CN104561036B - Plant salt tolerance related gene PpSIG1 and its encoding proteins and application - Google Patents

Abstract

The invention discloses a kind of plant salt tolerance related gene PpSIG1 and its application, its encoding amino acid sequence of plant salt tolerance related gene such as SEQ ID NO:Shown in 1, its nucleotide sequence of the gene of the albumen such as SEQ ID NO are encoded:Shown in 2, or its degenerate sequence.Channel genes rice of the present invention is overexpressed, it is possible to increase survival rate, growth rate and biomass under the salt stress of transgenic rice plant.Plant salt tolerance GAP-associated protein GAP PpSIG1 genes of the present invention provide the foundation to cultivate the crop that salt tolerance improves.

Description

Plant salt tolerance related gene PpSIG1 and its encoding proteins and application

Technical field

The invention belongs to biological technical field, is related to a kind of plant salt tolerance related gene PpSIG1 and its application.

Background technology

The soil salinization is a worldwide problem, according to UNESCO（UNESCO）And food and agricultural organization （FAO）Incomplete statistics, about 1,000,000,000 hectares of the saline and alkaline land area in the whole world.At present, with being distributed in NORTHWEST CHINA, northeast and strand The saline-alkali wasteland in area and saline and alkaline obstacle total cultivated area are more than 500,000,000 mu, wherein nearly 200,000,000 mu with agricultural use potentiality, account for me More than the 10% of state total cultivated area.It is saline and alkaline that there is material impact for growth and development of plants, it is the important of limitation crop yield Factor.Thus cultivate the important goal that salt tolerant crop new varieties are crop breedings.

An important means for improving plant salt endurance is that salt-resistant related gene is imported into plant by transgenic method, from And improve the salt tolerance of transfer-gen plant.It is resistance to by genetic engineering means raising crop to excavate new salt-resistant related gene resource The basis of salt.Halophytes obtains the resistance to environment salt stress in long-term evolutionary process, has in halophytes Outstanding adversity gene resource.

The content of the invention

It is an object of the invention to provide one from be grown in China Fujian, Coast of Guangdong Province intertidal zone halophytes it is red Tree Indian beech (Pongamia pinnata(L.) Pierre), the salt stress expression profile of salt tolerance of crop can be improvedPongamia pinnata Salt Induced Gene 1, abbreviation PpSIG1.

Plant salt tolerance related gene provided by the inventionPongamia pinnata Salt Induced Gene 1, referred to as PpSIG1, from legume Indian beech (Pongamia pinnata(L.) Pierre), its encoding proteins amino acid sequence Row such as SEQ ID NO:Shown in 1.

SEQ ID NO:1 sequence is made up of 362 amino acid residues.

The present invention is also provided SEQ ID NO:1 amino acid sequence passes through the substitution of one or several amino acid residues And/or missing and/or addition and it is related to plant stress tolerance by SEQ ID NO:Protein derived from 1 sequence.

In order that described plant salt tolerance GAP-associated protein GAP PpSIG1 is easy to purify, can be by SEQ ID NO:Ammonia shown in 1 The amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of base acid sequence composition.

The sequence of the label of table 1

It is above-mentioned by SEQ ID NO:Protein can be artificial synthesized derived from 1 sequence, also can first synthesize its encoding gene, then enter Row biological expression obtains, and its encoding gene can be by by SEQ ID NO:One or several amino are lacked in DNA sequence dna shown in 2 The codon of sour residue, and/or the missense mutation of one or several base-pairs is carried out, and/or connected at its 5 ' end and/or 3 ' ends The coded sequence of label shown in table 1 obtains.

The present invention also provides a kind of coding the plant salt tolerance related gene PpSIG1, its nucleotide sequence such as SEQ ID NO:Shown in 2, or its degenerate sequence.

Meanwhile the present invention also provides such as SEQ ID NO:DNA sequence dna shown in 2 has more than 90% homology, and encodes resistance to The DNA molecular of inverse property GAP-associated protein GAP.

The present invention be additionally provided under strict conditions with SEQ ID NO:DNA sequence dna shown in 2 hybridizes and encodes the egg White DNA molecular；Also provide simultaneously has more than 90% homology with the DNA molecular, and encodes the DNA of stress tolerance correlative protein Molecule；The stringent condition can be in 6 × SSC, 0.5% SDS solution, 65^oHybridize under C, then with 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS respectively wash film once.

The present invention also provides the recombinant expression carrier of the gene.

The recombinant expression carrier of the gene can be contained with existing plant expression vector construction.

The plant expression vector includes double base agrobacterium vector and the carrier available for plant micropellet bombardment etc..The plant Thing expression vector can also include 3 ' end untranslated regions of foreign gene, i.e., comprising polyadenylation signals and any other participation MRNA is processed or the DNA fragmentation of gene expression.The bootable polyadenylic acid of polyadenylation signals is added to the 3 ' of mRNA precursor End, such as Agrobacterium crown gall nodule induction (Ti) plasmid gene (such as kermes synzyme Nos genes), plant gene（Such as soybean storage egg White gene）The non-translational region of 3 ' end transcriptions is respectively provided with similar functions.

During using the gene constructed recombinant plant expression vector, any one can be added before its transcription initiation nucleotides Enhanced promoter or constitutive promoter, such as cauliflower mosaic virus（CAMV）The ubiquitin promoter of 35S promoter, corn （Ubiquitin）, they can be used alone or are used in combination with other plant promoters；In addition, the gene using the present invention When building plant expression vector, enhancer, including translational enhancer or transcriptional enhancer also can be used, these enhancer regions can To be ATG initiation codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, it is whole to ensure The correct translation of individual sequence.The source of the translation control signal and initiation codon is extensive, can be natural, also may be used Be synthesis.Translation initiation region can come from transcription initiation region or structural gene.

For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out Processing, the enzyme of color change or the gene of luminophor can be produced as added the coding that can be expressed in plant（Gus gene, Luciferase genes etc.）, resistant antibiotic marker（Gentamicin label, kanamycins label etc.）It is or anti- Chemical reagent marker gene（Such as anti-herbicide gene）Deng.From the security consideration of genetically modified plants, any selectivity can be not added with Marker gene, transformed plant is directly screened with adverse circumstance.

The recombinant expression carrier is the recombinant plasmid for obtaining gene insertion pCXUN multiple cloning sites.

Contain gene described in any of the above（PpSIG1）Expression cassette, transgenic cell line and recombinant bacterium belong to the present invention Protection domain.

Expand the gene（PpSIG1）The primer pair of total length or any fragment falls within protection scope of the present invention.

The present invention also provides the gene（PpSIG1）Salt resistance ability enhancing genetically modified plants in application, its be by Recombinant expression carrier containing the gene imports the genetically modified plants that purpose plant obtains salt resistance ability enhancing.

Using any carrier that foreign gene can be guided to be expressed in plant, the gene of encoding said proteins is led Enter plant cell, the transgenic cell line and transfer-gen plant of salt resistance ability enhancing can be obtained.Carry the expression of the gene Carrier can be by using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated Plant cell or tissue are converted Deng conventional biology methods, and the plant tissue of conversion is cultivated into plant.The plant being converted Host both can be monocotyledon or dicotyledon, such as：Tobacco, crowtoe, arabidopsis, rice, wheat, jade Rice, cucumber, tomato, willow, turfgrass, lucerne place etc..

Experiment shows, by the encoding regulator plant salt tolerance related gene of the present inventionPpSIG1 DNA sequence dna Introduced into Rice in It is overexpressed, it is possible to increase transgenic paddy rice salt tolerance, improve survival rate, growth rate under the salt stress of transgenic rice plant And biomass.The coded plant salt-resistant related gene of the present inventionPpSIG1Gene has the crop of salt tolerance raising to cultivate other Provide the foundation.

Brief description of the drawings

Fig. 1 identifies to be overexpressed transfer-gen plant PCR.

Fig. 2 identifies to be overexpressed transfer-gen plant real-time quantitative PCR.

Fig. 3 is that water planting control and salt stress handle wild type and PpSIG1 transgenic lines ox1.

Fig. 4 is that water planting control and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2 survival rate.

Fig. 5 is that water planting control and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2 plant height.

Fig. 6 is that earth culture experiment contrast and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2.

Fig. 7 is that earth culture experiment contrast and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2 survival Rate.

Fig. 8 is that earth culture experiment contrast and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2 plant height.

Fig. 9 is that earth culture experiment contrast and salt stress handle wild type and PpSIG1 transgenic lines ox1 and ox2 overground part Divide single-strain fresh weight.

Embodiment

Below by embodiment detailed description come the present invention is furture elucidated, but be not to the present invention limit System, is only illustrated.

Experimental method in following embodiments, it is conventional method unless otherwise specified, test material used, such as nothing Specified otherwise, it is to be commercially available from routine biochemistry reagent shop, experiment is respectively provided with to be repeated three times, results averaged.

Embodiment 1：PpSIG1 cDNA clone

Because currently without Indian beech Genomic sequence information, we utilize Illumina Genome Analyzer IIx carries out high-flux sequence to salt stress processing and untreated control Indian beech transcript profile, and the original series obtained filter off After joint and low quality sequence, using SOAPdenovo programs (http://soap.genomics.org.cn) carry out sequence Assembling, non repetitive sequence (Unigenes) is obtained, BLASTx program search Nr, Swiss-Prot, KEGG are used to Unigenes With COG databases, using annotation of the similitude highest Sequence annotation as the Unigene.Each base is calculated by RPKM values The expression of cause, we, which screen, obtains 1050 genes induced by salt stress.These genes overwhelming majority does not enter at present Row functional analysis, the new gene resource of key function is played to excavate Indian beech in salt stress response, from unknown function In gene, we select rapid elevated 10 genes of induced expression in salt stress processing, are cloned and pass through transgenosis Function in method validation its transgenic paddy rice salt stress reaction.Functional analysis confirms that one of them is yellow by the water of salt stress induction Skin Unigene can improve the salt tolerance of transgenic paddy rice after being overexpressed in rice, we are named as PpSIG1ORF.Experiment Method is as follows, according to the following special primer of deduction sequences Design：

Sense primer：5’-ATGGAAGAGATCAAGAAACC-3’；

Anti-sense primer：5’-TCAGCTTCGATTGTGATGTTC-3’.

With 200 mM NaCl handle a monthly age Indian beech (Pongamia pinnata(L.) Pierre) plant 6 hours Afterwards, 0.2g Indian beech blades, liquid nitrogen grinding, TRIzol methods extraction total serum IgE are taken.Take 2 μ g total serum IgE SuperScript II RT reverse transcriptase carries out reverse transcription, synthesizes the chains of cDNA first, as template, enters performing PCR reaction with above-mentioned special primer.PCR Product is cloned into pEASY-T after electrophoretic separation reclaims（Beijing Quanshijin Biotechnology Co., Ltd）, it is named as pEASY-T- PpSIG1ORF, it is sequenced.

Sequencing result shows, the nucleotide sequence such as SEQ ID NO of the fragment:Shown in 2, SEQ ID NO are encoded:Shown in 1 Protein.

Embodiment 2：Overexpressions of the PpSIG1 in rice

First, the structure of pCXUN-PpSIG1 expression vectors

1st, using recombinant plasmid pEASY-T-PpSIG1ORF in implementing 1 as masterplate, using HiTaq archaeal dna polymerases and following Row primer amplified obtains PpSIG1 sequences.

Sense primer：5’-ATGGAAGAGATCAAGAAACC-3’；

Anti-sense primer：5’-TCAGCTTCGATTGTGATGTTC-3’.

After PCR primer electrophoretic separation, the bp DNA fragmentations of gel extraction 1089.

2nd, with restriction enzyme XcmI digestions pCXUN (Ubiquitin promoters) (Plant Physiol. 2009 July; 150(3):1111-1121) skeleton, is reclaimed.

3rd, the fragment that step 1 obtains is connected with the fragment that step 2 obtains, converts bacillus coli DH 5 alpha, sequencing confirms just Really, pCXUN-PpSIG1 is obtained.

2nd, the acquisition of genetically modified plants

1st, recombinant expression carrier pCXUN-PpSIG1 is imported into Agrobacterium AGL0 (ATCC BAA-100 using electric shocking method , www.atcc.org).

5th, the Agrobacterium AGL0 containing pCXUN-PpSIG1 is infected into embryonic type callus group caused by the induction of Nipponbare wild type Knit, then the screening resistant calli in MS culture mediums (containing 30mg/L hygromycin), per 15 days generations, altogether 3 generation, then Resistant calli is induced into intact plant, rice transplanting and harvests the T1 of genetically modified plants for seed in crop field.

3rd, the Molecular Detection of genetically modified plants

CTAB methods extract leaf DNA, with-the AAGAGTTGCTCGTCACTG-3 ' of pCXUN-PpSIG1 specific primers 5 ' and 5 '-TCGGGTTGTGGAATGTTG-3 ' PCR are detected, the results showed that there is specific amplification band in transfer-gen plant, and wild type For DNA without specific amplified band (Fig. 1), this shows that PpSIG1 has been introduced into transfer-gen plant.

TRIzol methods extract 2 week old wild types and PpSIG1 transfer-gen plants (ox1,2) blade total serum IgE, take 2 μ g total RNA, with 2 u RQ1 RNase-free DNase (Promega), 37 °C handle 30 min, after adding terminating reaction liquid, with PolyA is primer, and the chains of cDNA first are synthesized within 1 hour using M-MLV reverse transcriptase 42 °C, then using cDNA as template, with Ubiquitin (primer 5 '-CCATCCTCAAGCTGCTTACC-3 ' and 5 '-GACTGGCAAGACCATTACCC-3 ') is internal reference, PCR method detection PpSIG1 genes (- the TATTTCCACAGCTCCCTCTTC-3 ' and 5 ' of primer 5 '- CCCACCACAACATCTTGTTTC-3 ') expression, as a result as shown in Figure 2.PpSIG1 is can't detect in Wild type control plants The expression of gene, and pCXUN-PpSIG1 transfer-gen plants (ox1,2) PpSIG1 gene expression doses are about Ubiquitin tables Up to horizontal 0.8-0.9 times.

Embodiment 3：PpSIG1 transgenic rice plants salt tolerance detects

First, water planting rice plant salt tolerance detects

After wild type and the processing 3 days of 42 °C of PpSIG1 transgenic paddy rice seeds, soaked seed 24 hours in water, 37 °C of vernalization, The consistent seed of rudiment is chosen in following nutrient solutions, 28 °C are cultivated 4 days, are then located in the nutrient solution containing 100 mM NaCl Reason 4 days.As a result as in Figure 3-5, wild type and PpSIG1 transgenic rice plants are not significantly different in untreated control, After 100 mM NaCl processing, WT lines survival rate is 35%, the cm of plant height 13.2, substantially less than PpSIG1 transgenosis Survival rate 60-65% and plant height, 19.2-19.5 cm.This show water planting salt stress processing in, PpSIG1 transgenic rice plants Salt tolerance is significantly higher than wild type control.

Cultivate formula of liquid：The mg/L of four water-calcium nitrate 945, the mg/L of potassium nitrate 506, the mg/L of ammonium nitrate 80, phosphoric acid The mg/L of potassium dihydrogen 136, magnesium sulfate 493 mg/L, 2.5 ml of iron salt solutions/L, micro- mother liquor 5 ml/L, pH= 6.0。

Iron salt solutions：Ferrous sulfate heptahydrate 2.78g, disodium ethylene diamine tetraacetate（EDTA.Na）3.73g distilled water 500ml, pH=5.5.

Micro- mother liquor：The mg/l of KI 0.83, boric acid 6.2 mg/L, manganese sulfate 22.3mg/L, sulfuric acid Zinc 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L.

2nd, earth culture rice plant salt tolerance detects

After wild type and the processing 3 days of 42 °C of PpSIG1 transgenic paddy rice seeds, soaked seed 24 hours in water, 37 °C of vernalization, The consistent seed kind of rudiment is chosen in the small basin equipped with Nutrition Soil, covers one layer of preservative film moisturizing, after coming up, is removed Preservative film, after 2 week, the small basin of rice cultivation seedling is put into the water containing 100 mM NaCl and soaked 1 week, then pours out salt solution, A running water is changed daily, is taken a picture after 1 week, counts physiological phenotype.As Figure 6-9, in the processing of no salt stress to according to the facts In testing, PpSIG1 transgenic paddy rice strains ox1 and ox2 grow no notable difference, and 1 week is handled then by 100 mM NaCl Rehydration, the survival rate of Wild type control plants only has 24%, and PpSIG1 transgenic paddy rice strains ox1 and ox2 survival rate are distinguished For 39% and 45%, it is significantly higher than wild type control.It is similar as shown in FIG. 8 and 9, PpSIG1 transgenic paddy rice strains ox1 and ox2 Two physical signs of plant height and fresh weight be also significantly greater than Wild type control plants.Result above shows, is tested in earth culture In, the salt tolerance of PpSIG1 transgenic paddy rice strains significantly improves.

<110>Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>Plant salt tolerance related gene PpSIG1 and its encoding proteins and application

<160> 2

<210> 1

<211> 362

<212> PRT

<213> Pongamia pinnata

<400> 1

Met Glu Glu Ile Lys Lys Pro Phe Gly Thr Ser Leu Leu Val Pro Ser

Val Gln Glu Leu Ala Glu Gly Lys Ile Ser Asn Val Pro Asp Arg Tyr

Ile Gln Pro Gln Gln His Glu Glu Leu Leu Val Thr Glu Ala Asp Tyr

His Val Leu Glu Ile Pro Val Ile Asp Met Gln Asn Leu Leu Ser Leu

Glu Ser Gly Ala Ser Glu Leu Thr Lys Leu His Leu Ala Cys Arg Tyr

Trp Gly Phe Phe Gln Leu Val Asn His Gly Val Ser Ser Leu Leu Leu

Glu Lys Val Lys Leu Glu Ile Gln Asn Phe Phe Asn Leu Pro Met Leu

Glu Lys Lys Lys Phe Trp Gln Ser Pro Gln His Met Glu Gly Phe Gly

Gln Gly Phe Val Val Ser Glu Asp Gln Lys Leu Asp Trp Ala Asp Met

Phe Tyr Met Thr Thr Leu Pro Thr Lys Gln Arg Ile Pro His Leu Phe

Pro Gln Leu Pro Leu Pro Phe Arg Asp Thr Met Glu Leu Tyr Ser Gln

Asp Val Lys Asn Ile Ala Leu Ile Ile Ile Ala His Ile Glu Lys Ala

Leu Lys Met Glu Glu Met Glu Ile Met Lys Leu Phe Glu Asp Leu Arg

Gln Thr Met Arg Ile Asn Tyr Tyr Pro Pro Cys Pro Glu Pro Glu Lys

Val Ile Gly Leu Thr Pro His Ser Asp Gly Thr Gly Leu Thr Ile Leu

Leu Gln Val Asn Glu Val Glu Gly Leu Gln Ile Lys Lys Asp Gly Met

Trp Val Pro Ile Met Pro Leu Pro Asn Ala Phe Ile Val Asn Ile Gly

Glu Ile Leu Glu Ile Ile Thr Asn Gly Ile Tyr Gln Ser Ile Glu His

Arg Ala Thr Val Asn Ser Glu Lys Glu Arg Leu Ser Ile Ala Thr Phe

His Asn Pro Lys Gln Asp Val Val Val Gly Pro Ala Ala Ser Leu Ile

Thr Glu Gln Ile Pro Ala Gln Phe Lys Arg Ile Arg Ile Glu Glu Tyr

Leu Arg Gly Ile Phe Ala Arg Lys Leu Asn Gly Lys Ser Tyr Leu Asp

Thr Phe Gly Ile Glu His His Asn Arg Ser

<210> 2

<211> 1089

<212> DNA

<213> Pongamia pinnata

<400> 2

atggaagaga tcaagaaacc atttgggact tctcttctgg tgccatcagt tcaagaattg 60

gctgaaggga aaatatcaaa tgttccagac agatacattc agcctcaaca acatgaagag 120

ttgctcgtca ctgaagctga ttatcatgtc cttgagattc cagttattga catgcagaac 180

ttgctttctc tagaatctgg tgcttctgag ttgaccaagc ttcaccttgc ttgcagatat 240

tggggattct tccagctggt aaaccatgga gttagttctt tattgctgga aaaggtaaag 300

ttggagattc agaatttttt caaccttcca atgctggaaa agaaaaaatt ttggcagagt 360

ccgcaacata tggagggatt tggacaagga tttgttgtta gtgaagacca aaaacttgat 420

tgggctgaca tgttctatat gacaaccctt ccaacaaagc agaggatccc ccacttattt 480

ccacagctcc ctcttccgtt cagggacact atggagcttt actcacaaga cgtgaaaaat 540

atagccttga ttattattgc acacattgag aaagctctta agatggagga aatggaaata 600

atgaagttat ttgaagactt gagacagact atgaggataa actattaccc tccatgtcca 660

gaaccggaga aggttattgg ccttactccc cattcagatg gaactggtct cactatcctt 720

cttcaagtta atgaggtgga agggctccag ataaagaaag atggaatgtg ggttcctatt 780

atgcccctgc ctaatgcatt cattgttaac attggcgaga tacttgagat tataaccaat 840

ggtatatacc aaagtattga acacagagca acagtgaact ctgaaaaaga aaggctttca 900

attgcaacat tccacaaccc gaaacaagat gttgtggtgg gtcctgcggc tagcttaatc 960

actgagcaaa taccagcaca gtttaaaaga ataagaattg aagaatattt gaggggcata 1020

tttgctcgta aacttaacgg aaagtcttac ctagatacct ttggaataga acatcacaat 1080

cgaagctga 1089

Claims (10)

1. A kind of 1. plant salt tolerance related gene PpSIG1, it is characterised in that：Its protein amino acid sequence such as SEQ ID encoded NO:Shown in 1.
2. 2. gene as claimed in claim 1, it is characterised in that：Its protein amino acid sequence such as SEQ ID NO encoded:1 institute Show, further comprise that coding is connected with sequence label in its amino terminal or carboxyl terminal.
3. 3. gene as claimed in claim 2, it is characterised in that：Described sequence label be Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc.
4. 4. the expression cassette containing the gene as described in any one of claims 1 to 3, it is characterised in that the gene can with promoter Operation connection.
5. 5. the recombinant expression carrier containing the gene as described in any one of claims 1 to 3.
6. 6. recombinant expression carrier as claimed in claim 5, it is characterised in that：It is double base agrobacterium vector.
7. 7. recombinant expression carrier as claimed in claim 6, it is characterised in that：The carrier is the load for plant micropellet bombardment Body.
8. 8. recombinant expression carrier as claimed in claim 5, it is characterised in that：The recombinant expression carrier is to insert the gene Enter the recombinant plasmid that pCXUN multiple cloning sites obtain.
9. 9. the recombinant host cell containing the gene as described in any one of claims 1 to 3.
10. 10. application of the gene in the genetically modified plants that salt resistance ability strengthens as described in any one of claims 1 to 3, it is Recombinant expression carrier containing the gene is imported to the genetically modified plants of purpose plant acquisition salt resistance ability enhancing, the plant It is rice.