The LaCAD gene, protein, recombinant vector, genetic transformation methods and applications of *Luffa cylindrica*

Abstract

This invention relates to a *LaCAD* gene, protein, recombinant vector, genetic transformation method, and its application in *Luffa cylindrica*, belonging to the fields of plant tissue culture and transgenic technology. The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the recombinant vector is pCAMBIA1300-GFP-CAD. This invention also provides a genetic transformation method for *Luffa cylindrica* and the application of the *Luffa cylindrica* cinnamyl alcohol dehydrogenase gene in increasing the lignin content of *Luffa cylindrica*. The method of this invention can increase the lignin content in *Luffa cylindrica* by 4-6 times.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture and plant transgenic technology, and relates to a lagenaria siceraria laCAD gene, protein, recombinant vector, genetic transformation method and application thereof. BACKGROUND

[0002] Lagenaria siceraria is a crop with a long cultivation history, is an annual climbing plant of the Cucurbitaceae family, is rich in nutrients and has medical and health care functions, has strong adaptability and disease resistance, high yield and high-temperature cultivation tolerance, and is one of main supply vegetables in the market in high-temperature seasons in China. During the development of lagenaria siceraria fruits, there are obvious morphological changes and complex physiological and biochemical changes, including fruit elongation, thickening, fibrosis, and synthesis of polysaccharides, lignin, phenols and flavonoids, which all affect the postharvest life and quality of the fruits.

[0003] Due to the fact that the planting range of old varieties and the yield and quality are not ideal, and a new variety has been lacked for a long time, how to cultivate lagenaria siceraria with high-quality fruits is still a problem to be solved at present. Transgenic technology can target the genome of a species for effective change, can improve crops, and has been used in molecular breeding of various plants. Through Agrobacterium-mediated genetic transformation technology, a target gene is introduced into a plant to obtain a transgenic line with stable expression of an exogenous gene, which is one of main technical means for studying the function of plant genes and genetic improvement. In addition, most plants induced by Agrobacterium are derived from a single plant cell, which ensures high genetic stability and prevents the generation of chimeras.

[0004] Lignin is a complex polyphenol polymer synthesized from monolignols through the phenylpropanoid pathway. Lignin polymers mainly include three types: p-hydroxyphenyl lignin, guaiacyl lignin and syringyl lignin. There are 12 known enzymes involved in lignin synthesis, and cinnamyl alcohol dehydrogenase (CAD) can reduce coniferyl aldehyde, sinapyl aldehyde and p-coumaric aldehyde to produce coniferyl alcohol, sinapyl alcohol and p-coumaric alcohol, etc. lignin monomers in the presence of NADPH as a cofactor, which is a key enzyme involved in lignin monomer synthesis. TaCAD1 of wheat shows a unique organ-specific expression pattern, which is highly expressed in stems and very low in leaves. The expression changes of different organs of the same plant are variable in space and time. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a lagenaria siceraria laCAD gene, protein, recombinant vector, genetic transformation method and application thereof, which can improve the content of lignin in lagenaria siceraria by overexpressing the laCAD gene in lagenaria siceraria.

[0006] The application is realized by the technical scheme as follows:

[0007] A lagenaria CAD dehydrogenase (LaCAD) gene, the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0008] The application also provides a protein encoded by the gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

[0009] The application also provides a recombinant vector containing the gene, and the recombinant vector is pCAMBIA1300-GFP-CAD.

[0010] The application also provides a genetic transformation method of lagenaria, and the method specifically comprises the following steps: obtaining a recombinant strain containing the LaCAD gene, transforming the strain into an agrobacterium liquid to obtain an agrobacterium infection liquid; placing cotyledons of aseptic seedlings of lagenaria as explants into the agrobacterium infection liquid, oscillating and incubating for 60 minutes, vacuumizing for 10 minutes, ultrasonicating for 10 minutes, vacuumizing for 10 minutes, and then placing the explants on sterile filter paper to dry, selecting the explants with stable growth state, subculturing on a rooting culture medium, and performing PCR detection to obtain positive plants.

[0011] The application also provides application of the gene in improving the lignin content of lagenaria, and the application is that the LaCAD gene is overexpressed in lagenaria plants by constructing a genetic transformation system.

[0012] Compared with the prior art, the application has the following beneficial effects:

[0013] 1. The application amplifies the CAD gene of lagenaria by means of genetic engineering, and constructs an overexpression vector.

[0014] 2. A stable transgenic strain is obtained by agrobacterium-mediated genetic transformation, an overexpression CAD gene lagenaria is obtained by introducing an exogenous gene, a high-efficiency screening system is established, the transformation efficiency is 11.66%, and the lignin content of lagenaria is increased by 4-6 times by overexpressing the CAD gene of lagenaria in lagenaria. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is an agarose gel electrophoresis graph of PCR amplification results;

[0016] Figure 2 It is a schematic diagram of the vector pCAMBIA1300-GFP-CAD skeleton;

[0017] Figure 3 It is an agarose gel electrophoresis graph of molecular identification of the overexpression CAD lagenaria;

[0018] Figure 4RT-PCR analysis histogram for CAD overexpression in Luffa cylindrica;

[0019] Figure 5 Flow chart for Luffa cylindrica transformation;

[0020] Figure 6 Lignin content determination histogram of transgenic materials. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further explained below by way of examples, but the scope of protection of the present application is not limited in any form by the examples.

[0022] Example 1: In this study, a CAD gene of Luffa cylindrica was cloned and identified using degenerate primers, as shown in Table 1, and the function of Arabidopsis thaliana CAD genes has been relatively fully studied, and the group of AtCAD4 and AtCAD5 clusters is considered to be CAD genes related to lignin synthesis, so the CAD of Luffa cylindrica was cloned as a candidate LaCAD using degenerate primers. Figure 1

[0023] Total RNA was extracted from the tender leaf parts of Luffa cylindrica seedlings using the TransZol method (Fullgen Biotech Co., Ltd., Catalog No: ET101-01), and the content and purity of the total RNA were detected using a nucleic acid analyzer, and the total RNA was used for reverse transcription reaction, and the kit used was One-Step gDNA Removal and cDNA Synthesis SuperMix (Fullgen Biotech Co., Ltd., Catalog No: AE311). The specific steps are as follows:

[0024] 1 Homogenization: Take the tissue sample of Luffa cylindrica stored at -80℃ and transfer it to a mortar pre-cooled with liquid nitrogen, and continuously grind the plant tissue with a pestle until the sample is ground into powder. Put it into a 2mL fully pre-cooled centrifuge tube. The liquid nitrogen in the tube should be evenly removed to prevent collapse, and take 50-100mg (about 1 / 5 of the tube) of the ground sample, and then add 1mL of TransZol and shake well.

[0025] 2 Layering: Place the homogenized sample on ice for 5min (to completely separate the nucleoprotein substances). Add 200mL of chloroform, tightly cap the tube, and shake vigorously for 3s, and place it on ice for 3min. Use a refrigerated centrifuge at 4℃ to centrifuge at high speed for about 15min to separate the layers, the colorless supernatant, the white protein layer in the middle, and the red organic phase at the bottom.

[0026] ​​3. Precipitation of RNA: At room temperature, transfer about 550-650 mL of the supernatant (as much as possible under the premise of ensuring quality) to a new 1.5 mL centrifuge tube (the centrifuge tube needs to be fully pre-cooled). Add 500 μL of isopropanol, mix well by inverting, and place at -20°C for 10 min. At 4°C, centrifuge at high speed for 10 min (during which time a 1% agarose gel can be run), and discard the supernatant (try to remove the isopropanol).

[0027] 4. RNA rinsing: Add at least 1 mL of 75% ethanol, vortex to mix, and then centrifuge at 7500 g at 4°C for 5 min.

[0028] 5. Resolubilization of RNA: Discard the supernatant, absorb the ethanol, and open the cap to air dry at room temperature for 10 min. Add 30-40 μL of RNase-free ddH2O to dissolve the RNA for 2-3 min.

[0029] 6. Use agarose gel electrophoresis and a nucleic acid analyzer to detect the content and purity of total RNA.

[0030] 7. Reverse transcription of RNA: Add the following system to a microcentrifuge tube in order:

[0031]

[0032]

[0033] 7.1 First mix the RNA template, Anchored Oligo(dT), and RNase-free Water, incubate at 65°C for 5 min, then ice bath for 2 min, and then add the other reaction components.

[0034] 7.2 Mix gently, and incubate at 42°C for 15 min.

[0035] 7.3 Inactivate Trans Script RT and gDNA Remover by heating at 85°C for 5 s.

[0036] Example 2: Cloning and construction of an overexpression vector for the LaCAD gene

[0037] Using the luffa cDNA as a template, use the degenerate forward primer and reverse primer: F: 5'-ATGGGAATAATGGAGGCAGA-3', R: 5'-TCAAGCGTCGAGATTGCTTCCTTC-3', perform PCR amplification, and after 10 min, use a gel imager to observe the gel map, as shown in Figure 2, the amplified fragment is about 1068bp. The reaction system is 20μL: forward primer 1μL, reverse primer 1μL, cDNA 1μL, water 7μL, phanta enzyme 10μL.

[0038] The construction method is as follows:

[0039] 1. The vector selects pCAMBIA1300-GFP vector as the skeleton, uses HindIII restriction endonuclease to cut pCAMBIA1300-GFP vector, and uses Novagen gel recovery kit to recover the obtained linearized vector, which is recorded as pCAMBIA1300-GFP-HindIII.

[0040] Enzyme cutting reaction system:

[0041]

[0042]

[0043] Hind III enzyme cutting reaction program:

[0044] 37℃ 60min

[0045] 2. The cDNA obtained by reverse transcription of the RNA of the luffa is used as a template to amplify the CAD fragment with the pCAMBIA1300-GFP-HindIII adapter, wherein P1300-F: tacaaatctatctctATGGGCAGCGTCGAAGG; P1300-R: aatgtttgaacgatcttaAAACTTGCTTCCTTCAACATCC;

[0046] Amplification reaction system:

[0047]

[0048] Amplification CAD fragment reaction program:

[0049]

[0050] The amplified product is subjected to gel recovery.

[0051] 3. The linearized vector pCAMBIA1300-GFP-HindIII obtained in 1 and the gel recovery fragment obtained in 2 are subjected to infusion connection using ClonExpress IIOne Step Cloning Kit kit (purchased from Novagen company);

[0052] Infusion reaction system:

[0053]

[0054] Infusion reaction procedure: 37°C for 60 min.

[0055] The ligation product was transformed into E. coli competent cells, identified by PCR with M13F and M13R primers, and sequenced by Genscript Company. Detection primers: M13F: TGTAAAACGACGGCCAGT; M13R: CAGGAAACAGCTATGACC.

[0056] 4. Detection reaction system:

[0057]

[0058] Detection reaction procedure:

[0059]

[0060]

[0061] 5. The correct bacterial liquid was detected pCAMBIA1300-GFP-HindIII-CAD plasmid (Novozyme Biotech Co., Ltd.), and the competent cells of Agrobacterium were transformed by freeze-thaw method: the competent cells of Agrobacterium EHA105 were melted on ice, and 5 μL (200 ng / ul) of plasmid DNA was added after melting on ice for 30 minutes, and frozen in liquid nitrogen for 2-3 minutes. Then 400 μL of LB medium without antibiotics was added in a 37°C water bath for 3 minutes, and cultured at 28°C, 200 rpm, for 2-4 hours. The bacterial liquid was concentrated by centrifugation at 4500 rpm for 10 minutes, and about 200 μL of bacterial liquid was taken and inoculated on LB medium containing 50 mg·L -1 kanamycin and rifampicin, and cultured at 28°C under inversion. After single colonies were grown, the CAD gene was identified using M13F / R primers, and positive colonies were saved.

[0062] Detection reaction system:

[0063]

[0064] Detection reaction procedure:

[0065]

[0066]

[0067] The saved positive bacterial liquid was inoculated into 50 mL of Agrobacterium liquid medium containing kanamycin resistance (LB base medium), and cultured at 28°C in the dark at a shaking speed of 200 rpm. When the bacterial liquid concentration OD 600When the value is between 0.6 and 0.8, centrifugal enrichment of bacterial bodies is performed. The bacterial liquid is resuspended with 35 mL of liquid medium to OD 600 When the value is equal to 0.2-0.3, the infection liquid is used to induce the infection of Momordica charantia explants.

[0068] Example 3: Genetic transformation of overexpression vector

[0069] 1. Obtain Momordica charantia sterile seedlings by tissue culture:

[0070] After the seed coat is directly stripped in the clean bench, the seed is first sterilized with 75% anhydrous ethanol for 2 min, washed with sterile water for 3-4 times, then sterilized with 3% sodium hypochlorite for 15 min, and washed with sterile water for 4 times. The sterilized seed is placed on sterile filter paper for 10 minutes. When the surface of the seed is dry, the radicle is inserted into the germination medium (1 / 2MS medium) with the lower half. Dark culture for the first 4 days, then move to normal light culture.

[0071] 2. Obtain Momordica charantia explant material: when the seed germinates and grows into sterile seedlings, the cotyledon node (remove 1 / 2 cotyledon and 1-1.5 cm stem segment from the growth point) is cut. The appropriate seedling age is 7-9 days after sowing (when the cotyledon is green and not flat), which is used as the explant for transformation infection.

[0072] 3. Put the cut explants into the infection liquid, shake and incubate for 60 minutes, vacuum for 10 min, ultrasonic for 10 min, vacuum for 10 min, and then place on sterile filter paper to dry.

[0073] 4. Rooting culture of Momordica charantia: select the explants with stable growth state, subculture in rooting culture medium, and the whole process is as shown in Figure 5

[0074] 5. Extraction of genomic DNA of Momordica charantia tissue culture seedlings: use tweezers to take the material from the culture medium into a 1.5 ml centrifuge tube, add 1 ml 2×CTAB extraction solution, crush and mix thoroughly, 65°C water bath for 30 min, gently invert the centrifuge tube several times during the water bath, after the water bath, when the liquid cools to room temperature, add equal volume of chloroform, shake vigorously for 15 seconds, centrifuge at room temperature for 10 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, add equal volume of isopropanol and shake well, precipitate in -20°C refrigerator for 30 minutes, centrifuge at room temperature for 10 minutes. Then wash the precipitate with 1 mL 70% ethanol, centrifuge for 5 minutes, discard the supernatant. Repeat the washing once, and dry naturally. Add 20 μL deionized water to dissolve the DNA, and measure the DNA concentration.

[0075] 6. Molecular biology identification of positive samples: use primers M13F / R for amplification. As shown in Figure 3

[0076] ​​Four positive seedlings were screened out by PCR detection, and the transformation efficiency was 11.66%, which improved the transformation efficiency.

[0077] Example 4: Fluorescent quantitative PCR detection

[0078] The qPCR analysis method of the present study adopts the SYBR Green method.

[0079] The original cDNA of each sample was mixed to establish a standard curve model, and three technical repeats and three biological repeats were set. The luffa reference gene 18S rRNA was used as the reference gene (Zhu Haisheng et al., 2015). The cDNA stock solution was diluted 4 times and used as the template for the fluorescent quantitative PCR reaction. Double standard curve method was used for data analysis.

[0080] The qPCR reaction system is as follows:

[0081]

[0082] The qPCR reaction program is as follows:

[0083]

[0084] The melting curve was collected, as shown in Figure 4 The expression of CAD gene after overexpression increased by about 3-23 times.

[0085] Example 5: Determination of total lignin content of overexpression material

[0086] Harvest mature overexpression luffa different tissues (leaves, roots, stems) for mixed sampling, and dry at 65℃ for 1-2 weeks. Then grind the dry sample.

[0087] 1. Weigh 20±0.03mg of dried sample into a 10mL spin glass tube, add 5mL 25% acetyl bromide (acetyl bromide: glacial acetic acid, volume ratio 1:3, freshly prepared), immediately cover the lid, mix well, and incubate at 50℃ for 4h;

[0088] 2. After taking out, cool to room temperature, centrifuge at 3500rpm for 15min;

[0089] 3. Prepare a 10mL plastic tube, add 2.5mL 2M NaOH, 3mL acetic acid, mix well, then add the centrifugal supernatant in step 2), mix well, finally add 0.25mL 0.5M hydroxylamine, mix well;

[0090] 4. Take 2mL solution, measure the absorbance at 280nm by ultraviolet spectrophotometer.

[0091] 5. Calculate total lignin content.

[0092] As shown in Figure 6 The total lignin content of the plants after overexpression increased by about 4-6 times.

[0093] The present application establishes an efficient screening system by Agrobacterium EHA105 mediation, by introducing exogenous genes and obtaining CAD gene overexpression luffa, which is conducive to subsequent gene function research of luffa.

[0094]

[0095] LaCAD protein (SEQ ID NO. 1):

[0096] MGSVEGERTVTGFAARDPSGFLSPYTYELRETGAEDVYIRVICCGLCHTDIHQVKNDLGMSNYPMVPGHEIVGEVMEIGKNVKRFRVKDVVGVGCIVGSCRSCERCESDREQYCNKRIWTNNDVYSDGRPTQGGFATAMVVHQKFVVKVPDGMAPEQAAALLCAGVTVYSPLKHFGLKKSGLRGGILGLGGVGHMGVKLAKALGHHVTVISSSNKKREEALDHLGADEYLVGSDEAQMQQAFDSLDYIIDTIPAFHPLEPYLSLLKLDGKLIMLGVVNNPLQFVSPMVILGRKTITGSFIGSMTETQEILDFFQEKELSSMVEVVKMDYINKAIERLEKNNVRYRFVVDVEGSKF*.

Claims

1. A Luffiacoralol dehydrogenase (LaCAD) gene, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The protein encoded by the Luffyl Dehydrogenase gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. A recombinant vector comprising the Luffyl Dehydrogenase gene of claim 1, characterized in that, The recombinant vector is pCAMBIA1300-GFP-CAD.

4. A genetic transformation method of luffa, which is specifically obtaining a recombinant strain containing the luffa cinnamyl alcohol dehydrogenase gene of claim 1, transforming the recombinant strain into an agrobacterium liquid to obtain an agrobacterium infection liquid; placing cotyledons of aseptic seedlings of luffa as explants into the agrobacterium infection liquid, oscillating and incubating for 60 minutes, vacuumizing for 10 minutes, ultrasonicating for 10 minutes, vacuumizing for 10 minutes, and then placing on sterile filter paper to dry, selecting explants with stable growth state, subculturing on rooting culture medium, and detecting positive plants by PCR.

5. The use of the F. hederacea lignin content increasing gene of claim 1 in increasing the lignin content of F. hederacea, characterized in that, The application is to make the luffa cinnamyl alcohol dehydrogenase gene overexpress in luffa plants by constructing a genetic transformation system.

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