CN107022553A

CN107022553A - Chinese herbaceous peony HSP70 genes and its plant expression vector and application

Info

Publication number: CN107022553A
Application number: CN201710431200.6A
Authority: CN
Inventors: 陶俊; 赵大球; 夏星; 苏江洪; 魏梦苒; 孙静
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2017-06-08
Filing date: 2017-06-08
Publication date: 2017-08-08

Abstract

The invention belongs to bioengineering field, and in particular to Chinese herbaceous peony HSP70 gene orders and its plant expression vector and application.The Chinese herbaceous peony HSP70 genes, its sequence is as shown in SEQ ID NO.1.The gene can be used for the heat-resisting ability for improving plant and prepare high temperature resistant new germ plasm.Chinese herbaceous peony ' purple phoenix feather ' PlHSP70 gene plant expression vectors that the present invention is built can be directly used for Agrobacterium-mediated genetic transformation, improve heat-resisting ability, the initiative high temperature resistant new germ plasm of plant.

Description

Chinese herbaceous peony HSP70 genes and its plant expression vector and application

Technical field

The invention belongs to bioengineering field, and in particular to Chinese herbaceous peony HSP70 gene orders and its plant expression vector and should With.

Background technology

Chinese herbaceous peony (Paeonia lactiflora Pall.), Paeoniaceae Paeonia persistent root herbs are the tradition of China Famous flower, is also always flourishing symbol.Chinese herbaceous peony is long because of its cultivation history, various in style, and flower pattern is enriched, large flower and brilliant color, Transplant extensively in private savings garden and urban garden and green space system.Chinese herbaceous peony property likes Cold and cool climate, but with Urbanization in China not The disconnected fast development accelerated with gardens gardening cause, many southern areas such as Nanjing, Suzhou, Yangzhou, Changsha, Zhejiang Chinese herbaceous peony plantation is all introduced in Hangzhou one after another, expands the cultural area and area of Chinese herbaceous peony, and cultivar is all introduced in Henan substantially The northern areas such as Luoyang, Shandong Heze.Because the summer temperature of southern area is high, high-temperature duration is long, most of north Chinese herbaceous peony Medicine kind shows, to altithermal inadaptable, to have had a strong impact on appreciation effect, constrained Chinese herbaceous peony in South China Urban Promote and application.In order to solve this difficulty, the Chinese herbaceous peony material for adapting to southern area Summer High Temperature weather is obtained, Chinese herbaceous peony is improved Medicine heat-resisting ability has turned into the vital task that current Chinese herbaceous peony produces.

Heat shock protein (HSPs, heat shock proteins) be different kind organism cell high temperature, arid, heavy metal from The class stress protein that the adverse circumstances such as son stress are synthesized extensively after stimulating, they participate in cytoprotection in the form of molecular chaperones.Its In, high temperature is the principal element for inducing HSPs synthesis, is required constituent of the plant to environment stress short-term regulation.According to point Heat shock protein, can be divided into 6 families by the difference of son amount, i.e. HSP110, HSP90, HSP70, HSP60, small molecule smHSP and general Element.In the research to heat shock protein family member, current HSP70 families are of greatest concern.HSP70 is most protected in HSPs families Keep, a most important albuminoid, HSP70 can prevent protein degradation under the conditions of high temperature stress, be conducive to the renaturation of albuminate, Therefore, it is closely related with the heat-resisting ability of plant.But HSP70 family members are a lot, on different plants it is specific which Member can regulate and control its high temperature resistant stress ability and not yet know.At present, HSP70 report focuses primarily upon field crop, vegetables Deng on, and on Chinese herbaceous peony, the HSP70 gene families member that can improve high temperature resistant stress ability has no report always.

In early-stage Study, we have found that Chinese herbaceous peony ' purple phoenix feather ' heat-resisting ability relatively strong (Zhao great Qiu, Han Chenxia, pottery Pretty differences Cultivars of Chinese Herbaceous Peony Heat tolerance identification Yangzhou Universitys journal (agricultural and life science version), 2015,36 (4):105-109), But the relative HSP70 gene family members that can improve high temperature resistant stress ability do not appear in the newspapers.

The content of the invention

It is an object of the invention to the heat-resisting ability for improving plant, there is provided new Chinese herbaceous peony ' purple phoenix feather ' high temperature resistant base Because of PlHSP70 DNA sequence dna.

The present invention also provides high temperature resistant gene PlHSP70 plant expression vector and its construction method and application.Institute's structure The plant expression vector built can be directly used for agriculture bacillus mediated Genetic Transformation in Higher Plants, improve heat-resisting ability, the initiative of plant High temperature resistant new germ plasm.

The technical solution adopted by the present invention：

A kind of high temperature resistant gene PlHSP70, the DNA sequence dna of the gene is SEQ ID NO.1.It is ' purple that the gene is derived from Chinese herbaceous peony Phoenix plumage '.

It is ' purple by Chinese herbaceous peony of the present invention the invention also discloses the plant expression vector containing above-mentioned PlHSP70 genes Phoenix plumage ' high temperature resistant gene PlHSP70 cDNA sequence (SEQ ID NO.2) and mesophyte expression vector pCAMBIA1301 structures Into.

Chinese herbaceous peony ' purple phoenix feather ' high temperature resistant gene PlHSP70 plant expression vectors, its construction method is as follows：

(1) Chinese herbaceous peony ' purple phoenix feather ' high temperature resistant gene PlHSP70 cDNA sequence clone：With Chinese herbaceous peony ' purple phoenix feather ' young leaflet tablet For material, total serum IgE is extracted, reverse transcription is cDNA, design primer expands the cDNA sequence of PlHSP70 genes, sense primer PlHSP70-CF：5 '-ATGGCAGGCAAAGGAGAAGGACCG-3 ' (SEQ ID NO.3), anti-sense primer PlHSP70-CR：5′- TTAGTCCACCTCTTCAATCTTGGG-3 ' (SEQ ID NO.4), using the cDNA of reverse transcription as template, enters performing PCR amplification, production Thing is connected to pEASY^TM- T5 Zero Cloning Vector carriers, convert Trans-T1 competent cells, then additional Screening positive clone carries out sequencing on the LB flat boards of 0.1% ampicillin；

(2) plant expression vector pCAMBIA1301-PlHSP70 structure：Design primer using Chinese herbaceous peony ' purple phoenix feather ' cDNA as Template, enters performing PCR amplification, and BamH I and Kpn I digestions position is introduced respectively in the upstream and downstream of PlHSP70 gene cDNA sequences Point, sense primer PlHSP70-MF：5 '-CGCGGATCCATGGCAGGCAAAGGA-3 ' (SEQ ID NO.5), anti-sense primer PlHSP70-MR：5′-CGGGGTACCTTAGTCCACCTCTTCAA-3′(SEQ ID NO.6).PCR primer is connected to pEASY^TM- T₅Zero Cloning Vector carriers, convert Trans-T₁Competent cell, then in additional 0.1% ampicillin Screening positive clone on LB flat boards, extract positive plasmid, the PlHSP70 fragments of BamH I and Kpn I double digestions and BamH I and The pCAMBIA 1301 of Kpn I double digestions is connected, converted, and extracts positive plasmid, digestion, electrophoresis detection and sequence verification, plant Expression vector pCAMBIA1301-PlHSP70 is successfully constructed.

(3) Chinese herbaceous peony high temperature resistant gene PlHSP70 plant expression vector is used for agriculture bacillus mediated Genetic Transformation in Higher Plants, carries High plant heat-resisting ability, method is as follows：Agrobacterium strains EHA105 competence is prepared and freeze-thaw method conversion；Arabidopsis floral is soaked Dye and seed screening；Transfer-gen plant is identified and heat-resisting ability evaluation.

Beneficial effects of the present invention are embodied in：

1st, Chinese herbaceous peony ' purple phoenix feather ' PlHSP70 that the present invention is provided is a new adversity gene on Chinese herbaceous peony, and the gene can be carried The heat-resisting ability of high plant.

2nd, Chinese herbaceous peony ' purple phoenix feather ' the PlHSP70 gene plants expression vector that the present invention is built can be used directly to report first In Agrobacterium-mediated genetic transformation, heat-resisting ability, the initiative high temperature resistant new germ plasm of plant are improved.

Brief description of the drawings

In the agarose gel electrophoresis detection of the DNA sequence dna amplified production of Fig. 1 PlHSP70 genes, figure：M：DNA Marker DL2000；1：The DNA sequence dna amplified production of PlHSP70 genes.

In the agarose gel electrophoresis detection of the cDNA sequence amplified production of Fig. 2 PlHSP70 genes, figure：M：DNA Marker DL2000；1：The cDNA sequence amplified production of PlHSP70 genes.

In the agarose gel electrophoresis detection of Fig. 3 pCAMBIA1301-PlHSP70 plasmid double digestion products, figure：M：DNA Marker DL2000；1：PCAMBIA1301-PlHSP70 plasmid double digestion products.

Fig. 4 plant expression vector pCAMBIA1301-PlHSP70 collection of illustrative plates.

In the identification of Fig. 5 transfer-gen plants, figure：A：GUS dyeing identifications；B：PCR is identified.

The phenotype of Arabidopsis plant after Fig. 6 high temperature stress.

Arabidopsis plant index of correlation is determined after Fig. 7 high temperature stress, in figure：A：The relative expression levels of PlHSP70 genes； B：Relative conductivity；C：DAB decoration methods observe H₂O₂Accumulating level；D：Fluorescence probe method observes superoxide anion accumulating level.

Embodiment

Involved pCAMBIA1301 is commercial vectors.

Embodiment 1.PlHSP70 DNA sequence dna clone

From Chinese herbaceous peony ' purple phoenix feather ' young leaflet tablet as material, with reference to MiniBEST Plant Genomic DNA Extraction Kit kit (TaKaRa) specifications method extracts DNA.

Using the leaf DNA of extraction as template, design primer PlHSP70-DF and PlHSP70-DR enter performing PCR reaction：Upstream Primer PlHSP70-DF：5 '-CTCTTACTTTTCTTCTCTCGACCCCTTCCG-3 ' (SEQ ID NO.7), anti-sense primer PlHSP70-DR：5′-GAAGACCAATTACTTAGTCGACCTCTTCAA-3′(SEQ ID NO.8)；25 μ L reaction systems：10 ×RCR Buffer(Mg²⁺Plus) 2.5 each 1.25 μ L (10mM) of μ L, PlHSP70-DF, PlHSP70-DR primers, dNTP 2.0 μ L, Taq DNA Polymerase of mixture (2.5mM each), 0.25 μ L, cDNA templates 2.0 μ L, ddH₂O 15.75μ L；Response procedures：94 DEG C of pre-degeneration 3min, then 94 DEG C of denaturation 30sec, 61.7 DEG C of annealing 30sec, 72 DEG C of extension 4min, react 35 circulations, 72 DEG C of extension 10min；PCR detects (Fig. 1) after terminating using 1% agarose electrophoresis, and product uses TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 gel reclaims kits (TaKaRa) reclaim pure Change, product is connected to pEASY^TM- T5 Zero Cloning Vector carriers (Trans), convert Trans-T1 competent cells, Then screening positive clone is sequenced on the LB flat boards of additional 0.1% ampicillin, and sequencing is SEQ ID NO.1 (wherein 282-1721bp is introne).

Embodiment 2.PlHSP70 cDNA sequence clone

From Chinese herbaceous peony ' purple phoenix feather ' young leaflet tablet as material, with reference to MiniBEST Plant RNA Extraction Kit kits (TaKaRa) specification method extracts total serum IgE, according to PrimerScript^TM RT reagent Kit with GDNA Eraser kits (TaKaRa) take 1 μ g total serum IgEs reverse transcriptions into cDNA.

Using the leaf cDNA of extraction as template, design primer PlHSP70-F and PlHSP70-R enter performing PCR reaction：Draw upstream Thing PlHSP70-CF：5 '-ATGGCAGGCAAAGGAGAAGGACCG-3 ' (SEQ ID NO.3), anti-sense primer PlHSP70-CR： 5′-TTAGTCCACCTCTTCAATCTTGGG-3′(SEQ ID NO.4)；25 μ L reaction systems：10×RCR Buffer(Mg²⁺ Plus) 2.5 each 1.25 μ L (10mM) of μ L, PlHSP70-F, PlHSP70-R primers, dNTP mixture (2.5mM each) 2.0 μ 0.25 μ L, cDNA templates of L, Taq DNA Polymerase 2.0 μ L, ddH₂O 15.75μL；Response procedures：94 DEG C of pre-degenerations 3min, then 94 DEG C of denaturation 30sec, 56.7 DEG C of annealing 30sec, 72 DEG C of extension 2min, react 35 circulations, 72 DEG C of extensions 10min；PCR detects (Fig. 2) after terminating using 1% agarose electrophoresis, and product uses TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 gel reclaims kits (TaKaRa) recovery purifying, product is connected to pEASY^TM- T5 Zero Cloning Vector carriers (Trans), convert Trans-T1 competent cells, then additional Screening positive clone is sequenced on the LB flat boards of 0.1% ampicillin, and sequencing is SEQ ID NO.2.

The plant expression vector pCAMBIA1301-PlHSP70 of embodiment 3. structure

Design primer PlHSP70-MF, PlHSP70-MR enter performing PCR reaction, in the upstream of PlHSP70cDNA sequences with Trip introduces restriction enzyme site BamH I and Kpn I respectively, and PCR primer is connected to pEASY^TM- T5 Zero Cloning Vector are carried Body, converts Trans-T₁Competent cell, then on the LB flat boards of additional 0.1% ampicillin screening positive clone, carry Take positive plasmid, the PlHSP70 fragments of BamH I and Kpn I double digestions and the pCAMBIA1301 of BamH I and Kpn I double digestions Connection, conversion, extraction positive plasmid, digestion, simultaneously sequence verification is SEQ ID NO.2 to electrophoresis detection, is comprised the following steps that：Upstream Primer PlHSP70-MF：5 '-CGCGGATCCATGGCAGGCAAAGGA-3 ' (SEQ ID NO.5), anti-sense primer PlHSP70- MR：5′-CGGGGTACCTTAGTCCACCTCTTCAA-3′(SEQ ID NO.6).(1) made with Chinese herbaceous peony ' purple phoenix feather ' leaf cDNA For template, enter performing PCR reaction, 25 μ L reaction systems：10×RCR Buffer(Mg²⁺Plus) 2.5 μ L, PlHSP70-MF, Each 1.25 μ L (10mM) of PlHSP70-MR primers, μ L, the Taq DNA Polymerase of dNTP mixture (2.5mM each) 2.0 0.25 μ L, cDNA template 2.0 μ L, ddH₂O 15.75μL；Response procedures：94 DEG C of pre-degeneration 3min, then 94 DEG C are denatured 30sec, 57.6 DEG C of annealing 30sec, 72 DEG C of extension 2min, react 35 circulations, 72 DEG C of extension 10min；PCR primer uses TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 gel reclaims kits (TaKaRa) reclaim pure Change.

(2) expression vector pCAMBIA1301 plasmids and the PlHSP70 glue reclaim products containing restriction enzyme site is taken to use BamH respectively I and Kpn I double digestions, 20 μ L double digestion reaction systems：0.5 × K Buffer 2.0 μ L, plasmid pCAMBIA1301 or 10 μ L, BamH I of PlHSP70 glue reclaims product, 1.0 μ L, Kpn I 1.0 μ L, ddH₂O 6.0μL；37 DEG C of reaction 1.5h；Double enzymes Cut product and enter row agarose gel electrophoresis analysis, use TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 gel reclaims kits (TaKaRa) recovery purifying plasmid pCAMBIA1301 large fragments and PlHSP70 are large stretch of Section.The product of two recovery, 20 μ L coupled reaction systems are connected with T4 DNA ligases (TaKaRa)：10×T4 ligase The μ L of 2.0 μ L, PlHSP70 large fragment of Buffer 2.0 μ L, T4 DNA ligase, 1.0 μ L, pCAMBIA1301 large fragments 10, ddH₂O 5μL；65 DEG C of water-bath 10min after 22 DEG C of room temperature connection 15min, take 5 μ L connection products to convert Trans-T1 competence thin Born of the same parents, then 37 DEG C of incubated overnights on the LB flat boards of additional 0.05% kanamycins, the expansion culture of picking positive monoclonal, are extracted Plasmid pCAMBIA1301-PlHSP70, double digestion (Fig. 3) is carried out to plasmid, and sequence verification is SEQ ID NO.2.Plant table Successfully constructed (Fig. 4) up to carrier pCAMBIA1301-PlHSP70.

The plant expression vector pCAMBIA1301-PlHSP70 genetic transformations arabidopsis of embodiment 4. and its heat-resisting ability mirror It is fixed

(1) agrobacterium strains EHA105 competence is prepared and freeze-thaw method conversion

From picking EHA105 single bacterium colonies on YEB (50 μ g/mL rifampins) flat board, it is inoculated in 50mL and contains 50mg/L rifampins YEB fluid nutrient mediums in, 28 DEG C, 200rpm cultivated to OD₆₀₀Value 0.5, then ice bath bacterium solution 30min, goes to the 50mL of precooling In centrifuge tube, 4 DEG C, 5000rpm, centrifugation 10min collect thalline, be suspended in the 50mM CaCl of 2mL precoolings₂(15% glycerine) is molten In liquid, it is divided in after mixing with 100 μ L/ pipes in 1.5mL sterile centrifugation tubes, -80 DEG C of preservations are stand-by after liquid nitrogen frozen.

10 μ L pCAMBIA-miR156e-3p vector plasmids are taken, 200 μ L EHA105 competent cells, ice bath are added 30min, liquid nitrogen frozen 5min, 37 DEG C of 5min, add 800 μ L YEB fluid nutrient mediums, 28 DEG C, 200rpm preculture 4h, bacterium solution Coated plate is on YEB (the μ g/mL kanamycins of 50 μ g/mL rifampins+50) solid medium, 28 DEG C of light cultures 2 days, picking monoclonal PCR is detected, is chosen positive colony and is shaken bacterium, for arabidopsis floral conversion.

(2) arabidopsis floral is contaminated and seed screening

Positive monoclonal is connected in 50mL YEB (the μ g/mL kanamycins of 50 μ g/mL rifampins+50) fluid nutrient medium, 24h, 5000rpm centrifugation 20min are cultivated, then (1/2MS, adds 50g/L sucrose, and it is 5.8, Ran Houjia to adjust pH with conversion fluid 200 μ L/L Silwet L-77 mixing) acutely suspend to be precipitated to and hang completely.

Arabidopsis aerial part in full-bloom stage is directly soaked in 1min in above-mentioned suspension, preservative film sealed bundle is used Preservative film is opened after wrapping up in plant, light culture 12h, continued growth in suitable environment is placed in, harvested when seed maturity.

Seed disinfection and sowing：Seed is put into 1.5mL sterile centrifugation tube, addition 1mL thimerosals (75% alcohol+ 0.1% TritonX-100) 15min is uninterruptedly vibrated, then with 100% alcohol washes 1min, be repeated twice, draw arabidopsis Seed waits for peacefully air-dried on double-layer filter paper disk, then seed is uniformly seeded in screening and culturing medium (1/2MS+30g/L sucrose+ 6.5g/L agar+25mg/L ampicillin+25mg/L hygromycin, pH 5.8) screening and culturing 10 days, then by resistance transplantation of seedlings Into soil, seedling is covered 1 week with moisturizing with preservative film.

(3) transfer-gen plant identification and heat-resisting ability evaluation

(1) transfer-gen plant is identified：

1. GUS dyeing identification：229 μ L X-GlcA solvent are added in 80mg X-GlcA, its concentration is reached 350mg/mL, gently vibration is mixed, as X-GlcA Solution；According still further to GUS Buffer A 2.5mL, GUS Buffer B 10μL、GUS Buffer C 10μL、ddH₂O 5.5mL, the μ L proportions 10mL of methanol 2mL, X-GlcA Solution 20 GUS dyeing liquors；Arabidopsis thaliana Seedlings then are taken in 1.5mL sterile centrifugation tubes, add appropriate GUS dyeing liquors submergence complete stool children Seedling, 37 DEG C of incubation 24h, is decolourized, time interval 30min is finally gently pressed from both sides with tweezers using 100%-50% gradient concentrations alcohol Decolouring seedling is taken to take pictures observation.As can be seen that wild type Col-0 Arabidopsis plants are transparent whites from Fig. 5 A, and turn PlHSP70 plant are then dyed to blueness, show that expression vector pCAMBIA1301-PlHSP70 successfully imports arabidopsis.

2. PCR is identified：7~8 leaves of resistance seedling are treated, collection Arabidopsis leaf is material, with reference to RNAiso Plus (Total RNA is extracted) kit (TaKaRa) specification method extraction total serum IgE, according to PrimerScript^TM RT reagent Kit With gDNA Eraser kits (TaKaRa) take 1.0 μ g total serum IgEs reverse transcriptions into cDNA.CDNA obtained by reverse transcription is carried out PCR amplifications, electrophoresis detection, using arabidopsis Actin as reference gene, design primer is：Sense primer AtActin-F：5′- TCTCCCGCTATGTATGTCGC-3 ' (SEQ ID NO.9), anti-sense primer AtActin-R：5′- TAAGGTCACGTCCAGCAAGG-3′(SEQ ID NO.10)；The sense primer PlHSP70-CF of PlHSP70 amplifications：5′- ATGGCAGGCAAAGGAGAAGGACCG-3 ' (SEQ ID NO.3), anti-sense primer PlHSP70-CR：5′- TTAGTCCACCTCTTCAATCTTGGG-3′(SEQ ID NO.4)；25 μ L reaction systems：10×RCR Buffer(Mg²⁺ Plus) 2.5 μ L, each 1.25 μ L (10mM) of sense primer, anti-sense primer, dNTP mixture (2.5mM each) 2.0 μ L, LA0.25 μ L, cDNA template of high-fidelity enzyme 2.0 μ L, ddH₂O 15.75μL；Response procedures：94 DEG C of pre-degeneration 3min, then 94 DEG C denaturation 30sec, 62 DEG C (Actin) or 56.7 DEG C (PlHSP70) annealing 30sec, 72 DEG C extension 30sec, react 35 circulation, 72 DEG C of extension 10min；Detected using 1% agarose gel electrophoresis.As can be seen that wild type Col-0 arabidopsis is planted from Fig. 5 B PlHSP70 fragments are not amplified in strain, and turn that 1 band clearly become clear can be amplified in PlHSP70 plant, again show that table Arabidopsis is successfully imported up to carrier pCAMBIA1301-PlHSP70.

(2) plant heat-resisting ability is evaluated：

1. plant phenotype：It will identify that successful transgenic seedling is preposition under 40 DEG C of hot conditions at stress in bolting through PCR 48h is managed, the growing state of plant is then observed.Fig. 6 be growing state of the plant after high temperature stress, wild type Col-0 and turn The high temperature injury symptom that the blade of pCAMBIA1301 plant occurs in that yellow, crispaturaed, and turn the blade table of PlHSP70 plant It is now good, there is not above-mentioned high temperature injury symptom.

2. PlHSP70 gene expression doses：Using the blade of arabidopsis as material, with reference to RNAiso Plus (Total RNA Extract) kit (TaKaRa) specification method extraction total serum IgE, according to PrimerScript^TM RT reagent Kit with GDNA Eraser kits (TaKaRa) take 1.0 μ g total serum IgEs reverse transcriptions into cDNA.By the cDNA obtained by reverse transcription according toTip Green qPCR SuperMix kits (Trans) carry out qRT-PCR detections, with arabidopsis Actin For reference gene, design primer is：Sense primer AtActin-F：5′-TCTCCCGCTATGTATGTCGC-3′(SEQ ID NO.9)；Anti-sense primer AtActin-R：5′-TAAGGTCACGTCCAGCAAGG-3′(SEQ ID NO.10)；Design PlHSP70 Gene primer is：Sense primer PlHSP70-QF：5′-GAATGCTTTGGAGAACTATGCTTAC-3′(SEQ ID NO.11)； Anti-sense primer PlHSP70-QR：5′-CCACTGAATAGCCTGATCAATAGA-3′(SEQ ID NO.12)；25 μ L reaction systems：12.5 μ L, cDNA templates of Tip Green qPCR 2.0 μ L, each 1.0 μ L of sense primer, anti-sense primer (10mM), ddH₂O 8.5μL；Response procedures：94 DEG C of pre-degeneration 30sec, then 94 DEG C denaturation 5sec, 62 DEG C (Actin) or 57.2 DEG C (PlHSP70) annealing 30sec, 72 DEG C of extension 30sec, react 45 circulations, 65 DEG C -95 DEG C of solubility curve, per 5sec 0.5 DEG C of heating；Using formula 2^-△ΔCTMethod calculates the relative expression quantity of gene.As can be seen that PlHSP70 is wild from Fig. 7 A Expression in type Col-0 Arabidopsis plants differs larger with turning PlHSP70 plant, turns the expression of PlHSP70 plant For 11 times of Col-0.

3. other physical signs：When determining relative conductivity, weigh 1g blades and taken out with the syringe containing appropriate distilled water Vacuum is added in test tube after being sunk to the bottom to blade, is placed in test tube under distilled water cumulative volume 20mL, normal temperature and conductivity meter is used after 3h (thunder magnetic DDS-307A, China) determines electrical conductivity E1, while determining the electrical conductivity E0 of distilled water (blank)；Then test tube is sealed After boiling 30min in boiling water bath, electrical conductivity E2 is measured after cooling again, blade relative conductivity is calculated by following equation：Relatively Electrical conductivity (%)=(E1-E0)/(E2-E0) × 100%.As can be seen that the phase of wild type Col-0 Arabidopsis plants from Fig. 7 B Electrical conductivity is significantly higher than and turns PlHSP70 plant, about turns 1.9 times of PlHSP70 plant.Then, on the one hand dyed using DAB Method observes H₂O₂Accumulating level, blade is immersed in using 50mM Tris- acetate buffers (pH5.0) 0.1mg/ that is made into In mL DAB dyeing liquors, 25 DEG C of placement 24h, then boil 15min in 95% alcohol and are taken pictures again under dark condition； On the other hand, with reference to active somatic cell oxidative stress active oxygen (ROS) native staining kit (superoxide anion) (breathing out spirit in Shanghai) Specification method observes the accumulating level of superoxide anion.Wild type Col-0 Arabidopsis plants are can be seen that from Fig. 7 C DAB dye levels and ROS fluorescence signal are relatively strong, H₂O₂It is all remarkably higher than with superoxide anion accumulating level and turns PlHSP70 plants Strain.

In summary, the present invention constructs the plant expression vector containing Chinese herbaceous peony adversity gene PlHSP70 PCAMBIA1301-PlHSP70, the wherein DNA sequence dna of PlHSP70 genes are to report first.Constructed carrier can import plant In, improve growth potential of the Arabidopsis plant under high temperature stress.

SEQUENCE LISTING

<110>Yangzhou University

<120>Chinese herbaceous peony HSP70 genes and its plant expression vector and application

<130>

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 3473

<212> DNA

<213>Chinese herbaceous peony

<400> 1

ctcttacttt tcttctctcg accccttccg cccttttccg cctctttgtt cagatcaaga 60

acaaacaatg gcaggcaaag gagaaggacc ggccattggc atcgaccttg gtacaaccta 120

cccctgtgta ggagtttggc agcatgatcg ggttgagatc atagccaatg accaaggaaa 180

caggactaca ccttcttatg ttgctttcac cgattctgag cgtttgattg gtgatgcagc 240

caagaatcag gtggccatga accccaccaa caccgttttc ggtaagattc tctcttcaga 300

tctctaagct tgtttatgaa tttgttctgt ttgtaatgta catactctgt attagtaatt 360

cacaatattt tggatctcag attcgttatt ttcaatgttt ataatatcta tatggtggat 420

ctgaacctgg gtttgtaaag ttgataactt tgtagatcca tctagatttc tcttattttc 480

gttgtctcaa atttgtatct ttagctcacg agtttcgttt tacccccaaa tttgttaatt 540

tgtttaatgt ttattagctc atgagttaat ttaatttgtt gttttatgag ggctctgaaa 600

gcaagtagaa accaaattgt agctctagta gaatgaaaca ggtgttgagc cgacatgtaa 660

ggccagatgt atagatcgaa acaatgaaac aggggtttta cccgtatgta aggccgggag 720

tacagatccg gttctgattg atatcgtggg tttaaagtta gtcataaaga ttctactttt 780

ggatccaatc tatattagcc ttgcttttta gatccaatct attatagcgt gctgtacaga 840

tcttgatttt agtatactca aatatggggt cttgcttatc agagtcgatt tctgattctt 900

ggattcagtt tgcagcgtaa gaatcagtga atctcttttt ttgaattcct aaaaaaggat 960

cttatggggt ggattcaaca tgacttgagt tgaatgactc tgttgaactc aagggtacaa 1020

tatagaggag gcacagccat atgcatcaca tataatatga aactttagca caccagctct 1080

gcaagtcgtt aacagatggc taagtgaatg gaattatttg gtctttgttt aggtttctaa 1140

tttatattac aacttgctta tatgttactt atcaacttgc ttataagtta cttataatta 1200

ttttttataa tattatttca tgaaattata tgcattactt tgcttatatt ttacttatca 1260

atttgtttat aagttactta taattatttt ttataatatt atttcatgaa attatatgca 1320

ttactgctca tatgttatta ttaatgtgat gtccacttgt tacatgctac atttttaatg 1380

aagtgtctac ttgtatttat ttatagggct atacatttat atatgacgat ttcttttacg 1440

atttacttag tttataatga ttgtagatgt taattatgat ttttttatgt agtgtccact 1500

tgttgttaca tgctacgttt atatattatg cgtcttttgg agatgtgagt ttgtaatagg 1560

gttatttgca tgatctgttg tgttctgtgt actttcaaga tatctgggtg agtcttttgt 1620

ttataatgac tgtagatgtt aattagattt cttttaatgg ttaaattttt tttatttaaa 1680

tgattgtttg ttcttacatt ttttggcttt acaattgtca gatgcaaaac ggttgattgg 1740

caggagattt agtgatgcat cagtccagag tgatatcaag ttgtggccat tcaaacttat 1800

ccctggtcct gctgagaagc ccatgattgc tgtcaactac aagggtgaag agaagcagtt 1860

tgctgctgag gagatctctt caatggttct gatgaagatg cgcgaaattg cagaggcctt 1920

tcttgggtcc actgtgaaga atgctgttgt gactgtgcct gcatacttca acgactctca 1980

gagacaggct accaaggatg ctggtgtcat tgctggtctc aatgtcatgc gtattatcaa 2040

tgagccaaca gctgctgcca ttgcttatgg tcttgacaag aaagccacca gtgttggaga 2100

gaagaatgtg ctcatctttg atctgggagg tggtactttt gatgtctccc ttctaaccat 2160

tgaagagggt atctttgaag tgaaagccac agctggtgac actcatcttg gtggagagga 2220

ctttgacaac agaatggtta accactttgt ccaggagttc aagcgtaaga acaagaagga 2280

catcagcggt aaccccagag ctcttaggag attgaggaca tcttgcgaga gggcaaagag 2340

gactctctct tccactgctc aaaccaccat tgagattgat tctttatttg agggtattga 2400

cttttattcc actgtcaccc gtgccagatt tgaggagctc aacatggatc tcttcagaaa 2460

gtgtatggac cctgttgaga agtgtttaag agatgctaaa atggataaga gtaccgtcca 2520

tgaagttgtt cttgttggtg gatctactag aattcccaag gtgcaacaac tcttgcagga 2580

cttcttcaat ggtaaggagc tttgcaagag catcaaccct gatgaggctg ttgcctatgg 2640

tgctgctgta caagctgcta ttctgagcgg tgaaggaaat gagaaggttc aagacttgtt 2700

gctgttggat gtcacacctc tctcacttgg attggaaact gccggagggg tcatgactgt 2760

cctgatccaa agaaacacca ccattccaac caagaaggaa caggtcttct ctacctactc 2820

agataaccaa cccggtgtgt tgattcaggt atacgagggt gaaagaacaa ggacccgaga 2880

caacaattta ttgggcaagt ttgagctgtc tggcattcct cctgccccca ggggtgttcc 2940

tcagatcact gtctgctttg atattgatgc taatggtatc ctcaatgtct ctgcggagga 3000

caagacaacc gggcagaaga acaagatcac catcaccaac gacaagggca ggctctctaa 3060

agaggagatt gagaagatgg ttcaggaagc agagaagtac aagtcagagg atgaagaaca 3120

caagaagaaa gctgaatcca agaatgcttt ggagaactat gcttacaaca tgaggaacac 3180

aatcaggggt gacaagattg gtgccaagct tgacccagct gacaagaaga agattgagga 3240

ttctattgat caggctattc agtggcttga tgctaaccaa cttgccgaag ctgatgagtt 3300

tgaggacaag atgaaggagc ttgagagcgt ttgtaatcca atcattgcta agatgtacca 3360

gggtgctggt ggtgacatgg gtggtgctgc catggatgat gatgatgctg ttccagctgg 3420

tggaagcggt gctggtccca agattgaaga ggtcgactaa gtaattggtc ttc 3473

<210> 2

<211> 1953

<212> DNA

<213>Chinese herbaceous peony

<400> 2

atggcaggca aaggagaagg accggccatt ggcatcgacc ttggtacaac ctacccctgt 60

gtaggagttt ggcagcatga tcgggttgag atcatagcca atgaccaagg aaacaggact 120

acaccttctt atgttgcttt caccgattct gagcgtttga ttggtgatgc agccaagaat 180

caggtggcca tgaaccccac caacaccgtt ttcgatgcaa aacggttgat tggcaggaga 240

tttagtgatg catcagtcca gagtgatatc aagttgtggc cattcaaact tatccctggt 300

cctgctgaga agcccatgat tgctgtcaac tacaagggtg aagagaagca gtttgctgct 360

gaggagatct cttcaatggt tctgatgaag atgcgcgaaa ttgcagaggc ctttcttggg 420

tccactgtga agaatgctgt tgtgactgtg cctgcatact tcaacgactc tcagagacag 480

gctaccaagg atgctggtgt cattgctggt ctcaatgtca tgcgtattat caatgagcca 540

acagctgctg ccattgctta tggtcttgac aagaaagcca ccagtgttgg agagaagaat 600

gtgctcatct ttgatctggg aggtggtact tttgatgtct cccttctaac cattgaagag 660

ggtatctttg aagtgaaagc cacagctggt gacactcatc ttggtggaga ggactttgac 720

aacagaatgg ttaaccactt tgtccaggag ttcaagcgta agaacaagaa ggacatcagc 780

ggtaacccca gagctcttag gagattgagg acatcttgcg agagggcaaa gaggactctc 840

tcttccactg ctcaaaccac cattgagatt gattctttat ttgagggtat tgacttttat 900

tccactgtca cccgtgccag atttgaggag ctcaacatgg atctcttcag aaagtgtatg 960

gaccctgttg agaagtgttt aagagatgct aaaatggata agagtaccgt ccatgaagtt 1020

gttcttgttg gtggatctac tagaattccc aaggtgcaac aactcttgca ggacttcttc 1080

aatggtaagg agctttgcaa gagcatcaac cctgatgagg ctgttgccta tggtgctgct 1140

gtacaagctg ctattctgag cggtgaagga aatgagaagg ttcaagactt gttgctgttg 1200

gatgtcacac ctctctcact tggattggaa actgccggag gggtcatgac tgtcctgatc 1260

caaagaaaca ccaccattcc aaccaagaag gaacaggtct tctctaccta ctcagataac 1320

caacccggtg tgttgattca ggtatacgag ggtgaaagaa caaggacccg agacaacaat 1380

ttattgggca agtttgagct gtctggcatt cctcctgccc ccaggggtgt tcctcagatc 1440

actgtctgct ttgatattga tgctaatggt atcctcaatg tctctgcgga ggacaagaca 1500

accgggcaga agaacaagat caccatcacc aacgacaagg gcaggctctc taaagaggag 1560

attgagaaga tggttcagga agcagagaag tacaagtcag aggatgaaga acacaagaag 1620

aaagctgaat ccaagaatgc tttggagaac tatgcttaca acatgaggaa cacaatcagg 1680

ggtgacaaga ttggtgccaa gcttgaccca gctgacaaga agaagattga ggattctatt 1740

gatcaggcta ttcagtggct tgatgctaac caacttgccg aagctgatga gtttgaggac 1800

aagatgaagg agcttgagag cgtttgtaat ccaatcattg ctaagatgta ccagggtgct 1860

ggtggtgaca tgggtggtgc tgccatggat gatgatgatg ctgttccagc tggtggaagc 1920

ggtgctggtc ccaagattga agaggtcgac taa 1953

<210> 3

<211> 24

<212> DNA

<213>Artificial sequence

<400> 3

atggcaggca aaggagaagg accg 24

<210> 4

<211> 24

<212> DNA

<213>Artificial sequence

<400> 4

ttagtccacc tcttcaatct tggg 24

<210> 5

<211> 24

<212> DNA

<213>Artificial sequence

<400> 5

cgcggatcca tggcaggcaa agga 24

<210> 6

<211> 26

<212> DNA

<213>Artificial sequence

<400> 6

cggggtacct tagtccacct cttcaa 26

<210> 7

<211> 30

<212> DNA

<213>Artificial sequence

<400> 7

ctcttacttt tcttctctcg accccttccg 30

<210> 8

<211> 30

<212> DNA

<213>Artificial sequence

<400> 8

gaagaccaat tacttagtcg acctcttcaa 30

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<400> 9

tctcccgcta tgtatgtcgc 20

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<400> 10

taaggtcacg tccagcaagg 20

<210> 11

<211> 25

<212> DNA

<213>Artificial sequence

<400> 11

gaatgctttg gagaactatg cttac 25

<210> 12

<211> 24

<212> DNA

<213>Artificial sequence

<400> 12

ccactgaata gcctgatcaa taga 24

Claims

1. Chinese herbaceous peony HSP70 genes, its sequence is as shown in SEQ ID NO.1.

2. the plant expression vector containing the cDNA sequence of Chinese herbaceous peony HSP70 genes described in claim 1, it is characterised in that described CDNA sequence is as shown in SEQ ID NO.2.

3. plant expression vector as claimed in claim 2, it is characterised in that by Chinese herbaceous peony PlHSP70 cDNA sequence and centre Plant expression vector pCAMBIA1301 is constituted.

4. the Chinese herbaceous peony HSP70 genes described in claim 1 are in improving the heat-resisting ability of plant and preparing high temperature resistant new germ plasm Application.