CN117143892A - A key gene GbSNAT that promotes ginkgo melatonin synthesis and enhances plant heat tolerance and its application - Google Patents

Abstract

The invention discloses a key gene GbSNAT that promotes melatonin synthesis in ginkgo and enhances plant heat resistance and its application. The invention clones a brand-new gene GbSNAT from ginkgo for the first time, and overexpresses it by transferring the GbSNAT gene into ginkgo. The melatonin content in transgenic Ginkgo and Arabidopsis thaliana with GbSNAT gene increased significantly, indicating that GbSNAT is the key gene to promote melatonin synthesis in Ginkgo. Combined with the results of in vitro enzymatic experiments, it was shown that GbSNAT can promote the synthesis of melatonin. In addition, the high temperature tolerance of transgenic Arabidopsis was significantly improved, indicating that melatonin can effectively alleviate the damage caused by high temperature to plants and improve the heat tolerance of plants. Therefore, regulating the expression of GbSNAT has important application value in increasing melatonin synthesis in Ginkgo and enhancing plant heat tolerance.

Description

A key gene that promotes ginkgo melatonin synthesis and enhances plant heat tolerance GbSNAT and its applications

Technical field

The invention belongs to the technical fields of plant genetic engineering and molecular biology, and specifically relates to a key gene GbSNAT that promotes ginkgo melatonin synthesis and enhances plant heat resistance and its application.

Background technique

Ginkgo (Ginkgo biloba), belonging to the genus Ginkgo of the Ginkgo family, is an ancient relict tree species native to my country and is currently the only surviving species in the Ginkgo family. Ginkgo is a tall tree with unique leaf shapes and golden autumn leaves. It has extremely high garden ornamental value and is widely introduced and cultivated around the world. Ginkgo leaves also have high medicinal value and are rich in phenolic acids, flavonoids (kaempferol, isorhamnetin, quercetin, etc.) and ginkgolides and other medicinal active ingredients. They are an important raw material for Ginkgo extract GBE. , plays an important role in antioxidant, nerve protection, cardiovascular system enhancement and anti-inflammation. Extremely high temperatures in summer often cause the leaves of large ginkgo trees to turn yellow and wither, causing the tree to weaken or even die. In the production of leaf ginkgo, extreme high temperatures can easily cause leaf yellowing and necrosis of seedlings or even the death of the entire plant, significantly reducing leaf yield and medicinal ingredient content. Therefore, the death of seedlings and heat damage to large trees caused by high temperatures have seriously harmed the growth of Ginkgo biloba for different purposes such as leaf use, ornamental use, and fruit use, and have become one of the important issues that the Ginkgo industry needs to solve urgently.

Melatonin (N-acetyl-5-methoxytryptamine) is a structurally stable amine substance commonly found in animals and plants. Tryptophan is the precursor substance for its synthesis. In animals, the biosynthetic pathway of melatonin is now very clear, and its main physiological functions are to regulate the body's circadian rhythm, antioxidant, immune enhancement and anti-aging functions. In plants, melatonin synthesis involves the participation of multiple enzymes. First, tryptophan is decarboxylated by tryptophan decarboxylase (TDC) to form tryptamine, and then hydroxylated by tryptamine-5-hydroxylase (T5H). Serotonin. The last two enzymes involved in melatonin biosynthesis are serotonin-N-acetyltransferase (SNAT) and N-acetyl-5-hydroxytryptamine methyltransferase/caffeic acid O-methyltransferase (ASMT/COMT). Key rate-limiting enzymes, their catalytic sequence is closely related to environmental conditions. Under normal growth conditions, 5-hydroxytryptamine is first acetylated by SNAT enzyme to form N-acetyl-5-hydroxytryptamine, and then O-methylated by ASMT/COMT enzyme to form melatonin. Under adverse conditions, 5-hydroxytryptamine in plants has a higher affinity for ASMT, and is more likely to be O-methylated to 5-methoxytryptamine by ASMT/COMT enzymes, and finally acetylated by SNAT enzymes to form melatonin. As a new type of plant growth regulator, melatonin not only regulates growth and development and delays organ aging, but also uses its powerful antioxidant ability to remove reactive oxygen species (ROS) and RNS produced by stress in the body, helping plants resist the environment. Coercion. Therefore, the 5-hydroxytryptamine-N-acetyltransferase gene (SNAT) is an indispensable key enzyme in multiple melatonin synthesis pathways. Especially when plants suffer abiotic stress, SNAT is mainly the last step to produce melatonin. One-step rate-limiting enzyme.

Due to the large genome and complex genetic background of Ginkgo, it is difficult to identify and study genes involved in Ginkgo stress resistance and their specific biological functions using conventional biotechnology and genetic methods. At present, most studies related to stress in Ginkgo are limited to the physiological, biochemical and transcriptional metabolism levels, without in-depth exploration and analysis of important functional genes and metabolites. In particular, the role of the Ginkgo SNAT enzyme gene in melatonin synthesis and its application in plant resistance to high temperatures have not been reported. Therefore, in-depth research on the Ginkgo SNAT gene can not only analyze the endogenous melatonin biosynthesis of the plant, but is also of great significance for studying the genetic mechanism of Ginkgo under high temperature stress, and has broad application prospects.

Contents of the invention

Purpose of the invention: In view of the shortcomings in the prior art, the present invention provides a key gene GbSNAT that promotes the synthesis of ginkgo melatonin and enhances plant heat resistance. By promoting the expression of this gene, it can improve the production of melatonin in ginkgo and other plants. content, and effectively improve the high temperature tolerance of plants.

The present invention also provides the application of the key gene GbSNAT that promotes ginkgo melatonin synthesis and enhances plant heat tolerance.

Technical Solution: In order to achieve the above objectives, the present invention proposes a key gene GbSNAT that promotes ginkgo melatonin synthesis and enhances plant heat tolerance. The nucleotide sequence of the gene GbSNAT is shown in SEQ ID NO. 1.

Among them, the primer pair used to amplify the gene GbSNAT is SEQ ID NO.2: ATGGCAGTGGGGATGGCG and SEQ ID NO.3: TTTTTTTGTCGATCGAGGTCTTT.

The protein expressed by GbSNAT, a key gene that promotes ginkgo melatonin synthesis and enhances plant heat resistance according to the present invention, is characterized in that its amino acid sequence is shown in SEQ ID NO. 4.

The prokaryotic expression vector of the key gene GbSNAT that promotes ginkgo melatonin synthesis and enhances plant heat resistance according to the present invention is characterized in that the prokaryotic expression vector uses the vector pGEX-6P-1 as the starting plasmid, and pGEX-6P The -1 vector plasmid was linearized by double enzyme digestion. The enzyme digestion sites were BamH I and EcoR I. The key gene GbSNAT was inserted into pGEX-6p-1 to construct the GbSNAT prokaryotic expression vector pGEX-6p-GbSNAT.

As a preference, the length of the constructed plasmid pGEX-6p-GbSNAT is 5704 bp; plasmid pGEX-6p-GbSNAT only has a BamHI restriction site at position 945 and an EcoRI restriction site only at position 1674. .

The overexpression vector of the key gene GbSNAT, which promotes melatonin synthesis in ginkgo and enhances plant heat resistance, according to the present invention, the overexpression vector assembles a constitutive strong promoter CaMV35S at the 5' end of the gene GbSNAT, and at the 5' end of the gene GbSNAT The NOS-terminator is assembled at the 3' end. Among them, the strong promoter CaMV35S can enable the efficient expression of GbSNAT gene in Ginkgo biloba, and the NOS-terminator can effectively terminate the transcription of GbSNAT gene in Ginkgo biloba.

Further, the expression vector is assembled with an NPTII gene expression cassette, which serves as a screening marker for transgenic Arabidopsis, and kanamycin is used to screen transgenic Ginkgo Arabidopsis; the overexpression vector is assembled with LB (T- Border left) and RB (T-Border right) sequences, which promote the integration of the gene GbSNAT expression framework and the screening marker gene NPTII assembled into the Ginkgo chromosome.

The key gene GbSNAT or the host bacteria of the prokaryotic expression vector or the overexpression vector, which promotes ginkgo melatonin synthesis and enhances plant heat resistance according to the present invention, generally uses Agrobacterium as the starting strain.

The application of the key gene GbSNAT or the protein or the prokaryotic expression vector or the overexpression vector of the present invention in the synthesis of plant melatonin and improving the heat tolerance of plants.

Preferably, the plants include Ginkgo biloba, Arabidopsis thaliana, etc.

Among them, the gene GbSNAT prokaryotic expression vector was used to increase the GbSNAT protein content. In vitro enzymatic experiment results showed that GbSNAT can catalyze the synthesis of melatonin from the substrate 5-methoxytryptamine.

A genetically engineered bacterium. The genetically engineered bacterium uses Escherichia coli as a starting strain and introduces the gene GbSNAT, which can catalyze 5-methoxytryptamine to produce melatonin.

Application of the key gene GbSNAT or the protein or the prokaryotic expression vector or the overexpression vector of the present invention in cultivating plants with high melatonin content and heat resistance.

Among them, the process of cultivating plants with high melatonin content and heat resistance is as follows: using ginkgo leaves as materials, cloning the gene GbSNAT, constructing the gene into the overexpression vector pRI 101, constructing a recombinant vector, and transforming the recombinant vector into Agrobacterium, and then soak the ginkgo callus in Agrobacterium resuspension for genetic transformation of Ginkgo. Driven by the promoter CaMV35S, GbSNAT can be efficiently expressed in Ginkgo, thereby promoting the synthesis of melatonin; using Agrobacterium resuspension was used to infect Columbia wild-type Arabidopsis thaliana inflorescences, and positive transgenic Arabidopsis of the T3 generation was screened and identified. Under high temperature treatment, the heat resistance was significantly improved compared to wild-type Arabidopsis.

The present invention determines and clones a brand-new gene GbSNAT from Ginkgo biloba for the first time through gene family analysis and expression analysis, constructs a prokaryotic expression vector of the gene GbSNAT and conducts in vitro enzymatic experiments. The prokaryotic expression vector of GbSNAT constructed by the present invention is used. It can significantly increase the GbSNAT protein content. In vitro enzymatic experiment results show that GbSNAT can catalyze the synthesis of melatonin from the substrate 5-methoxytryptamine, indicating that GbSNAT is the key gene for ginkgo melatonin synthesis, which is conducive to the development of GbSNAT for targeted and Melatonin is synthesized in large quantities for the purpose of developing commercial uses of this compound and its application in abiotic stresses. The present invention also constructs an overexpression vector and transfers it into ginkgo callus and Arabidopsis. The melatonin content in the obtained transgenic ginkgo callus is significantly increased, and the heat resistance of the transgenic Arabidopsis is significantly higher than that of wild type and Arabidopsis. The mutants prove that the gene of the present invention has broad application prospects and can provide a reference for using excellent resistance genes to improve the stress resistance of plants. In addition, the present invention provides important theoretical guidance for Ginkgo melatonin to alleviate high temperature damage to plants.

For the first time, this invention uses a specific prokaryotic expression system to directly synthesize plant melatonin in large quantities in vitro, which has broad application prospects. At the same time, the gene GbSNAT of the invention has the ability to synthesize plant melatonin and give plants the ability to resist high temperatures, which is completely new. Its functions and effects can provide new genetic resources for cultivating high-temperature resistant transgenic lines.

Beneficial effects: Compared with the existing technology, the present invention has the following advantages:

The present invention is based on the fact that exogenous melatonin can improve plant stress resistance. Through gene mining and experiments, a brand-new gene GbSNAT was cloned from Ginkgo biloba for the first time. It was found that the gene in Ginkgo only contains one exon. The GbSNAT gene was transferred into Ginkgo, and the melatonin content in transgenic Ginkgo and Arabidopsis overexpressing the GbSNAT gene increased significantly, indicating that GbSNAT is a key gene that promotes melatonin synthesis in Ginkgo. Combined with the in vitro enzymatic experiment results, GbSNAT can Promote the synthesis of melatonin. In addition, the high temperature tolerance of transgenic Arabidopsis was significantly improved, indicating that melatonin can effectively alleviate the damage caused by high temperature to plants and improve the heat tolerance of plants. Therefore, regulating the expression of GbSNAT has important application value in increasing melatonin synthesis in Ginkgo and enhancing plant heat tolerance.

Cloning and functional studies of the GbSNAT gene provide a theoretical basis for using gene regulation technology to promote the synthesis and accumulation of melatonin in Ginkgo to improve heat resistance, provide a reference for research on the mechanism of high-temperature heat resistance in Ginkgo, and also provide a basis for cultivating high melatonin levels. Genetically modified strains with high content and high temperature resistance provide new genetic resources.

In the present invention, the GbSNAT enzyme expressed through prokaryotic expression has high activity. The gene GbSNAT is used as the target gene of the engineering bacteria, and the recombinant enzyme is prepared as a catalyst, which can catalyze the substrate 5-methoxytryptamine in vitro to produce melatonin, which can be used as anti-tumor, anti-aging and It is resistant to stress and has good application prospects.

Description of the drawings

Figure 1 is a phylogenetic tree analysis of GbSNAT and SNAT proteins from other species;

Figure 2 shows GbSNAT cloning (A), E. coli bacterial liquid detection (B) and sequence alignment (C);

Figure 3 is a schematic structural diagram of the constructed prokaryotic expression vector pGEX-6p-GbSNAT;

Figure 4 shows the protein induction and enzymatic reaction of GST-tagged GbSNAT.

Figure 5 is a diagram of the vector enzyme digestion gel (A), a schematic structural diagram of the constructed overexpression vector 35S::GbSNAT (B), and Agrobacterium bacteria liquid detection (C);

Figure 6 shows the detection of overexpressed GbSNAT ginkgo callus (A), the determination of melatonin content in callus (B), and the identification of Arabidopsis mutants (C);

Figure 7 shows the screening and identification of GbSNAT transgenic Arabidopsis (A-C), the root length phenotype and data statistics of transgenic Arabidopsis and mutant Arabidopsis (D-E);

Figure 8 is the phenotype and survival rate analysis of transgenic Arabidopsis and mutant Arabidopsis after short-term high temperature treatment (A-C);

Figure 9 shows the phenotypes and chlorophyll and carotenoid contents of transgenic Arabidopsis and mutant Arabidopsis after long-term high temperature treatment (A-D);

Figure 10 shows the changes in MDA content and antioxidant enzyme activity in mutant and overexpressed GbSNAT Arabidopsis thaliana under long-term high temperature treatment (A-D);

Figure 11 shows the changes in endogenous melatonin content in transgenic Arabidopsis and mutant Arabidopsis before and after high temperature treatment.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1

Screening and evolutionary tree analysis of GbSNAT

Based on the ginkgo genome and gene expression analysis, a gene was identified and cloned from Ginkgo. Unlike other plants, this gene in Ginkgo only consists of one long exon. Based on the phylogenetic tree analysis of the conserved region of SNAT in Ginkgo, rice, Arabidopsis and Begonia biloba, the gene in Ginkgo was named GbSNAT. The SNAT family contains two types of conserved domains, namely acetyltransferase 1 (Acetyltransf_1) and acetyltransfase 7 (Acetyltransf_7), both belonging to the NAT_SFsuperfamily (Figure 1).

Example 2

Cloning GbSNAT gene

(1) Based on the above evolutionary tree analysis and Ginkgo genome, Primer Premier 5.0 software was used to manually design the amplification primers SEQ ID NO. 2 and 3 of GbSNAT as shown below. The target gene GbSNAT was amplified through the Ginkgo leaf cDNA template. The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the gene GbSNAT obtained from the nucleotide sequence is shown in SEQ ID NO. 4.

(2) Use the high-fidelity enzyme PrimeSTAR Max DNAPolymerase (Takara, Japan) to perform PCR amplification. The PCR system is as follows:

PrimeSTAR Max Premix(2×) 25μL PCR Forward Primer(10μM) 2μL PCR Reverse Primer(10μM) 2μL Ginkgo leaf cDNA template 2μL ddH ₂ O 19μL Total volume 50μL

Gently mix the above mixture by pipetting, centrifuge briefly at low speed and place it in an ordinary PCR reactor. Set the following program:

Gel running: After the PCR amplification program is completed, put the large-hole 1% agarose gel plate into the electrophoresis tank, and add the PCR reaction solution and marker mixed with the loading buffer into the gel hole, turn on the switch of the electrophoresis tank, and proceed Run rubber. After 15 minutes, close the electrophoresis tank, put the gel under UV irradiation, obtain the target fragment and verify the band size (Figure 2A). Then use Kangwei Century Rapid Agarose Gel DNA Recovery Kit (Kangwei Century Biotechnology Co., Ltd.) to recover the gel.

(3) Ligation reaction between purified fragment and cloning vector

Refer to the pEASY-Blunt Simple Cloning Kit (Quanjin, China) instruction manual to connect the gel recovery product to the cloning vector. The specific system is as follows:

pEASY-Blunt Simple Cloning Vector 1μL PCR Product 2μL ddH ₂ O 2μL Total volume 5μL

(4) E. coli transformation

Transfer the ligation product to the Vidy E.coli DH5α large intestine competent cell (Shanghai Vidy Biotechnology Co., Ltd.). For specific operations, refer to the instruction manual for the Vidy DH5α large intestine competent cell. Centrifuge the bacterial solution, discard the supernatant, and leave 100 μl for pipetting and mixing. Spread into the corresponding resistant solid medium and incubate upside down at 37°C for 12 hours.

(5) Positive clone screening and sequencing analysis

Select a single colony from the screening culture plate and inoculate it into LB liquid culture medium. Shake the culture at 37°C and 200rmp until the bacterial liquid becomes turbid. Use 2×Taq Master Mix (Norvizan) for PCR amplification and use agarose gel electrophoresis. Perform testing.

reaction system:

2×Taq master mix 12.5μl Primer F 1μl Primer R 1μl Bacterial liquid 3μl ddH ₂ O 7.5μl Total volume 25μl

Reaction procedure:

The clones that were positive in the bacterial liquid PCR test (Figure 2B) were sent to Sangon Biotechnology Company (Shanghai) for sequencing and identification, and the sequencing results were compared (Figure 2C). The sequence is shown in SEQ ID NO.1, which was used for subsequent experiments. The amino acid sequence of the expressed protein is shown in SEQ ID NO. 4.

SEQ ID NO.1

ATGGCAGTGGGGATGGCGATGGCACCTTCAATTATGTTATCATCTACCCATAT

TCATCGTTTGAATTCTAACGGCTTTTTCACGCCCAAATTAGGTAACATTTCAT

ACAAATTTTGCACTTGGGAGATACCCTGTAGACGTCAAACTACTAGATTTAA

GCAATTTGTTTTGCCAAGGAACTTCTGAATCCGTCGTCGCGTCTTCGGCCATA

TCTGAAGATAATAAAATCTCTGTAACAGAATCTGGGCAATTTTCTATTAGCG

ATTCCCAGCTAGATTCTCGGTGGGTTTGAGCTTCATAAATCCTTAGAGGATCT

TAATTTGGATCAACTGAATGCCCTCTTCGTCAAAGTTGGGTTCCCCCGCAG

ACAGAAGGACAAGATGGAGCGCGCTCTGCATAACACACCGTCTATGCTTTG

GTTTGAGGAGAAGAAATCCGGTAAGCTTGTTGCATTTGCCAGAGCCACTGG

GGATGATGTGTTCAATGCCATCATTTGGGATGTTGTTGTTGATCCTTCCTTTC

AGGGTTTTGGATTGGGAAAGGCTATTATGGAGCGTTTGATGGCTCATCTCTT

GGGGAAAGGTATTACAAATATTGCCCTGTATGCTGAACCTCACGTATTGGGT

TTCTATAGGCCATTGGGGTTCACTGCAGATCCTGATGGCATCAAGGCTATGG

TCTATTCCAAGAAACCTGAAAGACCTCGATCGACAAAAAAATAASEQ ID NO.4

MAVGMAMAPSIMLSSTHIHRLNSNGFFTPKLGNISYKFCTWEIPCRRQTTRFK

QFVCQGTSESVVASSAISEDNKISVTESGQFSISDSQLDSRGFELHKSLEDLNLD

QLNALFVKVGFPRRQKDKMERALHNTPSMLWFEEKKSGKLVAFARATGDDV

FNAIIWDVVVDPSFQGFGLGKAIMERLMAHLLGKGITNIALYAEPHVLGFYRP

LGFTADPDGIKAMVYSKKPERPRSTKK

Example 3

Construction of GbSNAT gene prokaryotic expression vector

1) Use TaKaRa QuickCut restriction endonuclease (TaKaRa, Japan) to perform an enzyme digestion reaction experiment on the prokaryotic expression pGEX-6P-1 vector (TaKaRa, Japan). The specific reaction system is as follows:

QuickCut Buffer 5μL pGEX-6P-1plasmid 8μL QuickCut BamHI 1μL QuickCut EcoR I 1μL ddH ₂ O 35μL Total volume 50μL

After mixing each solution in the system, perform instant centrifugation. The PCR reaction procedure is as follows:

37℃ 30min

85℃ 20min

4℃ to end

Conduct agarose gel electrophoresis on the digested product to observe the digested bands. If there are obvious band differences, it means that the vector has been cut. Place the cut vector in a -20°C refrigerator for subsequent vector ligation reactions.

(2) Design of homologous recombination primers

Use the CE Design (Nanjing Novezan Biotechnology Co., Ltd.) online website or software to manually design the homologous recombination amplification primers SEQ ID NO. 5 and 6 of GbSNAT according to the enzyme cutting site of the vector as shown below.

According to the positive plasmid extracted from E. coli in the above Example 2 as a template, the homologous recombination GbSNAT gene was cloned according to the method of Example 2, gel running and gel recovery, and the obtained gel recovery product was placed in a -20°C refrigerator for subsequent use. Vector ligation reaction.

(3)Homologous recombination ligation overexpression vector

Referring to the ClonExpress II One Step Cloning Kit (Nanjing Novezan Biotechnology Co., Ltd.) homologous recombination operating instructions, connect the prokaryotic expression vector enzyme digestion product and the GbSNAT homologous recombination gel recovery product to each other, and prepare the following reaction system on ice:

Next, put the mixture into a PCR machine, react at 37°C for 30 minutes, and store at 4°C for subsequent experiments.

(4) E. coli transformation, positive clone screening and sequencing analysis are the same as in Example 2 above.

Through PCR detection, it was confirmed that the prokaryotic expression vector of GbSNAT was successfully constructed. Named pGEX-6p-GbSNAT. The length of the constructed recombinant plasmid is 5704 bp; plasmid pGEX-6p-GbSNAT only has a BamHI restriction site at position 945 and an EcoRI restriction site only at position 1674 (Figure 3).

Example 4

GbSNAT gene enzymatically produces melatonin in vitro

1. In vitro enzymatic experimental methods

The recombinant plasmid of pGEX-6p-GbSNAT constructed in Example 3 was transferred into the ViDe BL21 (DE3) large intestine competent cell (Shanghai ViDe Biotechnology Co., Ltd.). For specific operations, refer to the instruction manual of the ViDe BL21 (DE3) large intestine competent cell. After 1 hour of activation, spread the bacterial solution evenly on a solid LB culture dish containing ampicillin resistance, place it in a 37°C incubator for overnight growth, observe the colony growth, and pick single clones for verification. Add the successfully verified monoclonal bacteria to 5 ml of liquid culture medium containing ampicillin resistance for propagation at 37°C, and add 2 ml of the propagated bacterial solution to 200 ml of liquid LB. Shake the OD600 value of the expanded bacterial solution to about 0.8, and take 1 ml. The bacterial liquid is marked as 0h for later use. Add IPTG to the remaining bacterial liquid to a final concentration of 1mM and induce at 28°C for 6h. After induction, first take 1 ml of bacterial liquid and mark it for 6 hours for later use. Then centrifuge the remaining bacterial liquid at 4°C and 5000g for 5 minutes. Discard the supernatant to collect the cells, and then add 10 ml of cell lysis buffer [50 mmol/L Tris-Cl (pH8.0) ), 1mM EDTA (pH8.0), 100mmol/LNaCl] vortex to resuspend the pellet, add 100μl PMSF (1:1000), 10μl MgCl2 (1M) and 20μl Lysozyme (100mg/ml), place on ice, and sonicate Crush the lysate for 30 minutes, centrifuge the lysate at 4°C and 12,000 rpm for 20 minutes, collect the supernatant, vortex and resuspend the precipitate in 8M urea solution, and store the supernatant and precipitate in a -20°C refrigerator temporarily for subsequent experiments. Take 50 μl of 0h bacterial liquid, 6h bacterial liquid, supernatant, and precipitate resuspension, add 10 μl of 5×SDS-PAGE Loading Buffer, boil in boiling water for 10 minutes, and then run a protein gel to examine the distribution of the target protein. When the protein is precipitating, pour the precipitate resuspension into the dialysis bag, and place it in 8M, 6M, 4M, and 2M concentrations of urea, Buffer A, and PBS to complete the denaturation and renaturation of the protein for subsequent experiments. Use GST agarose purification resin to purify GST-tagged proteins by gravity flow-through method. The steps are as follows:

(1) Equilibrate the column with 10ml Binding/Wash Buffer;

(2) Centrifuge the renatured protein, collect the supernatant and transfer it to an equilibrium column to completely flow out;

(3) Wash the column 3-5 times with Binding/Wash Buffer to remove impurities;

(4) Prepare glutathione (0.184g glutathione + 6ml Elution Buffer);

(5) Add 500 μl glutathione eluent, elute three times, and put the eluates into new 1.5 ml centrifuge tubes;

(6) After denaturing the protein, perform SDS polyacrylamide gel electrophoresis;

(7) Use Coomassie blue to stain the protein gel for 40 minutes;

(8) Prepare a decolorizing solution (100ml glacial acetic acid + 50ml absolute ethanol + 850ml distilled water), decolorize the protein gel, and observe the length of the protein band.

Add 60ng of the recombinant GbSNAT purified protein to 100μl of a solution mixed with 0.5mM 5-methoxytryptamine (substrate), 0.5mM acetyl CoA (acetyl CoA) and 100mM potassium phosphate buffer, incubate at 38°C for 30 minutes, and add Stop the reaction with 25 μl of methanol (MeOH), and detect the melatonin content by high-performance liquid chromatography (HPLC). For specific operation steps, see the melatonin (MT) content kit instructions (HPLC method) of Suzhou Keming Biotechnology Co., Ltd.

2. GbSNAT can catalyze 5-methoxytryptamine to produce melatonin

In this example, through an in vitro enzymatic experiment, 5-methoxytryptamine is used as a substrate, purified GbSNAT protein with a GST tag is added to perform an enzymatic reaction, and incubated at 38°C for 30 minutes. The content of melatonin generated is empty. About 7 times that of the control (Figure 4), indicating that GbSNAT can catalyze 5-methoxytryptamine to produce melatonin, further demonstrating that the GbSNAT gene is involved in the biosynthesis of melatonin in Ginkgo.

Example 5

Construction of GbSNAT gene overexpression vector

(1) This experiment uses TaKaRa QuickCut restriction endonuclease (TaKaRa, Japan) to perform an enzyme digestion reaction on the overexpression pRI101-AN vector (TaKaRa, Japan). The specific reaction system is as follows:

QuickCut Buffer 5μL pRI 101-AN plasmid 8μL QuickCut BamHI 1μL ddH ₂ O 36μL Total volume 50μL

After mixing each solution in the system, perform instant centrifugation. The PCR reaction procedure is as follows:

37℃ 30min

85℃ 20min

4℃ to end

Conduct agarose gel electrophoresis on the digested product to observe the digested bands. If there are obvious band differences (Figure 5A), it means that the vector has been cut. Place the cut vector in a -20°C refrigerator for subsequent use. Vector ligation reaction.

(2) Design of homologous recombination primers

Use the CE Design (Nanjing Novezan Biotechnology Co., Ltd.) online website or software to manually design the homologous recombination amplification primers SEQ ID NO. 7 and 8 of GbSNAT according to the enzyme cutting site of the vector as shown below. Compared with ordinary primers, homologous recombination primers have an extra homology arm, which can improve the efficiency of vector connection.

According to the positive plasmid extracted from E. coli in the above Example 2 as a template, the homologous recombination GbSNAT gene was cloned, gel run and gel recovery was performed. The obtained gel recovery product was placed in a -20°C refrigerator for subsequent vector ligation reactions.

(3) Homologous recombination to connect the overexpression vector, E. coli transformation, positive clone screening and sequencing analysis are the same as in Example 2 and Example 3 above.

Through PCR detection, it was confirmed that the overexpression vector of GbSNAT was successfully constructed. Named 35S::GbSNAT, as shown in Figure 5B, the constructed overexpression vector is assembled with the constitutive strong expression promoter CaMV35S at the 5' end of GbSNAT, the terminator NOS-terminator is assembled at the 3' end, and the expression vector is loaded with NPTⅡ The gene expression cassette serves as a selection marker for transgenic Arabidopsis. The LB and RB sequences are assembled on the expression vector at the same time, which promotes the integration of the gene expression framework and the selection marker gene NPTII assembled into the chromosome of the Ginkgo receptor cell.

(5) Agrobacterium transformation

According to the instructions for transformation of Shanghai Vidy GV3101 (Agrobacterium), mix the constructed 35S::GbSNAT overexpression vector plasmid with competent cells, and then proceed through the steps of standing in ice for 5 minutes, quick freezing in liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and rapid After incubating on ice for 5 minutes, add 700 μL liquid anti-antibody LB culture medium and incubate with shaking for 2 hours. After centrifugation at 6000 rpm for 1 min, take 100 μL of the supernatant and mix gently by pipetting, spread on an LB plate containing kanamycin and rifampicin antibiotics, and incubate in a 28°C incubator for 2 to 3 days with an inversion. Pick the single clone on the plate, add an appropriate amount of LB liquid medium containing kanamycin and rifampicin antibiotics, culture it at 28°C, 220rpm for 48 hours, and detect positive clones by PCR (Figure 5C), and obtain 35S::GbSNAT. vector Agrobacterium tumefaciens.

Example 6

Genetic transformation and positive identification of GbSNAT gene

1. Genetic transformation of Arabidopsis thaliana

(1) Plant Colombian ecotype wild-type Arabidopsis thaliana (Col-0) in a normal growth environment (16h light/8h dark and 23℃ day/18℃ night);

(2) Select and cultivate Arabidopsis plants that have just bloomed for about four weeks, and use scissors to cut off the flowers and existing siliques for Agrobacterium transformation;

(3) Inoculate the Agrobacterium containing the 35S::GbSNAT vector obtained in Example 5 into the LB liquid culture medium containing Kana and Rif antibiotics, place it in a shaker at 28°C, and culture it overnight for 18-24h until OD600=0.8- 1.0;

(4) Put the bacterial solution that meets the requirements into a centrifuge tube, centrifuge at 25°C, 6000rpm for 10 minutes and remove the supernatant;

(5) Add 50 mL of Arabidopsis transformation solution (5% sucrose + 0.02% Silwet L-77) to the pellet in step (4), and resuspend the pellet;

(6) After centrifugation at 6000 rpm for 10 minutes, remove the supernatant, add transformation solution and resuspend the pellet;

(7) Soak the entire Arabidopsis thaliana inflorescence prepared in step (2) in the transformation solution for 30 seconds;

(8) After watering the infected Arabidopsis, cover it with a plastic bag to keep it moisturized. After culturing it in a dark environment for 24 hours, remove the plastic bag and move the Arabidopsis to a light incubator (16h light/8h dark). Normal culture growth;

(9) After 7 days, soak again according to the above method, and culture normally until the seeds mature.

2. Genetic screening of Arabidopsis thaliana

(1) After drying the harvested mature seeds of Arabidopsis thaliana, put an appropriate amount into a centrifuge tube, add 15% mass fraction of sodium hypochlorite solution and soak for sterilization for 3 minutes. During this period, repeatedly shake it up and down, and then put in 70% volume fraction of sodium hypochlorite solution. Continue soaking in alcohol for 3 minutes (repeat twice), then rinse with sterile water three times;

(2) Spread the seeds onto a 1/2MS solid medium plate containing kanamycin and seal with parafilm;

(3) Place the petri dish in a 4°C environment for vernalization for 2 days, and then transfer it to a 23°C artificial incubator for cultivation. The conditions are: 16h light/8h dark;

(4) After the seeds grow in the culture medium for 10 days, move the kanamycin-resistant seedlings into nutrient soil and continue to grow in a light incubator (16 hours of light/8 hours of darkness) to obtain T1 generation Arabidopsis plants. .

3. Ginkgo callus transformation

(1) The Agrobacterium containing the 35S::GbSNAT vector obtained in Example 5 was spread on an LB plate containing kanamycin and rifampicin antibiotics using the plate streaking method. After culture, pick the single Agrobacterium clone on the LB plate, inoculate it into LB liquid culture medium with Kana and Rif antibiotics, and culture it at 28°C for 24 hours until the OD ₆₀₀ is 0.5-0.6;

(2) Put the bacterial solution into a centrifuge tube, centrifuge at 25°C, 4000rpm for 10 minutes and remove the supernatant;

(3) Add resuspension solution (100 mL MS liquid medium containing 100 μM acetosyringone) to the centrifuge tube to resuspend the bottom bacterial cells and leave it at room temperature for 2 hours;

(4) Put small pieces of ginkgo leaf callus of the same size into the Agrobacterium resuspension, let it sit at room temperature and soak for 15-20 minutes, then gently pinch it out with tweezers, and use sterile filter paper to absorb the resuspension on the surface. liquid;

(5) Place the infected callus tissue on the callus medium (MS+4.0mg/L NAA+2.0mg/LKT+100μM acetosyringone), culture it in the dark at 25°C for 2-3 days, take out the medium Quick-frozen in nitrogen, stored in an ultra-low temperature refrigerator, and used for subsequent determination of melatonin content.

4. Detection and content determination of genetically modified materials

(1) The Ginkgo callus overexpressing the GbSNAT gene was subjected to fluorescence quantitative detection to detect the expression of exogenous genes at the RNA level, and Primer Premier 5.0 software was used to manually design fluorescent quantitative primers for GbSNAT. Among them, the forward primer is SEQ ID NO. 9: 5'-CCGCAGACAGAAGGACAAGATGG-3', and the reverse primer is SEQ ID NO. 10: 5'-GTGGCCTCTGGCAAATGCAACAAG-3'. The forward primer of the ginkgo internal reference gene (Actin) is SEQ ID NO. 11: 5'-ATCCACGGGAGTCTTCAC-3', and the reverse primer is SEQ ID NO. 12: 5'-GACCTTCAACAATGCCAAAC-3'. Thus, the calli whose GbSNAT expression was significantly higher than that of the control were screened out (Figure 6A), and the endogenous melatonin content was measured. For the specific measurement method, see step 5 (1) of Example 7 below.

Compared with the empty control, the endogenous melatonin content of Ginkgo calli overexpressing the GbSNAT gene increased significantly (Figure 6B). This result shows that GbSNAT is a key enzyme in the melatonin synthesis pathway of Ginkgo and promotes the synthesis of endogenous melatonin in Ginkgo.

(2) Positive identification of Arabidopsis overexpressing GbSNAT gene.

When the T1 generation Arabidopsis plants obtained in step 2 of this example were cultured and grown to 5-6 true leaves under 16 hours of light/8 hours of darkness and 23°C day/18°C night, the TPS method was used to extract the genomic DNA of the Arabidopsis seedlings. Verified by PCR. The method is as follows: First prepare the TPS solution. Measure 50ml Tris-HCl (1M, PH=8.0), 10ml EDTA (0.5M, PH=8.0) and 37.27gKCl, add sterile ddH ₂ O to adjust the volume to 500ml, stir well with a glass rod and set aside. Next, use liquid nitrogen to grind the Arabidopsis leaves into powder, put them into a 1.5ml centrifuge tube, add 300μl TPS, seal the centrifuge tube, and put it in a 60°C water bath for 20-30 minutes. Invert it several times every 10 minutes; end of the water bath Then, centrifuge at 13,000 rpm for 10 min, add an equal volume of isopropyl alcohol to the supernatant, mix well, centrifuge again at 13,000 rpm for 10 min, discard the supernatant, dry the precipitate, add 50 μl of sterile ddH ₂ O, mix well and measure the concentration; use the extracted DNA Perform PCR reaction to amplify the target gene and use agarose gel electrophoresis to detect it. Those with the target band are T1 generation positive plants (Figure 7B). In addition, qRT-PCR was used to detect the positive plants of the T1 generation (the front primer sequence used in qRT-PCR was CCGCAGACAGAAGGACAAGATGG; the back primer sequence was GTGGCCTCTGGCAAATGCAACAAG), and the positive plants were identified from the RNA level. The fluorescence quantitative results showed that the expression level of the GbSNAT gene was significantly improved (Figure 7C). Finally, the tested positive lines were screened again using the method in step 2 of this example. Plants that can grow normally (with dark green leaves) on the 1/2MS solid medium plate containing kanamycin are positive plants (Figure 7A ), and obtain T2 generation positive strain seeds, and conduct sowing and propagation for later functional verification.

(3) Arabidopsis mutant verification: Purchase the Arabidopsis snat (At1g26220) mutant from the Arashare official website, and use the three-primer method for plant identification of the purchased Arabidopsis mutant (T-DNA insertion disruption). First, At1g26220 mutant Arabidopsis seeds were sown and maintained normally, with 16 hours of light/8 hours of darkness and 23°C day/18°C night. After 4-6 true leaves have grown, DNA is extracted for later use. Enter the T-DNAPrimer Design "http://signal.salk.edu/tdnaprimers.2.html" website to design primers. The primer sequences SEQ ID NO. 13, 14 and 15 are shown in the table below. Verification was performed by PCR, where LP and RP are the primers on both sides of the T-DNA insertion site on the plant genome, and BP is the primer on the T-DNA segment. The results of agarose gel electrophoresis show that there are two bands amplified by LP+RP primers and one band amplified by BP+RP primers, which are hybrid plants. There are no bands amplified by LP+RP primers and there is one band amplified by BP+RP primers. One band is a homozygous strain. The pure individual At1g26220-2 (snat-mutant) was selected for subsequent functional verification experiments (Figure 6C). This Arabidopsis mutant was used to better evaluate the role of the GbSNAT gene in improving plant heat tolerance.

Example 7

Functional verification of transgenic Arabidopsis and mutant Arabidopsis

1. The high-temperature treatment methods for transgenic Arabidopsis and mutant Arabidopsis are as follows:

Sterilize the transgenic Arabidopsis thaliana and Columbia wild-type seeds and sow them on MS medium in a sterile environment. When the seeds germinate and grow two true leaves, select plants with good growth and transfer them to square MS petri dishes (without (bacteria operation), the petri dishes were placed vertically in a 22°C incubator and maintained at 16h/8h (day/night) for two weeks, and the root length was counted using ImageJ software. In the short-term high temperature treatment experiment, transgenic Arabidopsis thaliana and Colombian wild-type seeds were sterilized in a clean bench, evenly spread on MS solid medium, and placed in a 22°C light incubator for 16h/8h (day/day). (night) for 7 days, placed in a 40°C light incubator for high temperature treatment for 180 minutes, and then returned to a 22°C light incubator to recover for seven days, and the survival rate was calculated. In the long-term high temperature treatment, Arabidopsis seeds are sown on the planting medium. When two true leaves sprout and grow, they are transplanted into small black square pots (7cm × 7cm), with 3 plants in each pot. After 20 days of maintenance, long-term treatment at 38°C is carried out. After high-temperature treatment, photosynthetic indicators were measured on 1d and 3d respectively; photos were taken and samples were taken on 0d, 1d, and 3d and stored in a -80°C refrigerator for later determination of antioxidant enzyme indicators. In addition, samples were taken at 0h, 6h, and 12h. Place in a -80°C refrigerator for later measurement of melatonin.

2. Through the Arabidopsis inflorescence dip dyeing method and the identification method in step 4 (2) of Example 6, three Arabidopsis thaliana GbSNAT gene overexpression positive plants were obtained, namely OE-SNAT-2, OE-SNAT-7 and OE-SNAT-8 (Figure 7A-C). Under normal growth conditions, it was found through phenotypic observation that the leaf growth and development of the overexpression strain was not significantly different from that of the wild type, and the root length statistical results showed that the snat-mutant obtained according to step 4 (3) of Example 6 The root system of the strain (At1g26220-2) was significantly shorter than Col-0, while the root system of the OE-SNAT-8 strain was significantly longer than Col-0, indicating that GbSNAT may promote the growth of the Arabidopsis root system (Figure 7D and E).

3. Short-term high temperature treatment of transgenic Arabidopsis and mutant Arabidopsis

The transgenic and mutant Arabidopsis thaliana seedlings obtained in Example 6 were treated at a high temperature of 40°C for 3 hours, and then placed under normal maintenance at 22°C for 7 days. Col-0, snat-mutant, OE-SNAT-2, OE- The survival rate of the seedlings of the five lines, SNAT-7 and OE-SNAT-8, was calculated. Among them, the survival rate of snat-mutant (At1g26220-2) was 55.6%, which was not significantly different from the survival rate of Col-0 54.3%. The survival rates of the three lines OE-SNAT-2, OE-SNAT-7 and OE-SNAT-8 overexpressing GbSNAT increased significantly, reaching 75.3%, 74% and 82.6% respectively (Figure 8A-C). This result shows that overexpression of GbSNAT in Arabidopsis can significantly improve the high temperature tolerance of seedlings, alleviate the damage caused by short-term high temperature to seedlings, and improve the plant survival rate.

4. Long-term high temperature treatment of transgenic Arabidopsis and mutant Arabidopsis

As shown in Figure 9A, after 3 days of high temperature treatment at 38°C, all strains showed varying degrees of leaf wilting. Among them, Col-0 had the most severe wilting, while the wilting of the GbSNAT overexpressing strain showed a certain degree of wilting. of relief. GbSNAT may be involved in regulating the high temperature tolerance of Arabidopsis thaliana and partially alleviate the damage caused by long-term high temperature to Arabidopsis.

Under high temperature treatment, the chlorophyll content of plants also changed to varying degrees. When plants suffer from high temperature stress, the chlorophyll a and chlorophyll b contents show an overall downward trend. Compared with Col-0, the chlorophyll a and carotenoid contents of the mutant snat strain were significantly reduced, while the chlorophyll b content was not significantly different. The chlorophyll a and chlorophyll b contents of the overexpression line were higher than those of the Col-0 wild type (Fig. 9B and C). At the same time, the carotenoid content of Col-0 wild type increased significantly under high temperature stress. In contrast, except for the significant increase in carotenoid content of OE-SNAT-2 in the overexpression line, the other two lines had no significant changes at high temperature (Figure 9D). High temperature seriously affects the total chlorophyll content of plants, causing the degradation of chlorophyll a and chlorophyll b. GbSNAT can alleviate the degradation of chlorophyll a and chlorophyll b induced by high temperature to a certain extent, maintain the photosynthetic capacity of plants, and alleviate the impact of high temperature on photosynthesis. harm.

Under high temperature stress, the MDA content in plants continues to accumulate. After 3 days of high temperature treatment, the MDA content accumulated rapidly. At this time, the accumulation of MDA content in the GbSNAT overexpression line was significantly lower than that in the Col-0 and sant-mutant lines, indicating that overexpression of GbSNAT can slow down high temperature-induced plants to a certain extent. accumulation of MDA (Fig. 10A). In addition, the activities of antioxidant enzymes SOD, CAT and POD showed an overall trend of first increasing and then decreasing under high temperature stress. The CAT and POD activities of the GbSNAT overexpression strain were slightly higher than those of the Col-0 strain after 3 days of high temperature treatment, while the sant-mutant CAT and POD activities were slightly lower than those of the Col-0 strain, but the differences were not significant (Figure 10B and C). Similarly, the SOD activity of the GbSNAT overexpression strain was slightly higher than that of Col-0 and sant-mutant strains after 3 days of high temperature treatment. Among them, the SOD activity of the OE-SNAT-7 strain was significantly higher than that of Col-0 after high temperature treatment for 1 day. After 3 days of high temperature treatment, the SOD activity of the OE-SNAT-7 and OE-SNAT-8 strains was significantly higher than that of Col-0, while the SOD activity of the sant-mutant strain was significantly lower than that of Col-0 (Figure 10D). The above results show that overexpression of GbSNAT can not only slow down the accumulation of MDA and alleviate the degree of plant cell membrane peroxidation, but also alleviate the oxidative damage caused by high temperature to plants by promoting the activity of SOD in the plant antioxidant enzyme system.

5. Determination of endogenous melatonin content in transgenic Arabidopsis and mutant Arabidopsis under high temperature

(1)Method for determination of endogenous melatonin content:

Cut the fresh ginkgo leaves into pieces, put them into a 10ml test tube, add 6ml of chloroform, and then place the test tube in a 4°C shaker for about 12 hours to extract melatonin. Put the sample into a centrifuge and centrifuge it at 4000g for 5 minutes. Collect the supernatant, transfer it to a C18 solid phase extraction (SPE) column for purification, and blow dry with nitrogen to leave the precipitate. The samples were sent to Suzhou Keming Biotechnology Co., Ltd. for analysis and detection using high-performance liquid chromatography (HPLC). For specific operation steps, please refer to the melatonin (MT) content kit instructions (HPLC method) of Suzhou Keming Biotechnology Co., Ltd. .

(2) Melatonin changes under high temperature

The detection of endogenous melatonin showed that under normal conditions, the average contents of endogenous melatonin in the two SNAT overexpression lines OE-SNAT-7 and OE-SNAT-8 were 5.61ng/g and 5.6ng respectively. /g, which is higher than the 4.95ng/g and 4.8ng/g of Col-0 and snat-mutant, and shows a significant difference (Figure 11, * in the figure indicates P value ≤ 0.05), indicating that GbSNAT enhances the endogenous function of the plant. Melatonin synthesis. After 6 hours of high temperature treatment, the endogenous melatonin content of the plants increased, indicating that high temperature induced an increase in melatonin. At the same time, the endogenous melatonin content of overexpressing GbSNAT plants was significantly higher than that of Col-0, while the content of snat-mutant was significantly higher. Lower than Col-0 (Figure 11), it shows that GbSNAT can increase the endogenous melatonin content under high temperature and increase the high temperature tolerance of plants.

The above results indicate that the GbSNAT gene is a key gene for melatonin synthesis in Ginkgo and can enhance the heat tolerance of plants.

Claims (10)

1. A key gene GbSNAT for promoting synthesis of ginkgo melatonin and enhancing heat resistance of plants is characterized in that the nucleotide sequence of the gene GbSNAT is shown as SEQ ID NO. 1.

2. The key gene GbSNAT for promoting the synthesis of ginkgo melatonin and enhancing the heat resistance of plants according to claim 1, wherein the primer pair for amplifying the gene GbSNAT is SEQ ID No.2: ATGGCAGTGGGGATGGCG and SEQ ID No.3: TTTTTTTGTCGATCGAGGTCTTT.

3. A protein for promoting the synthesis of ginkgo melatonin and enhancing the expression of a plant heat resistance key gene GbSNAT according to claim 1, wherein the amino acid sequence is shown in SEQ ID No. 4.

4. A prokaryotic expression vector containing the key gene GbSNAT for promoting the synthesis of ginkgo melatonin and enhancing the heat resistance of plants, which is disclosed in claim 1, is characterized in that the prokaryotic expression vector takes a vector pGEX-6P-1 as a starting plasmid, the pGEX-6P-1 vector plasmid is subjected to double-enzyme tangential digestion, and the key gene GbSNAT is inserted into the pGEX-6P-1 to construct the GbSNAT prokaryotic expression vector pGEX-6P-GbSNAT.

5. An over-expression vector containing the key gene GbSNAT for promoting the synthesis of ginkgo melatonin and enhancing the heat resistance of plants according to claim 1, wherein the over-expression vector is assembled with a constitutive strong promoter CaMV35S at the 5 'end of the gene GbSNAT and an NOS-terminator at the 3' end of the gene GbSNAT.

6. The over-expression vector according to claim 5, wherein the expression vector is assembled with an NPT ii gene expression cassette as a selection marker for transgenic arabidopsis thaliana, and kanamycin is used for selection of transgenic ginkgo arabidopsis thaliana; the over-expression vector is assembled with LB (T-Border left) and RB (T-Border right) sequences, so that the gene GbSNAT expression frame and the screening marker gene NPTII assembled between the sequences can be integrated into ginkgo chromosomes.

7. A host bacterium comprising the key gene GbSNAT for promoting the synthesis of ginkgo melatonin and enhancing the heat resistance of plants according to claim 1, or the prokaryotic expression vector according to claim 4, or the overexpression vector according to claim 5.

8. Use of the key gene GbSNAT of claim 1 or the protein of claim 3 or the prokaryotic expression vector of claim 4 or the overexpression vector of claim 5 in the synthesis of melatonin and for improving plant heat tolerance.

9. Use according to claim 8, characterized in that the prokaryotic expression vector is preferably used to increase the GbSNAT protein content, in vitro enzymatic catalysis of the synthesis of melatonin from the substrate 5-methoxy tryptamine.

10. Use of the key gene GbSNAT of claim 1 or the protein of claim 3 or the prokaryotic expression vector of claim 4 or the overexpression vector of claim 5 for the cultivation of plants with high melatonin content and heat resistance.