CN1116417C - Method for improving plant salt resistance by using transgenic technology - Google Patents

Abstract

The present invention provides a method using transgenic technology for improving plant salt tolerance, which comprises the following steps: a sucrose-6-fructosyl transferase gene SacB and the vacuole leading peptide (cpy) sequence of carboxypeptidase A from bovine pancreas are cloned from the DNA of bacillus subtilis and yeast; the obtained two kinds of genes are used for building a chimeric gene which is used for building a plant expression vector; the obtained plant expression vector is used for converting plants to filter out resistance seedlings. The method is used for obtaining the plants of drought resistance and salt tolerance. The PCR and northern analysis on the transgenic plants show that the exogenous gene is integrated into the transgenic plants of salt resistance.

Description

Improve the method for plant salt endurance with transgenic technology

The present invention relates to utilize transgenic technology to improve the method for plant salt endurance.

Saline and alkaline and arid is the principal element that influences plant-growth and crop production reduction.They can both cause the variation of osmotic pressure in the organism.In order to survive, many organisms can accumulate some Osmolyte regulators usually to adapt to the osmotic stress of outside atmosphere.Polylevulosan in the vegetable cell is exactly wherein a kind of natural osmoregulation thing, and its a large amount of accumulation in vacuole has no adverse effect to metabolism, and evidence suggests that the Polylevulosan accumulation is relevant with drought tolerance in plants (trembling with fear) ability in the angiosperm evolutionary process.Polylevulosan content has increase in the plant materials under osmotic stress (comprising temperature and salt marsh), thereby the Polylevulosan synthase gene is changed over to plant and accumulates the ability that its expression product may improve the drought tolerance in plants salt tolerant.

The biosynthesizing of Polylevulosan may be at least needs 2 kinds of enzymes in plant, sucrose-sucrose fructosyl transferase and Polylevulosan-Polylevulosan fructosyl transferase, and still fail from plant, to clone the gene of relevant enzyme so far.In microorganism, particularly in Bacillus subtilus the biosynthesizing of Polylevulosan by the catalysis of a kind of enzyme institute.Therefore Bacillus subtilus Polylevulosan synthase gene is transferred in the plant, it is expressed in this plant, thereby the ability that improves the drought-enduring salt tolerant of plant will be a good approach.Though existing in this respect people made trial (Van der Meer IM, Ebskamp MJM, Visser RGF, et al., The Plant Cell, 1994,6:561-570.), but it fails to construct the mosaic gene that does not have sudden change, and the report of success of the test is not arranged so far yet.

The purpose of this invention is to provide a kind of method of utilizing transgenic technology to improve the drought tolerance in plants salt resistance ability.

The inventor is according to the sequence data (SteinmetzM of the Polylevulosan synthase gene of Bacillus subtilus, Le Coq D, Aymerich S, et al., Mol Gen Genet, 1985,200:220-228), design special primer, adopting PCR method to clone this enzyme is levansucrase gene SacB.Because the synthetic and accumulation position of Polylevulosan is vacuole in the vegetable cell, the inventor guides peptide (cpy) sequence at the vacuole that SacB gene 5 ' end connects from the zymic Carboxypeptidase A, and it can also synthesize Polylevulosan with levansucrase gene SacB directional guide in vacuole.

Thereby the invention provides a kind of method that improves the drought tolerance in plants salt resistance ability, comprising: vacuole guiding peptide (cpy) sequence of 1, from Bacillus subtilus and zymic DNA, cloning levansucrase gene SacB and Carboxypeptidase A respectively; 2, with two kinds of gene constructed mosaic genes that obtain in above-mentioned 1; 3, make up plant expression vector with the mosaic gene in 2; 4, with the plant expression vector transformed plant that obtains, filter out the resistance seedling.

Wherein, described Bacillus subtilus can be Bacillus subtilis 168, and described yeast can be cereuisiae fermentum Saccharomyces cerevisiae X8.

Vacuole to levansucrase gene SacB and Carboxypeptidase A from Bacillus subtilus and zymic DNA guides the clone of peptide (cpy) sequence at first to extract its DNA with currently known methods, carry out with the PCR method amplification then, the primer of amplification Bacillus subtilus levansucrase gene (SacB) is:

5 ' end primer gatctagAAAGAAACGAACCAAAAGGCCATATAAG

3 ' end primer cagtcgaccTATTTGTTAACTGTTAATTGTCC amplification yeast Carboxypeptidase A vacuole leader peptide sequence (cpy) primer is:

5 ' end primer taggatccgaccATGAAAGCATTCACCAGTTTAC

3 ' end primer ctgaatattGACACGAAGCTGATAGTTTTC

Other step of PCR reaction, and the structure of mosaic gene, the structure of plant expression vector, the conversion of plant, the screening of resistance seedling is all undertaken by currently known methods.

Obtained drought-enduring salt tolerance plant with aforesaid method, and to the PCR and the Northern analysis revealed of transfer-gen plant, foreign gene has been integrated in the salt tolerant transfer-gen plant.

Brief Description Of Drawings: Fig. 1 represents the electrophoretic analysis (A is cpy, and B is that λ DNA EcoRI and HindIII enzyme are cut molecular weight standard, and C is SacB) of SacB and cpy gene fragment pcr amplification product; Fig. 2 represents the structure of mosaic gene; Fig. 3 represents the construction strategy of subclone; Fig. 4 represents the nucleotide sequence (arrow shows SacB and cpy gene tie point) of mosaic gene; Fig. 5 represents the plant expression vector structure of mosaic gene; Fig. 6 represent tobacco Kn resistant buds with to impinge upon on the saliferous substratum growth fraction; Fig. 7 represents that the PCR of transgenic tobacco plant detects; Fig. 8 represents that the Northern of transgenic tobacco plant detects.

Embodiment

The clone of embodiment one levansucrase gene and Carboxypeptidase A vacuole guiding peptide gene and the preparation of sequential analysis 1, Bacillus subtilus and zymic DNA

The DNA of DNA of Bacillus subtilus (Bacillus subtilis 168) and cereuisiae fermentum (Saccharomycescerevisiae X8) all extracts (Ausubel FM by methods such as Ausubel, Brent R, Kingston RE, et al., Current Protocols in Molecular Biology.New York, 1987, John Wiley and Sons.).2, design of primers and gene amplification

According to the sequence data of Bacillus subtilus levansucrase gene (SacB) (Steinmetz M, Le Coq D, Aymerich S, et al., Mol Gen Genet, 1985,200:220-228), designed a pair of primer:

5 ' end primer ga TctagAAAGAAACGAACCAAAAGGCCATATAAG

XbaI

3 ' end primer ca GtcgacCTATTTGTTAACTGTTAATTGTCC

SalI

According to yeast Carboxypeptidase A vacuole leader peptide sequence (cpy) (Valls LA, Hunter CP, Rothman JH, et al., Cell, 1987,48:887-897.), designed another to primer:

5 ' end primer ta GgatccGaccATGAAAGCATTCACCAGTTTAC

BamHI

3 ' end primer ctg AatattGACACGAAGCTGATAGTTTTC

SspI

Amplified reaction carries out in 50 μ l systems, contains about 50ng template, 5 μ l, 10 * PCR damping fluid, 4 μ ldNTP (2.5mM), 5 ' end and 3 ' each 2 μ l (10 μ m) of end primer, 0.4 μ l Taq enzyme (5U/ μ l) and ultrapure H ₂O.Amplification condition is 94 ℃, 1min; 56 ℃, 1min; 72 ℃, 2min; Totally 30 circulations are extended 10min at 72 ℃ at last.

Product is through 1.2% agarose gel electrophoresis analysis (Fig. 1), and SacB is similar with 0.3kb to the 1.3kb of expection respectively with the cpy clip size.3, the structure of mosaic gene and complete sequence analysis

Clone's process of mosaic gene is seen Fig. 2.After the SacB gene amplification product was used XbaI and SalI double digestion, directed cloning obtained recombinant plasmid pUB in XbaI and the SalI site of PUC19.Use XbaI enzyme cutting pUB plasmid then, under the condition of gentleness, handle again with the S1 enzyme, remove the remaining unnecessary strand base of XbaI enzyme cutting (4 bases), cut with the Smal enzyme again after reclaiming fragment, remove the BamHl site in the original vector, be connected (referring to Fig. 2) at last with through mending cpy gene fragment flat and that the Sspl enzyme is cut, behind the transformed into escherichia coli, adopt the PCR screening to contain the recombinant plasmid pUYB of mosaic gene (cpy+SacB).The primer has obtained 3 recombinant clones that contain mosaic gene to being 5 ' end primer of cpy gene fragment and 3 ' end primer of SacB gene, and wherein 1 clone confirms that through the forward order-checking cpy gene fragment is connected errorless with the SacB gene in the recombinant plasmid (pBYU).

Adopt respectively BamHI-EcoRI, EcoRI-KpnI, KpnI-SalI totally 3 groups of enzyme enzymes cut pUYB, 3 subclone PSBE have been obtained, PSEK and PSKS (see figure 3), check order respectively after extracting plasmid, the result shows mosaic gene open reading district total length 1664bp, 558 amino acid (Fig. 4) of encoding, and because design of primers and mosaic gene building process precision, being connected of SacB gene and cpy accurately meets former sequence.The conversion of embodiment two SacB genes in the model plant tobacco and the conversion of salt tolerance detection 1, plant expression vector construction and Agrobacterium

With BamHI and SalI enzymolysis recombinant plasmid (pUYB), mosaic gene obtains plant expression vector pBYB in the binary vector pBin438 that Geneclean recovery rear clone to same enzyme is cut, its structure is seen Fig. 5, and by (Horsch method Horsch RB, Fry JE, Hoffman NL., Science 1985,227:1229-1232.) transform Agrobacterium.2, tobacco transforms and the salt tolerance evaluation

Tobacco (Nicotiana tabaccum) product are K326, and program is cultivated aseptic seedling routinely.The leaf garden dish method of Agrobacterium (A.tumefaciens) mediation transforms (Horsch method Horsch RB such as pressing Horsch, Fry JE, Hoffman NL., Science 1985,227:1229-1232.), go up the screening resistant buds at the division culture medium that contains kantlex 200 μ g/ml (MS), through 3 take turns screening after, in 300 transformants, obtain 61 green resistant budses (the long 2-3cm of bud) altogether, it is transferred to the MS root media that contains 1%NaCl, not only normally take root and grow fine through 17 strains were arranged after 20 days, (screen and contrast without kantlex, directly go to and contain in the salt culture medium) indivedual can taking root only arranged, and root is long and radical all is significantly less than the transfer-gen plant (see figure 6).

The transgene tobacco seedling moves into the flowerpot nurse of containing vermiculite and cultivates (Hoagland nutritive medium) after 5 days, handled 17 days with the Hoagland nutritive medium that contains 1%NaCl, its upgrowth situation is significantly better than unconverted tobacco regrowth, and do not have the sign of obviously being injured, and serious the wilting appears in the bottom leaf of unconverted contrast.3, the PCR of transfer-gen plant and Northern analyze

The trace of rotaring gene plant blade DNA extracts and the PCR operation, according to Edwards etc. (Edwards K, Johnstone C, Thomson C Acids Res 1991,19:1349.), PCR detects with 3 ' of 5 ' the end primer of cpy and SacB and holds primer to carry out.The DNA that extracts transgene tobacco (5 strain) and contrast (2 strain) blade increases, transfer-gen plant has all provided the band with the plasmid amplification fragment same molecular amount (1.7kb) that contains mosaic gene, and adjoining tree does not amplify respective strap, illustrates that foreign gene has been integrated into (Fig. 7) in the salt tolerant transfer-gen plant.

The extraction of RNA and Northern operation with Jinsong ZHANG etc. (Jinsong ZHANG, all coursers, relaxation etc., Chinese science B collects, 1995,25:1172-1177).Northern result shows that all foreign genes that can stand the transgenic plant of salt stress selection all have transcribe (Fig. 8) in various degree.

Claims (1)

1. method that improves the Tobacco Salt ability comprises that apparatus has the mosaic gene of following sequence to make up plant expression vector, and with the plant expression vector transformation of tobacco that obtains, filters out the step of resistance seedling:

ATGAA?AGCAT?TCACC?AGTTT?ACTAT?GTGGA?CTAGG?CCTGT?CCACT?ACACT?CGCTA?AGGCC 60

ATCTC?ATTGC?AAAGA?CCGTT?GGGTC?TAGAT?AAGGA?CGTTT?TGCTG?CAAGC?TGCGG?AAAAA 120

TTTGG?TTTGG?ACCTC?GACCT?GGATC?ATCTC?TTGAA?GGAGT?TGGAC?TCCAA?TGTAT?TGGAC 180

GCTTG?GGCCC?AAATA?GAGCA?TTTGT?ACCCA?AACCA?GGTTA?TGAGC?CTTGA?AACTT?CCACT 240

↓

AAGCC?AAAAT?TCCCT?GAAGC?AATCA?AAACG?AAGAA?AGACT?GGGAC?TTTGT?GGTCA?AGAAT 300

GACGC?AAGTG?AAAAC?TATCA?GCTTC?GTGTC?AATAA?AGAAA?CGAAC?CAAAA?GCCAT?ATAAG 360

GAAAC?ATACG?GCATT?TCCCA?TATTA?CACGC?CATGA?TATGC?TGCAA?ATCCC?TGAAC?AGCAA 420

AAAAA?TGAAA?AATAT?CAAGT?TCCTG?AATTC?GATTC?GTCCA?CAATT?AAAAA?TATCT?CTTCT 480

GCAAA?AGGCC?TGGAC?GTTTG?GGACA?GCTGG?CCATT?ACAAA?ACGCT?GACGG?CACTG?TCGCA 540

AACTA?TCACG?GCTAC?CACAT?CGTCT?TTGCA?TTAGC?CGGAG?ATCCT?AAAAA?TGCGG?ATGAC 600

ACATC?GATTT?ACATG?TTCTA?TCAAA?AAGTC?GGCGA?AACTT?CTATT?GACAG?CTGGA?AAAAC 660

GCTGG?CCGCG?TCTTT?AAAGA?CAGCG?ACAAA?TTCGA?TGCAA?ATGAT?TCTAT?CCTAA?AAGAC 720

CAAAC?ACAAG?AATGG?TCAGG?TTCAG?CCACA?TTTAC?ATCTG?ACGGA?AAAAT?CCGTT?TATTC 780

TACAC?TGATT?TCTCC?GGTAA?ACATT?ACGGC?AAACA?AACAC?TGACA?ACTGC?ACAAG?TTAAC 840

GTATC?AGCAT?CAGAC?AGCTC?TTTGA?ACATC?AACGG?TGTAG?AGGAT?TATAA?ATCAA?TCTTT 900

GACGG?TGACG?GAAAA?ACGTT?ACAAA?ATGTA?CAGCA?GTTCA?TCGAT?GAAGG?CAACT?ACAGC 960

TCAGG?CGACA?ACCAT?ACGCT?GAGAG?ATCCT?CACTA?CGTAG?AAGAT?AAAGG?CCACA?AATAC 1020

TTAGT?ATTTG?AAGCA?AACAC?TGGAA?CTGAA?GATGG?CTACC?AAGGC?GAAGA?ATCTT?TATTT 1080

AACAA?AGCAT?ACTAT?GGCAA?AAGCA?CATCA?TTCTT?CCGTC?AAGAA?AGTCA?AAAAC?TTCTG 1140

CAAAG?CGATA?AAAAA?CGCAC?GGCTG?AGTTA?GCAAA?CGGCG?CTCTC?GGTAT?GATTG?AGCTA 1200

AACGA?TGATT?ACACA?CTGAA?AAAAG?TGATG?AAACC?GCTGA?TTGCA?TCTAA?CACAG?TAACA 1260

GATGA?AATTG?AACGC?GCGAA?CGTCT?TTAAA?ATGAA?CGGCA?AATGG?TACCT?GTTCA?CTGAC 1320

TCCCG?CGGAT?CAAAA?ATGAC?GATTG?ACGGC?ATTAC?GTCTA?ACGAT?ATTTA?CATGC?TTGGT 1380

TATGT?TTCTA?ATTCT?TTAAC?TGGCC?CATAC?AAGCC?GCTGA?ACAAA?ACTGG?CCTTG?TGTTA 1440

AAAAT?GGATC?TTGAT?CCTAA?CGATG?TAACC?TTTAC?TTACT?CACAC?TTCGC?TGTAC?CTCAA 1500

CGAAA?GGAAA?CAAAT?GTCGT?GATTA?CAAGC?TATAT?GACAA?ACAGA?GGATT?CTACG?CAGAC 1560

AAACA?ATCAA?CGTTT?GCGCC?AAGCT?TCCTG?CTGAA?CATCA?AAGGC?AAGAA?AACAT?CTGTT 1620

GTCAA?AGACA?GCATC?CTTGA?ACAAG?GACAA?TTAAC?AGTTA?ACAAA?TAG 1668 。

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