CN104004747A

CN104004747A - Method for improving insect resistance and active component of salvia miltiorrhiza by using tomato gene and endogenous gene of salvia miltiorrhiza

Info

Publication number: CN104004747A
Application number: CN201410234203.7A
Authority: CN
Inventors: 王喆之; 陈尘; 张璇
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2014-05-29
Filing date: 2014-05-29
Publication date: 2014-08-27
Anticipated expiration: 2034-05-29
Also published as: CN104004747B

Abstract

The invention discloses a method for improving insect resistance and active components of salvia miltiorrhiza by using a tomato gene and an endogenous gene of salvia miltiorrhiza. The method comprises the following steps: cloning by using a tomato prosystemin gene to establish a plant expression carrier containing the tomato prosystemin gene and a salvia miltiorrhiza caffeoyl-O-methyl transferase gene interference fragment, preparing a agrobacterium tumefaciens stain containing the plant expression carrier containing the tomato prosystemin gene and the salvia miltiorrhiza caffeoyl-O-methyl transferase gene interference fragment, and preparing a transgenic salvia miltiorrhiza plant. As the tomato prosystemin gene is transferred into the salvia miltiorrhiza plant, the salvia miltiorrhiza caffeoyl-O-methyl transferase gene is interfered, the insect resistance of salvia miltiorrhiza is remarkably higher than that of wild salvia miltiorrhiza, in the root of the transgenic salvia miltiorrhiza growing for 100 days, the contents of rosmarinic acid, danshinolic acid B and tanshinone IIA are respectively 1.31 times, 1.69 times and 2.62 times of those in the root of non-transgenic salvia miltiorrhiza in the same period.

Description

With tomato dna and red sage root Inner source gene, improve the method for the pest-resistant and activeconstituents of the red sage root

Technical field

The invention belongs to plant gene engineering technology field, be specifically related in the red sage root, introduce and express the method for tomato original system plain gene.

Background technology

The red sage root (Salvia miltiorrhiza Bunge) is Labiatae (Labiatae) Salvia per nnial herb, with its root and rhizome, is used as medicine, and is one of conventional bulk medicinal materials of China.The main active ingredient of the red sage root is fat-soluble tanshinone and water miscible phenolic acids, has promoting blood circulation and removing blood stasisly, inducing meastruation to relieve menalgia, and the functions such as relieving restlessness that clear away heart-fire, are widely used in the treatment of cardiovascular and cerebrovascular diseases, cancer and various inflammation clinically.Be accompanied by the expanding day of red sage root crude drug demand and the demand of GAP, cultivating the high good salvia new variety of active component content has become one of key issue urgently to be resolved hurrily in the production of red sage root crude drug.The initial water soluble component of separating from the red sage root is Salvianic acidA, and its energy platelet aggregation-against has antithrombotic and forms, promotes fibrin degradation and the effect resisting myocardial ischemia.In addition; salvianolic acid is also the important class in red sage root water soluble ingredient the inside; total salvianolic acid not only has provide protection to the damage of the organs such as the heart, brain, liver, kidney; wherein salvianolic acid B, also as one of the strongest natural product of current known antioxygenation, is being brought into play in vivo removing free radical, is being improved the important physiological actions such as antioxidant ability of organism.Salvianolic acid B is also one of the water-soluble index composition of the red rooted salvia quality control of < < Pharmacopoeia of People's Republic of China > > (version in 2010) regulation.

In the red sage root, representative composition Tanshinone II A has participated in multiple biochemical reaction and has shown the biological activitys such as natural anti-oxidation, antitumor, antisepsis and anti-inflammation.By inhibition tumor cell increase, the mechanism such as inducing apoptosis of tumour cell realizes its antitumor action; By vasodilation, improve the effect such as microcirculation, antithrombotic formation and remove oxyradical, improve energy metabolism, improve cardio and vascular function.So TANSHINONES is as antitumor, antimicrobial antiphlogistic and improve the aspects such as cardio and vascular function and all have good potential applicability in clinical practice.

Along with the further investigation of effective component in red sage and pharmacologically active thereof, the medicine that the red sage root of take is in recent years main raw material, preparation, makeup and serial health product emerge in an endless stream, and supply falls short of demand for red sage root resource.Be accompanied by the minimizing gradually of expanding day and the wild resource of red rooted salvia demand, artificial growth area increases year by year.Yet as often cross-pollinated plant, make a variation larger in red sage root kind, proterties is complicated.Planted rooted salvia is only planted the situation of not selecting and is made each place of production red sage root quality uneven, and deterioration of strains is serious, and active constituent content is unstable, becomes the red sage root and counts one of biggest obstacle of world market as high-quality plant amedica.Improve the content of effective constituent, especially rosmarinic acid and salvianolic acid B in the red sage root, cultivate excellent red sage root resource significant.

Because artificial cultivation condition puts in poison, species diversity is poor in the ecotope of field, species richness is low, cause that red sage root disease and pest dominant population is outstanding, disease and pest takes place frequently, bollworm, nematode and red sage root maize ear rot etc. have become serious threat red rooted salvia quality, have a strong impact on output and the quality of medicinal material, caused the unsettled phenomenon of the red sage root underproduction and the market supply and demand.Administer at present disease and pest and still adopt spraying insecticide, not only caused that the chemical substance of medicinal material is residual has also caused impact to a certain degree to environment.These problems have been exposed in Chinese Medicine Industryization development highlightedly, become the bottleneck of salviamiltiorrhizabung medicinal material industry development.

The problem that solves red sage root disease and pest, people have made a lot of explorations and effort.The content that utilizes in recent years the modern biotechnology means such as genetically engineered, cell engineering to improve to improve its effective constituent to the red sage root is more and more subject to people's attention.In all multi-methods, utilize genetic engineering technique to transform the hereditary property of medicinal plant secondary metabolism approach, improve active components in medicinal plant content, cultivation can accumulate the new variety of target secondary metabolite in a large number, more and more receives people's concern.The modern molecular breeding technology that the transgenic technology of take is core at improvement medicinal plant, enrich natural resources of Chinese medicinal materials, improve disease resistance and resistance, the novel transgenosis medicinal material of the high natural drug content of cultivation has wide practical use.But the technology of utilizing at present transgenic technology to improve red sage root insect-resistance has no report.At existing employing transgenic method, improve in the technology of red sage root medicinal ingredients, only be confined to proceed to the liposoluble ingredient content that foreign gene improves the red sage root, and the means of utilizing genetic manipulation are introduced synthetic this material that also weakens of foreign gene raising metabolism source material stream to other competition bypasses losses simultaneously, utilize the principle of this " increasing income and decreasing expenditure " to regulate and control pathways metabolism, thereby the technological method that has improved salvianolic acid and tanshinone component content there is no report.

Summary of the invention

Technical problem to be solved by this invention is to overcome the deficiency of above-mentioned technology, and a kind of method with tomato dna and interference red sage root Inner source gene raising red sage root insect-resistance and activeconstituents that insect-resistance is high, active component content is high is provided.

Solving the problems of the technologies described above adopted technical scheme is comprised of following step:

1, tomato prosystemin gene clone

Utilize the E.Z.N.A. of OMEGA company ^tMplant RNA Kit extracts test kit, and the total RNA of separation and Extraction tomato, with the PrimeScript RT reagent Kit of Takara company reverse transcription test kit, becomes cDNA standby the total RNA reverse transcription of tomato to specifications to specifications, adopt Primer Premier5.0 software, the upstream primer of design tomato prosystemin genes encoding frame, downstream primer, take tomato cDNA as template, carry out polymerase chain reaction, reaction system is: take tomato cDNA as template, carry out polymerase chain reaction, reaction system is: get tomato cDNA and archaeal dna polymerase, DNA polymerase buffer liquid, dNTP, upstream primer, downstream primer, the volume ratio of redistilled water is 1:0.5:5:5:1:1:37.5, according to following conditioned response: 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, obtain tomato prosystemin gene, and before the upstream primer of tomato prosystemin gene, introduce Spe I restriction enzyme site and protect bases G ACTAGT, before downstream primer, introduce BstE II restriction enzyme site and protection base CAGGGTAACC, with tomato prosystemin gene primer:

Upstream primer prosystemin-F5 '-GACTAGTATGGGAACTCCTTCATATGATATC-3 ',

Downstream primer prosystemin-R5 '-CAGGGTAACCCGTTTGTCTGTTATTATTTGAG-3 ',

Take tomato cDNA as template, by following reaction system, mix: tomato cDNA and archaeal dna polymerase, DNA polymerase buffer liquid, dNTP, prosystemin-F, prosystemin-R, the volume ratio of redistilled water is 2:0.5:5:5:1:1:36.5, and according to 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, carry out polymerase chain reaction (PCR) amplification, the tomato prosystemin gene that must contain restriction enzyme site, the amplified production obtaining carries out electrophoretic separation, reclaiming amplified production is connected by ligase enzyme working instructions with T4DNA ligase enzyme (being purchased from precious biotechnology Dalian company limited) with pMD19-T carrier (being purchased from precious biotechnology Dalian company limited), form prosystemin-pMD19-T carrier and adopt heat shock conversion method to proceed in bacillus coli DH 5 alpha competent cell, the volume ratio of prosystemin-pMD19-T carrier and bacillus coli DH 5 alpha is 1:9.5～10.5, select gained positive colony, through bacterium colony polymerase chain reaction, detect, sequence verification obtains tomato prosystemin gene order, this gene order is as follows:

2, build the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment

With Spe I and BstE II, distinguish double digestion prosystemin-pMD19-T carrier, plant expression vector pCAMBIA-1302, the enzyme reclaiming respectively after prosystemin gene fragment and pCAMBIA-1302 double digestion is cut product, fragment is connected with T4DNA ligase enzyme with product, screening, obtains pCAMBIA-1302-prosystemin intermediate carrier;

Choose and be positioned at red sage root caffeoyl-O-methyl transferase gene, be called for short COMT, the target fragment that the fragment of coding region 3 ' end 345bp is interfered as RNA, introduces respectively restriction enzyme site Cla I/Kpn I and Xho I/BamH I in this fragment, application Primer Prime5.0 design of amplification primers:

iCOMT-F：5′-CC ATCGAT? GGTACCACCCTCCCCGACGGCGGCGTCCA-3′

iCOMT-R：5′-GCTCTAGA CTCGAG? GGATCCCACACCACCTACAACCACCTCCT-3′；

Use polymerase chain amplified reaction, Cla I and two restriction enzyme sites of Kpn I are introduced in the upstream of this 345bp, in its downstream, introduce Xho I and two restriction enzyme sites of BamH I; Through the amplification of polymerase chain amplified reaction, must contain the product C OMTi of Cla I/Kpn I and Xho I/BamH I, connect p-MD19T carrier and obtain COMTi-pMD19T carrier; With Xho I and Kpn I respectively enzyme cut COMTi-pMD19T carrier, pKannibal carrier, by cut the fragment of COMTi-pMD19T carrier and the fragment of cutting pKannibal carrier through Xho I, Kpn I enzyme with Xho I, KpnI enzyme, with DNA ligase, be connected to pKannibal+ intermediate carrier; With BamH I and Cla I respectively enzyme cut COMTi-pMD19T carrier, pKannibal+ carrier, with containing the fragment that BamH I cuts COMTi-pMD19T carrier with Cla I enzyme, be connected with DNA ligase with the fragment of cutting pKannibal+ with Cla I enzyme through BamH I, the novel vector of acquisition is pKANNIBAL/COMTi.

With Sac I and Pst I double digestion pKANNIBAL/COMTi, pCAMBIA1302-prosystemin carrier, frame is transcribed in the interference that double digestion pKANNIBAL/COMTi is obtained, be connected on the pCAMBIA1302-prosystemin carrier after double digestion, obtain pCAMBIA1302/prosystemin+pKANNIBAL/COMTi carrier, and proceed in bacillus coli DH 5 alpha and increase, extract plasmid, with enzyme blanking method, identify, obtain the plant expression vector pCAMBIA1302/prosystemin+pKANNIBAL/COMTi that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment, called after pCPKC carrier.

The agrobacterium tumefaciens bacterial strain of the plant expression vector that 3, preparation contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment

PCPKC carrier is transformed to agrobacterium tumefaciens EHA105 competent cell, adopt frozen-thawed method to transform, product after conversion is inverted and is cultivated 36～72 hours in 27.5 ℃～28.5 ℃ incubators, picking resistance list bacterium colony carries out polymerase chain reaction with prosystemin gene-specific primer prosystemin-F and prosystemin gene-specific primer prosystemin-R respectively, screening, with Auele Specific Primer on pCPKC carrier, sequence is:

35S-F5′-TACAAAGGCGGCAACAAACG-3′

35S-R5′-GCAATGGAATCCGAGGAGGT-3′

Carry out polymerase chain reaction, screening, obtains the agrobacterium tumefaciens bacterial strain that contains pCPKC carrier.

4, preparation transgenosis red sage root plant

Adopt conventional organization cultural method to obtain aseptic red sage root test-tube plantlet, adopt leaf disc transformation method, the agrobacterium tumefaciens that contains pCPKC carrier and aseptic red sage root test-tube plantlet are contaminated, obtain the red sage root plant that contains tomato prosystemin gene and interfere the endogenous caffeoyl-O-of red sage root methyl transferase gene.

In the step 2 of the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment at structure of the present invention, described use Spe I and BstE II double digestion prosystemin-pMD19-T carrier, plant expression vector pCAMBIA-1302 is: get and contain prosystemin-pMD19-T carrier, 10 * M Buffer, restriction enzyme Spe I, restriction enzyme BstE II, redistilled water was in 37 ℃ of reactions 2 hours 20 minutes, restriction enzyme Spe I and restriction enzyme BstE II, 10 * M Buffer, contain prosystemin-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get plant expression vector pCAMBIA-1302,10 * M Buffer, restriction enzyme Spe I, restriction enzyme BstE II, redistilled water in 37 ℃ of reactions 3 hours, the volume ratio of restriction enzyme Spe I and restriction enzyme BstE II, 10 * M Buffer, plant expression vector pCAMBIA-1302, redistilled water is 1:1:2:6:10.

In the step 2 of the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment at structure of the present invention, described with Xho I and Kpn I respectively enzyme cut COMTi-pMD19T carrier, pKannibal carrier is: get COMTi-pMD19-T carrier, 10 * M Buffer, restriction enzyme Xho I, restriction enzyme Kpn I, redistilled water, 37 ℃ are reacted 2 hours 40 minutes, restriction enzyme Xho I and restriction enzyme Kpn I, 10 * M Buffer, COMTi-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pKannibal carrier, 10 * M Buffer, restriction enzyme Xho I, restriction enzyme Kpn I, redistilled water, 37 ℃ are reacted 2 hours 40 minutes, and the volume ratio of restriction enzyme Xho I and restriction enzyme Kpn I, 10 * M Buffer, pKannibal carrier, redistilled water is 1:1:2:6:10.

In the step 2 of the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment at structure of the present invention, described with BamH I and Cla I respectively enzyme cut COMTi-pMD19T carrier, pKannibal+ carrier is: get and contain COMTi-pMD19T carrier, 10 * K Buffer, restriction enzyme Cla I, restriction enzyme BamH I, redistilled water, 37 ℃ are reacted 2 hours 30 minutes, restriction enzyme Cla I and restriction enzyme BamH I, 10 * K Buffer, COMTi-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pKannibal+ carrier, 10 * K Buffer, restriction enzyme Cla I, restriction enzyme BamH I, redistilled water, 37 ℃ are reacted 2 hours 30 minutes, and the volume ratio of restriction enzyme Cla I and restriction enzyme BamH I, 10 * K Buffer, pKannibal+ carrier, redistilled water is 1:1:2:6:10.

In the step 2 of the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment at structure of the present invention, described use Sac I and Pst I double digestion pKANNIBAL/COMTi, pCAMBIA1302-prosystemin carrier is: get pKANNIBAL/COMTi carrier, 10 * M Buffer, restriction enzyme Sac I, restriction enzyme Pst I, redistilled water, 37 ℃ are reacted 3 hours, restriction enzyme Sac I, restriction enzyme Pst I, 10 * M Buffer, pKANNIBAL/COMTi carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pCAMBIA-1302-prosystemin carrier, 10 * M Buffer, restriction enzyme Sac I, restriction enzyme Pst I, redistilled water in 37 ℃ of reactions 3 hours, the volume ratio of restriction enzyme Sac I and restriction enzyme Pst I, 10 * M Buffer, pCAMBIA1302-prosystemin carrier, redistilled water is 1:1:2:6:10.

The present invention proceeds to red sage root plant by tomato prosystemin gene, has interfered red sage root caffeoyl-O-methyl transferase gene, and screening has obtained insect-resistance apparently higher than the wild red sage root, and transgenosis salvianolic acid content significantly improves.In the growth genetically modified red sage root dry root of 100 days, the content of rosmarinic acid is that the content of 16.96 ± 0.90mg/g, salvianolic acid B reaches 57.64 ± 1.94mg/g, Tanshinone II A content is 2.04 ± 0.19mg/g, is respectively rosmarinic acid (12.90 ± 0.48mg/g) in non-transformed common red sage root dry root of contemporaneously, salvianolic acid B (34.04 ± 0.89mg/g), Tanshinone II A (0.78 ± 0.11mg/g) 1.31 times, 1.69 times, 2.62 times.For improving all kinds of active constituent contents in the red sage root, provide a kind of novel method, can in the cultivation of the red sage root, promote the use of.

Accompanying drawing explanation

Fig. 1 detects tomato prosystemin genetic expression spirogram in transgenosis red sage root plant.

Fig. 2 detects red sage root caffeoyl-O-methyl transferase gene in transgenosis red sage root plant to express spirogram.

Fig. 3 is that bollworm bites the experiment photo of stinging transgenosis red sage root strain after 3 days 2 and the wild red sage root blade extent of damage.

Fig. 4 is the color atlas of active component content in the wild red sage root of high-performance liquid chromatogram determination and transgenosis red sage root strain 2.

Fig. 5 is active component content figure in the wild red sage root and transgenosis red sage root strain 2.

Embodiment

Below in conjunction with drawings and Examples, the present invention is described in more detail, but the invention is not restricted to these embodiment.

Embodiment 1

It is pest-resistant as follows with method steps activeconstituents that the present embodiment improves the red sage root with tomato dna and red sage root Inner source gene:

1, tomato prosystemin gene clone

Utilize the E.Z.N.A.TM Plant RNA Kit of OMEGA company to extract test kit, the total RNA of separation and Extraction tomato to specifications, with the PrimeScript RT reagent Kit of Takara company reverse transcription test kit, become cDNA standby the total RNA reverse transcription of tomato to specifications; Adopt Primer Premier5.0 software, upstream primer, the downstream primer of design tomato prosystemin genes encoding frame, take tomato cDNA as template, carries out polymerase chain reaction.Reaction system is: tomato cDNA1 μ l, archaeal dna polymerase 0.5 μ l, DNA polymerase buffer liquid 5 μ l, dNTP5 μ l, upstream primer 1 μ l, downstream primer 1 μ l, redistilled water 37.5 μ l, according to following conditioned response: 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, obtain tomato prosystemin gene, and before the upstream primer of tomato prosystemin gene, introduce Spe I restriction enzyme site and protect bases G ACTAGT, before downstream primer, introduce BstE II restriction enzyme site and protection base CAGGGTAACC, with tomato prosystemin gene primer:

Upstream primer prosystemin-F5 '-GACTAGTATGGGAACTCCTTCATATGATATC-3 '

Downstream primer prosystemin-R5 '-CAGGGTAACCCGTTTGTCTGTTATTATTTGAG-3 '

Take tomato cDNA as template, by following reaction system, mix: tomato cDNA2 μ l, archaeal dna polymerase 0.5 μ l, DNA polymerase buffer liquid 5 μ l, dNTP5 μ l, prosystemin-F1 μ l, prosystemin-R1 μ l, redistilled water 36.5 μ l, and according to 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, carry out polymerase chain reaction (PCR) amplification, the tomato prosystemin gene that must contain restriction enzyme site, the amplified production obtaining carries out electrophoretic separation, with the E.Z.N.A.TM GelExtraction Kit of OMEGA company test kit, reclaiming amplified production is connected by ligase enzyme working instructions with T4DNA ligase enzyme (being purchased from precious biotechnology Dalian company limited) with pMD19-T carrier (being purchased from precious biotechnology Dalian company limited), form prosystemin-pMD19-T carrier and adopt heat shock conversion method to proceed in bacillus coli DH 5 alpha competent cell, get prosystemin-pMD19-T carrier 10 μ l, bacillus coli DH 5 alpha competent cell 100 μ l, the volume ratio of prosystemin-pMD19-T carrier and bacillus coli DH 5 alpha is 1:10, select 10 of gained positive colonies, through bacterium colony polymerase chain reaction, detect, sequence verification obtains tomato prosystemin gene order, this gene order is as follows:

With Spe I and BstE II, distinguish double digestion prosystemin-pMD19-T carrier, plant expression vector pCAMBIA-1302, the method of double digestion is: get prosystemin-pMD19-T carrier 6 μ l, Spe I1 μ l, BstE II1 μ l, 10 * M Buffer2 μ l, redistilled water 10 μ l, mix, in 37 ℃ of reactions 2 hours 20 minutes, electrophoretic separation, with the E.Z.N.A. of OMEGA company ^tMgel Extraction Kit test kit reclaims endonuclease bamhi, obtains prosystemin gene and reclaims fragment.Get plant expression vector pCAMBIA-13026 μ l, Spe I1 μ l, BstE II1 μ l, 10 * M Buffer2 μ l, redistilled water 10 μ l, mix, and in 37 ℃ of reactions 3 hours, electrophoretic separation, with the E.Z.N.A. of OMEGA company ^tMgel Extraction Kit test kit reclaims endonuclease bamhi, obtains pCAMBIA-1302 and reclaims fragment.It is that 1:7 is connected with T4DNA ligase enzyme with pCAMBIA-1302 recovery fragment according to mol ratio that prosystemin gene is reclaimed to fragment, get and connect heat shock conversion method conversion bacillus coli DH 5 alpha competent cell 100 μ l for product 10 μ l, 10 of picking mono-clonals, 37 ℃ of 180rpm shaking culture 16～18 hours, with prosystemin special primer prosystemin-F and prosystemin-R, through bacterium colony polymerase chain reaction, detect, reaction conditions is 95 ℃, 10 minutes; Then 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 40 seconds, 35 circulations, obtained positive strain; With alkaline lysis method of extracting positive strain plasmid, by Spe I and BstE II double digestion method, carry out endonuclease reaction, screening, obtains pCAMBIA-1302-prosystemin intermediate carrier.

Choose and be positioned at red sage root caffeoyl-O-methyl transferase gene, be called for short COMT, the target fragment that the fragment of coding region 3 ' end 345bp is interfered as RNA, introduces respectively restriction enzyme site Cla I/Kpn I and Xho I/BamH I in this fragment, application Primer Prime5.0 software design amplimer:

iCOMT-F：5′-CC ATCGAT? GGTACCACCCTCCCCGACGGCGGCGTCCA-3′

iCOMT-R：5′-GCTCTAGA CTCGAG? GGATCCCACACCACCTACAACCACCTCCT-3′；

Cla I and two restriction enzyme sites of Kpn I are introduced in the upstream of this 345bp, in its downstream, introduce Xho I and two restriction enzyme sites of BamH I; Through polymerase chain amplified reaction amplification red sage root cDNA, reaction conditions is 95 ℃, 3 minutes; Then 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃, 25 seconds, 35 circulations, caffeoyl-O-methyltransgerase that must contain Cla I/Kpn I and Xho I/BamH I, called after COMTi reclaims COMTi fragment according to test kit specification sheets after electrophoretic separation, with p-MD19T carrier, be connected with the volume ratio 1:9 of COMTi fragment, obtain COMTi-pMD19T carrier.

With Xho I and Kpn I respectively enzyme cut COMTi-pMD19T carrier, pKannibal carrier, get COMTi-pMD19-T carrier 6 μ l, 10 * M Buffer2 μ l, restriction enzyme Xho I1 μ l, restriction enzyme Kpn I1 μ l, redistilled water 10 μ l in 37 ℃ of reactions 2 hours 40 minutes; Get pKannibal carrier 6 μ l, 10 * M Buffer2 μ l, restriction enzyme Xho I1 μ l, restriction enzyme Kpn I1 μ l, redistilled water 10 μ l in 37 ℃ of reactions 2 hours 40 minutes; The two enzyme is cut to product and carry out electrophoretic separation, according to test kit specification sheets, reclaim COMTi gene fragment and pKannibal carrier segments, get COMTi gene and reclaim fragment 1 μ l, pKannibal intermediate carrier recovery fragment 7 μ l, T4DNA ligase enzyme 1 μ l, 10 * T4DNA ligase enzyme Buffer1 μ l, 16 ℃ are reacted 3 hours, obtain pKannibal+ intermediate carrier.

With BamH I and Cla I respectively enzyme cut COMTi-pMD19T carrier, pKannibal+ carrier, get COMTi-pMD19T carrier 6 μ l, 10 * K Buffer2 μ l, restriction enzyme Cla I1 μ l, restriction enzyme BamH I1 μ l, redistilled water 10 μ l, mix in 37 ℃ of reactions 2 hours 30 minutes, get pKannibal+ intermediate carrier 6 μ l, 10 * K Buffer2 μ l, restriction enzyme Cla I1 μ l, restriction enzyme BamH I1 μ l, redistilled water 10 μ l, mix in 37 ℃ of reactions 2 hours 30 minutes, these two kinds of enzymes are cut to product and carry out electrophoretic separation, according to test kit specification sheets, reclaim COMTi gene fragment and pKannibal+ intermediate carrier fragment, get COMTi gene and reclaim fragment 1.5 μ l, pKannibal+ intermediate carrier reclaims fragment 6.5 μ l, T4DNA ligase enzyme 1 μ l, 10 * T4DNA ligase enzyme Buffer1 μ l, 16 ℃ are reacted 3 hours, the novel vector obtaining is pKANNIBAL/COMTi.

The new pKANNIBAL/COMTi and the pCAMBIA1302-prosystemin carrier that by Sac I and the two enzyme previous steps of Pst I, obtain, get pKANNIBAL/COMTi6 μ l, Sac I1 μ l, Pst I1 μ l, 10 * M Buffer2 μ l, redistilled water 10 μ l, mix, in 37 ℃ of reactions 3 hours; Get pCAMBIA-1302-prosystemin carrier 6 μ l, Sac I1 μ l, Pst I1 μ l, 10 * M Buffer2 μ l, redistilled water 10 μ l, 37 ℃ are reacted 3 hours; Electrophoretic separation, according to the E.Z.N.A. of OMEGA company ^tMgel Extraction Kit test kit specification sheets recovery pKANNIBAL/COMTi enzyme cuts product and pCAMBIA-1302-prosystemin carrier enzyme is cut product, get pKANNIBAL/COMTi enzyme and cut product 2 μ l, pCAMBIA-1302-prosystemin carrier recovery fragment 6 μ l, T4DNA ligase enzyme 1 μ l, 10 * T4DNA ligase enzyme Buffer1 μ l, 16 ℃ are reacted 3 hours, obtain pCAMBIA1302/prosystemin+pKANNIBAL/COMTi carrier.This carrier is proceeded in bacillus coli DH 5 alpha and increased, use alkaline lysis method of extracting plasmid, by the method for Sac I and Pst I double digestion, identify, obtain the plant expression vector pCAMBIA1302/prosystemin+pKANNIBAL/COMTi that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment, called after pCPKC carrier.

PCPKC carrier is transformed to agrobacterium tumefaciens EHA105 competent cell, adopt frozen-thawed method to transform, product after conversion is inverted and is cultivated 48 hours in 28 ± 0.5 ℃ of incubators, 20 of picking resistance list bacterium colonies, 28 ± 0.5 ℃, 160rpm concussion cultivate in 24 hours, with prosystemin gene-specific primer prosystemin-F and prosystemin gene-specific primer prosystemin-R, carry out polymerase chain reaction respectively, reaction conditions is 95 ℃, 10 minutes; Then 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 40 seconds, 35 circulations, obtained positive strain; Use again Auele Specific Primer on pCPKC carrier:

35S-F5′-TACAAAGGCGGCAACAAACG-3′

35S-R5′-GCAATGGAATCCGAGGAGGT-3′

Positive strain is carried out to polymerase chain reaction, and reaction conditions is 94 ℃, 3 minutes; 94 ℃, 30 seconds, 59 ℃, 30 seconds, 72 ℃, 50 seconds, 35 circulations, screening, the final agrobacterium tumefaciens bacterial strain that contains pCPKC carrier that obtains.

4, preparation transgenosis red sage root plant

The acquisition of aseptic red sage root test-tube plantlet can adopt conventional organization cultural method, and concrete grammar is as follows:

Salvia seeds is rinsed with flowing water, by volume fraction, is 75% aqueous ethanolic solution surface sterilization 20 seconds, and redistilled water rinses 2 times, each 4 minutes, the mercuric chloride solution surface sterilization that is 0.1% with massfraction 10 minutes, redistilled water rinses 5 times, and salvia seeds blots the remaining moisture in surface with filter paper, be placed on MS minimum medium and sprout, 25 ℃, illumination in 16 hours, intensity of illumination is 3000Lux, in 8 hours dark culturing casees, cultivate 2 weeks, obtain in vitro cuttings.

The concrete grammar that the agrobacterium tumefaciens that contains pCPKC carrier and aseptic red sage root test-tube plantlet are contaminated is as follows:

Choose the growth red sage root in vitro cuttings of 30 days, its blade is cut into the fritter of 0.5 centimetre of 0.5 cm x, proceed to that in preculture substratum, (pre-culture medium is that every 1LMS substratum contains the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, 1mL concentration is the substratum of the a-naphthylacetic acid of 1.0mg/mL), under normal culture condition, preculture is 1 day, a part of blade that preculture is crossed proceeds to containing in pCPKC carrier agrobacterium tumefaciens bacterium liquid of having diluted, 100 revs/min of dip-dyes of 28 ℃ of constant temperature 25～30 minutes, take out blade, with aseptic filter paper, blot, transfer to dark cultivation 2～3 days on preculture substratum, the pre-incubated blade of another part does not carry out the dip-dye of Agrobacterium, after being directly cultured on preculture substratum and sprouting, bud is peeled off, and is placed on 1/2MS substratum and takes root, as the unconverted blank plant of experiment, dark cultivation after 2～3 days, blade is taken out from former substratum, be transferred to and select on substratum (selecting substratum is the substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL in 1LMS substratum), within every 10～15 days, change and once select substratum, when resistant buds grows to approximately 0.5～1 centimetre of left and right, resistant buds is cut, proceed on 1/2MS substratum and take root.1/2MS substratum (every 1L contains the cephamycin that 10mL concentration is 200mg/mL, the Totomycin that 10mL concentration is 3mg/mL) screening and culturing 3～4 times, within every 10～15 days, change a subculture, the plant that can normally take root is cut off from internode, stem segment cuttage succeeding transfer culture on MS substratum with 2 axillalry buds, within 4～6 weeks, a subculture is changed in left and right, the positive transformation plant of screening and contrast strain are transplanted to equal conditions in scientific research greenhouse and are cultivated, and transplant and cultivate the transgenosis red sage root plant of 250 days for the mensuration of rosmarinic acid and content of danshinolic acid B.

Embodiment 2

In the tomato prosystemin of the present embodiment gene clone step 1, get prosystemin-pMD19-T carrier 10 μ l, bacillus coli DH 5 alpha competent cell 95 μ l, the volume ratio of prosystemin-pMD19-T carrier and bacillus coli DH 5 alpha is 1:9.5, select 10 of gained positive colonies, through the detection of bacterium colony polymerase chain reaction, sequence verification, obtain tomato prosystemin gene order.Other step in this step is identical with embodiment 1.Other step is identical with embodiment 1.

Embodiment 3

In the tomato prosystemin of the present embodiment gene clone step 1, get prosystemin-pMD19-T carrier 10 μ l, bacillus coli DH 5 alpha competent cell 10.5 μ l, the volume ratio of prosystemin-pMD19-T carrier and bacillus coli DH 5 alpha is 1:10.5, select 10 of gained positive colonies, through the detection of bacterium colony polymerase chain reaction, sequence verification, obtain tomato prosystemin gene order.Other step in this step is identical with embodiment 1.Other step is identical with embodiment 1.

Embodiment 4

In the preparation of above embodiment 1～3, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the agrobacterium tumefaciens bacterial strain step 3 of plant expression vector of fragment, pCPKC carrier is transformed to agrobacterium tumefaciens EHA105 competent cell, adopt frozen-thawed method to transform, product after conversion is inverted and is cultivated 72 hours in 27.5 ℃ of incubators, picking resistance list bacterium colony carries out polymerase chain reaction with prosystemin gene-specific primer prosystemin-F and prosystemin gene-specific primer prosystemin-R respectively, other step in this step is identical with embodiment 1.Its step is identical with embodiment 1.

Embodiment 5

In the preparation of above embodiment 1～3, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the agrobacterium tumefaciens bacterial strain step 3 of plant expression vector of fragment, pCPKC carrier is transformed to agrobacterium tumefaciens EHA105 competent cell, adopt frozen-thawed method to transform, product after conversion is inverted and is cultivated 36 hours in 28.5 ℃ of incubators, picking resistance list bacterium colony carries out polymerase chain reaction with prosystemin gene-specific primer prosystemin-F and prosystemin gene-specific primer prosystemin-R respectively, other step in this step is identical with embodiment 1.Its step is identical with embodiment 1.

In order to verify beneficial effect of the present invention, contriver adopts the transgenosis red sage root plant of the embodiment of the present invention 1 preparation to carry out the expression amount of tomato dna and red sage root native gene, the insect-resistance of transgenosis red sage root plant and the content of activeconstituents, carried out a large amount of experiments, various experiment situations are as follows:

1, the expression amount of prosystemin and caffeoyl-O-methyl transferase gene in detection transgenosis red sage root plant

Use PremierPrimer5.0 software, the synthetic primer that is applicable to required tomato prosystemin gene, red sage root caffeoyl-O-methyl transferase gene and red sage root house-keeping gene Actin muscle-β gene of real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification of design, is labeled as respectively qProsystemin-F, qProsystemin-R; QCOMT-F, qCOMT-R; QSmACT-F, qSmACT-R.Primer sequence is respectively:

qProsystemin-F5′-TAGTTGAAAAAGAGACTCCATCCCA-3′

qProsystemin-R5′-ACGACAAGTTAGTTTTGGAGGT-3′

qCOMT-F5′-TCCAGGTGTGGAGCATGTGG-3′

qCOMT-R5′-GTGTTTTACCCTTCCACTA-3′

qSmACT-F：5’-AGGAACCACCGATCCAGACA-3’

qSmACT-R：5’-GGTGCCCTGAGGTCCTGTT-3’

According to the E.Z.N.A. of OMEGA company ^tMthe specification sheets that Plant RNA Kit extracts test kit extracts the growth total RNA of the red sage root of 30 days, and the PrimeScript RT reagent Kit of Bing Yong Takara company reverse transcription test kit synthesizes red sage root cDNA the first chain, saves backup.

The non-transformed red sage root strain cDNA of take is blank, the red sage root plant that transforms pCPKC carrier of take is experimental group, measures the expression amount of tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene by real time fluorescent quantitative reverse transcription-polymerase chain reaction method.Real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification condition is: 94 ℃ of denaturations, 1 minute; 40 circulations (94 ℃ of sex change, 10 seconds, 60 ℃ of fluorescent signals of annealing and collect, 25 seconds), 95 ℃, 1 minute, 60 ℃, 1 minute, within 60～95 ℃, every 30 seconds, raise 0.5 ℃, collect first order fluorescence.Reaction finishes rear fluorescent signal value and adopts " the relatively relative quantification method of Ct value " to carry out the analysis of genetic expression.Concrete treatment process is according to " iQ ^tM5 multiple real time fluorescence quantifying PCR instrument specification sheetss " carry out.Detected result is shown in Fig. 1,2.In Fig. 1,2, X-coordinate is different transgenic line, and ordinate zou is relative expression quantity, and CK represents non-transformed red sage root strain, and * represents that experimental group and control group relatively have the significance difference opposite sex (P<0.05).As seen from Figure 1, compare with adjoining tree, external source goal gene tomato prosystemin gene all has expression in various degree in transformation plant 2,3,8,13, and there is compared with the control significant difference (P<0.5), show tomato prosystemin gene overexpression in the red sage root.In Fig. 2, the caffeoyl-O-methyltransgerase expression amount in strain 2,3,8,13,15,16 has contrasted obvious downward (P<0.05), illustrates that to the interference of red sage root Inner source gene caffeoyl-O-methyltransgerase be successful.The detected result of comprehensive tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene expression amount, chooses 2,13 two strains of transgenosis red sage root strain and carries out follow-up test.

2, the pest-resistant experiment of transgenosis red sage root strain

From 3 plant of difference of the wild red sage root of the transgenosis red sage root strain 2,13 of growing 30 days and isometric growth state, win 1, top respectively, be placed on the culture dish that is covered with moist absorbent cotton, in each culture dish, put 1 red sage root top.In each culture dish, inoculate in addition 3 cotton bollworm larvaes (for laboratory retains) in the third-instar larvae phase.Culture dish is placed in to 25 ℃ of illumination boxs and cultivates, after 3 days, observe blade degree of damage, the results are shown in Figure 3, as can be seen from Figure 3, left figure is the blade of experiment transgenosis red sage root strain 2 after 3 days, and blade is not almost snapped and bites, and bollworm is not because having food dead yet; Right figure is wild-type red sage root blade, and blade is snapped and bites seriously as can be seen from Figure, brownization of edge, necrosis.And the mortality ratio of adding up bollworm, in Table 1.

Table 1 is bitten and is stung the bollworm mortality ratio that is inoculated in transgenosis red sage root strain 2 and wild red sage root blade after 3 days

Strain title	Total borer population	Borer population after 3 days	Mortality ratio
				Transgenosis red sage root strain 2	9	2	77.78％
The wild red sage root	9	8	11.11％

As seen from Table 1, on wild red sage root blade, the mortality ratio of bollworm is only 11%, and the bollworm mortality ratio of inoculating on the blade of transgenosis red sage root strain 2 is up to 78%, show after having proceeded to anti insect gene prosystemin, the red sage root has had the pest-resistant character that is obviously better than Wild plant, for the pest-resistant evil of the wild red sage root provides feasible foundation.

3, use the content of activeconstituents in high-performance liquid chromatogram determination transgenosis red sage root strain

(1) preparation of high-efficient liquid phase chromatogram condition and system suitability and standardized solution

1. high-efficient liquid phase chromatogram condition

Adopt the Japanese SHIMADZU LC-2010 of company high performance liquid chromatograph, chromatographic column is Phenomenex silica matrix post (5 μ m C ₁₈reverse post, 4.6mm * 250mm), aqueous acetic acid, acetonitrile and the methyl alcohol that the massfraction of take is 0.4% is eluent gradient wash-out, eluent gradient elution program is 0.01～5 minute, massfraction is that 0.4% aqueous acetic acid volume reduces to 90% by 95%, and acetonitrile volume rises to 10% by 5%; 5～25 minutes, the aqueous acetic acid volume that massfraction is 0.4% reduced to 67% by 90%, and acetonitrile volume rises to 30% by 10%, and methyl alcohol volume rises to 3% by 0; 25～40 minutes, the aqueous acetic acid volume that massfraction is 0.4% reduced to 60% by 67%, and acetonitrile volume rises to 35% by 30%, and methyl alcohol volume rises to 5% by 3%; 30 ℃ of column temperatures, flow velocity 1.0mL/ minute, detects wavelength 280nm, sample size 20 μ L.

2. preparing standard solution

Preparing standard solution: salvianolic acid B (lot number A5502-5G), rosmarinic acid (lot number 536954-5G) are purchased from Sigma-ALDRICH, tanshinone IIA (lot number 110766-200417) is purchased from Nat'l Pharmaceutical & Biological Products Control Institute, and reference substance purity is all greater than 99%.Precision takes each 10.0mg of above reference substance, and the methanol solution that the volume fraction of take is 75% is joined to obtain the concentration standard substance mother liquor that is 10.0mg/mL as solvent, is placed in 4 ℃ of refrigerators and saves backup.During experiment, get respectively the standard solution that standard substance mother liquor is diluted to concentration gradient, sample introduction analysis after 0.22 μ m filtering with microporous membrane.

The eluent gradient elution program adopting in the present invention, rosmarinic acid retention time is 24.82 ± 0.08 minutes, and salvianolic acid B retention time is 28.17 ± 0.42 minutes, and the retention time of Tanshinone I I A is 98.84 ± 0.27 minutes.Peak shape is good, can guarantee the separated of above three kinds of materials and other materials in the red sage root.

(2) drawing standard curve

Draw respectively rosmarinic acid and salvianolic acid B standard substance mother liquor and be diluted to 2.0mg/mL, 1.0mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, a 0.0625mg/mL6 concentration gradient, sample introduction analysis after 0.22 μ m filtering with microporous membrane, drawing standard curve.According to above-mentioned corresponding chromatographic condition sample introduction, record collection of illustrative plates and chromatographic parameter, with rosmarinic acid peak area, standard substance concentration is carried out to regression analysis respectively, the equation of linear regression that obtains rosmarinic acid is:

y ₁＝6×10 ^-8x ₁-0.015

Correlation coefficient r ₂ ²be 0.9990, x in formula ₁the peak area that represents rosmarinic acid in sample, y ₁the concentration that represents rosmarinic acid, the mg/mL of unit.With salvianolic acid B peak area, standard substance concentration is carried out to regression analysis, the equation of linear regression that obtains salvianolic acid B is:

y ₂＝4×10 ^-8x ₂-0.006

Correlation coefficient r ₂ ²be 0.9990, x in formula ₂the peak area that represents salvianolic acid B in sample, y ₂the concentration that represents salvianolic acid B, the mg/mL of unit.

Draw Tanshinone II A standard substance mother liquor and be diluted to 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.05mg/mL, a 0.025mg/mL5 concentration gradient, sample introduction analysis after 0.22 μ m filtering with microporous membrane, drawing standard curve.According to above-mentioned corresponding chromatographic condition sample introduction, record collection of illustrative plates and chromatographic parameter, with Tanshinone II A, standard substance concentration is carried out to regression analysis, the equation of linear regression that obtains Tanshinone II A is:

y ₃＝1×10 ^-8x ₄+0.0122

Coefficient R ²be 0.9952.

(3) preparation sample solution

The growth red sage root of 100 days is placed in to 30 ℃ of baking ovens to dry to constant weight, with mortar, grind, take respectively transgenosis and each 25mg of contrast red sage root plant dried powder, be placed in 1.5mL centrifuge tube, add the methanol aqueous solution that 500 μ L volume fractions are 75%, 30 ℃ with the ultrasonic extraction of frequency 30KHz 20 minutes, and 12000 revs/min centrifugal 6 minutes.Shift supernatant stand-by to new centrifuge tube; Residue extracts twice by same procedure again, merge three times supernatant liquor, through 0.22 μ m filtering with microporous membrane, with high performance liquid chromatograph, analyze, record the peak area of rosmarinic acid, salvianolic acid B and Tanshinone II A in each sample, substitution equation of linear regression, calculates the content of rosmarinic acid, salvianolic acid B and Tanshinone II A.

Content with rosmarinic acid and salvianolic acid B in high-performance liquid chromatogram determination transgenosis red sage root strain, the results are shown in Figure 4,5.As seen from Figure 5, in the growth genetically modified red sage root dry root of 100 days, the content of rosmarinic acid is 16.96 ± 0.90mg/g, the content of salvianolic acid B is 57.64 ± 1.94mg/g, Tanshinone II A content is 2.04 ± 0.19mg/g, is respectively rosmarinic acid (12.90 ± 0.48mg/g) in non-transformed common red sage root dry root of contemporaneously, salvianolic acid B (34.04 ± 0.89mg/g), Tanshinone II A (0.78 ± 0.11mg/g) 1.31 times, 1.69 times, 2.62 times.

Claims

1. with tomato dna and red sage root Inner source gene, improve a method for the pest-resistant and activeconstituents of the red sage root, by following step, formed:

(1) tomato prosystemin gene clone

Use E.Z.N.A. ^tMplant RNA Kit extracts test kit, and the total RNA of separation and Extraction tomato, with PrimeScript RT reagent Kit reverse transcription test kit, becomes cDNA standby the total RNA reverse transcription of tomato to specifications to specifications, adopt Primer Premier5.0 software, the upstream primer of design tomato prosystemin genes encoding frame, downstream primer, take tomato cDNA as template, carry out polymerase chain reaction, reaction system is: get tomato cDNA and archaeal dna polymerase, DNA polymerase buffer liquid, dNTP, upstream primer, downstream primer, the volume ratio of redistilled water is 1:0.5:5:5:1:1:37.5, according to following conditioned response: 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, obtain tomato prosystemin gene, and before the upstream primer of tomato prosystemin gene, introduce Spe I restriction enzyme site and protect bases G ACTAGT, before downstream primer, introduce BstE II restriction enzyme site and protection base CAGGGTAACC, with tomato prosystemin gene primer:

Upstream primer prosystemin-F5 '-GACTAGTATGGGAACTCCTTCATATGATATC-3 '

Downstream primer prosystemin-R5 '-CAGGGTAACCCGTTTGTCTGTTATTATTTGAG-3 '

Take tomato cDNA as template, by following reaction system, mix: tomato cDNA and archaeal dna polymerase, DNA polymerase buffer liquid, dNTP, prosystemin-F, prosystemin-R, the volume ratio of redistilled water is 2:0.5:5:5:1:1:36.5, and according to 95 ℃ 3 minutes, 95 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 45 seconds, circulate 35 times, carry out polymerase chain reaction (PCR) amplification, the tomato prosystemin gene that must contain restriction enzyme site, the amplified production obtaining carries out electrophoretic separation, reclaiming amplified production is connected by ligase enzyme working instructions with T4DNA ligase enzyme with pMD19-T carrier, form prosystemin-pMD19-T carrier, and adopt heat shock conversion method to proceed in bacillus coli DH 5 alpha competent cell, the volume ratio of prosystemin-pMD19-T carrier and bacillus coli DH 5 alpha is 1:9.5～10.5, select gained positive colony, through bacterium colony polymerase chain reaction, detect, sequence verification obtains tomato prosystemin gene order, this gene order is as follows:

(2) build the plant expression vector that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment

iCOMT-F：5′-CC ATCGAT? GGTACCACCCTCCCCGACGGCGGCGTCCA-3′

iCOMT-R：5′-GCTCTAGA CTCGAG? GGATCCCACACCACCTACAACCACCTCCT-3′；

Use polymerase chain amplified reaction, Cla I and two restriction enzyme sites of Kpn I are introduced in the upstream of this 345bp, in its downstream, introduce Xho I and two restriction enzyme sites of BamH I; Through the amplification of polymerase chain amplified reaction, must contain the product C OMTi of Cla I/Kpn I and Xho I/BamH I, connect p-MD19T carrier and obtain COMTi-pMD19T carrier; With Xho I and Kpn I respectively enzyme cut COMTi-pMD19T carrier, pKannibal carrier, by cut the fragment of COMTi-pMD19T carrier and the fragment of cutting pKannibal carrier through Xho I, Kpn I enzyme with Xho I, KpnI enzyme, with DNA ligase, be connected to pKannibal+ intermediate carrier; With BamH I and Cla I respectively enzyme cut COMTi-pMD19T carrier, pKannibal+ carrier, with containing the fragment that BamH I cuts COMTi-pMD19T carrier with Cla I enzyme, be connected with DNA ligase with the fragment of cutting pKannibal+ with Cla I enzyme through BamH I, the novel vector of acquisition is pKANNIBAL/COMTi;

With Sac I and Pst I double digestion pKANNIBAL/COMTi, pCAMBIA1302-prosystemin carrier, frame is transcribed in the interference that double digestion pKANNIBAL/COMTi is obtained, be connected on the pCAMBIA1302-prosystemin carrier after double digestion, obtain pCAMBIA1302/prosystemin+pKANNIBAL/COMTi carrier, and proceed in bacillus coli DH 5 alpha and increase, extract plasmid, with enzyme blanking method, identify, obtain the plant expression vector pCAMBIA1302/prosystemin+pKANNIBAL/COMTi that contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment, called after pCPKC carrier,

(3) the agrobacterium tumefaciens bacterial strain of the plant expression vector that preparation contains tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interference fragment

35S-F5′-TACAAAGGCGGCAACAAACG-3′

35S-R5′-GCAATGGAATCCGAGGAGGT-3′

Carry out polymerase chain reaction, screening, obtains the agrobacterium tumefaciens bacterial strain that contains pCPKC carrier;

(4) prepare transgenosis red sage root plant

2. the method that improves the pest-resistant and activeconstituents of the red sage root with tomato dna and red sage root Inner source gene according to claim 1, it is characterized in that at structure of the present invention, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the step (2) of plant expression vector of fragment, described use Spe I and BstE II double digestion prosystemin-pMD19-T carrier, plant expression vector pCAMBIA-1302 is: get and contain prosystemin-pMD19-T carrier, 10 * M Buffer, restriction enzyme Spe I, restriction enzyme BstE II, redistilled water was in 37 ℃ of reactions 2 hours 20 minutes, restriction enzyme Spe I and restriction enzyme BstE II, 10 * M Buffer, contain prosystemin-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get plant expression vector pCAMBIA-1302,10 * M Buffer, restriction enzyme Spe I, restriction enzyme BstE II, redistilled water in 37 ℃ of reactions 3 hours, the volume ratio of restriction enzyme Spe I and restriction enzyme BstE II, 10 * M Buffer, plant expression vector pCAMBIA-1302, redistilled water is 1:1:2:6:10.

3. the method that improves the pest-resistant and activeconstituents of the red sage root with tomato dna and red sage root Inner source gene according to claim 1, it is characterized in that at structure of the present invention, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the step (2) of plant expression vector of fragment, described with Xho I and Kpn I respectively enzyme cut COMTi-pMD19T carrier, pKannibal carrier is: get COMTi-pMD19-T carrier, 10 * M Buffer, restriction enzyme Xho I, restriction enzyme Kpn I, redistilled water, 37 ℃ are reacted 2 hours 40 minutes, restriction enzyme Xho I and restriction enzyme Kpn I, 10 * M Buffer, COMTi-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pKannibal carrier, 10 * M Buffer, restriction enzyme Xho I, restriction enzyme Kpn I, redistilled water, 37 ℃ are reacted 2 hours 40 minutes, and the volume ratio of restriction enzyme Xho I and restriction enzyme Kpn I, 10 * M Buffer, pKannibal carrier, redistilled water is 1:1:2:6:10.

4. the method that improves the pest-resistant and activeconstituents of the red sage root with tomato dna and red sage root Inner source gene according to claim 1, it is characterized in that at structure of the present invention, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the step (2) of plant expression vector of fragment, described with BamH I and Cla I respectively enzyme cut COMTi-pMD19T carrier, pKannibal+ carrier is: get and contain COMTi-pMD19T carrier, 10 * K Buffer, restriction enzyme Cla I, restriction enzyme BamH I, redistilled water, 37 ℃ are reacted 2 hours 30 minutes, restriction enzyme Cla I and restriction enzyme BamH I, 10 * K Buffer, COMTi-pMD19-T carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pKannibal+ carrier, 10 * K Buffer, restriction enzyme Cla I, restriction enzyme BamH I, redistilled water, 37 ℃ are reacted 2 hours 30 minutes, and the volume ratio of restriction enzyme Cla I and restriction enzyme BamH I, 10 * K Buffer, pKannibal+ carrier, redistilled water is 1:1:2:6:10.

5. the method that improves the pest-resistant and activeconstituents of the red sage root with tomato dna and red sage root Inner source gene according to claim 1, it is characterized in that at structure of the present invention, containing tomato prosystemin gene and red sage root caffeoyl-O-methyl transferase gene interferes in the step (2) of plant expression vector of fragment, described use Sac I and Pst I double digestion pKANNIBAL/COMTi, pCAMBIA1302-prosystemin carrier is: get pKANNIBAL/COMTi carrier, 10 * M Buffer, restriction enzyme Sac I, restriction enzyme Pst I, redistilled water, 37 ℃ are reacted 3 hours, restriction enzyme Sac I, restriction enzyme Pst I, 10 * M Buffer, pKANNIBAL/COMTi carrier, the volume ratio of redistilled water is 1:1:2:6:10, get pCAMBIA-1302-prosystemin carrier, 10 * M Buffer, restriction enzyme Sac I, restriction enzyme Pst I, redistilled water in 37 ℃ of reactions 3 hours, the volume ratio of restriction enzyme Sac I and restriction enzyme Pst I, 10 * M Buffer, pCAMBIA1302-prosystemin carrier, redistilled water is 1:1:2:6:10.