CN102002505A - Plant high-pH saline-alkaline tolerance gene SucHP and application thereof - Google Patents

Abstract

The invention discloses a plant high-pH saline-alkaline tolerance gene SucHP and researches the expression of a high-pH saline and alkaline stress induction gene in a common seepweed herb seedling period by using suppression subtractive hybridization and high-density lattice film technology. The plant high-pH saline-alkaline tolerance gene SucHP can be prepared by the following steps of: firstly separating a gene of a coding tonoplast proton pump from common seepweed herbs; constructing a sense expression vector of the gene; and converting Arabidopsis. A transgenic plant has high high-pH saline-alkaline tolerance and can normally grow under the conditions of 100mM NaHCO3+200mM NaCl and 8.5 of pH. The plant high-pH saline-alkaline tolerance gene SucHP can be used for plant genetic transformation and increases plant saline and alkaline resistance.

Description

Anti-high pH salt alkali gene SucHP of one kind of plant and application

Technical field

The invention belongs to biological technical field, exactly anti-high pH salt alkali gene SucHP of a kind of plant and application.

Background technology

Because the salting of soil in the long-term effect of natural cause and human factor, global range is on the rise.At present, the whole world has 1,000,000,000 hectares of saltingss approximately, and China's saltings area is 9,913 ten thousand hectares (account for world's total area 10%).These saltingss mainly comprise with NaCl, Na ₂SO ₄In neutral salt is main solonchak, and with NaHCO ₃, Na ₂CO ₃Deng basic salt is main soda saline-alkali soil.In the landlocked saltings that China enlarges day by day, very major part is a soda saline-alkali soil.In this class soil, because a large amount of carbonate (NaHCO ₃, Na2CO ₃) hydrolytic action, soil pH is up to more than 8.0.Therefore, NaHCO in the soda saline-alkali soil ₃, Na ₂CO ₃To the factor of coercing of plant except with NaCl, Na ₂SO ₄Outside total ion murder by poisoning, the osmotic stress, the factors such as the obvious reduction of mineral element utilizability that also have high pH value and cause.More serious saline and alkaline area sporadicly is distributed in this soil only for the plant of several kinds in western part, China northeast, and other plant almost can not be survived, and has brought tremendous influence for the development of local agriculture production, economy and ecotope.Therefore, improve the salt tolerant alkali ability of crop, the ability of especially anti-high pH salt alkaline stress, it is significant to cultivate anti-high salinity crop new variety.Therefore, to plant salt tolerance base molecule Study on Mechanism, and a forward position content that has become international in recent years molecular biology of plants research field by genetic engineering technique cultivation salt tolerant alkali crop new variety.

Under high salinity, plant alleviates the murder by poisoning that the salt alkaline stress causes by producing stress protein and solubility osmoregulation material.But, both at home and abroad about plant salt tolerance base molecule mechanism and engineered research, mainly be research object (Sahuet al, 2009 at present with NaCl; Qudeimat et al, 2008; Rapala-Kozik et al., 2008; Wu et al., 2004), and to NaHCO in the soda saline-alkali soil ₃, Na ₂CO ₃Mechanism research Deng the effect of coercing less (Zhang et al., 2006).Recently, the proton pump gene that is positioned on the vacuole skin is separated from many plants.Result of study shows that the vacuole skin proton pump is with the H in the tenuigenin ⁺Pump in the vacuole, on the one hand, set up and to stride the vacuole skin electrochemical gradient,, can keep cell plasma balance and osmotic equilibrium, can alleviate some mineral ions again (such as Na for mineral ion and other solutes turnover vacuole provide motivating force ⁺And Cl ^-) murder by poisoning of pair cell matter and the effect of pH value (Queir ó s et al., 2009; Shi et al., 2003; Blumwald et al., 2000), help carrying out smoothly of biochemical reactions in the tenuigenin.This has promoted the research work of saline-resisting and alkaline-resisting gene engineering aspect greatly and has made a breakthrough.Calendar year 2001, the AVP1 gene that Gaxiola etc. will have 35S promoter changes Arabidopis thaliana over to, compares with the wild-type of homogenic type, and the transfer-gen plant salt tolerance and the drought tolerance of AVP1 overexpression significantly improve.Park etc. change the AVP1 gene in the commercial Cultivar (money maker) of tomato (Lycopersicon esculentum) over to, and transfer-gen plant shows following characteristics: root growth is comparatively strong, and biomass significantly increases; Accumulation depends on H in a large number in the root system vacuole ⁺-PPase drives and the positively charged ion of transportation, thereby the transfer-gen plant drought tolerance obviously strengthens (Park et al, 2005, Proc Natl Acad Sci USA, 102 (52): 18830-18835).But saline and alkaline research yet there are no report to Chinese scholars for anti-high pH.

Alkali fluffy (Suaeda corniculate) is annual chenopod, and is nutritious, is a kind of quality vegetables and oil crops.Alkali is fluffy to be again a kind of halophytes, is commonly called as " salt wormwood artemisia ", " seafood dish ", and happiness high humidity, salt tolerant alkali, impoverishment tolerant, few disease and pest are kind of natural pollution-free green food, are suitable for the plantation of coastland sandy soil or silty loam.Alkali is fluffy to be a kind of wild plant of high salt tolerant alkali, and it can be saline and alkaline from absorption in the saltings in the process of growth, improves the soil, and then attracts other grass growth, makes the saltings change into new grassland.

Summary of the invention

The purpose of this invention is to provide the anti-high pH salt alkali gene SucHP of a kind of plant.

One kind of plant anti-high pH salt alkali gene SucHP, its base sequence is as SEQ ID No, shown in 1;

Described SucHP, push away thus proteic aminoacid sequence as: shown in the SEQ ID No.2;

It is a full-length cDNA of isolating coding vacuole skin proton pump from alkali is fluffy;

The anti-high pH salt alkali gene SucHP of one kind of plant is to be obtained by following method:

1) with the fluffy seed of alkali with after the mercuric chloride sterilization, insert kind in the MS substratum, after 3 weeks seedling is transferred to hydroponic system, in hydroponic system, cultivate 4 week back 100mM NaHCO ₃200mM NaCl handles, and behind the processing 24h, takes whole strain plant, separates the fluffy total RNA of alkali;

2) adopt paramagnetic particle method separation and purification mRNA, utilize the reverse transcription of Library Construction Kit test kit, set up the fluffy cDNA of alkali library; Utilize PCR-Select ^TMCDNA Subt raction Kit test kit suppresses subtractive hybridization and makes up the SSH library; Be used in the fluffy salt tolerant alkali of the alkali of having identified in SSH library key gene then, i.e. plasma membrane type H ⁺-Ppase gene fragment is a probe, the fluffy cDNA of screening alkali library, thereby the full-length cDNA fragment of acquisition said gene.

Another object of the present invention provides the preparation method of the anti-high pH salt alkali gene SucHP of a kind of plant:

1) extract the fluffy total RNA of alkali, adopt paramagnetic particle method separation and purification mRNA, reverse transcription is cDNA, the synthetic following primer:

Primer 1:5 '-AAAAAGCAGGCTATGAGTGGCATTCTTCTTC-3 '

Primer 2: 5 '-AGAAAGCTGGGTTTAGAAGATCTTCAAGAGCA-3 '

Primer 3:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '

Primer 4:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '

2) utilizing primer 1 and primer 2, is template with the cDNA of the fluffy total RNA reverse transcription of alkali, carries out first round pcr amplification, obtains 2295bp SucHP gene fragment;

3) the PCR product dilution with the first round is a template for 100 times, uses primer 3, primer 4, carries out second and takes turns pcr amplification, obtains the 2295bp SucHP gene fragment of purifying.

Another object of the present invention is, a kind of plant expression vector is provided, and it is characterized in that it contains the SucHP gene fragment of above-mentioned purifying.

Another purpose of the present invention is the anti-high pH salt alkali gene SucHP of a kind of plant, the application in transgenic plant.

The anti-high pH salt alkali gene SucHP of the present invention's one kind of plant, be to utilize to suppress poor hybridization and the saline and alkaline stress-inducing expression of gene of high-density dot matrix membrane technique research alkali high pH in fluffy seedling stage of subtracting, from alkali is fluffy, isolate the gene of coding vacuole skin proton pump first, should gene constructed sense expression vector, arabidopsis thaliana transformation, the transfer-gen plant tool is from higher anti-high pH saline alkali is arranged, at 100mMNaHCO ₃+ 200mM NaCl, under the pH8.5 condition, the energy normal growth, this gene can be used for the genetic transformation of plant, improves the salt tolerant alkali ability of plant.

Description of drawings

Fig. 1 is that the salt alkaline stress is handled the fluffy RT-PCR detected result of alkali.

Fig. 2 is that 1% agarose gel electrophoresis detected the gene 2295bp fragment product that obtains after enzyme was cut carrier pCB35S-SucHP-GFP.

Fig. 3 is the salt and the salt alkaline purification transgenic arabidopsis plant picture of different concns.

Fig. 4 is the salt and the salt alkaline purification transgenic arabidopsis seed picture of different concns.

Concrete invention embodiment

Embodiment 1: the clone of the fluffy vacuole skin proton pump gene of alkali SucHP

1) extraction of total RNA: the fluffy seed of alkali picks up from the Baicheng Prefecture, Jilin Province.The fluffy seed of alkali with after the mercuric chloride sterilization, is seeded in the MS substratum, after 3 weeks seedling is transferred to hydroponic system, use 100mMNaHCO after in hydroponic system, cultivating for 4 weeks ₃+ 200mM NaCl handles; After handling 24h, take whole strain plant (comprising root system and blade), after liquid nitrogen was fixing, it was standby to store in-80 ℃ of refrigerator-freezers;

Adopt RNAeasy mini kit (promoga company product) to extract the fluffy total RNA of alkali;

2) the fluffy total RNA of alkali of the above-mentioned salt alkaline purification of separation adopts paramagnetic particle method (Promega company) separation and purification mRNA, utilizes the reverse transcription of Library Construction Kit (Clontech) test kit, sets up the fluffy cDNA of alkali library; Utilize PCR-Selecl ^TMCDNA Subt raction Kit (Clontech) test kit suppresses subtractive hybridization and makes up the SSH library:

3) by the SSH library of screening and cloning, obtain a plurality of positive colonies.With the ABI377 sequenator positive colony is checked order, obtain quality EST (Expressed Sequence Tag) sequence preferably altogether, to obtaining not repeat EST after the bit comparison; With do not repeat est sequence on NCBI with Genbank in the dbEST storehouse carry out homology relatively, find that some of them EST can find the higher cDNA of sequence homology; These genes all are the genes that direct or indirect pair cell is shielded by environment stress in organism;

4) utilize vacuole skin proton pump gene H ⁺-Ppase gene fragment is a probe, the fluffy cDNA of screening alkali library, thereby the full-length cDNA fragment of the fluffy vacuole skin proton pump gene of acquisition alkali SucHP;

5) gene clone: get 2 μ l PCR and be connected with the pMD18-T carrier, operation steps is undertaken by Promcga company product pMD18-T Vector system specification sheets.Connect product transformed into escherichia coli DH5 α south strain (preserve in this laboratory) then, scribble grow overnight on the LB flat board that contains penbritin (100 mcg/ml) of 5-bromo-4-chloro-3-indoles-(D-galactoside) and X-gal on the surface.The picking white colony, overnight incubation in the LB liquid nutrient medium;

6) extraction of plasmid DNA: alkaline process extracts plasmid DNA;

7) sequencing: originally be operated in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carry out, measurement result, its determined dna sequence, obtaining total length is the cDNA fragment of 2295bp, comprises initiator codon and terminator codon; Push away thus its 764 amino acid whose one section sequence, its base sequence as: shown in the SEQ ID No 1, aminoacid sequence as: shown in the SEQ ID No.2.

The alkali of embodiment 2RT-PCR detection salt alkaline stress is fluffy

RT-PCR detects primer:

Primer 5 (upstream sequence): 5 '-CTgCTggAAACACTACCgCT-3 '

Primer 6 (downstream sequence): 5 '-CATTgTCCCAAgCACCTCCT-3 '

Primer 7 (upstream sequence): 5 '-TACCACATCCAAggAAggCA-3 '

Primer 8 (downstream sequence): 5 '-ACCCAAAgTCCAACTACgAg-3 '

Primer 5 and primer 6 are primers of SucHP gene in the carrier, and amplified fragments is 569bp; Primer 7 and primer 8 are the primer of internal control gene Actin, and amplified fragments is 438bp; Alkali is fluffy respectively through 250mM NaCl and 200mM NaCl+100mMNaHCO ₃Handle 0h, 6h, 12h, 24h, 48h.With fluffy of alkali, the cDNA of stem and leaf is a template, detects expression of gene; After the PCR reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, detected result is seen Fig. 1, and the result shows that the SucHP expression of gene has certain saline and alkaline inducibility.

The structure of embodiment 3 plant expression vector pCB35S-SucHP

According to SEQ ID No 1 sequence, the design construction expression vector add ATTB1 and ATTB2 joint primer:

Primer 1:5 '-AAAAAgCAggCTATgAgTggCATTCTTCTTC-3 '

Primer 2: 5 '-AgAAAgCTgggTTTAgAAgATCTTCAAgAgCA-3 '

Primer 3:5 '-ggggACAAgTTTgTACAAAAAAgCAggCT-3 '

Primer 4:5 '-ggggACCACTTTgTACAAgAAAgCTgggT-3 '

1) be template with the fluffy total cDNA of alkali, utilize the primer 1, the primer 2 that add ATTB1 and ATTB2 joint to carry out first round pcr amplification, amplification system is as follows:

Amplification system and program are:

2×Taq?Mix 12.5μl

Primer 1 1.0 μ l

Primer 2 1.0 μ l

Template DNA 1.0 μ l

ddH2O 9.5μl

The PCR reaction conditions is:

After the PCR reaction finishes, get 5 μ l products and mix in 6 * Loading Buffer of 2 μ l, carrying out concentration is that 1% agarose gel electrophoresis detects, and obtains the purpose fragment of 2295bp;

2) be template for 100 times with the dilution of the PCR product of the first round, use primer 3, primer 4 is a primer, carry out second take turns its PCR reaction system of PCR and reaction conditions the same, annealing temperature is 58 ℃; PCR product utilization PEG sedimentation techniques are carried out the segmental purifying of purpose, obtain the purpose fragment of the 2295bp of purifying.

3) utilize the Gateway technology that the purpose fragment is building up to pCB35SR ₁R ₂On the GFP carrier, create the clone that crosses the threshold, goal gene is cloned the access door carrier by the BP reaction; The LR reaction mixes the ABC of clone and the pCB35SR that comprises goal gene ₁R ₂GFP carrier and Gateway LR Clonase enzyme make up plasmid;

4) with the plasmid transformation escherichia coli that builds, be applied on the kantlex LB flat board of 50mg/L, choose positive colony then and carry out the PCR detection, detect correct checking order, and extract phase is answered the positive colony plasmid, name pCB35S-SucHP;

5) enzyme is cut carrier pCB35S-SucHP, and reaction end back enzyme is cut product and carried out the detection of 1% agarose gel electrophoresis, sees Fig. 2, and wherein the left side is DNA marker 2000, and arrow is expressed the size of purpose fragment 2295bp, proves the expression vector establishment success.

The salt tolerant alkalescence of embodiment 4 transgenic arabidopsis is analyzed

1) titbit infestation method:

Utilize the CaCl2 method that plasmid pCB35S-SucHP is changed among the Agrobacterium EHA105, utilize titbit infestation method arabidopsis thaliana transformation;

2) screening of transgenic arabidopsis:

Transgenosis and wild Arabidopis thaliana seed kind on the MS substratum, when seedling grows 2-3 to true leaf, are transferred to the Arabidopis thaliana seedling and contained 0mmolL ^-1, 100mmolL ^-1, 150mmolL ^-1And 200mmolL ^-1The MS substratum of NaCl and contain 100mM NaCl+7.5mM NaHCO ₃(pH 8.0), 100mM NaCl+10.0mM NaHCO ₃On the MS substratum of (pH 8.5), observe after 10 days.As shown in Figure 3, the transfer-gen plant upgrowth situation is good, and non-transgenic plant leaf dehydration flavescence is wilted.Equally, transgenosis and wild Arabidopis thaliana seed kind are being contained 0mmolL ^-1, 100mmolL ^-1, 150mmolL ^-1And 200mmolL ^-1The MS substratum of NaCl and contain 100mMNaCl+7.5mM NaHCO ₃(pH 8.0) .100mMNaCl+10.0mM NaHCO ₃On the MS substratum of (pH 8.5), observe after 15 days.As shown in Figure 4, the transfer-gen plant percentage of germination is than the height of wild-type.Prove that thereby this gene has the salt tolerant alkalescence that anti-saline and alkaline ability strengthens transfer-gen plant.

Sequence table

Claims (7)

1. the anti-high pH salt alkali gene SucHP of a kind of plant, its base sequence as: shown in the SEQ ID No.1.

2. the anti-high pH salt alkali gene SucHP expressed proteins of a kind of plant, its aminoacid sequence is shown in sequence table SEQ ID No.2.

3. the anti-high pH salt alkali gene SucHP of a kind of plant according to claim 1 is characterized in that it is the cDNA that separates from alkali is fluffy.

4. the anti-high pH salt alkali gene SucHP of a kind of plant according to claim 3 is characterized in that, it is to be obtained by following method:

1) with the fluffy seed of alkali with after the mercuric chloride sterilization, be seeded in the MS substratum, after 3 weeks seedling is transferred to hydroponic system, in hydroponic system, cultivate 4 week back 100mM NaHCO ₃+ 200mM NaCl handles, and behind the processing 24h, takes whole strain plant, separates the fluffy total RNA of alkali;

2) adopt paramagnetic particle method separation and purification mRNA, utilize the reverse transcription of Library Construction Kit test kit, set up the fluffy cDNA of alkali library; Utilize PCR-Select ^TMCDNA Subt raction Kit test kit suppresses subtractive hybridization and makes up the SSH library; Be used in the fluffy salt tolerant alkali of the alkali of having identified in SSH library key gene then, i.e. plasma membrane type H ^--Ppase gene fragment is a probe, the fluffy cDNA of screening alkali library, thereby the full-length cDNA fragment of acquisition said gene.

5. the preparation method of the anti-high pH salt alkali gene SucHP of a kind of plant:

1) extract the fluffy total RNA of alkali, adopt paramagnetic particle method separation and purification mRNA, reverse transcription is cDNA, the synthetic following primer:

Primer 1:5 '-AAAAAGCAGGCTATGAGTGGCATTCTTCTTC-3 '

Primer 2: 5 '-AGAAAGCTGGGTTTAGAAGATCTTCAAGAGCA-3 '

Primer 3:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '

Primer 4:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '

2) utilizing primer 1 and primer 2, is template with the cDNA of the fluffy total RNA reverse transcription of alkali, carries out first round pcr amplification, obtains 2295bp SucHP gene fragment;

3) the PCR product dilution with the first round is a template for 100 times, uses primer 3, primer 4, carries out second and takes turns pcr amplification, obtains the 2295bp SucHP gene fragment of purifying.

6. a plant expression vector is characterized in that, it contains the SucHP gene fragment of the described purifying of claim 5.

7. the anti-high pH salt alkali gene SucHP of a kind of plant, the application in transgenic plant.

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