides a new method for molecular breeding. The method comprises introducing PCGP1The promoter is connected with a target gene and then transferred into cotton, and the synthesis, accumulation and extraction efficiency of secondary metabolites of pigment glands is improved by driving the expression of the gene in gland cells through the promoter. The cloned promoter is connected with a target gene and is transferred into a plant, so that adverse effects caused by the over-expression of the target gene by the plant can be avoided.
The present invention can further provide or utilize the above DNA fragments to obtain transgenic plants and corresponding seeds, as well as plants transformed with the promoters of the present invention or recombinants based on the promoters or seeds obtained from such plants. The promoter of the present invention can be transferred to other plants by sexual crossing.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows an embodiment of the present invention PCGP1And (3) a promoter PCR amplification result graph.
FIG. 2 shows cotton PCGP1The basic structure of the promoter, +1 is the predicted transcription start site; the predicted TATA box is the binding site for RNA polymerase and DNA single strand; the predicted CAAT box regulates the transcription initiation frequency.
FIG. 3 is PCGP1The promoter was ligated to a GUS (β -glucuronidase) reporter gene and constructed into a plasmid with kanamycin resistance.
FIG. 4 is an illustration of the expression pattern of GUS in genetically transformed plantsCGP1The promoter promotes the specific expression of the gene in the pigment gland cells.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The Taq DNA Polymerase and the Phanta Max Super-Fidelity DNA Polymerase related by the invention are purchased from Nanjing NooZan Biotech Co., Ltd; the Cycle-Pure Kit is available from Promega, Beijing Biotechnology Ltd; pEASY-Blunt Simple Cloning Kit available from Beijing Quanjin Biotechnology Ltd; the plant genome DNA extraction kit and the rapid plasmid small-scale extraction kit are purchased from Tiangen Biotechnology (Beijing) Co., Ltd; the X-Gluc staining solution is purchased from Ku Lagbo science and technology Limited company of Beijing; the primer is synthesized by Shanghai biological engineering company Limited, purified by PAGE method, and diluted to 10 μ M when in use; sequencing was performed by Shanghai Bioengineering, Inc.
Example 1: isolation of clone PCGP1Promoters
1. Obtaining cotton DNA template
Selecting good upland cotton TM-1 seeds, shearing off the tips of the seed shells on the premise of not damaging embryos to expose radicles, soaking the seeds in water for 4-6 h, wrapping the soaked seeds with wet gauze, placing the seeds in a 30 ℃ biochemical incubator for overnight germination, taking the seeds with consistent hypocotyl length, sowing the seeds in nutrient soil, covering a layer of mulching film on the surface of the seeds for moisturizing, placing the seeds in an illumination incubator at the temperature of 25 ℃, the humidity of 70%, illuminating for 16 h and culturing in darkness for 8 h, and removing the mulching film after the seeds expose cotyledons.
The cotyledons growing for about 5 days were taken and DNA was extracted using a plant genome DNA extraction kit. Taking about 100mg of young cotton cotyledons, quickly freezing by liquid nitrogen, putting the young cotton cotyledons into a mortar, fully grinding, and then extracting DNA according to the kit instructions. The DNA concentration was determined using a NanoDrop 2000c UV-visible spectrophotometer (Tbermo Fisber, USA) and the DNA purity was checked by 1.2% (w/V) agarose gel electrophoresis (150V, 15 min).
2. PCGP1Cloning of the promoter
Obtaining upland cotton TM-1 coverage full length P by amplificationCGP1A fragment of the promoter sequence, PCR system:
wherein the sequence of the used amplification primer is as follows: CGP1-PF 1: 5'-GAAATAAGGGTAAAAAGGGAAAACG-3', CGP1-PR 1: 5'-ATTTATAGCTTAGAATCTCATTGGG-3' are provided. The template is the cotton DNA extracted in the first embodiment, and the PCR program is 95 ℃ for 3 min; 35 cycles of 95 ℃ for 10 s, 58 ℃ for 30 s, 72 ℃ for 40 s; then storing at 72 deg.C for 10 min and 16 deg.C. The PCR product was detected by agarose gel electrophoresis (FIG. 1), purified with the Cycle-Pure Kit to recover the PCR product, ligated with the T vector using the pEASY-Blunt Simple Cloning Kit to transform E.coli DH 5. alpha. and tested by Taq DNA Polymerase to select positive clones for sequencing by Shanghai Biotechnology Ltd. The clone with correct sequencing is used for extracting the plasmid by using a rapid plasmid miniprep kit through sequence comparison and is named as PCGP1The sequence information is shown in SEQ ID NO. 1.
3. PCGP1Structural analysis of promoters
The promoter structure and cis-element prediction of the obtained DNA fragment was performed using the online bioinformatics software PlantCARE. The results of the analysis are shown in FIG. 2, indicating that the fragment contains the complete promoter structure, including the CAAT box, the TATA box, the transcription start site, and several predicted promoter cis-elements.
Example 2: pCGP1Genetic transformation vector construction to drive GUS expression
Obtaining a promoter fragment with a joint by PCR amplification, wherein the PCR system comprises:
wherein the sequence of the used amplification primer is as follows: CGP1-PAF 1: 5' -GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAATAAGGGTAAAAAGGGAAAACG
-3’，CGP1-PAR1：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTATTTATAGCTTAGAATCTCATTGGG-3', the underlined nucleotide sequence is the linker sequence for the BP reaction. Wherein the template is the template containing P in the first embodimentCGP1T vector of DNA fragment, PCR program of 95 ℃ for 3 min; 10 s at 95 ℃, 30 s at 58 ℃, 40 s at 72 ℃ and 28 cycles; then storing at 72 deg.C for 10 min and 16 deg.C. Purifying and recycling PCR products, and utilizing Gateway method to purify and recycle PCGP1The vector is connected to an entry vector pDNOR221, and the specific operation is as follows: 0.5 mu L of PCR product; pDNOR221 vector 0.5. mu.L; BP enzyme 0.5. mu.L; TE (8.0) Buffer 1. mu.L was reacted at 25 ℃ for 1.5 hours. And (3) transforming the ligation product into escherichia coli DH5 alpha, detecting by PCR, selecting positive clones, sending the positive clones to Shanghai Biotechnology Limited company for sequencing, and extracting plasmids by correct clones after sequence comparison. Preparing an LR reaction system: BP plasmid 0.5 u L; 0.5. mu.L of PGWB433 vector; LR enzyme 0.5 μ L; 10 × TE (8.0) Buffer 1 μ L, reacted at 25 ℃ for 1.5 h. And (3) transforming the ligation product into escherichia coli DH5 alpha, and selecting positive clone to extract plasmids through PCR detection. LR plasmid is transferred into agrobacterium EHA05 through electric shock transformation, and positive clone is selected for mixed sample preservation for standby through PCR detection. The correct plasmid and bacterial suspension were designated as PCGP1GUS, plasmid map is shown in figure 3.
Example 3: p isCGP1Functional verification of promoters
1. Creation of transgenic Material
P carried by EHA105 agrobacterium by agrobacterium-mediated cotton hypocotyl transformation methodCGP1GUS was introduced into cotton (Jin et al, 2006). Husking cotton to be transformed, and soaking in 0.1% mercuric chloride solution for 8 min; after mercuric chloride removal, rinse 4 times with sterile water, 5 min each. The sterilized seeds were placed in sterile shoot germination medium (1/2 MS) and cultured in the dark for 7 days. The activated carrier PCGP1Diluting EHA105 agrobacterium of GUS plasmid to OD value of 0.6 by MGL culture medium; cutting hypocotyls of the cotton aseptic seedlings after 7 days of growth into sections of 0.6 cm, and placing the sections into agrobacterium with OD =0.6 until the hypocotyl tissues are completely submerged; gently shaking for 7 min, filtering to remove bacteria solution, sucking off excess bacteria solution on the surface of plant tissue with sterile filter paper, and spreading on MSB culture medium. After culturing for 72 h in the dark, hypocotyl tissue is placed on an induction medium containing kanamycin to induce embryonic callus. Subculturing for 1 time every 30 days until callus embryogenic differentiation occurs to form transgenic cotton. The invention obtains 13 independent transgenic lines.
2. PCGP1Promoter-driven glandular cell specific expression GUS
At T0In the positive transgenic plants of the generations, GUS staining experiments are carried out on cotton materials of different tissues. The specific operation is as follows: rapidly adding pre-cooled 80% (v/v) acetone into cotton tissue, fixing for 30 min, removing acetone, and adding pre-cooled ddH2Transferring the washed O to a staining solution containing a reaction substrate X-Gluc, placing the mixture in an incubator at 37 ℃ for 8-16 hours until the mixture is completely developed, then adding 75% alcohol at 42 ℃ to fully decolorize the sample, and finally fixing the material by using FAA tissue fixing solution (100 mL, the formula is 90 mL of 70% ethanol, 5 mL of glacial acetic acid and 5 mL of formaldehyde). The stained specimens were observed and photographically decolorized using a stereomicroscope (Leica MZFL III). The stained tissue was then cut into sections of 8 μm in thickness, and observed and photographed by an optical microscope (OLYMPUS IX73, Japan). Tissue sections As shown in FIG. 4, GUS staining was concentrated in the sclerenchymal cells surrounding the central lysogenic cavity of the pigmented glands.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> university of Henan
<120> cotton promoter PCGP1 and application thereof
<141> 2021-01-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1862
<212> DNA
<213> Cotton genus (Gossypium spp)
<400> 2
gaaataaggg taaaaaggga aaacgaaatc tctagaagga aaaaaagtaa ataaatgaag 60
ttgtaaagca taacattgtt tgactgatta ccaacctaaa ttttcaaatg gtatacgtgg 120
aacccgaaac attaaaaaaa attctgaggt ctaaaaaaaa aactcaaaat tattattaga 180
ttaaattaat taaaactttt ttccatcaaa aaattattaa atctaaaatt aaattccaag 240
tgaaaaaaac aacaacaaat gtttattaga aagaaaaaga ccttttcagc attaaacttc 300
caccaaacat gaacgtgcaa atgttctttc tcaagacaca cacatatata taaataaacc 360
gaaaatacat atatgacatt ttaattctga agtctaaaaa aaatgttgtt ttcattaaaa 420
gtattttgta aaactgaaaa agtttaaaat aattattttt ttaatcttat ataaatatac 480
ttgaattgaa acccatctaa atgcaaaaaa tgttctttta tttcaataat tttcaaacat 540
caaacttttc accctcaaat ccatttaaat atatgaattt tttattttga ttatgatcct 600
tttattatag gaaaattacg tactagacat gttttcatgt ttttatcatt gaaattaaat 660
tttttttatt tagcaaactt gcttttcaat tttaaaaaat aaaaataaaa attagttttt 720
aaaaatgaaa ataaaaaata tttttagaaa ctaaatggag cctcaagact ttatttggct 780
ttaggaaata tatgtatttt tttttcattt tcttgaaaaa tggaaaatat tatagaaaac 840
atggttaaaa attaaaaaaa tgatttttaa aaaatgaaaa aaatatataa attataaact 900
aataaaaatt ttgttttcat tatcttactg aaaacacatt ataaaactta aaaaaaaaag 960
aaaagaaaga aagagagagt ttaaaacgcg ttattaaatc tattatcttg attcgatttt 1020
atataaatac aaaaaaaaat taaaatttcc tcatattatc attattggaa ataaattttt 1080
gtttctattt taccaaatgc attttttaat tattaaataa tataaacaat atatactcaa 1140
acaactataa tcggaattaa ctattacaaa gtatattgtg ctaaaaattg aaagatatca 1200
aaatctaaat tctggggcaa aggaaagggg taagcaaggc actcgcctcc ccttaaatag 1260
aaattttttg cttttaatct ttgaagtttt ataaaatttt aaataaataa atggtaaaat 1320
tctagtcaac cttttaaaaa tgataaaatt ttgattttat cctcttaaaa actacaaaga 1380
tttatatcaa tttagtgatg aaattacaat ttaactttca taaaacatat agctcaatct 1440
caccctcctc cggagaaaaa tttcggactt acctaaattg taaacttaca tataattcac 1500
acaaagtgaa aactggaact agaaaactta taacctctgc cttacatgta agctactacg 1560
tcagattgaa taatacacat atttgtacat aggcaacgtt gaaatgtttt ggaattatat 1620
agttggaatg gtagctgcaa tcaatacttt ttaaaacaga gccgtcacta cttactttct 1680
tttacgagga acttttctgc ggataagaaa ccatgcacgt cgccattgtt attaaggctt 1740
ttctatctcc caatgagatt ctaagctata aataaacgga gtttggccaa cacatgatat 1800
tatagaaaaa gtgacatttt ataaacacac ctagctagca agctagttat aacagcttct 1860
at 1862
Claims (5)
1. Cotton promoter PCGP1Characterized in that the promoter PCGP1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.
2. The cotton promoter P of claim 1CGP1The application of the promoter as a probe to screening a promoter with a nucleotide sequence shown as SEQ ID NO.1 or a homologous promoter thereof from a genome library.
3. The cotton promoter P of claim 1CGP1The application of the gene in driving gene transcription in cotton.
4. The cotton promoter P of claim 1CGP1The application of the gene in the specific expression of the regulatory gene in cotton pigment gland cells.
5. The application according to claim 4, characterized in that the steps of applying are: the cotton promoter PCGP1Transferred into plant cells after being connected with target genesIn (1), a transgenic plant is constructed.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450733A (en) * | 2014-11-12 | 2015-03-25 | 南京农业大学 | Cloning and application of cotton gland formation gene GoPGF |
| CN106754956A (en) * | 2016-11-30 | 2017-05-31 | 华中师范大学 | Cotton GhJAZ11 D identified for genes and application |
| CN108277224A (en) * | 2018-03-22 | 2018-07-13 | 上海交通大学 | A kind of reproductive organs and glandular hairs tissue specific promoter GhS and its application |
| CN111808860A (en) * | 2020-07-15 | 2020-10-23 | 河南大学 | Cotton breeding method utilizing exogenous self-incompatibility |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9163252B2 (en) * | 2011-09-28 | 2015-10-20 | Bayer Cropscience Nv | Fiber-preferential promoter in cotton |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450733A (en) * | 2014-11-12 | 2015-03-25 | 南京农业大学 | Cloning and application of cotton gland formation gene GoPGF |
| CN106754956A (en) * | 2016-11-30 | 2017-05-31 | 华中师范大学 | Cotton GhJAZ11 D identified for genes and application |
| CN108277224A (en) * | 2018-03-22 | 2018-07-13 | 上海交通大学 | A kind of reproductive organs and glandular hairs tissue specific promoter GhS and its application |
| CN111808860A (en) * | 2020-07-15 | 2020-10-23 | 河南大学 | Cotton breeding method utilizing exogenous self-incompatibility |
Non-Patent Citations (3)
| Title |
|---|
| "GhACT1 启动子驱动 Lc 基因在棉花幼胚纤维中特异表达";范小平 等;《棉花学报》;20191231;第31卷(第6期);第529-535页 * |
| "Gossypium turneri isolate D10-2 chromosome D10-07，GenBank：CP032577.1";Udall,J.A. et al.;《GenBank》;20190821;第1-2页 * |
| "The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton";Wei Gao et al.;《Plant Biotechnology Journal》;20201231;第18卷;第1573-1584页 * |
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