阅读说明：本技术 实时荧光定量PCR检测外源GhCAD6基因在棉花纤维中表达量的方法 (Method for detecting expression quantity of exogenous GhCAD6 gene in cotton fiber by real-time fluorescence quantitative PCR ) 是由 胡文冉 苏秀娟 李晓荣 周小云 杨洋 李波 范玲 樊国全 刘建喜 邓晓娟 于 2018-06-20 设计创作，主要内容包括：本发明公开了一种采用实时荧光定量PCR检测外源GhCAD6基因在棉花纤维中的表达量的方法,首先采用热硼酸-蛋白酶K法提取出棉花纤维的RNA,反转录成cDNA,利用特异性引物和内参基因,在特定的反应条件和反应程序下进行荧光RT-PCR,检测外源GhCAD6基因在不同发育阶段棉纤维中的表达量,该方法自动收集荧光信号,避免了肉眼判断的主观性,提高了实验的灵敏度,保证了结果的可靠性和重复性,免除了常规PCR中的电泳、定量扫描等后续繁琐步骤,大大缩短了实验时间；本发明中棉花GhCAD6基因实时荧光RT-PCR检测方法的建立,为研究棉花GhCAD6基因表达调控机理及利用其改良棉花纤维品质的研究奠定了基础。(The invention discloses a method for detecting the expression quantity of an exogenous GhCAD6 gene in cotton fibers by adopting real-time fluorescent quantitative PCR, which comprises the steps of firstly extracting RNA of the cotton fibers by adopting a hot boric acid-proteinase K method, carrying out reverse transcription to form cDNA, utilizing specific primers and reference genes, carrying out fluorescent RT-PCR under specific reaction conditions and reaction programs, and detecting the expression quantity of the exogenous GhCAD6 gene in the cotton fibers at different development stages, wherein the method automatically collects fluorescent signals, avoids the subjectivity of naked eye judgment, improves the sensitivity of an experiment, ensures the reliability and repeatability of a result, avoids the subsequent fussy steps of electrophoresis, quantitative scanning and the like in the conventional PCR, and greatly shortens the experiment time; the establishment of the real-time fluorescence RT-PCR detection method of the cotton GhCAD6 gene lays a foundation for researching the expression regulation mechanism of the cotton GhCAD6 gene and the improvement of the quality of cotton fibers by using the cotton GhCAD6 gene.)

1. A method for detecting the expression quantity of an exogenous GhCAD6 gene in cotton fibers by real-time fluorescent quantitative PCR is characterized in that specific upstream and downstream primers of a target gene and an internal reference gene which accord with the characteristics of fluorescent PCR reaction are adopted, and the expression quantity of the target gene in the cotton fibers at different development stages is detected by the real-time fluorescent quantitative PCR, and the method specifically comprises the following steps:

a. extracting total RNA of cotton fibers: extracting RNA of cotton fibers at different development stages by a hot boric acid-proteinase K method;

b. first strand cDNA Synthesis: synthesizing a first chain of cDNA by using cotton RNA as a template, wherein the reaction system is 20 mu L;

c. real-time fluorescent PCR: taking the synthesized cDNA first strand as a template, and taking the following primers as specific primers to perform real-time fluorescence PCR amplification reaction, wherein each sample is provided with 3 parallel tubes, and Ct values of the 3 parallel tubes obtained after amplification are averaged;

the sequence specificity upstream and downstream primers of the target gene GhCAD6 which accord with the characteristics of fluorescent PCR reaction are as follows:

GhCAD6-F：5’-GTTCCTGGGCATGAAGTGGT-3’

GhCAD6-R：5’-TGCAACATCCAACAAGACAACC-3’

and cotton GhUBQ7 gene specific upstream and downstream primers as reference genes:

GhUBQ7-F：5’-AGAGGTCGAGTCTTCGGACA-3’

GhUBQ7-R：5’-GCTTGATCTTCTTGGGCTTG-3’

the reaction system of the fluorescence quantitative PCR is as follows: the total volume is 20 μ L, including10 μ L of Select Master Mix (2X), 0.40 μ L of Forward primer (10 μ M), 0.40 μ L of Reverse primer (10 μ M), 1 μ L of cDNA, and up to 20 μ L of RNase-free water;

the reaction conditions of the real-time fluorescent quantitative PCR amplification are as follows: UDG Activation AmpliTaq DNA, 50 ℃, 2 min; polymerase, UP Activation, 95 deg.C, 2 min; PCR reaction of 40cycles, 95 ℃, 15sec, 60 ℃ for 1 min;

d. calculating the relative expression quantity of the GhCAD6 gene in cotton fibers at different developmental stages: after the real-time fluorescence PCR is finished, calculating delta Ct and delta Ct 2 of the GhCAD6 gene in the cotton fiber at each time period according to the Ct value^-ΔΔCtThe values are calculated as follows:

Δ Ct ═ GhCAD6(mean Ct) -GhUBQ7 (mean Ct at the same developmental stage)

Δ Δ Ct ═ Δ Ct (progeny sample of transgenic GhCAD6 gene) — Δ Ct (control sample at the same developmental stage)

By 2^-ΔΔCtThe values represent the expression level of the exogenous GhCAD6 gene relative to the control non-transgenic cotton GhCAD6 gene.

One, the technical field

The invention relates to the field of biotechnology, relates to an analysis method of exogenous gene expression of transgenic cotton, and particularly relates to a method for detecting the expression of exogenous GhCAD6 gene in cotton fiber by a real-time fluorescence quantitative PCR technology.

Second, background Art

Since the establishment of new China, the yield and the quality of cotton (G.hirsutum) in China are greatly improved. However, the problems of low specific strength of the fiber and unreasonable matching of various indexes of the fiber quality exist, and the fiber is difficult to meet various requirements of domestic and foreign markets and textile industries (Tangshurong et al, 2015). Sinkiang also has the same problem as the biggest high-quality cotton production base in China, and seriously influences the international competitiveness of cotton in China (permit silver, etc., 2017). The cotton fiber quality improvement effect is not obvious in the conventional breeding aspect due to the lack of excellent germplasm and parent materials. The current approach for improving cotton fiber has been shifted from conventional breeding to the combination of conventional breeding and biotechnological breeding, wherein transgenic research is an important aspect of the improvement of the quality of cotton fiber.

During the 'fifteen' period, the country starts a great special item for transgenosis according to the actual demand of crop production. The project group of fangling researchers has led to the development of important special items for new varieties of transgenic organisms, and in the process of project development, the independent research results are applied, and according to the fact that cinnamate Dehydrogenase (Cinnamyl Alcohol Dehydrogenase, CAD) is an important functional gene formed by a plant cell wall cross-linked structure, a related major gene GhCAD6(GenBank serial number: EU281305.1) for regulating the formation of the cell wall cross-linked structure is cloned from developed cotton fibers. CAD is an important gene family in the phenylpropanoid metabolic pathway, and plays an important role in the formation of cell wall cross-linking structures, the cessation of cell wall elongation, the initiation of secondary wall development, and the development of secondary walls. The ChCAD6 gene is an important functional gene in a CAD gene family, and the expression level of the gene is synchronous with the development of a secondary wall of cotton fiber. The project group transforms the ChCAD6 gene into a cotton receptor by an agrobacterium-mediated method to obtain a plurality of transgenic lines with the fiber length increased by 1.17-3.21mm (4.22-11.58%) compared with the receptor and the specific strength increased by 3.2-7.1cN/tex (12.26-27.20%), and the transgenic lines show stable inheritance after multi-generation planting in the field.

The length and specific strength of transgenic progeny fibers obtained after the exogenous ChCAD6 gene is transformed into a cotton receptor are obviously improved, but the expression condition of the exogenous ChCAD6 gene in transgenic progeny plant fibers, particularly in the development process of secondary walls of cotton fibers, is lack of systematic research. Therefore, in order to research the mechanism of exogenous GhCAD6 for improving the quality of cotton fibers, it is necessary to research the expression level of the GhCAD6 gene in the development process of the secondary wall of transgenic progeny cotton fibers.

The quantitative detection method of gene expression is many, and mainly starts from several aspects of protein expression level, mRNA expression level and exogenous DNA, including enzyme-linked immunosorbent assay (ELISA), protein hybridization (Western Blotting), High Performance Liquid Chromatography (HPLC), Southern hybridization, Northern hybridization, biochip technology (Biochips) and Polymerase Chain Reaction (PCR) technology, semi-quantitative RT-PCR, Real-Time fluorescent quantitative PCR (Real-Time PCR) and the like. Each detection method has the advantages and disadvantages when detecting gene expression quantity, wherein the real-time fluorescence quantitative PCR technology has the advantages of high specificity, high sensitivity, capability of quantifying, effective solving of the problem of PCR pollution, high automation degree and the like, ensures the reliability and repeatability of results and is widely applied to the research fields of molecular biology, medicine and the like. However, for the characteristics of the cotton fibers that the cotton fibers are rich in phenolic compounds, polysaccharides and secondary metabolites, the activity of endogenous RNA enzyme is high, and the like, a suitable cotton fiber RNA extraction method, a suitable primer and an internal reference gene are key points for ensuring the implementation of real-time fluorescence quantitative PCR, and a suitable real-time fluorescence quantitative PCR reaction condition is one of essential contents for the implementation of experiments. Therefore, no report is found for detecting the expression quantity of the exogenous GhCAD6 gene in cotton fibers at different development stages by using real-time fluorescent quantitative PCR after the exogenous GhCAD6 gene is transferred into cotton by an agrobacterium-mediated method.

Third, the invention

The invention aims to provide a method for detecting the expression quantity of an exogenous GhCAD6 gene in cotton fibers by adopting a real-time fluorescent quantitative PCR technology. The specially designed primers are used for carrying out real-time fluorescent quantitative PCR amplification on cotton fibers at different development stages under specific reaction conditions, and the influence of exogenous genes on the quality of the cotton fibers can be rapidly and accurately judged according to the relative expression quantity of the GhCAD6 genes.

The technical scheme adopted by the invention is as follows:

(1) sampling cotton fibers: picking 5-25DPA (day post anti-gossypium) bolls from a detected cotton sample, removing cotton hulls, wrapping with tin foil paper, marking, quick freezing with liquid nitrogen, and storing in a freezer at-80 deg.C;

(2) extraction of cotton fiber RNA and cDNA Synthesis: extracting RNA in cotton fibers at different development stages by a hot boric acid-proteinase K method, and completing cDNA synthesis by adopting a reverse transcription kit;

(3) selection of an internal reference gene and design of related primers of the internal reference gene and a target gene:

searching a proper reference gene according to related documents, searching sequence information of the reference gene and a target gene in an NCBI database, designing by using an NCBI Primer design tool and Primer-Blast, performing specificity test on a Primer sequence by using a related program, and synthesizing a Primer by Shanghai bioengineering company. The synthetic primer sequences were designed as follows:

GhCAD6-F：5’-GTTCCTGGGCATGAAGTGGT-3’

GhCAD6-R：5’-TGCAACATCCAACAAGACAACC-3’

GhUBQ7-F：5’-AGAGGTCGAGTCTTCGGACA-3’

GhUBQ7-R：5’-GCTTGATCTTCTTGGGCTTG-3’

(4) real-time fluorescent quantitative PCR detection: and performing fluorescent quantitative PCR by using the diluted cotton fiber cDNA as a template, GhCAD6-F and GhCAD6-R as primers, GhUBQ7 of the cotton fiber as an internal reference gene and GhUBQ7-F and GhUBQ7-R as internal reference gene primers to obtain respective Ct values (cycle threshold). All reactions included three biological replicates.

(5) And (3) data analysis: data sorting was performed using excel2003 with 2^-ΔΔCtThe method calculates the expression level of the cotton GhCAD6 gene, the expression level is repeated three times in each PCR reaction of each sample, the average value of the three times of repetition is taken as the final expression level of the cotton fiber GhCAD6 gene, 2^-ΔΔCtThe expression quantity is relative expression quantity of cotton fiber GhCAD6 gene, wherein delta Ct is delta Ct (descendant sample of GhCAD6 gene) -delta Ct (same development stage control sample), and delta Ct is GhCAD6(mean Ct) -GhUBQT (same development stage mean Ct). DPS7.05 was used for statistical data analysis, and the GraphPad Prism 5 software was used for mapping.

The invention discloses a method for quantitatively detecting the expression quantity of an exogenous GhCAD6 gene in cotton fibers by real-time fluorescence, wherein the reaction system of the fluorescence quantitative PCR is as follows: the total volume is 20 μ L, including10 μ L of Select Master Mix (2X), 0.40 μ L of Forward primer (10 μ M), 0.40 μ L of Reverse primer (10 μ M), 1 μ L of cDNA, and make up to 20 μ L of RNase-free water.

The invention relates to a method for quantitatively detecting the expression quantity of an exogenous GhCAD6 gene in cotton fibers by real-time fluorescence, which comprises the following reaction conditions of fluorescence quantitative PCR: UDG Activation AmpliTaq DNA, 50 ℃, 2 min; polymerase, UP Activation, 95 deg.C, 2 min; PCR was carried out for 40cycles, 95 ℃, 15sec, 60 ℃ and 1 min.

The technical key of the implementation of the technology is as follows: the method aims at the characteristics that the cotton fiber is rich in phenolic compounds, polysaccharides and secondary metabolites, high in activity of endogenous RNA enzyme and the like, and adopts the hot boric acid-proteinase K method to extract the RNA in the cotton fiber, so that the completeness and purity of the obtained RNA in the cotton fiber are ensured; selecting and designing primers of a target gene GhCAD6 and a proper reference gene; the suitable real-time fluorescent PCR amplification system and the setting of real-time fluorescent PCR amplification parameters are all conditions which need a great deal of creative experiments and can be realized by simple and simple technology combination.

The technique has the following advantages: (1) the fluorescence signals are automatically collected, so that the subjectivity of naked eye judgment is avoided, the sensitivity of the experiment is improved, and the reliability and the repeatability of the result are ensured; (2) subsequent complicated steps such as electrophoresis, quantitative scanning and the like in the conventional PCR are avoided, and the experimental time is greatly shortened; (3) the operation is simple, and the popularization is strong.

Description of the drawings

FIG. 1 is the new Luzao No. 36 and its GhCAD6 gene transferring high generation material fiber RNA electrophoresis picture.

FIG. 2 is a graph showing the amplification curve of the GhCAD6 gene of the new-land early 36 GhCAD6 gene transgenic high-generation material fiber.

FIG. 3 is the melting curve diagram of the new Luzao No. 36 GhCAD6 gene-transferred high-generation material fiber GhCAD6 gene.

FIG. 4 is a graph showing the amplification of the reference gene GhUBQ 7.

FIG. 5 is a melting curve diagram of the reference gene GhUBQ 7.

FIG. 6 shows the relative expression levels of the GhCAD6 gene in different developmental stages of Xinluzao No. 36 fiber. In the graph, Δ indicates a significant difference (P < 0.05) compared to 5 d; tangle-solidup indicates a significant difference (P < 0.05) compared to 10d was achieved;indicating a very significant difference (P < 0.01) compared to 15 d;indicating a significant difference (P < 0.05) compared to 20 d.

FIG. 7 shows the relative expression levels of the GhCAD6 gene in different developmental stages of the Xinluaozao No. 36 GhCAD6 gene high-generation material fiber. In the graph, Δ indicates a significant difference (P < 0.05) compared to 5 d; a means that a significant effect is achieved compared to 10dSignificant differences (P < 0.05);indicating a very significant difference (P < 0.01) compared to 15 d;indicating a significant difference (P < 0.05) compared to 20 d.

FIG. 8 is a comparison of the relative expression levels of the GhCAD6 gene in different developmental stages of the new-Luzao No. 36 and new-Luzao No. 36 GhCAD6 gene high-generation material fiber. The graph indicates that the expression level of the GhCAD6 gene in the cotton fiber reaches a very significant difference (P < 0.01) in the same development stage.

Fifth, detailed description of the invention

Instruments, reagents and solutions:

a Thermo mixer; GL-88B vortex mixer; a neogauge 15R bench top high speed refrigerated centrifuge; k5500 nucleic acid protein quantifier; DYCP-31DN horizontal electrophoresis apparatus; a Tanon2500 gel imaging system; a pipettor; Bio-Rad gradient PCR instrument; ABI Real Time PCR instrument; DKZ series electric heating constant temperature water tank; mitsubishi-86 ℃ DW-HL398S ultra-low temperature refrigerator.

Diethyl Coke acid (DEPC), sodium tetraborate decahydrate (Na)₂B₄O₇·10H₂O), LEGTA, SDS, Dithiothreitol (DTT), sodium deoxycholate, PVP40, ethylphenylpolyethylene glycol (NP-40), proteinase K, potassium chloride (KCl), lithium chloride (LiCl), Tris-HCl, potassium acetate (KAc), absolute ethyl alcohol, isopropanol, agarose, a TRUUCcript first strand cDNA reverse transcription kit, a real-time quantitative PCR kit (Roche), a nucleic acid dye (Gold view), a conventional PCR reagent and the like, wherein all the reagents are made in China or imported and analyzed pure.

0.1% (V/V) aqueous solution of diethyl cokenate (DEPC): dissolve 800. mu.L DEPC in 800mL distilled water, shake well, and let stand overnight. Sterilizing at 121 deg.C for 30 min. For configuring and extracting RNA related solution.

RNA extraction solution: 0.2mol/L sodium tetraborate decahydrate (Na)₂B₄O₇·10H₂O), 0.03mol/LEGTA, 0.1% (W/V) SDS, 0.01mol/L dithiothreoseAlcohol (DTT), 1% (W/V) sodium deoxycholate, 2% (W/V) PVP40, 0.5% (V/V) ethylphenyl polyethylene glycol (NP-40). The first three solutions are weighed and dissolved according to requirements, and are sterilized by moist heat at 105 ℃ for 20 min. DTT was added when the solution was cooled to about 40 ℃. Before each use, sodium deoxycholate, PVP40 and NP-40 were added in different amounts as required.

20mg/mL proteinase K: weighing a proper amount of proteinase K, dissolving the proteinase K with DEPC water, and preparing into a 20mg/mL proteinase K solution. The mixture was dispensed into 1.5mL RNase-free centrifuge tubes at 200. mu.L/tube and stored at-20 ℃.

1.6mol/L potassium chloride (KCl), 4mol/L lithium chloride (LiCl), 2mol/L lithium chloride (LiCl), 10mg/L Tris-HCl (pH7.5), 2mol/L potassium acetate (KAc, pH5.5), 70% (V/V) ethanol, all of the above reagents were prepared with 0.1% (V/V) DEPC water.

Experimental materials:

new Luzao No. 36 and its high generation material (generation 6 material) transformed with GhCAD6 gene.

RNA extraction and quality detection

The method for extracting RNA from cotton fibers at different development stages by adopting a hot boric acid-proteinase K method comprises the following specific steps:

(1) in the full-bloom stage of cotton, cotton stalks are marked by adopting different color yarns, the time is recorded, 20-30 cotton bolls, 20-25 cotton bolls, 15-20 cotton bolls, 10-15 cotton bolls and 5-10 cotton bolls are picked at 5DPA respectively, the cotton bolls are removed, the cotton bolls are wrapped by tinfoil paper, 1-2 cotton bolls in each cotton boll are marked, the tinfoil paper wrapping the cotton bolls is quickly frozen in liquid nitrogen, and the cotton bolls are stored in a freezer at minus 80 ℃ for later use;

(2) grinding a sample: taking 5-8 cotton bolls in the same development day, randomly taking 1 cotton petal in each cotton boll, mixing and placing in a mortar, grinding into powder under the condition of liquid nitrogen, and placing in an RNase-free centrifuge tube;

(3) adding 1mL of RNA extract preheated to 80 ℃ into the tube, and violently shaking for 2 min;

(4) adding 10 μ L proteinase K (20mg/mL) into each centrifuge tube, mixing, and incubating at 42 deg.C in Thermo mixer at 800rpm for 1.5 h;

(5) respectively adding 50 mu L of 1.6mol/L KCl into the centrifuge tube, and carrying out ice bath for 1 h;

(6) centrifuging at 4 deg.C and 12,000rpm for 20min, and transferring the supernatant into a new centrifugal tube without RNase;

(7) adding equal volume of 4mol/L LiCl, and standing overnight at-80 ℃;

(8) centrifuging at 12,000rpm at 4 deg.C for 20min, and retaining precipitate;

(9) washing the precipitate with 0.5mL of LiCl 2mol/L for 3 times, mixing uniformly each time, centrifuging at 12,000rpm for 20min, and discarding the supernatant;

(10) resuspending the pellet in 0.2mL of 10mmol/L Tris-HCl (pH7.5), centrifuging at 4 ℃ and 12,000rpm for 20min, and transferring the supernatant into a new RNase-free centrifuge tube;

(11) adding 40 μ L of 2mol/L KAc (pH 5.5) into the supernatant, and ice-cooling for 15 min;

(12) centrifuging at 4 deg.C and 12,000rpm for 20min, and transferring the supernatant into a new centrifugal tube without RNase;

(13) 1mL of absolute ethyl alcohol is respectively added into each tube, and after uniform mixing, the mixture is kept overnight at minus 80 ℃;

(14) centrifuging at 4 deg.C and 12,000rpm for 20min, and collecting precipitate;

(15) washing the precipitate twice with 0.5mL of pre-cooled 70% ethanol;

(16) air-drying the precipitate, and adding 20 μ L DEPC-ddH₂Dissolving O, taking 1 mu L of the solution to detect the concentration and purity of RNA at the absorption values of 260nm and 280nm of an ultraviolet spectrophotometer, taking another 1 mu L of the solution to further detect whether DNA pollution exists by utilizing 1% agarose gel electrophoresis, and storing the rest RNA at-80 ℃;

2. reverse transcription Experimental procedure

The preparation method comprises the following steps:

placing the solution in a PCR reaction tube without RNase, and gently mixing the solution uniformly; taking out at 25 ℃ for 10min, 42 ℃ for 30min and 85 ℃ for 5s, and finishing the reverse transcription experiment; the cDNA obtained by reverse transcription was stored at-20 ℃ for subsequent experiments.

3.Real-Time PCR

The fluorescent quantitative PCR reaction system is as follows:

ABI7500 fluorescent quantitative PCR cycling conditions:

4. statistical analysis

Data sorting was performed using excel2003 with 2^-ΔΔCtThe method calculates the expression level of the cotton GhCAD6 gene, the expression level is repeated three times in each PCR reaction of each sample, the average value of the three times of repetition is taken as the final expression level of the cotton fiber GhCAD6 gene, 2^-ΔΔCtThe expression quantity is relative expression quantity of cotton fiber GhCAD6 gene, wherein delta Ct is delta Ct (descendant sample of GhCAD6 gene) -delta Ct (same development stage control sample), and delta Ct is GhCAD6(mean Ct) -GhUBQT (same development stage mean Ct). DPS7.05 is adopted for data statistical analysis, GraphPad Prism 5 software is adopted for mapping, all data are expressed by mean +/-standard deviation (x +/-s), two groups of mean comparison are analyzed by independent sample T test, a multi-group mean comparison is analyzed by a one-factor variance analysis method, and P < 0.05 has statistical significance.