CN104894140A - Gene capable of improving oxidation resistance of tomato and application thereof - Google Patents

Abstract

The invention relates to a gene AntioxL1 capable of improving oxidation resistance of a tomato. The gene fragment is obtained form Arabidopsis. A specific method for separation, cloning, functional verification and application of the fragment is provided. A specific expression promoter E8-2 in the tomato is utilized, the gene for driving specific expression of the gene in the tomato, and thus the content of flavonoid and caffeoyl quinic acid in a tomato plant is greatly increased. According to the flavonoid, the rutin content is increased by 18.2 times, and the kaempferol rutin content is increased by 33.1 times; the 1-caffeoyl quinic acid content is increased by 18.1 times, the 2-caffeoyl quinic acid content is increased by 68.0 times, and the 3-caffeoyl quinic acid content is increased by 108.4 times. The oxidation resistance of the tomato is improved by over three times.

Description

A kind of gene and application thereof improving tomato antioxidant ability

Technical field

The present invention relates to field of plant genetic, provide a kind of gene fragment and the embody rule method thereof that improve tamato fruit resistance of oxidation, shown in the gene order thing SEQ ID NO:1 of this fragment, plant can be given more accumulate flavonoid and comprise rutin, kaempferol violaguercitrin etc., and the material such as caffetannic acid, and improve tomato antioxidant ability.

Background technology

Flavonoid (Flavonoids), refers to that two phenyl ring with phenolic hydroxyl group are by the interconnective a series of compound of central thricarbon atom.Distributed in nature is extensive, and being often present in higher plant and filiciform, stem, leaf, flower, fruit with the form of free state or glucosides, is many medium-height grass the effective elements of the medicines (Graf et al., 2005; Hertog et al., 1995).Different flavonoid class compounds has different biological functions in plant, is usually divided into flavonoid, flavonols, flavones ketone, cachou extract class, osajin, anthocyan.Owing to being extensively present in plant, physiologically active is various, causes extensive concern both domestic and external in recent years, has important using value in the field such as agricultural, medicine, feed, chemical industry.

Flavonoid is the natural Secondary metabolites of a class, plays promoter action, and can reduce the risk of some diseases to human health.As antioxidant, free-radical scavengers and divalent cation intercalating agent, can antisepsis and anti-inflammation, anticancer and suppress platelet aggregation (Simona et al., 2010; Zhen et al., 2010), every day, quantitatively edible flavonoid can reduce the cancer incidence of 7-31%, heart trouble incidence ((Hertog et al., 1993 of 30-40%; Soobrattee et al., 2006).

Flavonoid also to play a role (Mol et al., 1998 as signaling molecule, phytoalexin and allelochemical in plant and microbial interaction; Harborne et al., 2000; Pietta, 2000; Winkel-Shirley, 2001), anthocyan material also affects the color (Tang Chuanhe, 2005) etc. of plant.The biological activity of flavonoid is summarised as the following aspects substantially: (1) anti-oxidant and effect of scavenging radical; (2) antibacterial and antivirus action; (3) anti-inflammatory and anti-allergic effects.

Caffetannic acid (Caffeoyl quinic acid) is a class by quinic acid and different number coffic acid by esterification phenolic acids natural compounds comprehensively, is mainly present in plant of Solanaceae, and coffee, apple, and in pears.Over nearly 20 years, Chinese scholars conducts in-depth research with regard to the vegetable chemistry of caffetannic acid and pharmacology, finds that this compounds has some important biological activitys, has clinical value.

Chlorogenic acid (Chlorogenic acid), by coffic acid (Caffeic acid) and quinic acid (Quinic acid, QA) depside that generates, be plant materials in aerobic repiration process through a kind of phenylpropanoids that shikimic acid pathway produces.Different according to the number of caffeoyl on quinic acid, theoretically, a caffetannic acid can be divided into, cynarin and three caffetannic acids, different according to the position of caffeoyl on quinic acid, there is again many isomer.

In human body, chlorogenic acid is main directly by intestinal absorption (Williamson et al., 2000), limit the oxidation of low density fat thus reduce atherosclerotic possibility, toxicity in vivo is reduced by removing alkyl peroxy radical, tissue DNA destroys thus prevents cancer from (Sawa et al., 1999) occurring.It is reported, the primary bioactivity of chlorogenic acid has (1) to the restraining effect of Unidasa and G-6-Pase; (2) to removing and the lipoid peroxidization resistant of free radical; (3) anti-mutagenic activity; (4) hepatic cholagogic effect; (5) effect such as antibacterial, antiviral and spasmolysis.

The chemical process based on natural materials (Ginkgo Leaf, onion, lemon, various Chinese medicinal materialss etc.) is utilized to obtain mainly through conventional means at present, be subject to raw material sources deficiency, content be on the low side, the serious restriction of primary extract complicated and the factor such as relevant pharmacology toxicity is unclear, controlled the production and selling of derived product by fewer companies, hold at high price.

The tomato of extensively plantation is only containing a small amount of flavonoid and caffetannic acid at present.Tomato is utilized to produce as bio-reactor the focus that plant vaccine, compound etc. are just becoming genetically engineered research.Tomato is important transgene receptor plant, and agriculture bacillus mediated tomato leaf disc transformation method is used widely after report always.But how can improve the content of flavonoid and caffetannic acid in tomato body, and then raising tamato fruit resistance of oxidation is the insurmountable problem of existing routine techniques always.

Summary of the invention

The present inventor, for the situation of above-mentioned prior art, provides a kind of DNA fragmentation can giving tomato strong anti-oxidation ability.

Contriver provide firstly one and comprises antioxL1the DNA fragmentation of gene, its antioxL1the gene order of gene is as shown in SEQ ID NO:1, and above-mentioned DNA fragmentation has the effect improving tamato fruit resistance of oxidation.

First the present inventor passes through special primer antioxL1f1, its gene order as shown in SEQ ID NO:2, and antioxL1r1, its gene order, as shown in SEQ ID NO:3, adopts prior art high-fidelity amplification total length sEQ ID NO:1gene, obtains the gene fragment of gene order as shown in SEQ ID NO:1.

Contriver is by special primer E8PF1 afterwards, its gene order is as shown in SEQ ID NO:4, and E8PR1, its gene order is as shown in SEQ ID NO:5, with No. 4 genomic dnas of vegetables in tomato variety for masterplate, combination amplifies the DNA sequence dna of coding E8 promotor, and its gene order is as shown in SEQ ID NO:6.

After the above-mentioned two fragment gene fragments of acquisition, adopt xho I- spe Iremove GFP fragment after double digestion and replace the GFP gene in conventional plant expression vector pX6-GFP by above-mentioned two fragment gene fragments, finally can obtain plant expression vector pX6-E8:: antioxL1.

The plant expression vector pX6-E8-2: of final acquisition: antioxL1, import Agrobacterium tumefaciens attachment AGL1.By leaf disk method transformed into tomatoes kind CSL, obtain PCR transfer-gen plant, by PX6 vector primer PX6F1, its gene order is as shown in SEQ ID NO:7, and PX6F2, its gene order as shown in SEQ ID NO:8, amplification proceed to carrier E8-2 and antioxL 1overall sequence, checking transgenic positive plant.Utilize HPLC to carry out flavonoid and caffetannic acid content detection to the tamato fruit of transgenic positive strain, in the fruit rind of transgenic line, the content of above material is all significantly improved, and passes through ABTS ⁺the resistance of oxidation of method measurement tamato fruit is compared wild-type and is significantly improved.

On the basis of above-mentioned technology, according to what cloned antioxL1gene makes probe, and from cDNA and genomic library, screening can obtain gene of the present invention or homologous gene.Equally, adopt PCR(polymerase chain reaction) technology, also can increasing from genome, mRNA and cDNA, it is of the present invention to obtain antioxL1gene and any interested section of DNA or the section of DNA with its homology.Adopt above technology, can be separated and be comprised antioxL1what the sequence of gene comprised gene fragment is equivalent to the DNA sequence dna shown in SEQ ID NO:1 substantially, or its function is equivalent to the subfragment of sequence shown in SEQ ID NO:1, this sequence is connected with suitable carrier, can vegetable cell be proceeded to, produce transgenic plant.

In sum, the present inventor provides one first and comprises rutin, kaempferol violaguercitrin etc. by giving plant of Solanaceae accumulation flavonoid, and caffetannic acid ability and then increase substantially the DNA fragmentation of plant of Solanaceae resistance of oxidation, this fragment obtains from Arabidopis thaliana, comprises and improves tamato fruit resistance of oxidation gene antioxL1, inventor provide the concrete grammar of the separating clone of this fragment, functional verification and application; The present invention utilizes tamato fruit specific expressing promoter E8-2, drives antioxL1specifically expressing in tamato fruit, substantially increase flavonoid and caffetannic acid content in tamato fruit, wherein, flavonoid comprises that rutin improves 18.2 times, kaempferol violaguercitrin improves 33.1 times, and one caffetannic acid improve 18.1 times, cynarin improves 68.0 times, three caffetannic acids improve 108.4 times, and tamato fruit resistance of oxidation is improved more than 3 times; Thus demonstrate Arabidopis thaliana antioxL1gene has the function improving tamato fruit resistance of oxidation; Set up on this basis simultaneously, produce flavonoid and caffetannic acid biological products using cultivation type fruitlet tomato as bio-reactor, carry out industrialization; Also this engineering cell of tomato be can obtain, flavonoid and caffetannic acid produced by cell cultures mode.The conversion carrier simultaneously used in tomato of the present invention can be marked by the inducing action rejecting screening of estradiol, obtains the anti-oxidant tomato variety with edible safety, carries out breed of variety.

Accompanying drawing explanation

Fig. 1 is whole process schema of the present invention;

Fig. 2 a is E8-2 promotor pcr amplification rear electrophoresis figure;

In Fig. 2 a, M is TaKaRa DNA Marker DL 2,000,1 is E8-2 promotor;

Fig. 2 b is antioxL1gene PCR amplification rear electrophoresis figure;

In Fig. 2 b, M is TaKaRa DNA Marker DL 2,000, and 1 is antioxL1gene;

Fig. 3 is that E8-2 promotor TA clones warp ecoR Ienzyme cuts rear electrophoresis figure;

In figure, M is Trans2K DNA Marker, and 1 is E8-2 promotor positive colony;

Fig. 4 is transition vector pX6-E8-2 warp xho I-Spei enzyme cuts rear electrophoresis figure,

In figure, M is rans2K DNA Marker, and 2 is pX6-E8-2 negative clone, and 3,4 and 5 for building pX6-E8-2 positive colony;

Fig. 5 is pX6-E8-2:: antioxL1recombinant plasmid enzyme cuts qualification electrophorogram,

In figure, M is TaKaRa DNA Marker DL 2,000,1 is pX6-E8-2:: antioxL1positive colony

Fig. 6 is the schematic diagram of tomato genetic transformation different times;

In figure, A is schematic diagram after callus induction; B is schematic diagram during differentiation generation indefinite bud; C is that regrowth is taken root schematic diagram; D is that transgenic seedling transplants plant schematic diagram behind land for growing field crops; E is T ₀for transgenosis fruit schematic diagram;

Fig. 7 is goal gene in transfer-gen plant antioxL1pCR detected result schematic diagram,

In figure, M is TaKaRa DNA Marker DL 2,000, P1 is positive control, and P9 is negative control, and 2-8 is transfer-gen plant;

Fig. 8 is wild-type (CSL) and transgenosis fruit rind (CSL11-1) extracting solution HPLC analytical results schematic diagram,

Fig. 9 is Transgenic tomato fruit (CSL) and transgenosis fruit fresh (CSL11-1) extracting solution HPLC analytical results schematic diagram,

In figure, S1 is chlorogenic acid; S2 is rutin; S3 is 1,5-DCQA; S4 is kaempferol violaguercitrin; S5 is 3,4,5-tri-caffetannic acid;

Figure 10 is wild-type (CSL) transgenosis T different from three ₁for strain fruit rind and pulp (CSL11-1, CSL2-1, CSL13-1) extracting solution resistance of oxidation analytical results schematic diagram.

Embodiment

The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art;

Multiple substratum involved in the present invention is specific as follows:

MS substratum: 100 mL 10 × MSmax, 10 mL 100 × MSmin, 10 mL 100 × Fe2-EDTA, 10 mL 100 × Tomato Vitamin, 30 g sucrose, potassium hydroxide or hydrochloric acid adjust pH to 5.7-5.8, are settled to 1 L.If MS solid medium, add 0.8% agar powder.

Tomato is pre-, common, wild Oryza species: on above-mentioned MS medium base, add growth hormone (indoles nitrilotriacetic) and zeatin, final concentration is respectively 0.2 mg/L and 2 mg/L.

Tomato division culture medium: on above-mentioned MS medium base, add Pyocianil and kantlex before using, final concentration is respectively 400 mg/L and 30 mg/L.

Tomato root media: on MS medium base, add growth hormone and Pyocianil, final concentration is respectively 0.1 mg/L and 400 mg/L.

embodiment 1

1. tamato fruit specificity E8-2 promotor and antioxL1the clone of gene DNA

With No. 4 genomic dnas of vegetables in tomato variety for masterplate, primer E8PF1, its gene order is as shown in SEQ ID NO:4, and E8PR1, and its gene order is as shown in such as SEQ ID NO:5, and combination amplification, obtains expection 1.1 kb fragment (shown in Fig. 2 a).And then E8PF1/R1 is combined amplified production carry out TA clone, be connected to PCR primer cloning vector pGEM-T Easy carrier.Transformation of E. coli DH5 α, obtains and clones in a large number.Utilize specific primer sets E8PF1/R1 to carry out bacterium colony PCR qualification to 12 of random choose clones, obtain 8 clones having target fragment to insert.Random choose two clone extract plasmid and use EcoR I digestion verification, and confirm that it carries target fragment (shown in Fig. 3), this fragment is SEQ ID NO:6 through its sequence that checks order.

According to PCR method, the DNA originated with Arabidopis thaliana Col-1, for template, passes through special primer antioxL1f1, its sequence as shown in SEQ ID NO:2, and antioxL1r1, its sequence as shown in SEQ ID NO:3, high-fidelity amplification total length antioxL1gene, shown in about 1.8 kb(Fig. 2 b).Sequencing result shows its sequence as SEQ ID NO:1.

PX6 carrier selects existing PX6-GFP, by for subsequent use after removing GFP fragment after the Xho I-Spe I double digestion of routine, cloning with above-mentioned E8-2 promotor and antioxL1gene connects structure pX6-E8-2:: antioxL1carrier.

Shown in the primer sequence used in test and illustrated in table 1.

Table 1 primer instruction card

2. plant expression vector pX6-E8-2:: antioxL 1structure

To the E8-2 promoter fragment of TA clone be cloned into, warp xho I- spe Idouble digestion rear electrophoresis reclaims, and is connected with the pX6-GFP carrier that same enzyme cuts back to close.Random picking recon after transformation of E. coli, warp xho I- spe Idouble digestion rear electrophoresis detects, and recon enzyme is cut containing target fragment in result, and size about 1.1 kb, shows that transition vector pX6-E8-2 successfully constructs (as Fig. 4).This transition vector carries out PCR checking with E8 promotor special primer E8PF1/R1 further, reconfirms that transition vector pX6-E8-2 successfully constructs.

PX6-E8-2 transition vector warp spe Iafter enzyme is cut, dephosphorylation process, glue reclaims simultaneously antioxL1dNA fragmentation.Connect and to adopt bacterium colony PCR to screen 1 after transforming to carry target gene fragment and by the correct clone (as Fig. 5) of sequence verification direction of insertion.This recombinant vectors imports agrobacterium strains GV3101 and carries out tomato genetic transformation after order-checking repeated authentication.

embodiment 2 agriculture bacillus mediated tomato genetic transformation and transfer-gen plant PCR detect

1. agriculture bacillus mediated tomato genetic transformation

Utilize tomato cotyledon for explant, adopt the leaf disc transformation method that Agrobacterium (GV3101 bacterial strain) mediates, by plant expression vector pX6-E8-2:: antioxL1transform CSL acceptor material.Every operation all carries out under strict gnotobasis, and transgeneic procedure base program is as follows:

1) seed is through 75% ethanol disinfection 1 min, available chlorine 2% NaOCl process 15 min, sterile distilled water washing 4-5 time, is seeded in 1/2MS substratum, spacing is suitable for, and is cultured to cotyledon and launches completely after 24-26 DEG C of light culture 3-4 d under transferring to 16 h illumination/8 h dark.

2) blade excision cotyledon before and after two sections, leave and take centre about 0.5 cm blade, adaxial and its surface is placed in pre-culture medium upward, 24-26 DEG C of light culture 2 d.The while that explant being pre-incubated, adopt plate streak, 28 DEG C of large scale culturing contain the agrobacterium strains of recombinant expression vector.

3) the resuspended Agrobacterium of MS nutrient solution, concentration OD600=0.4-0.6 soaks and infects 25-30 min.Aseptic filter paper blots bacterium liquid, and adaxial and its surface is 24-26 DEG C of dark condition Dual culture 2 d upward.

4) sterile distilled water washing explant 4-5 time, each 30 min, be finally that 30 min washed by 250 ppm Pyocianils by concentration, aseptic filter paper blots, and adaxial and its surface cultivates 3 d after 24-26 DEG C of 16 h illumination/8 h dark conditions upward.

5) explant transfers to division culture medium, and every 2 weeks subcultures once break up to resistant buds.

6) when resistant buds grows to 2-3 cm, cut and insert root media.When there being a large amount of root to grow, cleaning root substratum and being transplanted to greenhouse.

CSL as acceptor material obtain 15 strains independently T0 for transfer-gen plant (Fig. 6).Regrowth take root after intermediate house, strengthen the management to tomato common pests such as plant hopper, aphid, red spider, Liriomyzas and control, period of bearing fruit suitably imposes fertilizer.

2. transfer-gen plant PCR detects

Choose young tender tomato leaf, utilize CTAB method to extract transgenosis and nontransgenic plants genomic dna, to comprise antioxL 1full length gene DNA carries out PCR detection at interior outside vector primer PX6F1/R1.PCR primer, through 1% agarose gel electrophoresis analysis, amplifies and pX6-E8-2:: antioxL1the onesize about 3 kp fragments (Fig. 7) of plasmid amplification are positive plant, and positive plant has 10 strains, and positive rate is: 67%.

embodiment 3 tamato fruit flavonoid and caffetannic acid content HPLC analyze

Transgenic Fructus Lycopersici esculenti is at vegetative growth stage and nontransgenic plants and no significant difference, from the variable color phase, the pericarp of transgenosis fruit tends to orange or deep yellow gradually, contrast fruit still maintains corresponding redness, and the increase (Fig. 8, Fig. 9) of the flavonoid substances such as the colour-change of follow-up HPLC analytical proof transgenosis fruit and rutin and caffetannic acid content exists and necessarily contacts.Tamato fruit flavonoid and caffetannic acid content HPLC analytical procedure as follows: until obtained by above-mentioned steps Tomato Ripening results after, with grinding powder after freeze drier (EYELA FDU-1100, TOKOYO RIKAKIAI CO.LTD) lyophilize.Be dissolved in the ratio dissolving of 30 μ l 70% methyl alcohol with every mg lyophilized powder after, be placed in-20 DEG C of 3h and carry out flavonoid extraction, extracting solution uses 0.45 μm of membrane filtration supernatant liquor after 1600g is centrifugal, get 20 μ l filtered liquids to analyze for HPLC, according to (2009) such as Luo, HPLC condition is set, standard substance are rutin, chlorogenic acid and kaempferol violaguercitrin (respectively purchased from Sigma and Extrasynthese company), 1,5-cynarin and 3,4,5-tri-caffetannic acid is purchased from Chengdu Purification Technology Development Co., Ltd..

Utilize HPLC to carry out flavonoid and caffetannic acid content detection to positive transgenic strain, transgenic line is all significantly improved.

Transgenosis fruit rind chlorogenic acid content is up to 304.44 μ g/g DW, improves 18.1 times compared with the control; Cynarin and three caffetannic acids improve 68.0 and 108.4 times respectively compared with the control; In addition, the content of rutin and kaempferol violaguercitrin also improves 18.2 times and 33.1 times respectively, as shown in table 2.

Table 2 CSL transgenic Fructus Lycopersici esculenti T0 is for flavonoid and caffetannic acid content statistics

In 10 strain transgenic lines, have chosen 3 strains (CSL-2, CSL-11, CSL-13) utilizes HPLC to carry out T ₁for flavonoid in positive transgenic strain and caffetannic acid content detection, transgenic positive material content and T ₀in generation, compares equal genetic stability, is specifically shown in Table 3.

Table 3.CSL transgenic Fructus Lycopersici esculenti T ₁for flavonoid and caffetannic acid content statistics

embodiment 4 transgenic tomato fruit resistance of oxidation is analyzed

The increase (Fig. 8, Fig. 9, table 2, table 3) of flavonoid substances and caffetannic acid content in above-mentioned HPLC analytical proof transgenosis fruit.Tamato fruit antioxidant assay method is as follows: after the Tomato Ripening results obtained by above-mentioned steps, with grinding powder after freeze drier (EYELA FDU-1100, TOKOYO RIKAKIAI CO.LTD) lyophilize.After being dissolved in the dissolving of 70% methyl alcohol with every 50mg lyophilized powder, with the ABTS measuring anti-oxidant activity ⁺method carries out antioxidant assay ability mensuration respectively to transgenosis and wild-type tomatoes fruit rind and pulp.Enzyme plate is standard control with Trolox, microplate reader (SYNERGY MX all-wave long multi-functional microplate reader, BIOTEK) can be crossed and measure polyphenoils and to verify previously prepared free radical ABTS ⁺scavenging activity, the ability of the Scavenging ability of test substance and Trolox scavenging free radicals compared, determine relative anti-oxidant activity, unit is anti-oxidant amount TEAC, can reflect in fruit the efficiency extracting anti-oxidation active substance.

Utilize ABTS ⁺method measures T ₁for positive transgenic fruit rind and pulp resistance of oxidation, aobvious enhancing during transgenic line pericarp resistance of oxidation, wherein the positive strain resistance of oxidation of CSL11-1 rises about 3 times, and other two transgenic lines improve multiples at 2-2.5 doubly (Figure 10).The content difference that this and HPLC measure rutin in positive transgenic strain has corresponding relation (table 3) clearly.

Bai Sen bio tech ltd, <110> Anhui

<120> mono-kind improves gene and the application thereof of tomato antioxidant ability

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tcagttgggc acaaaaatat cgaaacgatg gctgatgatt tcgttgactg ggactttgta 1440

tggagagaag gtcaaaccct ttgggacgaa aaagaggatc ttgattcggt tttgtcgagg 1500

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ttgtctcttt cttgttccca tttctctctt agatttcaaa aagtgaacta ctttatctct 1020

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Claims (6)

1. improve a gene for tomato antioxidant ability, its nucleotide sequence is as shown in SEQ ID NO:1.

2. a tomato fruit-specific promoter E8-2 for gene described in claim 1, its nucleotide sequence is as shown in SEQ ID NO:6.

3. the tamato fruit expression vector pX6-E8-2: containing the nucleotide sequence described in claim 1: antioxL1.

4. a kind of gene improving tomato antioxidant ability according to claim 1 is improving the application in tomato in flavonoid and multiple caffetannic acid content.

5. a kind of gene improving tomato antioxidant ability according to claim 1 is improving the application in tomato antioxidant ability.

6. expression vector pX6-E8-2: according to claim 3: antioxL1improving the application in tomato antioxidant ability.