CN117965464A - Hydroxy cinnamoyl spermidine biosynthesis and transport gene cluster and application thereof - Google Patents

Abstract

The invention discloses a hydroxy cinnamoyl spermidine biosynthesis and transport gene cluster and application thereof, and belongs to the field of bioengineering, wherein the gene cluster codes four proteins, namely, sl4Cl1.2, slCV86, slDH29 and SlDTX29, wherein the amino acid sequence of Sl4Cl1.2 is shown as SEQ ID No.1, the amino acid sequence of SlCV86 is shown as SEQ ID No.2, the amino acid sequence of SlDH29 is shown as SEQ ID No.3, and the amino acid sequence of SlDTX29 is shown as SEQ ID No. 4. The over-expression gene cluster can enhance the drought tolerance of tomatoes, lays a foundation for genetic improvement on tomato resistance by utilizing the gene cluster, and provides a technical reference for excavation of related metabolic gene clusters in other crops and on resistance breeding.

Description

Hydroxy cinnamoyl spermidine biosynthesis and transport gene cluster and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a hydroxy cinnamoyl spermidine biosynthesis and transport gene cluster.

Background

Tomato (Solanum lycopersicum l.) is an annual herb of the solanaceae family, native to south america, and widely cultivated in north and south china. The fruit is nutritious and has special flavor, and has various eating methods, such as raw food, cooking, tomato sauce processing, or whole fruit canning. Besides eating, the tomato has medicinal value, has the functions of promoting the production of body fluid to quench thirst, invigorating stomach to promote digestion, clearing heat and relieving summer heat, tonifying kidney and promoting urination, and can treat diseases such as fluid impairment and thirst, inappetence, summer heat and internal abundance. However, tomatoes do not have movable ability like animals, so that the tomatoes are seriously affected when abiotic stress (such as drought, waterlogging, high temperature, low temperature, saline alkali and the like) comes, and the growth and the yield of the tomatoes are restricted. Thus, tomatoes, in order to counteract or adapt to these adverse factors, develop a complex series of stress response regulatory mechanisms during long-term evolution, where plant secondary metabolites play a vital role in the adaptation of plants to abiotic stress.

The phenolic amine is also called hydroxycinnamayl phenolic amine, and is a special metabolite generated by one branch of the phenylpropionamide biosynthesis pathway in plants; they may exert protective effects in the face of abiotic and biotic stresses (Chen et al, 2018; saha et al, 2015). For example, feruloyl putrescine (Fer-Put) is the phenolic amine described earliest, which has been found in the middle of the 20 th century, and has recently been found to accumulate significantly in rice plants prey on by laodelphax striatellus (Tanabe et al, 2016). Recently, high levels of caffeoyl putrescine (Caf-Put), coumoyl agmatine (Cou-agm) and coumoyl putrescine (Cou-Put) have proven to be particularly effective in inhibiting late blight growth in potatoes (Dobritzsch et al, 2016). Also, under drought stress, caf-Put and Cou-Put accumulate in tobacco and false purple in Rohdea (Li et al, 2022; torras-Claveria et al, 2012). Furthermore, the metabolism of phenolamines often interweaves with metabolic pathways involved in defense signaling, such as abscisic acid ABA. Thus, phenolamines offer a promising approach to improving environmental tolerance in crops. However, to date, few studies have addressed the effects of phenolamine on tomato abiotic stress resistance, particularly on drought tolerance.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: a metabolic gene cluster is provided for controlling spermidine biosynthesis and transport in tomato.

The technical scheme of the invention is as follows: an isolated hydroxycinnamoyl spermidine biosynthesis and transport gene cluster encoding four proteins, the four proteins being sil 4cl1.2, slCV86, slDH, and SlDTX29, wherein the amino acid sequence of sil 4cl1.2 is shown as SEQ ID No.1, the amino acid sequence of SlCV86 is shown as SEQ ID No.2, the amino acid sequence of SlDH29 is shown as SEQ ID No.3, and the amino acid sequence of SlDTX29 is shown as SEQ ID No. 4.

Further, the nucleotide sequence of the coding gene of the protein SL4CL1.2 is shown as SEQ ID No.5, the nucleotide sequence of the coding gene of the protein SlCV is shown as SEQ ID No.6, the nucleotide sequence of the coding gene of the protein SlDH29 is shown as SEQ ID No.7, and the nucleotide sequence of the coding gene of the protein SlDTX29 is shown as SEQ ID No. 8.

An expression vector comprising the gene cluster described above.

The application of the expression vector in improving drought resistance of tomatoes.

The expression vector is applied to the synthesis of hydroxy cinnamoyl spermidine.

Compared with the prior art, the invention has the following beneficial effects:

The invention clones a metabolic gene cluster for controlling biosynthesis and transportation of hydroxycinnamoyl spermidine in tomatoes for the first time, verifies the biosynthesis and transportation components of the hydroxycinnamoyl spermidine encoded by the gene cluster by means of in vitro enzyme activity measurement, transient expression of tobacco, subcellular localization, stable isotope labeling, transgenic tomato overexpression and the like, greatly improves the phenolic amine content in tomatoes by utilizing the transient expression gene cluster of the tobacco, enhances the drought resistance of tomatoes by utilizing the overexpression gene cluster, lays a foundation for genetic improvement in tomato resistance by utilizing the gene cluster, and provides technical references for excavation of related metabolic gene clusters in other crops and in resistance breeding.

Drawings

Fig. 1: identification of hydroxycinnamamide clusters. A shows a manhattan plot of GWAS results for diCaf-Spd (n=401). The horizontal dashed line represents the genome-wide significance threshold. The genomic structure of candidate metabolic gene clusters is shown at the bottom of the panel, sl4cl1.1 (dark red), slCV, slDH (light blue) and SlDTX (earthy yellow). Metabolic pathways of biosynthesis and transport of the B tomato putrescine derived HCAAs gene cluster.

Fig. 2: gene expression patterns of hydroxycinnamamide gene cluster components after different tissues, drought and ABA treatment.

Fig. 3: in vitro enzyme activity identifies the synthetic components of the gene cluster. FIG. A shows a schematic structural diagram of the prokaryotic protein expression vector pGEX-6P-1. Panel B shows the chromatographic peak which in turn shows the results of the enzyme activity of the recombinant proteins SlCV and SlDH29 reacted with the substrate Spd. Panel C shows a chromatographic peak showing the result of the enzyme activity of recombinant protein Sl4CL1.2 reacted with donor CoA and substrate Caf. Caf-Spd, caffeoylspermidine; diCaf-Spd, secondary caffeoylspermidine; caf-CoA, caffeoyl-CoA; spd, spermidine; standard stands for Standard for substrates CoA, cou-CoA and Put.

Fig. 4: the transient expression of tobacco verifies the synthetic components of the gene cluster. Panel A shows a schematic representation of the structure of the tobacco transient expression vector pEAQ-HT. Panel B shows the results of tobacco transient expression SlDH for Caf-Spd synthesis. Panel C shows the results of tobacco transient expression SlCV, 86, synthesis of diCaf-Spd.

Fig. 5: tomato subcellular localization vector and overexpression vector profile. Panel A shows a schematic representation of the structure of tomato subcellular localization vector pH7WGF 2-N-eGFP. Panel B shows a schematic structural diagram of tomato overexpression vector pBI 121.

Fig. 6: tomato over-expression plant construction of transporter SlDTX and transport analysis. Panel A shows subcellular localization images of SlDTX obtained using a laser scanning confocal microscope. All experiments were repeated twice with similar results. Ruler, 25 μm. EV, empty vector; ERM, endoplasmic reticulum membrane. Panel B shows the identification of the expression level of the T2 generation SlDTX over-expressed strain. Panel C shows that the T2 generation SlDTX29 overexpressed line absorbs the stable isotope 8D-Spd marker. Panel D shows the Spd post-treatment sensitivity phenotype of SlDTX gene-overexpressing lines. Panel E shows SlDTX gene over-expression strain metabolite analysis. The control variety was tomato Micro Tom, labeled WT. Standard deviation was based on three biological replicates, each sample being a mix of 3 strains of material. Significance analysis was expressed as P <0.05, P <0.01 and P <0.001, respectively (t-test).

Fig. 7: tomato over-expression plant construction of acylases SlCV and SlDH29 was analyzed for metabolic diversity with phenolamine. Panel A shows the identification of the expression level of the T2 generation SlDH over-expressed strain. B shows the identification of the expression quantity of the T2 generation SlCV86 over-expression strain. Panels C and D show SlDH and SlCV86 overexpression lines phenolic amine metabolite analysis. The control variety was tomato Micro Tom, labeled WT. Standard deviation was based on three biological replicates, each sample being a mix of 3 strains of material. Significance analysis was expressed as P <0.05, P <0.01 and P <0.001, respectively (t-test).

Fig. 8: tobacco transient coexpression gene cluster components (SlCV, slDH, 29, sl4cl1.2 and SlDTX). Panel A shows the results of the combined co-expression of four genes to synthesize Caf-Spd. Panel B shows the results of the combined co-expression of four genes to synthesize Fer-Spd. Panel C shows the results of combining the co-expression of four genes to synthesize diCaf-Spd.

Fig. 9: phenotype statistics data, metabolite analysis and expression quantity analysis of T2 generation SlCV86,86 gene over-expression strain after drought. Panel A shows the phenotype of the SlCV86 gene-overexpressed strain after 16 days of drought treatment and the phenotype of the rehydrated strain after 16 days of drought treatment, and panels B and C show the survival statistics and the relative water content statistics of the SlCV86 gene-overexpressed strain after drought, respectively, with standard deviations based on three biological replicates. D is the detection of the content of phenolamine metabolites of SlCV and 86 gene over-expression lines before and after drought. E graph shows ABA content detection of SlCV86,86 gene over-expressed strain before and after drought. F and G graphs show the detection of the expression level of ABA related genes (NCED 3 and ABA 3) after drought of SlCV gene over-expression strain.

The control variety was tomato Micro Tom, labeled WT. Significance analysis was expressed as P <0.05, P <0.01 and P <0.001, respectively (t-test).

Detailed Description

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.

Example 1: discovery and definition of hydroxycinnamamide cluster

To analyze the natural variation of tomato phenolamine and its genetic basis, the inventors performed genome-wide association analysis on phenolamine metabolites detected in 401 tomato natural varieties (mGWAS). The results show that the variation in content of secondary acylated caffeoylspermidine (diCaf-Spd) in tomato populations is closely related to SNP sf0703267875 on chromosome 7 of tomato. In combination with this region of gene annotation information in the tomato genome (tomato genome database SGN), four candidate genes involved in spermidine and phenolamine synthesis and transport, a 4-hydroxycinnamoyl coa ligase (sl 4cl1.2 encoding), 2 BAHD acyltransferases (SlCV, slDH encoding) and a MATE transporter (SlDTX encoding) were determined. Taken together, the proteins encoded by these candidate genes belong to three distinct enzyme families, a feature that corresponds to the definition of a plant gene cluster, and therefore the inventors named this metabolic gene cluster as the "hydroxycinnamospermidine biosynthesis and transport gene cluster" according to its final product, hydroxycinnamospermidine (fig. 1).

Example 2: cloning of the Hydroxycinnamoyl spermidine Gene Cluster component (Sl4CL1.2, slCV, 86, slDH, 29 and SlDTX 29) genes

Applicant extracted total RNA from the sequenced tomato variety Micro Tom leaves using TRIZOL reagent (available from Invitrogen company) (extraction method according to the TRIZOL reagent instructions described above), reverse transcribed RNA into cDNA using reverse transcription kit Supermix (available from beijing holo gold company), reaction conditions: 42 ℃ for 30min and 80 ℃ for 5sec. To obtain the coding sequences of the genes sl4cl1.2, slCV, slDH29 and SlDTX, the applicant used the cDNA as a template to predict the full length open reading frame sequences of these candidate genes based on their position and structure in the tomato genome (tomato genome database SGN), designed PCR specific primers with homologous recombination junctions (table 1, synthesized by the prime biosystems) to amplify the full length CDS fragments of the 4 genes using the ultra-fidelity KOD enzyme (date Lian Bao, physical engineering ltd). PCR reaction conditions: pre-denaturation at 94℃for 5min;98℃10sec,60℃30sec,68℃2min,32 cycles; extending at 68℃for 5min. And connecting the amplified PCR product into pDonr207,207 entry vector through BP reaction of Gateway cloning technology, and transforming E.coli DH5 alpha competent cells. Colony PCR was verified to obtain positive clones, and sequencing gave correct clones (Haikovia engine Co.) containing the candidate gene without mutation, and entry vectors for pDonr207 sl4CL1.2, slCV86, slDH29 and SlDTX genes, respectively, were obtained.

Example 3: analysis of expression patterns of the components of the hydroxycinnamoyl spermidine gene cluster

RNA extraction was performed on different tissues (roots, stems, leaves, flowers and fruits) of the tomato variety Micro Tom using the method of example 2, and in order to verify whether drought or ABA would induce expression of the hydroxycinnamoyl spermidine gene cluster components, the researchers of the present invention performed the following experimental study: (1) Drought treatment (blank control, labeled Mock) was performed on Micro tom tomato seedling plants grown for about 4 weeks, and total RNA was extracted from tomato leaf tissue 7d after treatment. (2) Microdom tomato seedling plants were exogenously treated with abscisic acid at a concentration of 100 μm for about 4 weeks. After 24 hours of treatment, the tomato leaf tissue was sampled to extract total RNA.

Reverse transcription was performed on the total RNA extracted as described above. The reverse transcription specifically comprises the following steps: 3-5 g of total RNA was treated with DNaseI (Invitrogen, U.S.A.) for 15min to remove genomic DNA contamination, and reverse transcribed using oligo (dT) 18 oligo primer and M-MLV reverse transcriptase (Promega, U.S.A.). Real-time quantitative PCR reactions were performed on an ABI7500REAL TIME PCR SYSTEM instrument (America Applied Biosystems) using a real-time quantitative PCR assay kit GR EEN PCR MASTER Mix (Takara Bio Inc.) according to the instructions for use of the kit. Gene expression was quantified using a relative quantification method (LIVAK AND SCHMITTGEN, 2001). The RNA content of the sample was measured and homogenized by using the expression level of tomato housekeeping gene Ub iquitin gene.

QRT-PCR analysis shows that the 4 component genes of the hydroxycinnamoyl spermidine gene cluster are highly expressed in roots, leaves and flowers (A in figure 2) and are induced to express drought and abscisic acid hormone to different degrees (B in figure 2 and C in figure 2), which indicates that the 4 component genes can be involved in the defense process of tomatoes against drought.

TABLE 1 primers used in the present invention

Example 4: construction of hydroxycinnamate cluster synthesis component (SlCV, slDH, and Sl4CL1.2) protein expression vector and prokaryotic protein expression

The protein expression vector construction method comprises the following steps: the plasmids of positive clones pDonr207_ SlCV, slDH29 and sl4cl1.2 in example 2 were first ligated into the protein expression vector pGEX-6P-1 vector (fig. 3a, invitrogen, usa) by LR reaction of Gateway cloning technology. Then respectively transforming into competent cells BL21 (Shanghai Weidi organism) of the escherichia coli, and respectively expressing proteins encoded by candidate genes of the escherichia coli by induction.

The method comprises the following specific steps:

(1) E.coli culture: plasmid containing target gene fragment was transformed into competent cell of E.coli BL21, single colony was picked and inoculated into 5ml LB medium containing 50. Mu.g/ml ampicillin antibiotic, and cultured overnight at 37 ℃. The next day was inoculated in a ratio of 1:50 into 250ml of LB medium containing 50. Mu.g/ml ampicillin antibiotics, and the culture was expanded at 37℃and 220rpm for 3 hours until the OD600 of the bacterial liquid became about 0.5, and then 1mol/L IPTG (isopropyl thiogalactoside, shanghai Biotechnology Co., ltd.) was added to the bacterial liquid to induce protein expression. After addition of IPTG, the bacterial solutions were subjected to shaking culture at 16℃and 160rp m overnight.

(2) E.coli collection: the cells were collected by a 500ml centrifuge tube and centrifuged at 5000rpm at 4℃for 8min. The supernatant was removed, 50ml of Lysis buffer was added to suspend the cells, and the cells were placed on ice. Cell disruption was performed on the suspended cells using a high pressure cell disruptor (nanotechnology, inc. Of ampere, su).

(3) SDS-PAGE: the broken cells were detected to be always kept on an ice-water mixture, 20. Mu.l of total protein was added to 5X SDS Loading buffer. Mu.l, 1ml of total protein was further centrifuged at 12000rpm at 4℃for 10min, the supernatant and the precipitate were separated, 20. Mu.l of supernatant was added to 5X SDS Loading buffer. Mu.l, and the mixture was heated on a dry bath at 100℃for 10min. After the heated sample was cooled to room temperature, 15. Mu.l of the sample was spotted and SDS-PAGE was performed to see whether the target protein was present in the total protein and the supernatant. The remaining protein was stored at-80℃until use.

(4) Protein purification: the purified column containing glutathione sepharose 4B sepharose (company GE HEALTHCARE of America) was washed thoroughly with Lysis buffer, the supernatant containing the target protein was added, and the effluent was collected and passed through the column again. The column was washed thoroughly with Lysis buffer (5 column volumes). After washing sufficiently, adding 15mmol of reduced glutathione eluent (Shanghai H Co., ltd.) into the purification column, 1ml each time, and collecting 1ml of the flow-through solution and repeating for 5-6 times. The presence or absence of the target protein in the collected flow-through was confirmed by SDS-PAGE. The inventor sequentially obtains recombinant proteins of SlCV, slDH29 and Sl4Cl1.2 fusion GST tag protein with good purification, and the proteins are packaged and stored in a refrigerator at the temperature of minus 80 ℃ for standby.

Example 5: in vitro enzyme activity assay of the hydroxy cinnamoyl spermidine Gene cluster Components (SlCV, slDH29 and Sl4CL1.2)

In vitro enzyme Activity assay for SlCV86 and SlDH proteins

To verify the function of candidate genes SlCV and SlDH, the inventors used the purified SlCV86 and SlDH29 recombinant proteins described above to perform in vitro enzyme activity assays. The in vitro enzyme activity reaction system of the acylase was 100. Mu.l containing 1mM of amine substrate 1. Mu.l, 200. Mu.M hydroxycinnamate CoA 1. Mu.l, 500ng of purified protein, 1. Mu.l of 100mM Tris-HCl buffer (pH 7.4) and 2.5mM MgCl ₂ 1μl,ddH₂ O were made up to 10. Mu.l, and after incubation at 37℃for 30min, 50. Mu.l of precooled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 12000rpm at 4℃for 10min, and 50. Mu.l of the supernatant was used for LC-MS detection analysis. LC-MS detection showed that primary hydroxycinnamoyl spermidine was detected in the reaction product of DH29 protein and secondary acylated hydroxycinnamoyl spermidine was detected in the detection of SlCV in vitro enzyme activity, indicating that both enzymes have the function of catalyzing synthesis of hydroxycinnamoyl spermidine with hydroxycinnamate coa (fig. 3B).

In vitro enzyme activity assay for the sl4cl1.2 protein

To verify the function of the candidate gene sl4cl1.2, the inventors performed an in vitro enzyme activity assay using the purified sl4cl1.2 protein described above. The in vitro enzyme reaction system for the SL4CL1.2 protein was 100. Mu.l, 100mM Tris-HCl buffer (pH 7.4) 1. Mu.l, 2.5mM MgCl ₂. Mu.l, 2.5mM ATP 1. Mu.l, 0.2mM CoA 1. Mu.l, 1mM caffeic acid1. Mu.l and 500ng purified protein, ddH ₂ O was supplemented to 10. Mu.l, and after incubation at 37℃for 30min, 50. Mu.l of pre-chilled methanol was added to terminate the reaction. The reaction mixture was centrifuged at 12000rpm at4℃for 10min, and 50. Mu.l of the supernatant was used for LC-MS detection analysis. LC-MS detection showed that caffeoyl-coa was detected in the reaction product of the sl4cl1.2 protein, indicating that the enzyme encoded by the sl4cl1.2 gene is responsible for the first step catalytic reaction of the hydroxycinnamate spermidine gene cluster (C in fig. 3).

Example 6: tobacco transient expression verification SlCV, function of SlDH29

To further verify the function of the gene cluster components SlCV, 86 and SlDH genes. Applicant linked pDonr207_ SlCV86 and SlDH29 obtained in example 2 to pEAQ-ht_ SlCV86 and SlDH29 by LR reaction of Gateway cloning technology, and used for transient transformation of tobacco after plasmid sequencing was correct (fig. 4 a). pEAQ-HT containing either candidate tomato gene or GFP (negative control) was transformed into Agrobacterium tumefaciens (GV 3101), respectively, and incubated for 3d at 30℃on Luria-Bertani (LB) plates containing 50mg/mL kanamycin and 30mg/mL rifampicin. Positive clones were selected, cultured in LB medium containing 50. Mu.g/mL kanamycin to an OD600 of 2.0, washed with wash buffer (10 mM MES, pH 5.6), resuspended in MMA buffer (10 mM MES (pH 5.6), 10mM magnesium chloride, 100mM acetosyringone) and OD600 of 1.0. Cultures were incubated at room temperature for 2h and 1ml of the culture was infiltrated into the bottom surface of 6 week old Nicotiana benthamiana leaves using a 1ml needleless syringe. Leaves of different plants (n=3) were harvested 3d after infiltration, frozen in liquid nitrogen and stored at-80 ℃, after which metabolite analysis found SlDH that Caf-Spd was synthesized by SlDH and diCaf-Spd by SlCV (B and C in fig. 4).

Example 7: subcellular localization analysis of transporter SlDTX29

The plasmid of positive clone pDonr207_ SlDTX in example 1 was first ligated into the subcellular localization vector pH7WGF2-GFP vector (a in fig. 5, invitrogen, usa) by LR reaction of Gateway cloning technology and fused with GFP under the control of Ca MV 35S promoter. Endoplasmic reticulum localization marker 35S: HDELn-OFP for Co-location analysis Agrobacterium strain GV3101 containing the corresponding construct was infiltrated into four-week-old Nicotiana benthamiana leaves. Confocal microscopy images (a in fig. 6) were acquired using a laser scanning confocal microscope (LEICA TCS SP).

Example 8: verification of the Transporter SlDTX Gene

To further verify the function of SlDTX genes. Applicant connects pDonr _ SlDTX obtained in example 2 to pbi121_ SlDTX29 (B in fig. 5) by LR reaction of Gateway cloning technology, and plasmid sequencing was used for tomato genetic transformation after correct. Firstly, introducing the tomato genetic transformation system mediated by agrobacterium LBA4404 (Shanghai Weidi biological company) into a tomato variety MicroTom, and obtaining transgenic plants after preculture, infection, co-culture, selection of callus with kanamycin resistance, differentiation, rooting and transplanting, and identification. Agrobacterium-mediated tomato genetic transformants are based mainly on the methods reported by Zhang Yang et al (Li et al 2020).

And detecting the expression quantity of the genes in the plant body by adopting real-time quantitative PCR analysis. And breeding the obtained over-expression plants to obtain T2 generation transgenic plants. The results showed that the amount of SlDTX gene expression was significantly increased in positive transformed plants compared to control wild type (non-transgenic tomato) Micro Tom (fig. 6B).

The uptake function was further verified by stable isotope Spd-8D labelling experiments, i.e.wild-type (WT) grown on MS agar plates for 14 days and transgenic seedlings transferred to MS liquid medium. After overnight incubation, plants were immersed/immersed in 4.5mL of treatment buffer (MS+0.5 mM CaCl ₂ +2.85mM MES) and then 0.5mL of MS medium containing 100. Mu.M 8D Spd (Sigma, USA) was added to initiate the reaction. After 3 hours of incubation, seedlings were washed three times with 40mL of wash buffer (MS+0.5 mM CaCl ₂ containing 10 times the concentration of substrate) and then subjected to metabolic extraction. The results showed that SlDTX gene over-expression strain was able to take up more Spd than WT (C in fig. 6).

To test the sensitivity of SlDTX-OE lines to Spd, 7 day old plants were transferred to MS medium with or without 1mM Spd and incubated for 7d. As a result, it was found that the growth of plant roots was significantly inhibited in the Spd-added medium (D in FIG. 6). LC-MS detection and analysis was further performed on hydroxycinnamoyl spermidine in tomato leaves. The assay showed that the accumulation of spermidine derived phenolamine was higher in the SlDTX over-expressed plants compared to the wild type material (E in fig. 6).

Example 9: hydroxycinnamate cluster components SlCV, 3836 and SlDH contribute to the metabolic diversity of spermidine-derived phenolamines

To further verify the function of SlCV, 86 and SlDH29 genes. The inventors connected pDonr207_ SlCV86 and SlDH obtained in example 2 to pbi121_ SlCV86 and SlDH29 (B in fig. 5) by LR reaction of Gateway cloning technique, and performed tomato genetic transformation by the method in example 8 after plasmid sequencing was correct.

And detecting the expression quantity of the genes in the plant body by adopting real-time quantitative PCR analysis. And breeding the obtained over-expression plants to obtain T2 generation transgenic plants. The results showed that the expression levels of SlCV and SlDH29 genes were significantly increased in positive transformed plants compared to control wild type (non-transgenic tomato) Micro Tom (B in fig. 7a and 7).

In order to further verify the function of the candidate genes in regulating and controlling metabolite synthesis in tomato bodies, the inventor selects strains with higher over-expression multiples in T2 generation plants of SlCV, slDH transgenic materials respectively, and performs LC-MS detection and analysis on hydroxycinnamoyl spermidine in tomato leaves. The test results showed that SlDH-OE showed higher accumulation of primary acylated spermidine derivative phenolamines, e.g., caf-Spd, and SlCV-OE showed higher accumulation of secondary acylated spermidine derivative phenolamines, e.g., diCaf-Spd (D in FIGS. 7C and 7), in over-expressed plants of SlCV86 and SlDH29 compared to wild-type material.

Example 10: transient expression of spermidine derived phenolic amine gene clusters in tobacco for biosynthesis of phenolic amine metabolites

To further verify whether the gene cluster has overall regulated phenolic amine function. The applicant linked pDonr _sl4cl1.2, slCV86, slDH and SlDTX obtained in example 2 to pEA Q-ht_s4cl1.2, slCV86, slDH29 and SlDTX29 by LR reaction of Gateway cloning technology, performed tobacco transient transformation after plasmid sequencing was correct by the method in example 6 and co-expressed all components of the gene cluster using tobacco transient expression system.

1. Separately transforming the four genes into tobacco; 2. Sl4CL1.2 co-transformed tobacco with SlCV86 or SlDH, respectively, the concentrations of Agrobacterium were adjusted to OD=1 at co-expression, at 1:1, mixing the materials in proportion; 3. three genes of Sl4CL1.2, slCV h and SlDH are simultaneously co-transformed into tobacco, and the concentration of agrobacterium is regulated to OD=1 during co-expression, and the ratio is 1:1:1, mixing the materials in proportion; 4. four genes of Sl4CL1.2, slCV, 86, slDH, and SlDTX, namely tobacco were co-transformed simultaneously, and the concentration of Agrobacterium was adjusted to OD=1 at the time of co-expression, and the ratio was 1:1:1:1, and mixing the components according to the proportion.

Applicants found that when all four genes in the gene cluster were co-expressed, caf-Spd accumulated approximately 35-fold more than when three genes SlCV-SlDH 29-sl4cl1.2 were co-expressed, whereas expression of each component alone induced Caf-Spd accumulation only 3-9-fold (fig. 8 a). The results for Fer-Spd and diFer-Spd are also similar (fig. 8B and C).

Example 11: drought treatment of transgenic lines overexpressing the Acyltransferase SlCV86 gene

To verify whether candidate gene SlCV86 has the effect of improving drought tolerance of tomatoes, researchers of the invention performed drought treatment on wild type plants and SlCV86 over-expression lines at 4 weeks of seedling stage.

The specific operation method of tomato drought treatment is as follows: first, slCV transgenic tomato seeds and wild tomato seeds (Micro tom) are sown in plug trays, seedlings with consistent growth vigor are selected after germination, and transplanted in the same pot. Culturing under the conditions of 80% of water, constant temperature of 24 ℃ and 16 hours of light and 8 hours of darkness until the seedlings are four-week old. After RWC is sampled and measured, drought treatment is carried out on RWC, days are counted and states are observed, and measured parameters are sampled and measured.

The calculation formula of the Relative Water Content (RWC) of tomato plants is as follows: RWC (%) = (fresh weight-dry weight)/(rehydrated weight-dry weight). Leaves were cut from each group and their fresh weight was immediately scored. After soaking in deionized water at 4 ℃ for 24 hours, the rehydration weight was determined. Finally, the leaves were dried at 70 ℃ for 48h and the dry weight of the leaves was measured (Wang et al, 2015).

And (3) survival rate measurement: each group of plants was continuously dehydrated for 15-19 days without adding water, then dehydrated with sufficient water, and after 15 days, representative pictures were taken and survival rates were calculated.

The results showed that the SlCV86 over-expressed lines all exhibited a stronger drought tolerant phenotype (a in fig. 9) after 16 days of drought treatment and 19 days of rehydration compared to the wild-type plant control. RWC statistics showed that the relative moisture content on leaves of the tomato over-expression plants after drought was far higher than that of wild tomato plants, indicating a stronger water retention (B in fig. 9). Similarly, slCV86 over-expressed plants showed significantly enhanced survival compared to wild-type tomato plants (C in fig. 9).

To investigate the changes in metabolites of tomato plants during drought, slCV-OE and WT plants were grown under normal and drought conditions and leaf tissue samples were analyzed for phenolamine content using LC-MS. The content of diCaf-Spd, caf-Spd and other substances of the Sl CV86-OE strain which shows stronger drought resistance is obviously higher than that of the WT strain. Therefore, diCaf-Spd and Caf-Spd may play a key role in drought stress response (D in FIG. 9). Further analysis of ABA content revealed that ABA accumulation in SlCV-OE lines after drought was significantly increased compared to wild type (E in fig. 9) and that the expression levels of key synthetic genes NCED3 and ABA3 were significantly induced (F and G in fig. 9).

The result shows that the over-expression of the hydroxycinnamamide cluster component SlCV gene can obviously enhance the accumulation of hydroxycinnamamide in tomato plants so as to enhance the drought resistance, so that the drought-resistant tomatoes can be cultivated by adopting the method.

Claims (5)

1. An isolated hydroxy cinnamoyl spermidine biosynthesis and transport gene cluster, wherein the gene cluster encodes four proteins, the four proteins being sl4cl1.2, slCV86, slDH29 and SlDTX, wherein the amino acid sequence of sl4cl1.2 is shown in SEQ ID No.1, the amino acid sequence of SlCV86 is shown in SEQ ID No.2, the amino acid sequence of SlDH29 is shown in SEQ ID No.3, and the amino acid sequence of SlDTX29 is shown in SEQ ID No. 4.

2. The gene cluster of claim 1, wherein the nucleotide sequence of the coding gene of the protein Sl4CL1.2 is shown in SEQ ID No.5, the nucleotide sequence of the coding gene of the protein SlCV86 is shown in SEQ ID No.6, the nucleotide sequence of the coding gene of the protein SlDH 29 is shown in SEQ ID No.7, and the nucleotide sequence of the coding gene of the protein SlDTX29 is shown in SEQ ID No. 8.

3. An expression vector comprising the gene cluster of claim 1 or 2.

4. Use of the expression vector of claim 3 for improving drought resistance of tomatoes.

5. Use of the expression vector of claim 3 for the synthesis of hydroxycinnamospermidine.