CN100465276C

CN100465276C - Method for improving stress resistance of plant

Info

Publication number: CN100465276C
Application number: CNB2006101649823A
Authority: CN
Inventors: 李银心; 周树峰
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2006-12-11
Filing date: 2006-12-11
Publication date: 2009-03-04
Anticipated expiration: 2026-12-11
Also published as: CN1970768A

Abstract

The invention discloses an improving method of plant inhibiting-proof property, which is characterized by the following: transplanting SeNHX1 gene and BADH gene into plant through plant expressive carrier; sieving to obtain high-inhibiting plant; obtaining gene-transmitting tobacco; improving wild-typed plant production.

Description

A kind of method that improves stress resistance of plant

Technical field

The present invention relates to a kind of method that improves stress resistance of plant.

Background technology

The salinization problem of soil has had influence on more than 100 countries in the world, is a bottleneck that limits all semiarid zone agricultural developments.According to statistics, global solonchak accounts for 1/4 to 1/3 of land area, and the arable land in the whole world nearly 20% is subjected to saliferous influence.From the strand to the inland, dissimilar salt affected soils is all distributing from the lowland to the plateau in China.Saline-alkali soil is other country in the world not only, and also is an important land resources in China.The total area of China's saline-alkali soil has more than 500,000,000 mu approximately, and that has wherein opened up wasteland has more than 100,000,000 mu, also has more than 300,000,000 mu of salt wasteland (the accounting for 80%) utilization that awaits development.Since the influence of weather condition and human unreasonable tillage method, the degree of salting of soil and area and the trend that increases year by year in addition.China has more than 100,000,000 mu of this secondary salinization of land approximately, accounts for 10% of total cultivated area.Improvement and utilization to the salt marsh soil both at home and abroad launched extensive studies, and dropped into great amount of manpower and material resources and financial resources.Past utilizes engineering measure to improve more, though obtain certain achievement, has some unsurmountable shortcomings in these measures, can not wait lastingly as expense costliness, effect.The fresh water salt discharge also can be drained other nutrients in the soil, is to lose more than gain.And China's Freshwater resources are also not enough, so the application of this method is very limited.The difficulty in improvement saltings makes that the researchist begins to attempt from other angle, promptly plant itself is carried out the transformation of salt tolerant alkali, to improve the salt tolerance of existing plant.

Plant usually can synthesize and accumulate the balance permeate substance of some high densitys under salt stress, be used for regulating the osmotic potential of cell, the seepage water pressure that causes to alleviate a large amount of salt ions.These permeate substances have little, the good water solubility of molecular weight; Be electric neutrality in the physiological pH scope; Do not change characteristics such as enzymatic structure.These osmoregulation materials comprise amino acid, polyvalent alcohol and sugared three major types.Wherein more important, research is maximum is proline(Pro) and trimethyl-glycine.

Trimethyl-glycine is that research at present comparatively deeply, extensively is present in a kind of tenuigenin intermiscibility material in many higher plants, animal and the bacterium, the trimethyl-glycine that studies show that in the past mainly participates in the osmoregulation effect of cell, keeps the osmotic equilibrium of cell and external environment; Also has the effect of the proteic 26S Proteasome Structure and Function of stabilized cell endoenzyme simultaneously.Plant that some are important such as tomato, tobacco and Arabidopis thaliana do not accumulate trimethyl-glycine, and trimethyl-glycine is synthetic by way of simple relatively, this allow naturally people consider trimethyl-glycine synthetic by way of in key gene import in some plants that do not accumulate trimethyl-glycine, make these plants also accumulate trimethyl-glycine, to improve its resistance.

In recent years, the plant salt tolerance biotechnology research had been obtained encouraging progress, particularly by overexpression and accumulation in transgenic plant such as lower molecular weight non-toxic organic solute and osmotic protection albumen, had given transgenic plant certain salt tolerance.It is generally acknowledged that plant salt endurance is the compound inherited character that is subjected to controlled by multiple genes, depends on the interaction between a plurality of genes, is the comprehensive embodiment of multiple salt tolerant physiological character.Therefore, just individual gene is imported the resistance that plant obtains and still far can not reach satisfied effect.

Summary of the invention

The purpose of this invention is to provide a kind of method that improves stress resistance of plant.

The method of raising stress resistance of plant provided by the present invention is that the encoding gene of SeNHX1 and the encoding gene of BADH are changed in the plant by plant expression vector, and screening obtains the plant that resistance improves.

In the described method, the encoding sequence of described SeNHX1 gene is to have from GENBANK number to hold the nucleotide sequence of 492-2174 position for 5 ＇ of AY131235 hold the 5th ＇; The encoding sequence of described BADH gene is to have from GENBANK number to hold the nucleotide sequence of 4-1512 position for 5 ＇ of X69770.

Described SeNHX1 gene can import in the plant by expression vector pBI121-Se.Described BADH gene can import in the plant by expression vector pBin438.The encoding gene of described SeNHX1 and the encoding gene of BADH can change in the plant jointly by bivalent expression carrier pBinBS.

Described resistance is salt resistance and/or oxidation-resistance.

Method of the present invention both can improve monocotyledons, also can improve the resistance of dicotyledons, as: tobacco, tomato, paddy rice, wheat, corn, cucumber, willow, turfgrass or lucerne place etc.Carry the encoding gene of SeNHX1 and/or BADH encoding gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and plant transformed become plant through tissue cultivating, obtain the plant that resistance improves.

Experiment showed, the transgene tobacco of the encoding gene of encoding gene that contains SeNHX1 that method of the present invention obtains and BADH, under adverse environmental factor, all have stronger resistance, show as and have stronger salt resistance and oxidation-resistance than wild-type tobacco.SeNHX1 and BADH function have had bigger additive effect in changeing SeNHX1 gene and BADH genetic tobacco, and the linkage inheritance that SeNHX1 and BADH can be stable among its offspring.

The transgene tobacco of encoding gene that contains SeNHX1 that the inventive method obtains and the encoding gene of BADH, not only resistance strengthens, and the single plant yield under adverse circumstance (particularly at salt and/or oxidative stress) also obviously improves than wild-type plant.

Description of drawings

Fig. 1 is the pBin438 plasmid map that contains BADH

Fig. 2 is the pBI121-Se plasmid map that contains SeNHX1.

Fig. 3 is the structure schematic flow sheet of bivalent expression carrier pBinBS

Fig. 4 cuts for Xho I enzyme and identifies the pRTL2-Se electrophorogram

Fig. 5 cuts the electrophorogram of identifying pRTL2-Se for Hind III enzyme

Fig. 6 is the PCR qualification result of plasmid pBinBS

Fig. 7 is for changeing the Southern hybridization qualification result of BADH gene T0 for tobacco

Fig. 8 is for changeing the Northern hybridization qualification result of BADH gene T0 for tobacco

Fig. 9 is for changeing the Southern hybridization qualification result of SeNHX1 gene T0 for tobacco

Figure 10 is for changeing the Northern hybridization qualification result of SeNHX1 gene T0 for tobacco

Figure 11 is for changeing BADH and the SeNHX1 gene T0 Southern hybridization qualification result for tobacco

Figure 12 is for changeing BADH and the SeNHX1 gene T0 Northern hybridization qualification result for tobacco

Figure 13 is that tobacco leaf disc Paraquat (MV) is handled the photo after 24 for changeing BADH and/or SeNHX1 tobacco T0 for strain

Figure 14 is tobacco leaf disc is handled 16h at 10 μ M Paraquats conductivity variations for changeing BADH and/or SeNHX1 tobacco T0 for strain

Figure 15 is tobacco leaf disc is handled 16h at 20 μ M Paraquats conductivity variations for changeing BADH and/or SeNHX1 tobacco T0 for strain

Figure 16 ties up to 200mM NaCl for strain and coerces down superoxide-dismutase (SOD) activity change for changeing BADH and/or SeNHX1 tobacco T0.

Figure 17 ties up to 200mM NaCl for strain and coerces down glutathione reductase (GR) activity change for changeing BADH and/or SeNHX1 tobacco T0.

Figure 18 ties up to 200mM NaCl for strain and coerces down catalase (CAT) activity change for changeing BADH and/or SeNHX1 tobacco T0.

Figure 19 identifies kalamycin resistance for seed for changeing BADH and/or SeNHX1 tobacco T1

Figure 20 is for changeing the SeNHX1 gene and changeing BADH gene T1 and detect for plant PCR

Figure 21 is that SeNHX1 and BADH gene cotransformation T1 detect for plant PCR

Figure 22 is that T1 is for changeing BADH and the dual-gene tobacco seed BS15 of the SeNHX1 situation in 3 weeks of 200mM substratum upper seeding wheel

Figure 23 is that T1 is for the growing state of tobacco BS23 after 200mM N.F,USP MANNITOL coerced for 1 week

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

Acquisition and the functional verification thereof of embodiment 1, commentaries on classics SeNHX1 and/or BADH plant

The commodity of microbiotic Timentin are called " Ticarcillin/Clavulanate Acid " (injection Ticarcillin Disodium one Clavulanic Potassium), manufacturers: SmithKline Beecham Pharmaceuticals, UK is available from the 3rd affiliated hospital of Peking University.

One, the transgenosis structure of recombinant expression vector

1, the structure of recombinant vectors that contains the synthetic key gene BADH of trimethyl-glycine of prunella asiatica

Extract total RNA of prunella asiatica, (available from Takara company) carries out reverse transcription to total RNA with the AMV ThermoScript II, obtain cDNA as template, carry out pcr amplification with primer 15 ' TTACCAAGCTTATGGCGTTCCCAATTCCT 3 ' and primer 25 ' TTACCGAGCTCTCAAGGAGACTTGTACCA 3 ', amplification system is 50 μ l, contain 10mmol/LTri-HCl pH8.3,50mmol/L KCl, 1.5mmol/L Mg ₂Cl, the various dNTP of 200 μ mmol/L, every kind of primer 2 5pmol, the cDNA of 200-500ng, and the Taq polysaccharase of 1.25U, amplification condition are 95 ℃ of pre-sex change, enter 93.5 ℃ behind the 10min, 1min, 55 ℃, 1min, 72 ℃, 30 circulations of 1.5min, in 72 ℃ of insulation 10min, amplified production reclaims target gene fragment through 1% gel electrophoresis afterwards, through order-checking show this fragment have from EMBL number for 5 ＇ of X69770 hold the 1st to the 1513rd nucleotide sequence, be the sequence of BADH gene.With the BADH gene fragment that this PCR obtains, carry out HindIII and SacI double digestion.This fragment is inserted between pUC19 (available from the prosperous company that helps) HindIII and the SacI restriction enzyme site, will be shown the reorganization pUC19 carrier called after pUC19B of the encoding sequence that contains the BADH gene through order-checking.PUC19B is cut with BamHI and KpnI enzyme again, obtain containing the fragment of goal gene BADH, this fragment is inserted between the BamHI and KpnI restriction enzyme site of pBin19 (available from Clonetech company), the detection of checking order will show the recombinant expression vector called after pBin438 that contains BADH through order-checking.The accession number of BADH gene in the EMBL database is X69770; The sequence of BADH gene is to have from the EMBL number of landing to hold the 1st to the 1513rd nucleotide sequence for 5 ＇ of X69770.

2, contain salicornia europaeal vacuole skin Na ⁺/ H ⁺The structure of reverse transport protein (anitiporter) gene SeNHX1 recombinant vectors

Extract total RNA of salicornia europaeal over-ground part, the synthetic first chain cDNA of reverse transcription is as template, the primer (primer 35 ' GAGTCTAGATGGAGGGAATTTGGAGGAGC 3 ' and primer 45 ' TCTTCTAGACTGCCTAACTGCCTCGGATT 3 ') that has Xba I restriction enzyme site by adding carries out pcr amplification, amplification system is that 50 μ l are respectively 10 * buffer, 5 μ l; 10 * dNTP (2.5mmol/L each), 5 μ l; Each 5 μ l of primer 3 and primer 4 (10pmol/ μ l); Taq polysaccharase (1U/ μ l), 3 μ l; CDNA template 5 μ l; Distilled water 22 μ l, amplification system are 95 ℃ of pre-sex change 4min; 95 ℃, 1min; 55 ℃, 1min; 72 ℃, 1.5min; 35 circulations are 72 ℃ at last, 10min.The fragment that obtains of amplification through order-checking show have from GENBANK number for 5 ＇ of AY131235 hold the 298th to 2201 nucleotide sequence, this sequence contains the expressed intact frame of SeNHX1.After the fragment of above-mentioned amplification cut with Xba I enzyme, be inserted into the Xba I enzyme recognition site of pBI121 (available from Clonetech company), after order-checking, order-checking detected the recombinant vectors called after pBI121-Se (Fig. 2) that shows the expressed intact frame that contains SeNHX1.

3, the structure (the building process synoptic diagram as shown in Figure 3) that contains BADH and SeNHX1 bivalent expression carrier pBinBS

With the goal gene band that reclaims 1.9kb behind the XbaI enzyme cutting plasmid pBI121-Se, handle back and the XbaI enzyme cutting site that is inserted into pRTL2 (available from Clonetech company) with CIAP.Because carrier has all carried out identical single endonuclease digestion with the goal gene segment in connection procedure, forward and reverse situation appears in ease of connection, in order to choose the clone that forward connects, carries out enzyme with XhoI on the carrier and the XhoI on the goal gene and cuts evaluation.If the forward connection can cut out the fragment of 486bp, if reverse connection can cut out the fragment of 1741bp.To be accredited as the carrier called after pRTL2-Se that forward inserts, enzyme is cut the result as shown in Figure 4, and swimming lane 1 is the pRTL2-Se plasmid among Fig. 4; Swimming lane 2 is cut the result for pRTL2-Se Xho I enzyme, and swimming lane 3 is Maker D2000 (available from a Takara company).

Obtain two fragments of 3342bp and 2461bp after pRTL2-Se cut with Hind III enzyme, the result as shown in Figure 5, swimming lane 1 is pRTL2-Se among Fig. 5, swimming lane 2 is the pRTL2-Se after Hind III enzyme is cut, and swimming lane 3 is Maker IH (is Time Inc. available from sky, Beijing) (arrow among Fig. 5 is the 2nd band above electrophorogram).Wherein the 3342bp fragment contains encoding sequence and its expression regulation sequence of SeNHX1, reclaims this fragment, and the pBin438 that cuts with Hind III enzyme is connected.

Select the part clone and carry out the recombinant vectors that PCR detection connection obtains.

The primer of pcr amplification: the primer that detects SeNHX1:

Primer 5:5 ' TCTAGATGGAGGGAATTTGGAGGAGC3 ',

Primer 6:5 ' GGATCCTGCCTAACTGCCTCGGATT3 ';

Detect the primer of BADH:

Primer 7:5 ' AGAATGGCGTTCCCAATTCCTGCTC 3 ',

Primer 8:5 ' TTCAAGGAGACTTGTACCATCCCCA 3 '.

The result as shown in Figure 6, the result shows, primer 5 and primer 6 amplifications obtain the SeNHX1 full-length cDNA of 1904bp, primer 7 and primer 8 amplifications obtain the BADH full-length cDNA of 1513bp, show that goal gene SeNHX1 has been connected on the plasmid pBin438.Show that to detecting the further sequencing result of correct clone gene SeNHX1 reading frame is correct, target gene sequences also is correct, will identify correct recombinant vectors called after pBinBS.Swimming lane 1-7 is respectively the PCR result of recombinant vectors pBinBS clone 1-7SeNHX1 among Fig. 6; Swimming lane M is MakerIII (is Time Inc. available from sky, Beijing); Swimming lane 8-14 is respectively the PCR result of recombinant vectors pBinBS clone 1-7BADH.

Two, change acquisition and the evaluation of BADH and/or SeNHX1 tobacco

1, changes the acquisition of BADH and/or SeNHX1 tobacco

PBI121-Se, pBin438, pBinBS are changed among the tobacco bred W38 by the mediation of agrobacterium tumefaciens (Agrobacteriumtumefaciens) LBA 4404.Concrete grammar is as described below:

The preparation of tobacco aseptic seedling: tobacco bred W38 seed is used 10% clorox (adding two Tween 20) sterilization 15min then with 70% alcohol immersion 30s, during constantly shake, use aseptic water washing subsequently 3 times, be inoculated on the MS substratum.Place between cultivation and to germinate (26 ± 2 ℃ of temperature, humidity 60-80%), treat seed germination after, cultivated 25-30 days.

In order to determine that tobacco W38 wild-type blade selects minimum lethal concentration on the substratum at kantlex, improve transformation efficiency, the kantlex susceptibility of W38 is detected, to determine its threshold concentration that can tolerate.On 4 selection substratum that contain the different concns kantlex (50mg/L, 100mg/L, 150mg/L, 200mg/L), when finding that kantlex concentration is 50mg/L, the differentiation of tobacco leaf disc and growth are affected hardly.In the time of the concentration of 100mg/L, be subjected to certain inhibition, but major part still can survive.And after 2 weeks on the 150mg/L substratum, the tobacco leaf disc explant is all dead.Therefore carry out the screening of transformed plant with the substratum that contains the 150mg/L kantlex.

PBI121-Se, pBin438 and pBinBS are changed over to respectively among the Agrobacterium LBA 4404, obtain containing the Agrobacterium engineering bacteria of pBI121-Se, pBin438 and pBinBS respectively.Get tobacco bred W38 aseptic seedling blade as transforming explant, place the Agrobacterium engineering bacteria liquid that contains pBI121-Se, pBin438 and pBinBS respectively, infect 5-10min.Take out blade, place on the aseptic dry Whatman filter paper, blot residual bacterium liquid.The tobacco leaf that infected is placed MS ₀On the substratum (the MS substratum that does not contain any hormone), cultivate 48h in the dark altogether.With material transfer to the SIM substratum (the MS substratum that contains 1.0mg/L6-BA, 0.1mg/L NAA, 150mg/L kantlex (km) and 150mg/L Timentin) in 25 ± 2 ℃, illumination every day 12h.Per 2 weeks are changed a subculture.3-4 has green bud to grow after week.When treating bud length, it is downcut immigration SMS to 3-4cm ₀Root induction in the substratum (the MS substratum that contains 150mg/L kantlex and 150mg/L Timentin) can see about 10 days that young root grows.After treating that plant root is than prosperity, vessel port was opened wide hardening 3-5 days in room temperature.Take out plant afterwards, clean agar, transplant in greenhouse or field soil.Promptly changeed the T0 of pBI121-Se, pBin438 and pBinBS respectively for transfer-gen plant.Obtain changeing BADH gene plant (changeing the pBin438 plant) 23 strain T0 altogether for transfer-gen plant; Change SeNHX1 plant (changeing the pBI121-Se plant) 11 strain T0 for plant; BADH and SeNHX1 cotransformation plant (changeing the pBinBS plant) 32 strain T0 are for transfer-gen plant.

2, change the Molecular Detection of the T0 of BADH and/or SeNHX1 tobacco for transfer-gen plant

1) PCR detects

The T0 that extracts above-mentioned commentaries on classics pBI121-Se, pBin438 that obtains and pBinBS carries out following PCR respectively and detects for the DNA in the plant young leaflet tablet:

The pcr amplification of A, BADH gene

Respectively with the T0 that changes pBin438 and pBinBS for the DNA in the plant young leaflet tablet as template, carry out pcr amplification and detect BADH cDNA, be contrast with wild-type tobacco W38, the primer of detection is:

Primer 9:5 ＇ AGAATGGCGTTCCCAATTCCTGCTC 3 ＇,

Primer 10:5 ＇ TTCAAGGAGACTTGTACCATCCCCA 3 ＇.

Reaction system is: totally 50 μ l, 10 * buffer, 5 μ l, 10 * dNTP (2.5mmol/L each), 5 μ l, primer 9 (10pmol/ μ l) 5 μ l, primer 10 (10pmol/ μ l) 5 μ l, template DNA (0.1 μ g/ μ l), 5 μ l, Taq polymerase (1U/ μ l) 3 μ l, sterilized water 22 μ l.

PCR response procedures: 95 ℃ of 4min of elder generation; 95 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1.5min, totally 35 circulations; Last 72 ℃ of 10min.

The result shows that changeing pBin438 and pBinBS T0 all amplifies 1513bp for plant (from the BADH cDNA total length of holding the 1st nucleotide sequence to the 1513 for 5 ＇ of X69770 GENBANK number, and wild-type does not amplify any fragment.

The pcr amplification of B, SeNHX1 gene

Respectively with the T0 that changes pBI121-Se and pBinBS for the DNA in the plant young leaflet tablet as template, carry out pcr amplification and detect SeNHX1 cDNA, be contrast with wild-type tobacco W38, the primer of detection is:

Primer 11:5 ' TCTAGATGGAGGGAATTTGGAGGAGC3 ',

Primer 12:5 ' GGATCCTGCCTAACTGCCTCGGATT3 '.

PCR response procedures and reaction system are with step A.

The result shows that commentaries on classics pBI121-Se T0 all amplifies 1904bp (holding the 304th to the 2208th for 5 ＇ of AY131235 from GENBANK number) SeNHX1 cDNA total length for plant and commentaries on classics pBinBS T0 for plant), and the wild-type plant does not amplify any fragment.

2) Southern hybridization analysis

Part PCR is detected the male plant carried out the Southern hybridization analysis.

The detection of A, commentaries on classics BADH gene plant

The T0 that 23 strains that obtain is changeed pBin438 has carried out further Southern for 6 strains in the plant and has detected.Respectively change the genomic dna of pBin438 strain system and wild-type tobacco W38 contrast with KpnI and EcoRI double digestion after, be probe, carry out Southern hybridization with the BADH cDNA of α-32P mark.The result as shown in Figure 7, the result shows that the T0 that changes pBin438 is that B1 is two copies for strain; Its T0 is that B8, B15, B19, B23 are single copy for strain, and its T0 is that B5 does not have corresponding hybridization signal for strain.Swimming lane 1 is the contrast of pBin438 plasmid among Fig. 7; Swimming lane 2 is wild-type tobacco W38 contrast; It is B1, B5, B8, B15, B19 and B23 for strain that swimming lane 3-8 is respectively the T0 that changes pBin438.

Simultaneously Southern is detected male strain system and carried out the Northern detection, extracting the T0 that changes pBin438 is RNA for strain, BADH cDNA with α-32P mark is a probe, carry out Northern hybridization, the result as shown in Figure 8, the result shows that strain is that the expression of B1, B8 and B23 is higher, because B1 is two copies, has selected the B8 of single copy and B23 to study in the functional analysis afterwards.Among Fig. 8, swimming lane 1 is wild-type tobacco W38 contrast; It is B1 for strain that swimming lane 2-6 is respectively commentaries on classics pBin438 T0, B8, B15, B19 and B23; RRNA contrasts as applied sample amount.

The detection of B, commentaries on classics SeNHX1 gene plant

The 5 strain Southern that 11 strains that obtain are changeed in the SeNHX1 gene plant detect, change the genomic dna of pBI121-Se T0 generation 5 strain plant and wild-type tobacco W38 contrast with BamH I/EcoR I complete degestion after, be that probe has carried out Southern hybridization with the SeNHX1 cDNA of α-32P mark.The result as shown in Figure 9, the result shows that goal gene all has been incorporated in the tobacco gene group in these 5 plant, and is single copy and inserts.Among Fig. 9, swimming lane 1 is the plasmid contrast; Swimming lane 2 is the wild-type contrast; Swimming lane 3-7 is respectively changes pBI121-Se T0 for plant S1, S8, S11, S15 and S21.

Extract to change the total RNA of pBI121-Se T0, carry out Northern and detect for plant, the result as shown in figure 10, the result shows that goal gene has obtained correct expression at transcriptional level, but the expression amount of each transgenic line is different.Among Figure 10,1 is wild-type tobacco W38 contrast; 2-6 is respectively transgenic line S1, S8, S11, S15 and S21; RRNA contrasts as last sample.

The detection of the dual-gene cotransformation plant of C, BADH and SeNHX1 (changeing the pBinBS plant)

The Southern detection has been carried out in 5 strains in the positive seedling of 32 strains that BADH and SeNHX1 cotransformation are obtained.Using KpnI/EcoRI (A among Figure 11) or BamHI/EcoRI (B among Figure 11) enzyme to cut respectively the T0 plant of 5 strain PCR test positive commentaries on classics pBinBS and the genomic dna of wild-type tobacco W38 contrast, is that probe is hybridized with α-32P-dCTP mark SeNHX1 cDNA (B among Figure 11) or BADH cDNA (A among Figure 11) respectively.The result as shown in figure 11, the result shows that goal gene BADH and SeNHX1 have been incorporated in the tobacco gene group.Wherein strain is that two genes of BS1 (swimming lane 3 of A and B among Figure 11) all are two copies, shows it might is that multidigit point inserts, and all the other strains are that two genes are single copy.Reorganization does not take place or loses in the identical explanation of copy number goal gene on the T-DNA in conversion process of goal gene BADH and SeNHX1 in same strain system, as an integration in the tobacco gene group.The swimming lane 1 of A and B is respectively the contrast of pBinBS plasmid among Figure 11; Swimming lane 2 is respectively the wild-type contrast; Swimming lane 3-7 is respectively changes pBinBS T0 for plant BS1, BS5, BS15, BS21 and BS23.

Extract the T0 plant and the total mRNA of wild-type tobacco W38 contrast blade that change pBinBS and carry out the Northern detection, be that probe is hybridized with α-32P-dCTP mark SeNHX1 cDNA (B among Figure 11) or BADH cDNA (A among Figure 11) respectively, the result as shown in figure 12, the result shows changes pBinBS T0 for plant BS1, BS5, BS15, BS21 and BS23 all can detect expression signal, illustrate that foreign gene BADH in these several plant and SeNHX1 can normal expressions, but expression level has bigger difference, and be not directly proportional with copy number, selecting the high strain of two equal expression amounts of gene is that BS5 and BS15 carry out later functional analysis.Among the A and B of Figure 12, swimming lane 1 is respectively wild-type tobacco W38 contrast; Swimming lane 2-6 is respectively changes pBinBST0 for plant BS1, BS5, BS15, BS21 and BS23; RRNA is the applied sample amount contrast.

Detecting by Southern and Northern and to show and obtain 3 types transgene tobacco, is respectively to change SeNHX1 genetic tobacco (changeing the pBI121-Se tobacco), change BADH genetic tobacco (changeing the pBin438 tobacco) and SeNHX1 and BADH gene cotransformation tobacco (changeing the pBinBS tobacco).Goal gene has been incorporated in the tobacco gene group, and has obtained correct expression.

Three, change the Function Identification of BADH and/or SeNHX1 tobacco

1, the detection of BADH activity and beet alkali content

In order to detect the expression that external source BADH gene inserts the tobacco gene group, to the T0 that changes pBin438 (B8, B23) and pBinBS (BS5, BS15) for plant or wild-type tobacco (WT) BADH enzymic activity and the beet alkali content in no salt stress and 200mmol/L condition of salt stress lower blade measure.

1) BADH enzyme assay

A. the extraction of enzyme liquid

The T0 that gets the above-mentioned commentaries on classics pBin438 of 2g (B8, B23), commentaries on classics pBin438BS (BS5, S15) after the quick-frozen grinding, at room temperature adds albumen extraction buffer (solution that contains 50mmol/L Hepes/KOH (pH 8.0), 1.0mmol/L EDTA (pH8.0) and 5.0mmol/L DTT) extracting 5min respectively for plant and wild-type tobacco (WT) plant leaf in liquid nitrogen.4 ℃, the centrifugal 10min of 12000rpm gets supernatant and is stored in 4 ℃.Enzyme liquid is measured zymoprotein concentration in ultraviolet spectrophotometer 280nm place after suitably diluting

B. enzyme assay

Reaction system (1ml): extraction buffer (Extraction buffer) 0.85ml, 20 * betaine aldehyde chloride mother liquor (20mmol/L) 0.05ml, 20 * NAD ⁺Mother liquor (20mmol/L) 0.05ml, the zyme extract 0.05ml that steps A obtains.

The spectrophotometer wavelength is transferred to the 340nm reaction pick up counting the record initial OD from adding enzyme liquid ₃₄₀OD when reaching 5min (or 10min) ₃₄₀

The ratio of enzyme is lived

(nmol \cdot \min^{- 1} \cdot {mg}^{- 1} protein) = \frac{ΔOD \times 10^{- 3}}{ϵ_{\max} \times C} \times 10^{9}

In the formula: Δ OD is the mean value that the every min of light absorption value increases under the 340nm; ε _MaxFor the NADH molar extinction coefficient, equal 6.22 * 10 ³C is the amount of the zymoprotein that mensuration added, and unit is mg; 10 ^-3Be converted into the coefficient that rises for the 1ml reaction system; 10 ⁹Be converted into the coefficient of nmol for mol.

2) detection of beet alkali content

With reference to the method for (2000) such as Chen Shaoliang (.2000. reversed-phase HPLC paired ion chromatographies such as Li Jinke are measured the trimethyl-glycine in the plant tissue for Chen Shaoliang, Bi Wangfu. Botany Gazette, 42; 1014-1018) T0 that changes pBin438 (B8, B23), pBinBS (BS5, BS15) is carried out extraction and the purifying and the mensuration of trimethyl-glycine for plant or wild-type tobacco (WT).

Liquid chromatogram measuring condition: the liquid chromatographic system P4000 of U.S. TSP company pump, SCM1000 vacuscope, SN4000 controller, UV3000 UV-detector.With C18 reverse-phase chromatographic column (10 μ m, 4.6mm * 250mm, Irregular-H add material), moving phase is the K of 50mmol/L ₂HPO ₄(pH4.45) and 0.1% PIC-8 (perfluoroetane sulfonic acid, Waters company), flow velocity 0.7ml/min.

The result is as shown in table 1, and the result shows, no matter whether NaCl coerces existence, the BADH activity of wild-type tobacco is all very low, almost detect less than.And 4 plant (B8, B23, BS5, BS15) that have the BADH foreign gene can detect tangible BADH activity (5.63-8.87U) under salt stress, compare with the wild-type contrast to reach utmost point conspicuous level.But have only plant B8 and B23 to show relative higher BADH activity under normal operation; Strain is that the BADH activity of BS5 and BS15 and wild-type does not have the significance difference.Beet alkali content has also shown the active similarly trend with BADH.No matter whether salt stress, beet alkali content is all very low in the wild-type plant, almost detect less than, wild-type plant beet alkali content and each transgenic line content difference do not reach conspicuous level under the normal irrigation condition, and the beet alkali content of B8, B23, BS5, BS15 all is higher than wild-type extremely significantly but coerce down at salinity.

Above-mentioned experiment shows that the plant that has the BADH foreign gene coerces BADH activity and beet alkali content obviously to improve than wild-type plant at salinity.

Table 1.T0 is for BADH activity and the beet alkali content of each transgenic line under salinity is coerced

3 multiple mean numbers of numerical value in the table indicate different alphabetical persons and are LSD check significant difference on P≤0.01 level in every row.

2, the salt tolerance in T0 generation and oxidation-resistance are identified

Method by cuttage detects the positive T0 that changes pBI121-Se, pBin438 and pBinBS of a part that obtains to Southern and Northern and expands numerously for seedling, sets up clonal population.

1) mensuration of photosynthetic efficiency and fluorescence induction kinetic parameter

It is exactly the reduction of photosynthetic efficiency that salinity is coerced one of injury that plant is caused, thereby has reduced the accumulation of plant assimilation material, is unfavorable for the growth of plant.In addition, salinity is coerced the destruction that also can cause photosynthetic organ, is reflected at the active decline of PSII, is embodied in the decline of Fv/Fm.With the above-mentioned positive pBI121-Se that changes, the numerous strain of the expansion in T0 generation of pBin438 and pBinBS system is transplanted in the flowerpot in greenhouse, after transplanting for 4 weeks, normal irrigation is carried out in numerous strain system or wild nature type tobacco W38 contrast (WT) respectively or irrigation 200mM NaCl solution is coerced to expanding, after 6 weeks, measure photosynthetic speed and PSII activity, the result is as shown in table 2, the result shows changes pBI121-Se under normal operation, the photosynthetic speed of expansion numerous strain system in T0 generation of pBin438 and pBinBS and wild-type (WT) contrast does not reach significant difference, and salinity is coerced the photosynthetic speed that has reduced all strains systems.Wild-type (WT) contrast relatively, the numerous strain of expansion of changeing T0 generation of pBI121-Se, pBin438 and pBinBS is that photosynthetic speed is subjected to the influence of salt stress little, and coercing down at the 200mM salinity, the photosynthetic speed of each transgenic line all is significantly higher than wild-type contrast (WT).Wherein the photosynthetic speed of BS5 strain system is up to 8.5 μ mol CO ₂m ^-2S ^-1, and wild-type only is 4.9 μ mol CO ₂m ^-2S ^-1The numerous strain of expansion of changeing T0 generation of pBI121-Se, pBin438 and pBinBS is that salinity is coerced down higher photosynthetic speed, has guaranteed the accumulation of its assimilate, thus the growth that it can be continued.

The Fv/Fm that under the normal irrigation condition, changes the expansion numerous strain system in T0 generation of pBI121-Se, pBin438 or pBinBS and wild-type contrast about 0.86, and between each transgenic line and they with contrast between do not have significant difference; The Fv/Fm that coerces the numerous strain based material of expansion that changes down the T0 of pBI121-Se, pBin438 and pBinBS generation at salinity all is significantly higher than the wild-type contrast, and the Fv/Fm between the same type transgenic line does not reach significant difference.The Fv/Fm of wild-type under 200mM NaCl is 0.71, only be 83% under the normal condition, though each the numerous strain of expansion system that changes T0 generation of pBI121-Se, pBin438 and pBinBS coerces all normal down condition at salinity decline is arranged, all be higher than 0.8, strain is that the Fv/Fm of S15 is up to 0.84.Above result shows that the numerous strain of expansion of changeing the T0 of pBI121-Se, pBin438 and pBinBS generation ties up to salinity and coerces down the destruction that photosynthetic organ is subjected to and will be lower than wild-type, and PSII has higher relatively activity.

Wherein, WT is a wild-type in the table 2; S1, S15 is for changeing the pBI121-Se T0 numerous strain of the expansion system in generation; B8, B23 is for changeing the pBin438 T0 numerous strain of the expansion system in generation; BS5, BS15 is for changeing the pBinBS T0 numerous strain of the expansion system in generation.

Photosynthetic speed and the Fv/Fm of table 2. transgene tobacco under 200mM NaCl

Numerical value is 3 multiple mean numbers in the table, indicates different alphabetical persons in every row and is LSD check significant difference on P≤0.05 level

2) paraquat resistance detects

Paraquat (paraquat) has another name called methyl viologen, and (methyl viologen MV), is a kind of double pyridines compound.Numerous strain based material of expansion or wild-type tobacco W38 contrast (WT) to T0 generation of changeing pBI121-Se (S1, S15), pBin438 (B8, B23), pBinBS (BS5, BS15), with Paraquat (10 μ M or 20 μ M) or water treatment blade 24 hours, measure specific conductivity and mda (MDA) content after handling, to determine the anti-oxidant ability of coercing of different material sections.The result shows that wild-type leaf plate edge has occurred sallow, and begins to bleach after 10 μ M MV coerce 24h; Brownization occur though each transgene tobacco contrasts color, the degree of coercing that is subjected to is starkly lower than wild-type.After 20 μ M MV coerced 24h, wild-type almost all bleached, and is downright bad fully; And each transgenic line only more serious brownization (Figure 13) occurred in the edge section of leaf dish.

The MDA content of each material is along with the increasing of MV concentration presents the trend of tangible rising, the amplitude maximum that wild-type rises, and the MDA content under 0 μ M, 10 μ M, 20 μ M MV is respectively 98.50nmol/g.522.83nmol/g and 840.70nmol/g.Also have largely and rise though change the numerous strain of the expansion system in T0 generation of pBI121-Se, pBin438 and pBinBS, no matter in the amplitude that rises or on the MDA absolute content, all be less than wild-type.But dissimilar transgenic lines has bigger difference, and the MDA content that changes SeNHX1 and BADH dual-gene (changeing pBinBS strain system) strain system will be lower than single-gene transformation plant (changeing pBI121-Se and pBin438 strain system); Change SeNHX1 gene strain system and will be lower than commentaries on classics BADH gene strain system.Illustrated that to change the oxidative stress degree that dual-gene strain system (changeing pBinBS strain system) is subjected to minimum, secondly for changeing BADH gene strain system and changeing SeNHX1 gene strain system, the oxidative stress degree the most serious (concrete outcome is as shown in table 3) that the wild-type contrast is subjected to.

MDA content behind the table 3. transgene tobacco leaf dish Paraquat processing 24h

Numerical value is 3 multiple mean numbers in the table, indicates different alphabetical persons in every row and is LSD check significant difference on P≤0.05 level.

After Paraquat is handled, the specific conductivity of changeing the expansion numerous strain system in T0 generation of pBI121-Se (S1), pBin438 (B8), pBinBS (BS15) and wild-type contrast with in the relation of soak time among the MV shown in Figure 14,15, the prolongation that the specific conductivity of each strain system is all coerced along with MV presents tangible ascendant trend, but the speed that wild-type rises will be higher than transgenic line.Under 10 μ M MV handle (Figure 14), each conductivity of electrolyte materials does not have big difference in the 4h of beginning, begins wild-type from 8h and will be higher than each transgenic line.Under 20 μ M MV handle (Figure 15), 12h is a weight break point.12h in the past, each conductivity of electrolyte materials does not have big difference, after 12h, the specific conductivity of wild-type significantly is higher than each transgenic line.

BADH is improving the transgenic plant salt resistance, be likely the higher structure by stablizing some enzymes and keep the integrity of film to work, all cause the rapid rising of MDA content at the MV of 10 μ M and 20 μ M, but in the transgene tobacco that changes BADH, MDA content will significantly be lower than the wild-type contrast, and the rising of changeing BADH tobacco specific conductivity also will be lower than wild-type, illustrates that BADH is playing significant effect aspect the integrity that reduces peroxidation and maintenance film really.

3) activities of antioxidant enzymes detects

With the numerous strain of expansion in above-mentioned positive T0 generation of changeing pBI121-Se (S1, S15), pBin438 (B8, B23) and pBinBS (BS5, BS15) is to be transplanted in the flowerpot in greenhouse, after transplanting for 4 weeks, normal irrigation is carried out in numerous strain system and wild nature type tobacco W38 contrast (WT) respectively or irrigation 200mM NaCl solution is coerced to expanding, after handling for 6 weeks, measure the activity of the enzyme relevant (superoxide-dismutase (SOD), glutathione reductase (GR) and catalase (CAT)) under high-salt stress with oxidation.The active detected result of superoxide-dismutase (SOD) as shown in figure 16, the result shows, the intravital superoxide-dismutase of plant (SOD) specific activity is lower under the normal condition, all be lower than 6U, but each transgenic line SOD enzymic activity has significantly rising under 200mM NaCl, each transgenic line all is significantly higher than the wild-type contrast, wherein the BS5 of SeNHX1 gene and BADH gene cotransformation (changeing pBinBS strain system) and the enzyme of BS15 strain system are lived the highest, be respectively 28.2U and 28.5U, risen 4.9 times and 4 times than its normal processing contrast.The SOD enzyme work of changeing the strain of BADH gene and be B8 and B23 will be significantly higher than that to change the strain of SeNHX1 gene be S1 and S15.

The activity of glutathione reductase (GR) detects as shown in figure 17, and the result shows that the expansion numerous strain system that changes the T0 of pBI121-Se (S1, S15), pBin438 (B8, B23), pBinBS (BS5, BS15) generation and the activity of wild-type contrast (WT) glutathione reductase (GR) under the normal irrigation condition all do not reach conspicuous level; Coercing the numerous strain of the expansion system that changes down the T0 of pBI121-Se (S1, S15), pBin438 (B8, B23) and pBinBS (BS5, BS15) generation at salinity all is significantly higher than wild-type and contrasts.But the work of GR enzyme has big difference between different transgenic lines, changes SeNHX1 and the dual-gene strain of BADH and is BS5 and BS15 and will be significantly higher than and respectively change single-gene strain system; BADH gene strain system is significantly higher than changes SeNHX1 gene strain system.The active detected result of catalase (CAT) as shown in figure 18, the result shows, though the enzyme work of the catalase of wild-type tobacco (CAT) under salinity is coerced will be higher than normal condition, still significantly is lower than each transgenic line.The CAT enzyme that changes SeNHX1 and the dual-gene strain of BADH and be BS5 is lived the highest, is that to be significantly higher than the strain of the single SeNHX1 of commentaries on classics gene be S1 and S15 for the enzyme work of B8 and B23 but change the strain of BADH gene.

Among Figure 16-18, numerical value is 3 multiple mean numbers among the figure, and the strain that indicates different letters among the figure in the same treatment ties up to significant difference on P≤0.05 level.

Coerce down enzyme work with the closely-related 3 kinds of enzymes of oxidation from salinity, the enzyme work of changeing dual-gene strain system (changeing pBinBS strain system) will be significantly higher than the strain of commentaries on classics single-gene (BADH gene and SeNHX1 gene) and be, changes BADH gene strain system (changeing the pBin438 strain is) and is significantly higher than commentaries on classics SeNHX1 gene strain system (changeing pBI121-Se strain system).Illustrate that SeNHX1 and BADH gene all can improve the activity of antioxidase, two genes have additive effect, but BADH gene role will be significantly higher than the SeNHX1 gene.

Coerce the enzyme that down BADH accumulation significantly improved CAT, SOD, GR at 200mM NaCl and live, illustrate that changeing the BADH genetic tobacco will be higher than wild-type on the efficient of removing levels of reactive oxygen species.In view of this, BADH aspect be likely by the activity that improves some antioxidases and effectively kept the ortho states of going back in the cell, thereby reduce the peroxidation of plasma membrane, only need a spot of trimethyl-glycine just passable and will exercise this function.

4) ATPase and PPase determination of activity

The SeNHX1 gene is the Na that is positioned on the vesicular membrane ⁺/ H ⁺Reverse running albumen, its major function is against Na ⁺Concentration gradient is with intracytoplasmic Na ⁺Separating reduces the murder by poisoning of organoid film system and enzyme in the salt ion pair cell matter in vacuole, also reduced the osmotic potential of cell simultaneously, alleviates the osmotic stress of cell.Main with the H on the vacuole ⁺-ATPase (V-ATPase) and the membranous sub-gradient of striding that produces thereof are motivating force.Vesicular membrane pyrophosphorylase PPase is prevalent on the vesicular membrane of higher plant, and hydrolysis PPi is with the H in the tenuigenin ⁺Be transported in the vacuole; H on the plasma membrane ⁺-ATPase (P-ATPase) plays the similar function with V-ATPase.More than 3 kinds of enzymes the energy of cell transmembrane transportation are provided, be to keep Na and stride membranous sub-gradient ⁺/ H ⁺The normal function of reverse running albumen is necessary, external source Na ⁺/ H ⁺The importing of gene will have influence on the activity of these enzymes probably, for this reason, has measured the activity of V-ATPase, P-ATPase and PPase.The mensuration of the extraction of cytolemma and 3 kinds of related enzyme activities is carried out (Vera-Estrella R with reference to the method for (2005) such as Vera-Estrella, Barkla B J, Garc í a-Ram í rezLa.et al.2005.Salt stress in Thellungiella halophila activates Na ⁺Transport mechanismsrequired for salinity tolerance.Plant Physiol, 9:1507-1517).

According to the method described above normal irrigation is carried out in expansion numerous strain system in T0 generation of changeing pBI121-Se (S1, S15), pBin438 (B8, B23), pBinBS (BS5, BS15) or wild nature type tobacco W38 contrast (WT) respectively or irrigate 200mM NaCl solution and coerce, after handling for 6 weeks, measure the activity of V-ATPase, P-ATPase and PPase, the result is as shown in table 4, the result shows, under 200mM NaCl, each transgenic line and wild-type contrast vesicular membrane PPase specific activity normal irrigation condition rise to some extent, and it is big that the amplitude that each transgenic line rises is wanted.But, no matter coerce still that the PPase enzymic activity of each transgenic line and wild-type does not have significant difference under the normal irrigation condition at salinity.The difference of the P-ATPase enzymic activity of transgenic line and wild-type plant does not reach conspicuous level under the normal irrigation condition; The P-ATPase enzyme of all transgenic lines is lived and all is significantly higher than the non-transgenic contrast under 200mM NaCl, and the P-ATPase enzymic activity of all strain systems increases in normal irrigation.

In each transgenic line, V-ATPase has been subjected to inducing of salinity consumingly, wherein strain is that the enzymic activity of BS15 (changeing pBinBS strain system) under the normal irrigation condition is 14.13U, enzymic activity under salinity is coerced is 33.07U, 1.34 times have been risen, all the other each transgenic line enzymes are lived and have all been risen more than 1 times, but the normal irrigation conditions of the enzymic activity under the salt stress only slightly rises in wild-type tobacco.Compare without any difference with wild-type in the V-ATPase enzymic activity that does not have each transgenic line under the condition of salt stress, but under salt stress, all be significantly higher than wild-type.Above result shows that the importing intensive of SeNHX1 gene has strengthened the V-ATPase enzymic activity of each transgenic line under salinity is coerced.

Table 4.T0 coerces the enzymic activity of following ATPase and PPase at 200mM NaCl for transgene tobacco

5) ion content is measured

For most of plants, one of main injury that salinity is coerced is exactly a large amount of Na ⁺Ion accumulation causes the toxic action to enzyme in the destruction of plant membrane system and the kytoplasm in tenuigenin.Because the vacuoleization degree of root and tender leaf is lower, and root is the organ that at first contacts salt, and stem plays the function of underground part salt to the over-ground part transportation, the vacuoleization degree of Lao Ye is than higher, its salt content may have very big difference in these four kinds of organs according to a preliminary estimate, and the accumulation of excessive salinity often can have influence on K ⁺Accumulation.Therefore measured under the salt stress (200mM NaCl) and changeed Na among BADH gene (B8, B23) and SeNHX1 gene (S1, S15) tobacco root, stem, tender leaf, the Lao Ye ⁺And K ⁺Ionic content (table 5, table 6).

Na under the salt stress ⁺The result is as shown in table 5 for the ionic assay, and the result shows the Na that under normal circumstances only accumulates trace in the tobacco spire ⁺, but coerce Na down at salinity ⁺Content sharply rises, and has risen 38.6 times in wild-type, and each transgenic line has also shown identical trend.Under normal circumstances, the Na of each strain system ⁺The difference of content does not reach conspicuous level; But coerce two Na that strain is S1 and S15 that change SeNHX1 down at salinity ⁺Content is significantly higher than wild-type, and wherein strain is the Na of S1 ⁺Content reaches 17.85mg/g, changes two Na that strain is B8 and B23 of BADH gene ⁺Content significantly is lower than wild-type.

Under normal circumstances, the Na among the tobacco Lao Ye ⁺Content illustrates that than high in the tender leaf Lao Ye may be as the effect in Na+ storehouse more.Salinity is coerced down commentaries on classics SeNHX1 strain system and the BADH strain is Lao Ye Na ⁺Content will be higher than tender leaf, and is identical with the content trend of sodium in the tender leaf, and changeing the SeNHX1 strain is Na ⁺Content is significantly higher than wild-type, changes BADH gene strain system and significantly is lower than wild-type.

All strains tie up to the Na in the root under the normal irrigation condition ⁺Content difference does not reach conspicuous level; Each transgenic line all significantly is lower than the wild-type contrast but coerce down at 200mMNaCl, and changes the Na of SeNHX1 strain system and BADH strain system ⁺Content does not reach conspicuous level.Sodium ion in changeing SeNHX1 strain system in the root might be transported to the leaf of overground part, and then separates among the vacuole, has caused Na in the root ⁺The decline of content.Each strain ties up to the Na in the stem under the normal irrigation condition ⁺Content difference does not reach conspicuous level yet; But coercing down at salinity, commentaries on classics SeNHX1 strain is Na ⁺Content will be significantly higher than the wild-type contrast, and the difference of changeing BADH strain system and wild-type does not reach conspicuous level.Illustrate that changing over to of external source BADH gene can reduce the Na of plant salinity under coercing ⁺Content has reduced Na ⁺Toxic action, thereby improved salt tolerance.And the Na in changing SeNHX1 tobacco T0 generation leaf and stem over to ⁺As if content but is significantly higher than contrast, and this is conflicting with the raising of the salt tolerance of the strain system that changes these two genes respectively over to.But, the Na that in this test, is measured ⁺Content is the total content in each subcellular organelle of vegetable cell, for vegetable cell, and the Na in tenuigenin only ⁺The injury maximum that plant is become.BADH is probably at the Na that regulates cytolemma ⁺The ion transportation aspect plays a part positive, has reduced cytolemma to Na ⁺The ionic permeability, thus the total Na of plant reduced ⁺Content.And in changing the SeNHX1 tobacco over to, be positioned at the Na on the vesicular membrane ⁺/ H ⁺Reverse running albumen can be the Na in the tenuigenin ⁺Be transported among the vacuole, reduced the Na in the tenuigenin ⁺Content makes plant can tolerate higher salinity and coerces, because excessive Na ⁺Accumulation in the vacuole has caused total Na ⁺The rising of content.

Table 5.T0 is for the Na in the transgene tobacco different sites ⁺Content (mg/g DW)

K ⁺The assay result is as shown in table 6, and the result shows, the K in each transgenic line tender leaf ⁺The difference of content under the normal irrigation condition does not reach conspicuous level; Coerce each transgenic line K down at salinity ⁺Content slightly reduces than salt-free, but all is significantly higher than wild-type.Salinity is coerced K in the tobacco tender leaf ⁺Content influence is little, may be that the organic centre of plant is relevant with tender leaf, and plant will be kept the supply that the lasting growth of salinity under coercing must will guarantee tender tissue nutrition.Compare the K among the Lao Ye with tender leaf ⁺Content has been subjected to the influence that salinity is coerced largely.The decline maximum of wild-type drops to 11.17mg/g by the 24.60mg/g under the normal irrigation condition, has descended 54.6%, and strain is the K of S15 under the salt stress ⁺Content is the highest, for the 20.23mg/g. salinity is coerced down K among the Lao Ye ⁺The decline of content may with K ⁺Recycling relevant, the K among the Lao Ye ⁺Be transported in the tender leaf, make K among the Lao Ye ⁺Content is bigger, and the variation in the tender leaf is less.This may be that plant is coerced formed a kind of adaptation mechanism down at salinity.

Salinity is coerced and has also been reduced the K in the root ⁺Content, but compare K in the root with Lao Ye ⁺Content be not very big.But each transgenic line K ⁺Content be significantly higher than wild-type contrast equally, change the K of SeNHX1 gene strain system ⁺Content will be higher than changes BADH gene strain system.

Table 6.T0 is for the K in the transgene tobacco different sites ⁺Content (mg/g DW)

Compare K in the stem with tender leaf, Lao Ye and root ⁺The absolute value of content is the highest, and each strain is K under the normal irrigation condition ⁺Average content be 61.27mg/g, be respectively 2.26 times, 2.44 times and 2.51 times of average content in tender leaf, Lao Ye and the root.Even K in the tobacco stem under 200mM NaCl coerces ⁺Content also maintain higher level, its average content is 43.76mg/g DW, far above the average content at other position.Salinity is coerced down K higher in the stem ⁺Content may play an important role aspect the toughness of keeping the plant cane and the hardness.Each strain is the K in the stem under the salt stress ⁺Content all is significantly higher than the wild-type contrast, and each strain is K under normal operation ⁺The difference of content does not reach conspicuous level.The importing that foreign gene SeNHX1 and BADH be described has improved salinity significantly and has coerced down K in the tobacco plant ⁺Content, 4 position transfer SeNHX1 genetic tobacco K that measured ⁺Average content higher than corresponding commentaries on classics BADH genetic tobacco, show improving salinity and coerce down K ⁺Nutritional status aspect SeNHX1 bigger than BADH role, as if to participate in the ionic transhipment directly relevant with SeNHX1 for this.

6) T0 is for the mensuration of transgenosis single plant yield

The biomass of coercing the different transgenosis type tobaccos in 6 week backs (the numerous strain of expansion of changeing T0 generation of pBI121-Se (S1, S15), pBin438 (B8, B23) and pBinBS (BS5, BS15) is) under 200mM NaCl is measured, with wild-type tobacco W38 is contrast, the result is as shown in table 7, the result shows that the difference of the total dry weight between all strains under the normal irrigation condition are does not all reach conspicuous level; The total dry weight that salinity is coerced following 3 kinds of different transgenosis type plant all is significantly higher than the wild-type contrast, but single-gene conversion (change pBI121-Se strain system and change pBin438 strain system) and dual-gene cotransformation (commentaries on classics pBinBS strain system) and different single-genes have bigger difference again between transforming.The single plant yield of dual-gene transformed plant is the highest, secondly is SeNHX1 single-gene transformed plant (changeing pBI121-Se strain system).The total dry weight of the dual-gene transformation plant of SeNHX1 and BADH (change pBinBS strain system) BS5 is up to 13.91g, is 1.51 times of wild-type, and small difference is only arranged under the normal irrigation condition between the two.

The biomass (g/ strain dry weight) of table 7.T0 transgene tobacco under 200mM NaCl coerces

Numerical value is 6 multiple mean numbers in the table, and indicating little in every row is that LSD checks significant difference on P≤0.05 level with alphabetical person.

Simultaneously to all types of transfer-gen plants with respect to its relative underproduction rate under normal irrigation and the susceptibility that 200mMNaCl coerces compared (table 8) separately.(LSD) method of the relative underproduction rate (%) of individual plant=[1-(it is output under the normal irrigation that salinity is coerced down output/this strain)] * 100. usefulness multiple comparisons on the P=0.05 level each is handled and the significance of strain difference compares, and has carried out the variance analysis of whole test before comparing earlier.Used statistical software is SPSS 10.0.The result shows that commentaries on classics SeNHX1 and the dual-gene strain of BADH are that BS5 only is 8.4% with respect to the underproduction rate that its normal irrigation contrasts, and wild-type underproduction rate has reached 38.6%, the underproduction rate of single-gene transformed plant is placed in the middle, but underproduction rate also has bigger difference between the not homophyletic of same type of transgene tobacco system.Method (Jain R K with reference to (1990) such as Jain, Sunita J, Nainawatee H S.et al.1990.Salt-tolerance in Brassica juncea L.I.In vitro selection, agronomic evaluation and geneticstability.Euphytica, 48:141-152) total dry weight to tobacco has carried out data-switching, calculate its separately salt stress Sensitivity Index (Salinity stress susceptibility index, S).Concrete calculation formula is: S=(1-YD/YP)/D.Every concrete meaning in the formula: the average performance value under the YD=proterties salt stress, this character pair of YP=according under average performance value, D=1-(the average YD of all these proterties of genotype of participating in the experiment)/(the average YP of all these proterties of genotype of participating in the experiment).

The size of S has reflected the sensitivity of a certain genotype character pair salt stress, and the size of D has reflected the coercive intensity that a certain experiment is handled.This computing method has been considered the coercive intensity that a certain experiment is handled, and can comparatively objectively reflect between differing materials the sensitivity to same processing.If certain proterties has identical performance under salinity is coerced with under the control case, then the salt stress Sensitivity Index S of this proterties is 0, otherwise is a certain numerical value greater than 0.Result by table 8 shows that the salt stress Sensitivity Index S of wild-type tobacco is 2.04, each transfer-gen plant is 1.27 except B8, remaining salt stress Sensitivity Index is all less than 1, show that further wild-type tobacco is more responsive to the NaCl of 200mM, this coercive intensity intense influence has arrived its normal growth.From the salt tolerance power of each strain system of salt stress Sensitivity Index and underproduction rate reaction is consistent, and strain is that the salt tolerance of BS5 is the strongest, is followed successively by BS15, S1, S15, B23, B8, WT.

Table 8.T0 is for underproduction rate and the salt stress Sensitivity Index (S) of transgene tobacco under 200mM NaCl coerces

Plant	Underproduction rate (%)	Salt stress Sensitivity Index S
Plant	Underproduction rate (%)	Salt stress Sensitivity Index S	WT S1 S15 B8 B23 BS5 BS15	38.6 14.7 17.2 24.1 18.1 8.4 11.1	2.04 0.78 0.91 1.27 0.96 0.44 0.59

3, the Molecular Detection of the transfer-gen plant in T1 generation and the resistance preliminary evaluation

Transgenosis plantlet of transplant in the present age is gone into flowerpot and outdoor big Tanaka, fully guarantees the liquid manure supply, and carries out the control of disease and pest.Though tobacco is strict selfing species, but still need take certain quarantine measures.After treating seed maturity, collect seed and carry out filial generation (T1 generation) analysis.Because experimental scale limits, so be to analyze only to part strain wherein.

1) screening and the Molecular Detection in T1 generation

With commentaries on classics pBI121-Se, the pBin438 of results and the seed in pBinBS T0 generation, promptly T1 is for seed, after fully drying,, screens in the substratum of MS0+100mg/L Km (kantlex) according to the seed disinfection method kind of routine.Change pBI121-Se, pBin438 and pBinBST1 seed and wild type seeds and began simultaneously to germinate at the 5th day adding on the substratum of Km, all reached 100% percentage of germination by the 8th day basically.At this moment, change pBI121-Se, pBin438 and pBinBS seed and wild type seeds, on bud gesture and upgrowth situation, all do not see any difference.Change the kantlex of different batches, all obtained similar result, got rid of the kantlex failure reasons.But along with dissimilar seeds in the prolongation that adds growth time on the kantlex substratum, we find that the kantlex intensive has suppressed the wild-type growth of seedlings, as and if the transgenosis seedling is not subjected to any influence, situation such as Figure 19 after its 3 weeks of growing.A is for changeing pBinBS T1 for the seed situation in 3 weeks after the 100mg/L kantlex is cultivated upper seeding wheel among Figure 19; The situation in B was wild type seeds behind 100mg/L kantlex substratum upper seeding wheel 3 weeks; C is for changeing pBinBS T1 for the seed situation in 3 weeks behind no kantlex substratum upper seeding wheel; D is the situation that wild type seeds is sent out 3 weeks behind no kantlex substratum upper seeding wheel.

In order further to detect the reliability that foreign gene exists in generation at T1, the part plant to 3 kinds of transgenosis types (changeing pBI121-Se, pBin438 and pBinBS tobacco) tobacco has carried out the PCR detection, A among partial results such as Figure 20, B and shown in Figure 21 respectively.The result shows, the T1 of the S15, the B23 that detect and BS15 for strain system in, most of plant all expands the BADH gene that 1.5Kb or/and the SeNHX1 of 1.9Kb, and another part plant detects less than the purpose band.Wherein in the T1 of BADH gene and SeNHX1 gene cotransformation plant BS15 for strain is, two goal gene show identical separation trend, illustrate that these two genes have inserted the same site on the tobacco gene group jointly, this also when transforming these two genes on identical carrier, conform to.Wherein the swimming lane 1-7 of A is that swimming lane 8 is Marker III for strain for the T1 that changes SeNHX1 gene S15 among Figure 20; The T1 of the swimming lane 1-8 of B commentaries on classics BADH gene B23 is that swimming lane 9 is Marker III for strain among Figure 20.Swimming lane 1 and 11 among Figure 21; 2 and 12; 3 and 13; 4 and 14; 5 and 15; 6 and 16; 7 and 17; 8 and 18; The T1 of corresponding respectively same BADH gene and SeNHX1 gene cotransformation plant BS15 is an individual plant for strain, and swimming lane 1-9 is NHX1 gene PCR result; Swimming lane 10 is Marker III; Swimming lane 11-19 is BADH gene PCR result.

2) bud phase salt tolerance is identified

With the T1 that changes pBI121-Se, pBin438 and pBinBS for seed through be sowed at after the sterilization contain 0 or the MS substratum of 200mM NaCl on cultivated for 3 weeks, observe seed germination and growth of seedlings situation, be contrast with wild-type tobacco W38.The result as shown in figure 22, the result shows, 200mM NaCl intensive has suppressed the germination of wild-type tobacco seed, nearly all seed promptly stops growing after showing money or valuables one carries unintentionally, up to perish.By contrast, there is a spot of T1 that changes pBI121-Se, pBin438 and pBinBS can survive, and grows elongated root for seedling.This part seedling of getting off of surviving is carried out PCR detect, all can amplify goal gene, the T1 that commentaries on classics pBI121-Se, pBin438 and pBinBS be described is for being caused by the importing of foreign gene really of can growing on 200mM NaCl substratum of plant.And on the substratum of no NaCl transgenosis seedling and wild-type seedling and do not see any difference.T1 to single NHX1 of commentaries on classics (changeing pBI121-Se strain system), BADH gene (changeing pBin438 strain system) and SeNHX1 and BADH gene cotransformation (changeing pBinBS strain system) has carried out identical germination test for seed, all obtained similar result, quite a lot of but the seedling of dual-gene conversion (changeing pBinBS strain system) upgrowth situation on 200mM NaCl substratum transforms (change pBI121-Se and change pBin438 strain system) than single-gene.Because limited mensuration and the comparison of not carrying out the relative growth index of seedling.Wherein among Figure 22, A is wild-type (left side) and changes BADH and the sowing growing state of the dual-gene seed BS15 of SeNHX1 (right side) in no NaCl cultivation; B is wild-type (left side) and changes BADH and the dual-gene seed BS15 of SeNHX1 (right side) is contained in sowing growing state on the 200mM NaCl substratum.

3) osmotic stress in T1 generation test

N.F,USP MANNITOL is as a kind of multi-sugar alcohol, has very high water-solublely, is a kind of osmotic pressure regulator commonly used.In order further to verify the salt tolerance of each transfer-gen plant, the plant of 3 kinds of transgenosis types has been carried out the test of coercing of N.F,USP MANNITOL.

Commentaries on classics pBI121-Se, pBin438 that detection obtains to PCR and the T1 of pBinBS utilize method 2 weeks of water planting in 1/2 Hoagland nutritive medium of water planting for positive seedling or wild-type tobacco W38.Treat plant long to about the 4cm after, select the size seedling consistent with upgrowth situation, carrying out N.F,USP MANNITOL coerces, coerce the N.F,USP MANNITOL that in nutritive medium, adds 200mM when handling, coerce 1 Zhou Houzai and transfer in the 1/2 Hoagland nutritive medium to cultivate and 1 week saw its recovery situation under this concentration, contrast is cultivated in 1/2 Hoagland nutritive medium always.The result as shown in figure 23, the result shows, the N.F,USP MANNITOL of 200mM has been become serious injury to tobacco, all material all withered leaf occurred because of the osmotic stress dehydration, also having some white masses to separate out at the edge of some leaves, is that excessive N.F,USP MANNITOL has been transported to leaf surfaces with transpiration and caused in the plant body.But after forwarding 1 week of growing in the 1/2 Hoagland nutritive medium to, each T1 that changes pBI121-Se, pBin438 or pBinBS contrasts significantly better than wild-type for the seedling recovery situation, all there is new leaf to grow, and gone out to have the root of a small amount of white the foundation minister, and most of root of wild-type all rots, and does not have new root to grow.Illustrated after the T1 that changes pBI121-Se, pBin438 and pBinBS ties up to the osmotic stress elimination for strain and can recover growth faster, and wild-type can not.A is wild-type (left side) and changes BADH and the growing state of the dual-gene seed BS23 of SeNHX1 (right side) before N.F,USP MANNITOL is coerced among Figure 23; B be wild-type (left side) and change BADH and the dual-gene seed BS23 of SeNHX1 (right side) after 200mM N.F,USP MANNITOL coerced for 1 week, transfer to the situation after 1 week of growth in the 1/2 Hoagland nutritive medium again.

Detect the biomass each strain ties up to 1 week of growing after osmotic stress is eliminated in nutritive medium after, the result is as shown in table 9, and the result shows that the T1 that changes pBI121-Se, pBin438 and pBinBS all is significantly higher than wild-type for strain system.But difference is bigger between dissimilar transgenic lines, and dual-gene conversion is significantly higher than single-gene and transforms, and the SeNHX1 single-gene transforms and is significantly higher than the conversion of BADH single-gene.Single-strain fresh weight as dual-gene transformation plant BS5 (T1 that changes pBinBS for strain is) is 5.63g, is respectively 1.33 times, 1.55 times and 2.01 times that SeNHX1 single-gene transformation plant S15 (T1 that changes pBI121-Se is), BADH single-gene transform B23 (T1 that changes pBin438 is) and wild-type for strain for strain.But under control case, the biomass between each strain system does not have significant difference, and the importing that the external source salt resistant gene has been described has improved the ability that the impermeabilisation of tobacco is coerced really.

Table 9.T1 is for the biomass of transgenic line after 200mM N.F,USP MANNITOL coerced for 1 week

6 multiple mean numbers of numerical value in the table indicate different alphabetical persons and are LSD check significant difference on P≤0.05 level in every row.

Claims

1, a kind of method that improves stress resistance of plant is that SeNHX1 gene and BADH gene are changed in the plant jointly by plant expression vector, and screening obtains the plant that resistance improves.

2, method according to claim 1 is characterized in that: the encoding sequence of described SeNHX1 gene is from the GENBANK number nucleotide sequence for the 492nd to 2174 at 5 of AY131235 ' end;

The encoding sequence of described BADH gene be from GENBANK number for the 4th at 5 of X69770 ' end to the 1512nd nucleotide sequence.

3, method according to claim 2 is characterized in that: described SeNHX1 gene and BADH gene change in the plant jointly by a plant expression vector.

4, method according to claim 3 is characterized in that: described SeNHX1 gene and BADH gene are that the pBinBS by as shown in Figure 3 changes in the plant jointly.

5, according to arbitrary described method in the claim 1 to 4, it is characterized in that: described resistance is salt resistance.

6, according to arbitrary described method in the claim 1 to 4, it is characterized in that: described resistance is oxidation-resistance.

7, according to arbitrary described method in the claim 1 to 4, it is characterized in that: described resistance is salt resistance and oxidation-resistance.

8, according to arbitrary described method in the claim 1 to 7, it is characterized in that: described plant is a tobacco.