CN110656128B - Recombinant construct containing human keratinocyte growth factor gene expression and application thereof - Google Patents

Abstract

The invention provides a recombinant construct containing human keratinocyte growth factor gene expression and application thereof. The present invention uses recombinant DNA technology to insert the KGF2 gene into the pDONR207 plasmid as the basic cloning vector, constructs the pGWB-KGF2 vector for plant expression, and transforms it into cotton Shixiya (Gossypium spp. Chemical processing processes such as active protein emulsification filling and support reduce production costs. The invention provides usable new genetic resources for the medicinal use of cotton fibers.

Description

Recombinant construct containing gene expression of human keratinocyte growth factor and application thereof

technical field

The invention belongs to the field of plant biotechnology. The invention patent specifically relates to a recombinant construct containing human keratinocyte growth factor gene expression and its application, especially in cotton.

Background technique

Cotton is an important economic crop and natural fiber material planted in large areas in the world, and its production and application play an important role in the national economy. my country's cotton fiber quality is single, there are few high-quality fiber resources, and there are few researches on gene functions that control cotton fiber optimization. These greatly limit the development of my country's cotton textile manufacturing industry, and will lead to the long-term lack of international competitiveness of my country's cotton fiber products. Therefore, modern agriculture can not only successfully improve the quality of cotton fiber synthesis but also increase cotton germplasm resources by applying the transgenic technology that regulates cotton fiber development to the improvement of cotton fiber quality.

Human keratinocyte growth factor 2 (KGF2), also known as fibroblast growth factor-10, is a member of the growth factor related to the proliferation of epithelial cells in the fibroblast growth factor superfamily. The biological function of KGF2 growth factor is mainly to promote the proliferation, differentiation and migration of specific epithelial cells by binding to epithelial cell membrane receptors, and at the same time, it has the function of inducing the synthesis of new epidermal cells to replace aging epidermal cells. This specific effect has become a hotspot in the development of genetic engineering drugs as a new peptide drug in the treatment of skin burns, corneal repair, and ulcer healing, and even to maintain the skin's continuous tenderness. At present, the use of genetic engineering to produce KGF2 factor has become a national first and second class peptide new drug for clinical treatment. At the same time, biological products and gene beauty products containing KGF2 have become the earliest commercialized and successful profitable cases in the international genome project.

At present, with the penetration of biotechnology and the hot development of cosmetic products, genetic engineering technology is used to produce active protein factors that affect the growth of human cells, and through cell permeability, they are processed into a variety of beauty and skin care products. As a paracrine growth factor in the gene beauty growth factor family, KGF2 has great application prospects in the markets of skin beauty, sports injury repair and hair growth. For example, growth factors containing KGF2 can prevent the formation of red blood cells in the skin by inhibiting the growth of new blood vessels and repairing the stratum corneum ^[7,8] . Smith et al ^[1] found that KGF2 biological agents can be used to repair burns (scalds), various surgical traumas and cosmetic surgery wounds and inhibit the formation of scar tissue. In addition, Jun-Hyeog et al. used organ culture experiments to find that adding 10ng/ml KGF2 growth factor could stimulate 26-35% of human hair follicle cell proliferation ^[12] , indicating that KGF2 participates in regulating hair follicle regeneration and maturation to achieve hair regeneration by promoting the proliferation of human embryonic neural stem cells and inducing the activity and content of hair follicle growth factor.

Alopecia is one of clinically common hereditary intractable diseases, and there is no hair growth medicine with better effect, which has brought great pain and troubles to many patients so far. For such people with hair loss and thinning hair, there are many existing medical or cosmetic solutions, the most common of which are hair growth agents and hair transplantation. The principle of using hair growth medicine is to use the active ingredients of the medicine to nourish the hair follicles and the skin of the head to improve metabolism and promote hair regeneration. The method has practical problems such as long time, slow effect, and easy to cause androgen disturbance. In addition, the cost of hair implant repair is high, the operation risk is high, and the popularity rate is not high. So it is necessary to develop a hair-increasing fiber spray that not only contains the curative effect of the hair growth agent but also has the effect of concealing and beautifying hair. The hair-increasing fibers currently on the market are processed from plant fibers and keratin derivatives. The electrostatic effect is used to make countless fine hairs adhere closely to the hair, making the originally sparse hair instantly thicker and thicker visually, so that it looks as healthy and perfect as natural hair from the outside.

Our patent aims to construct an exogenous gene expression vector containing KGF2 growth factor through recombination, obtain transgenic cotton containing recombinant human KGF2 growth factor through Agrobacterium infection technology, and obtain transgenic cotton fiber containing KGF2 growth factor protein through the overexpression of KGF2 gene in cotton fiber organs, so that a large amount of pure plant cotton fiber powder containing human KGF2 growth factor protein from green sources can be harvested, which can be added to hair-increasing fiber sprays, and can also be used in medical products such as biological bandages for wound repair, and can even be used to make Beauty mask, etc. Then, the KGF2 protein contained in the fiber is released by a simple water solvent, which can be absorbed by scalp cells, hair follicle cells or epidermal wound cells, achieving the purpose of hair growth, beauty and even wound repair. Therefore, the pure plant fiber-derived gene activity products produced by such transgenic cotton varieties have good application prospects and attractive economic benefits.

The transgenic technology of DNA chromosomal integration established by introducing exogenous genes into the plant genome is an important technology successfully used in modern life sciences. Cotton fiber cells are one of the ideal choices for medicinal protein fiber reactors. Genetic transformation methods commonly used in cotton transgenic technology mainly include gene gun method and Agrobacterium transformation method. Since there are very few research and development cases of KGF2 factor protein expression in plant fibers, this patent now uses diploid cotton as planting material, and successfully introduces human KGF2 gene and 35s promoter into cotton seedlings, obtaining a total of 16 independent resistant transformed plants. The results of PCR technology and gene sequence sequencing experiments showed that the KGF2 gene was integrated into the cotton genome, laying the basic material for comprehensive promotion of cotton fibers stably expressing KGF2 factor from plant sources.

Contents of the invention

The object of the present invention is to provide a recombinant construct containing human keratinocyte growth factor gene expression and its application, especially the cotton fiber obtained by using it in cotton. The cotton fiber can be used as biological fiber for medical bandages and hair growth fibers.

To achieve the above object, the technical scheme adopted in the present invention is as follows:

The invention provides a recombinant construct containing human keratinocyte growth factor gene expression, which is characterized in that it includes the nucleotide sequence of SEQ ID NO1, and the vector used for the construct is the pDONR207 vector for cloning or the pGWB vector for expression.

The preparation method of the recombinant construct containing human keratinocyte growth factor gene expression includes amplifying the nucleotide sequence SEQ ID NO1, upstream primer KGF2B1: 5'-AAAAAGCAGGCTTCATGGTGTCACCAGAGGCCACCAA-3', downstream primer KGF2Bst2: 5'-AGAAAGCTGGGTCTCATGAGTGTACCACCATTGGAAGA-3'; PCR amplification product is purified by BP Clonase ^TM IIenz yme mix was cloned into the pDONR207 vector and transformed into E.coli DH5α competent cells to obtain the recombinant construct pDONR207-KGF2.

In the preparation method of the recombinant construct containing human keratinocyte growth factor gene expression, the plasmid DNA of the cloning vector pDONR207-KGF2 is recombined into the plant expression vector pGWB21, and transformed into E.coli DH5α competent cells to obtain the plant expression construct pGWB-KGF2.

The transgenic Agrobacterium containing the recombinant construct expressed by human keratinocyte growth factor gene is LBA4404.

The application of the recombinant construct containing human keratinocyte growth factor gene expression in plant tissue expression.

The application of the recombinant construct containing human keratinocyte growth factor gene expression in plant tissue expression is characterized in that: the plant tissue is root, stem, leaf, flower, fiber, tiller bud or seed.

The application of the recombinant construct containing human keratinocyte growth factor gene expression in plant tissue expression, and the plant is a monocotyledonous plant or a dicotyledonous plant.

The application of the recombinant construct containing human keratinocyte growth factor gene expression in plant tissue expression, the monocotyledonous plant is rice, wheat, barley, sorghum or corn, and the dicotyledonous plant is Arabidopsis thaliana, tomato, tobacco, soybean or potato.

A cotton fiber, which is a mature cotton fiber obtained by transforming the recombinant construct containing human keratinocyte growth factor gene expression into cotton crops.

The invention utilizes recombinant DNA technology to insert KGF2 gene into basic cloning vector pDONER207, constructs pGWB-KGF2 vector for plant expression, and transforms the plasmid DNA into cotton by using Agrobacterium infection technology. Finally, it inserts the gene whose nucleotide sequence is shown in the sequence table (SEQ ID No. 1) into the chromosomal sequence of the transgenic cotton recipient, and then uses the leaves and seeds of the plant to express the KGF2 gene in large quantities, and obtains a new cotton variety containing the expression product protein of KGF2. The cotton fiber containing recombinant human keratinocyte growth factor KGF2 produced by this new variety can be directly used as an active ingredient in the production of corresponding pharmaceutical fields and daily chemical fields, which simplifies the processing techniques such as purification of KGF2 active protein and addition of protein emulsifier (or protective agent) in the past when using bacterial strains to produce active protein, and reduces production costs.

Compared with the prior art, the present invention has the advantages of:

The method for producing cotton fiber containing keratinocyte growth factor KGF2 gene expression belongs to the first case in the world, and has the characteristics of simple operation, low cost, high expression level, green color, no allergies, no bacterial toxicity, easy preservation and portability of active protein, and no need for chemical processing. Since the cotton fiber itself contains the human keratinocyte growth factor KGF2 protein, the active KGF2 protein can be released through a simple solvent by utilizing the solubility of the KGF2 protein. If it is made into a medical bandage, beauty mask or hair implant fiber can fill the gap in the international market.

Description of drawings

Fig. 1 contains the nucleic acid electrophoresis figure of keratinocyte growth factor KGF2 gene fragment;

Figure 1 shows the result of nucleic acid electrophoresis of the KGF2-containing gene fragment obtained after PCR amplification, and the arrow indicates that the size of the gene fragment is about 500 bp.

Fig. 2 contains the nucleic acid electrophoresis figure of keratinocyte growth factor gene cloning vector pDONR207-KGF2 plasmid DNA and plant expression vector pGWB-KGF2 plasmid DNA;

Figure 2 shows the results of nucleic acid electrophoresis detection of the plasmid DNA of the cloning vector containing the KGF2 gene fragment and the plant expression vector obtained after genetic recombination, and the arrows indicate the size of the fragments corresponding to each vector.

Figure 3 Agrobacterium plasmid DNA nucleic acid electrophoresis pattern containing keratinocyte growth factor KGF2;

Figure 3 shows the result of nucleic acid electrophoresis detection of the plasmid DNA extracted after transformation of the plant expression vector containing the KGF2 gene fragment successfully recombined into Agrobacterium, and the arrow shows the size of the fragment corresponding to the vector.

Fig. 4 represents KGF2 transgenic cotton plant identification nucleic acid electrophoresis figure;

Figure 4: The upper figure shows the +swim image of the genomic DNA extracted from the transgenic cotton seedlings containing the KGF2 gene fragment successfully transformed with Agrobacterium. The figure below shows the PCR identification of positive transgenic cotton plants containing the KGF2 gene fragment. CK+: Plasmid DNA containing KGF2 gene as a positive control template. CK-: Non-transgenic cotton gDNA was used as a negative control template. 2, 10, 14, 17, 22, 3: corresponding to the number of positive transgenic plants. 21: corresponding to the number of negative transgenic plants. Arrows indicate the corresponding fragment sizes.

Fig. 5 is a graph showing the growth status of KGF2 transgenic cotton plants and control group cotton stone West Asia plants;

Fig. 5: The growth status of the successfully transformed KGF2-containing transgenic cotton plant OE and the non-transgenic cotton plant, that is, the control group Cottonia CK. KGF2 transgenic cotton had larger leaves, compact plant type and earlier flowering than the control group.

Fig. 6 is a graph showing the mature cotton fiber growth status of KGF2 transgenic plants and control group cotton Shisia plants;

Fig. 6A: Fiber length profile of harvested KGF2-containing transgenic cotton plants (OE) and non-transgenic, ie, control cotton Shisia (CK) plants. KGF2 transgenic cotton fiber was significantly longer than the control group. Fig. 6B: Statistical analysis of fiber length of transgenic cotton plants OE and non-transgenic cotton plants CK by Student-t test.

Figure 7 is a graph showing the relative expression of the KGF2 gene in the fiber organs of the control group cotton and KGF2 transgenic cotton lines;

Figure 7: RNA extracted from the cotton fibers of harvested KGF2-containing transgenic cotton plants (OE) and non-transgenic cotton plants (CK) in the control group, and the KGF2 gene expression was detected by RT-PCR. Upper panel: The results show that the KGF2 gene is highly expressed in the fibers of the transgenic plants. Bottom: 18sRNA was used as internal reference. Arrows indicate the fragment sizes of the corresponding genes.

Fig. 8 is the SDS-PAGE electrophoresis detection picture of the cotton fiber of KGF2 transgenic cotton plant and control group cotton Shisia plant;

Figure 8: The total protein extracted from the cotton fibers of the harvested KGF2-containing transgenic cotton plant (OE) and the non-transgenic cotton plant (CK) was detected by SDS-PAGE electrophoresis to detect the accumulation of KGF2 protein. The results showed that KGF2 protein was highly expressed in the fibers of transgenic plants. 20KD and so on represent the size of the marker band, and the arrow shows the fragment size corresponding to the KGF2 protein.

Detailed ways

The present invention will be further elaborated below by examples, the purpose of which is to better understand the content of the present invention rather than limit the protection scope of the present invention.

The present invention intends to further analyze the efficacy of KGF2 protein in the process of cell growth, and use cotton fiber as a carrier to stably express KGF2 protein, in order to achieve the purpose of green low-cost high-throughput production of medicinal protein, and finally provide new ideas and resources for green medicinal protein to serve human beings through genetic engineering.

Obtaining and performance analysis of cotton transgenic strains

1 Materials and methods

1.1 Plant materials and planting methods

The cotton variety tested is Cotton Shixia, which is kept in the Key Laboratory of Plant Genetics and Molecular Breeding of Zhoukou Normal University. Seeds were soaked at 37°C for more than 12h, planted in nutrient soil, lighted for 16h, dark for 8h, and cultivated in a greenhouse at 28-30°C. After flowering, they began to be listed, and the samples of fiber and young leaf tissues at different developmental stages were collected and quickly placed in liquid nitrogen, and stored in a -80°C refrigerator for later use.

1.2 Reagents and carriers used

本实验所采用的大肠杆菌为DH5α，农杆菌为LBA4404，这些菌株为周口师范学院植物遗传与分子育种重点实验室保存，Gateway重组酶BP和LR Clonase ^TM Ⅱenzyme mix、植物表达载体pGWB21和重组克隆载体pDONR207购自于Invitrogen公司；其中克隆载体PMD18-T载体、T4 DNA连接酶、Taq DNA聚合酶购自于TaKaRa生物公司；采用AXYGEN 公司的AxyPrepTM DNA Gel Extraction Kit 进行胶回收；RIPA （RIPA Lysis Buffer）蛋白提取液购自Thermo Scientific公司；考马斯亮蓝G-250, 甲醇，潮霉素（Hyg）、卡那霉素（Kan）和利福平（Rif）等购自北京鼎国生物技术有限公司，试验所有引物均由北京奥克鼎盛生物公司合成。 Sequencing was performed by BGI.

1.3 Synthesis of human keratinocyte growth factor KGF2 gene

The nucleotide sequence of human keratinocyte growth factor KGF2 was first found from GENBANK. According to the degeneracy of codons, the enzyme cutting sites used in this experiment were eliminated, and the open reading frame (SEQ ID No. 1) of the keratinocyte growth factor KGF2 gene was artificially synthesized by Shanghai Sangon Bioengineering Co., Ltd.

The gene open reading frame SEQ ID NO.1, its nucleotide sequence is as follows:

ATGGTGTCACCAGAAGGCCACCAACTCTTCTTCCTCCTCCTTCTCCTCTCCTTCCAGCGCGGGAAGGCATGTGCGGAGCTACAATCACCTTCAAGGAGATGTCCGCTGGAGAAAGCTATTTCTCTTCACCAAGTACTTTTCCAGATTGAGAAGAACGGGAAGGTCAGCGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCG GAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAG GAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCA

1.4. Cloning of recombinant construct pDONR207-KGF2

The nucleotide sequence of SEQ ID No. 1 was amplified in full length by the standard polymerase chain reaction (PCR) technology with designed primers to obtain a PCR product fragment with a length of 495 bp, as shown in Figure 1 for details. It was constructed into pDONR207 (Invitrogen ^TM ) vector by Gateway recombination technology and sequenced.

The nucleotide sequence of the open reading frame of the amplified KGF2 gene is SEQ ID NO1, the upstream primer KGF2B1: 5'-AAAAAGCAGGCTTCATGGTGTCACCAGAGGCCACCAA-3', the downstream primer KGF2Bst2: 5'-AGAAAGCTGGGTCTCATGAGTGTACCACCATTGGAAGA-3'. PCR reaction system (20 μL): 10×PCR reaction buffer 2 μL, dNTP mixture (2.5 mmol/L each) 1.6 μL, Mg ²⁺ (25 mmol/L ) 1.6 μL, primer (10pm/μL) 1 μL each, pfu high-fidelity enzyme (5U/μL) 0.2 μL, the nucleotide sequence fragment containing KGF2 synthesized by the company is the nucleotide sequence SEQ ID NO 1 is template 2 µL, ddH2O 10.6 µL. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 15 s, annealing at 56°C for 25 s, extension at 72°C for 40 min, 34 cycles; extension at 72°C for 10 min. After the PCR amplification product was purified, it was cloned into the pDONR207 vector by BP Clonase Ⅱ enzyme mix (Invitrogen ^TM ), and transformed into E.coli DH5α competent cells. After the plasmid PCR identified ^positive colonies, it was sequenced by Huada Gene Company, and finally the recombinant construct pDONR207-KGF2 was obtained.

1.5. Cloning of recombinant construct pGWB-KGF2

Using the plasmid DNA of the correctly sequenced cloning vector pDONR207-KGF2, it was recombined into the plant expression vector pGWB21 (AccessionNumber: AB289784) through LR Clonase ^TM Ⅱ enzymemix (Invitrogen ^TM ) reaction, and transformed into E.coli DH5α competent cells. After the plasmid was identified as a positive colony by PCR, it was sequenced by BGI, and finally the plant expression construct pGWB-K was obtained. GF2. See Figure 2 for the nucleic acid electrophoresis diagram of the keratinocyte growth factor gene cloning vector pDONR207-KGF2 plasmid DNA and the plant expression vector pGWB-KGF2 plasmid DNA.

1.6. Transformation of Agrobacterium LBA4404 with pGWB-KGF2 plasmid DNA

Take 100 μL of Agrobacterium LBA4404 competent cells and place them in an ice bath for 5 min to melt them for later use. (2) Take 2 μlpGWB-KGF2 plasmid DNA and add it to the Agrobacterium competent cells, mix slowly, and then ice-bath for 25 minutes. (3) After the ice bath, place it in liquid nitrogen and freeze it for 3 minutes, then take it out and put it in 37°C for incubation for 5 minutes. (4) Add 1 mL of YEP-free liquid medium without resistance to the centrifuge tube, and incubate the bacteria on a shaker at 180 rpm/min for 2 hours at 28°C. (5) Then use a centrifuge at 5000rpm, centrifuge for 5min, discard the supernatant, spread the precipitated bacterial liquid on an LB solid medium plate containing two antibiotics (100 μg/mLRif, 50 μg/mLKan), and cultivate it in a 28°C incubator for 2 days. Pick a single colony for liquid culture, and if it is positive by bacterial liquid PCR, it is the Agrobacterium strain containing the target gene pGWB-KGF2, or extract the Agrobacterium plasmid DNA to screen the transformants. Figure 3 shows the nucleic acid electrophoresis pattern of the Agrobacterium plasmid DNA containing keratinocyte growth factor KGF2. Strains were preserved with 70% glycerol for future use. The strain is now stored in a -80°C refrigerator in the Key Laboratory of Plant Genetics and Molecular Breeding of Zhoukou Normal University.

1.7. Agrobacterium-mediated genetic transformation

Pick a single Agrobacterium colony containing the pGWB-KGF2 plasmid, inoculate it into 5ml LB liquid medium, and culture it on a shaker at 28°C overnight. The next day, inoculate 100 microliters of the overnight activated bacterial solution into 50ml LB liquid medium, culture with shaking at 28°C and 180rpm until the OD600 is 0.8, then centrifuge and resuspend until the suspension concentration reaches OD=1. Infect the cotton shoot tip cells in the resuspension solution for 16 hours in the dark, rinse the bacterial solution the next day and cultivate it under the light for 20 days until green new leaves grow. Because the carrier contains the plant selection marker kanamycin, apply 2mg/L kanamycin to select resistance-positive seedlings.

1.8. Leaf genomic DNA extraction and specific primer PCR detection of transgenic plants

The transgenic lines were grown and developed in the greenhouse or in the field, and the genomic DNA of the young leaves of the control group and the young leaves of the transgenic plants were extracted respectively. Using the genome as a template, use specific gene primers: upstream primer K1: 5'-TCCTTCTCCTCCCCATCCCTCTGCT-3', downstream primer K2: 5'-GTGTTCTTCCGCCGGGTCTTCTGG-3' to amplify the KGF2 corresponding gene fragment. The target band (about 440bp) containing the KGF2 gene fragment can be amplified in the transgenic single plant, which is the transgenic positive plant. See Figure 5 for the growth status of the KGF2 transgenic cotton plants and the cotton Shisia plants of the control group.

Genomic DNA Extraction from Single Plant of KGF2 Transgenic Cotton Lines

The DNA of young cotton leaves was extracted by CTAB method. The test samples were KGF2 transgenic cotton (OE) and control cotton Shisia (CK). After growth culture, take 0.2 g of young leaves, add liquid nitrogen and grind them into powder in a mortar, then transfer to a 2.0 mL centrifuge tube, add 600 µL of preheated CTAB extract (0.1 mol/L Tris-HCl, 0.02 mol/L EDTA (pH8.0), 2% CTAB, 2% PVP40, 1mol/L sodium chloride, 0.2% β-mercaptoethanol) . Heated in a water bath at 65°C, then added pre-cooled chloroform:isoamyl alcohol:absolute ethanol (76:4:20) for extraction, then precipitated with ice isopropanol, cleaned impurities with 70% ethanol solution, and dried to obtain gDNA of cotton leaves. Then as a template and KGF2 specific primers K1/K2 for PCR amplification detection. The PCR amplification reaction system was 20 µL, including 2 µL of 10×LA TaqBuffer II (Mg ²⁺ plus), 0.5 µL of dNTPs (10 mM), 14.5 µL of ddH2O, 0.5 µL of upstream specific primers, 0.5 µL of downstream specific primers, 2 µL of templates, and 0.5 µL of Taq enzyme; the reaction conditions were pre-denaturation at 95°C for 5 minutes , denatured at 95°C for 15 s, annealed at 55°C for 20 s, extended at 72°C for 50 s, cycled 30 times, extended at 72°C for 10 min, and stored at 4°C. Take 10 µL of the PCR reaction product, detect it by electrophoresis on 1% agarose gel, stain with GelRED, and take pictures with the gel imaging system of BIO-RAD Company for preservation. AxyPrepTM DNA Gel Extraction Kit from AXYGEN was used for gel recovery. The recovered product was connected to the PMD18-T vector, the connection system was: pMD 18-T Vector 0.5 µL, Solution I 4.5 µL, and the recovered PCR product 5 µL. Ligated at 16°C for 12 hours, and detected by 1% agarose gel electrophoresis. Positive clones were sent to BGI for sequencing. The nucleotide sequence of the KGF2 gene is verified after the comparison of the clones, which means that the nucleotide fragment of the KGF2 gene is inserted into the genome chromosome of the transgenic plant. See Figure 4 for the nucleic acid electrophoresis diagram for the identification of KGF2 transgenic cotton plants.

1.9. Fiber detection of KGF2 transgenic cotton lines

After growing and developing in the greenhouse or field, then flower and set seeds, the transgenic T1 generation seeds are formed. After the seeds of the T1 generation were harvested, the mature cotton fibers of the transgenic cotton of KGF2 transgenic cotton and the control group cotton Shixia were picked, and 5 bolls were picked from the upper, middle, and lower three parts of each plant, and the bolls were exposed to the sun for 3 days until they completely dehydrated. Use a dissecting needle to separate and comb the fibers on both sides in the direction perpendicular to the seeds, take pictures and analyze the data statistically, and finally analyze the obtained data with the Student-t test. The growth status of the mature cotton fibers of the KGF2 transgenic plants and the control plants cotton Shisia plants is shown in Figure 6, CK in Figure 6 is the mature cotton fibers of the control cotton Shisia plants, and OE is the mature cotton fibers of the KGF2 transgenic cotton. The results showed that the average length of KGF2 transgenic cotton fibers was 2.905556 (±0.157624) cm longer than that of the control group, which was 0.945294 (±0.114843) cm. The results indicated that KGF2 gene played a very important role in the elongation of cotton fiber.

2.0. Identification of expression level of KGF2 gene in transgenic line fibers

The tested transgenic KGF2 cotton and the control cotton were grown and cultured, and the mature cotton fibers were taken for total RNA extraction and reverse transcription to obtain cDNA, which was used as a template for RT-PCR amplification reaction in order to detect the expression level of KGF2 gene. Among them, the target band (about 440 bp) containing the KGF2 gene fragment can be amplified in the transgenic single plant, which means that the KGF2 gene has stable expression, while the expression of the human KGF2 gene cannot be detected in the cotton fiber of the control group. The detection chart of the relative expression of KGF2 gene in the fiber organs of the control cotton and KGF2 transgenic cotton lines is shown in FIG. 7 . This result provides a new gene resource for further application of KGF2 gene in other plants such as tomato, cucumber and watermelon.

Cotton fiber RNA was extracted from the test samples by CTAB method. Take about 0.5 g of cotton fiber, add liquid nitrogen to grind it into powder, transfer it to a 2 mL centrifuge tube, use 1 mL of extraction buffer (1.4 mol/L NaCl, 0.1 mol/L Tris-HCl, pH8.0, 20 mmol/L EDTA, 2 % CTAB, 2 % PVP, 1 % β-mercaptoethanol), shake and mix, and put in a 65 ° C water bath for 20 min to make RNA Precipitate. After the solution is cooled, add chloroform and LiCl (8 mol/L) solution to stand in an ice bath for precipitation, and then wash the impurities with 70% ethanol, centrifuge and dry them. After dissolving, they can be purified by acid phenol: chloroform (1:1), and then precipitated by cold isopropanol and ethanol to collect RNA samples for later use or storage. Take 1 µL for electrophoresis to check the quality of RNA.

Then take the same amount of cotton fiber RNA from the control group and the KGF2 transgenic group for RT-PCR detection. Using 2 μg of RNA as a template, the first-strand cDNA was synthesized according to the operating instructions of the cDNA synthesis kit (TaKaRa). Using KGF2 gene cDNA, use specific gene primers: upstream primer K1: 5'-TCCTTCTCCTCCCCATCCCTCTGCT-3', downstream primer K2: 5'-GTGTTCTTCCGCCGGGTCTTCTGG-3' to amplify the KGF2 corresponding gene fragment. The expression of KGF2 in the control group and transgenic lines was detected by semi-quantitative RT-PCR. Semi-quantitative RT-PCR reaction system (20 μL): 2 μL of 10×PCR reaction buffer, 1 μL of dNTP (2.5 mmol/L), 1 μL of each primer (10 pm/μL), 0.2 μL of Taq polymerase (5 U/μL), 2 μL of template cDNA, and 12.8 μL of ddH ₂ O. PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 15 s, annealing at 56°C for 20 s, extension at 72°C for 50 s, 30 cycles; extension at 72°C for 10 min, storage at 4°C; the primer sequences for semi-quantitative RT-PCR are shown in Table 1.

2.1. Analysis of the stable accumulation of KGF2 protein in the fibers of transgenic lines

The tested KGF2 transgenic cotton and the control cotton were grown and cultured, and the mature cotton fibers were extracted for fiber total protein, and the differential protein expression between the samples was compared.

The test samples were extracted with RIPA (RIPA Lysis Buffer, Thermo Scientific) method to extract the total soluble protein of cotton fiber. Take about 1g of cotton fiber, add a small amount of quartz sand, grind it into powder in liquid nitrogen, transfer it to a 10 mL centrifuge tube, add RIPA buffer solution and centrifuge in an ice bath, and reprecipitate the precipitated protein with 5 times the volume of 100% acetone, freeze for two hours and then resuspend. The control group samples and transgenic samples were used to estimate the total protein concentration by Bradford method, and then adjust the amount of protein loaded at the same concentration for SDS-PAGE electrophoresis. After electrophoresis, the gel was stained and soaked in Coomassie brilliant blue solution for 2 hours, then slowly decolorized with double distilled water until the background was clear, and then photographed for analysis.

Since the band size of the target protein KGF2 protein is about 20KD, the results show that the target band of the expected size (about 20kD) can be detected in the transgenic fibers, while no human KGF2 protein bands can be detected in the cotton fibers of the control group. The results show that KGF2 protein is stably expressed in the cotton fibers of the KGF2 transgenic plants. The results provide new ideas and new gene resources for the further use of KGF2 protein expression in cotton fiber powder processing or in tomato, cucumber, watermelon and other plants.

The above embodiments are only for illustration, rather than limiting the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the present invention can still be modified or equivalently replaced, and any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the claims of the present invention.

SEQUENCE LISTING

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gtgttcttcc gccgggtctt ctgg 24

Claims (5)

1. Use of a recombinant construct comprising expression of the human keratinocyte growth factor KGF2 gene for promoting elongation of cotton fibers, characterized in that: the nucleotide sequence of the human keratinocyte growth factor KGF2 gene is shown in SEQ ID NO1, and the vectors used for the construct are a pDONR207 vector for cloning and a pGWB vector for expressing a water-soluble KGF2 protein.

2. The use according to claim 1, characterized in that: the preparation method of the recombinant construct containing the human keratinocyte growth factor KGF2 gene expression,

amplification nucleotide sequence SEQ ID NO1, upstream primer KGF2B1:5'-AAAAAGCAGGCTTCATGGTGTCACCAGAGGCCACCAA-3', downstream primer KGF2Bst2:5'-AGAAAGCTGGGTCTCATGAGTGTACCACCATTGGAAGA-3'; the PCR amplified product is purified and subjected to BP Clonase ^TM II enzyme mix is cloned to pDONR207 vector and transformed into E.coli DH5 alpha competent cells to obtain recombinant construct pDONR207-KGF2.

3. The use according to claim 2, characterized in that: the preparation method of the recombinant construct containing the human keratinocyte growth factor KGF2 gene comprises the steps of recombining plasmid DNA of a cloning vector pDONR207-KGF2 onto a plant expression vector pGWB21, and transforming into E.coli DH5 alpha competent cells to obtain the plant expression construct pGWB-KGF2.

4. The use according to claim 1, characterized in that: the transgenic agrobacterium containing the human keratinocyte growth factor KGF2 gene is LBA4404.

5. A cotton fiber, characterized in that: the recombinant construct containing the human keratinocyte growth factor KGF2 gene expression of claim 1 is transformed into cotton crops to obtain the mature cotton fiber with KGF2 protein and growing cotton fiber.