CN101906433B

CN101906433B - Application of Na+/H+ reverse transport protein and protein-coding gene thereof in culturing disease-resistant transgenic plant

Info

Publication number: CN101906433B
Application number: CN2009100865443A
Authority: CN
Inventors: 李银心; 陈显扬
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2009-06-05
Filing date: 2009-06-05
Publication date: 2012-05-30
Anticipated expiration: 2029-06-05
Also published as: CN101906433A

Abstract

The invention discloses application of Na+/H+ reverse transport protein and a protein-coding gene thereof in culturing a disease-resistant transgenic plant, wherein an amino acid sequence of the Na+/H+ reverse transport protein is as shown in Genbank No. AAN08157; and a nucleotide sequence of the protein-coding gene is as shown in Genbank No. AY131235. The Na+/H+ reverse transport protein and the protein-coding gene thereof can be used for improving disease resistance, salt tolerance, oxidative stress resistance and systemic acquired resistance of the plant.

Description

Utilize Na +/ H +Its encoding sox of Reverse transship albumens And is cultivated disease-resistant transgenic plant

Technical field

The present invention relates to biological technical field, particularly Na ⁺/ H ⁺The application of its encoding sox of Reverse transship albumens And in cultivating disease-resistant transgenic plant.

Background technology

Because plant has irremovable character, it develops during evolution and various mechanism, biochemically adapts to physiological regulation and tackles various environment and coerce with self.Wherein, no matter in halophytes still is glycophyte, all guard the ability that exists vacuole district separatedization sodium ion; This process comprises: through the Na on the vacuole skin ⁺/ H ⁺Reverse transport protein; With the sodium ion district separatedization entering vacuole in the kytoplasm, thereby reduce of the murder by poisoning (Blumwald E (2000) Sodium transport and salt tolerance inplants.Curr Opin Cell Biol.12:431-434) of deleterious sodium ion to various enzymes in the kytoplasm.

Up to now, the researchist has cloned the Na on the vacuole skin from many species ⁺/ H ⁺The reverse transport protein gene is for example from Arabidopis thaliana (AtNHX1 and AtNHX2) (Gaxiola RA, Rao R; Sherman A, Grisafi P, Alper SL; Fink GR (1999) The Arabidopsis thaliana protontransporters, AtNhx1 and Avp1, can function in cation detoxification inyeast.P Natl Acad Sci USA 96:1480-1485), wheat (TaNHX1 and TaNHX2) (BriniF; Hanin M; Mezghani I, Berkowitz GA, Masmoudi K (2007) Overexpression ofwheat Na ⁺/ H ⁺Antiporter TNHX1 and H ⁺-pyrophosphatase TVP1 improve salt-anddrought-stress tolerance in Arabidopsis thaliana plants.J Exp Bot.58:301-308), paddy rice (OsNHX1) (Fukuda A; Nakamura A, Tanaka Y (1999) Molecularcloning and expression of the Na ⁺/ H ⁺Exchanger gene in Oryza sativa.BBA-GeneStruct Exp 1446:149-155) and in the soybean species such as (GmNHX1) clone NHX1 gene (Li WYF; Wong FL; Tsai SN; Phang TH; Shao GH, Lam HM (2006) Tonoplast-located GmCLC1and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells.Plant Cell and Environ 29:1122-1137).

TAIR ( Http:// www.arabidopsis.org) on the website, we investigate the AtNHX1 of Arabidopis thaliana, find that the NHX1 gene of Arabidopis thaliana can be comprised by abiotic stress: salt, infiltration, up-regulated expression that oxidative stress is induced; Simultaneously also can be by pseudomonas syringae (Pseudomonas syringae) and gray mold biological stress-inducing up-regulated expressions such as (Botrytis cinerea).These evidence explanations NHX1 active response biology is coerced with abiotic stress and is reacted.In recent years, carried out very deeply to the NHX1 gene in the functional study aspect the raising salt resistance of plants.At Arabidopis thaliana (Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na ⁺/ H ⁺Antiportor in Arabidopsis.Science 285:1256-1258), tomato (Zhang HX; BlumwaldE (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliagebut not in fruit.Nat Biotechnol 19:765-768) and English ryegrass (Wu YY; ChenQJ; Chen M; Chen J, Wang XC (2005) Salt-tolerant transgenic perennialryegrass (Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediatedtransformation of the vacuolar Na ⁺/ H ⁺Antiporter gene.Plant Sci 169:65-73) all studied proof in: the NHX1 gene on the overexpression of tonoplast can obviously improve the salt resistance of plant.Simultaneously, research proves that also the NHX1 gene of plant has vital role (SunJ, the Chen S that regulates cell plasma balance and kytoplasm pH; Dai S, Wang R, Li N; Shen X, Zhou X, Lu C; Zheng X, Hu Z, ZhangZ; Song J, Xu Y (2008) NaCl-Induced Alternations of Cellular and Tissue IonFluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species.PlantPhysiol 149:1141-1153).

Although many reports prove that all NHX1 has anti-salt functional in the plant, seldom other functions of NHX1 gene are paid close attention in research, and particularly this gene is coerced middle role at biology.

Summary of the invention

The object of the present invention is to provide Na ⁺/ H ⁺The application in cultivating disease-resistant transgenic plant of reverse transport protein or its encoding sox.

Na provided by the invention ⁺/ H ⁺Reverse transport protein or its encoding sox can be used for cultivating disease-resistant transgenic plant, said Na ⁺/ H ⁺The aminoacid sequence of reverse transport protein is shown in GeneBank AAN08157.

The nucleotide sequence of said encoding sox is shown in Genbank AY131235.

Said disease-resistant transgenic plant is the transgenic plant of resistance to bacteria disease and/or fungal disease.

Said bacterial disease is a wildfire; Said fungal disease is a balck shank.

Said plant is a tobacco.

Na of the present invention ⁺/ H ⁺Reverse transport protein or its encoding sox also can be used to cultivate disease-resistant and anti-salt transgenic plant.

Said Na ⁺/ H ⁺The aminoacid sequence of reverse transport protein is shown in Genbank AAN08157; The nucleotide sequence of said encoding sox is shown in Genbank AY131235.

Na of the present invention ⁺/ H ⁺Reverse transport protein or its encoding sox also can be used to cultivate anti-oxidant stress transgenic plant.

Na of the present invention ⁺/ H ⁺Reverse transport protein or its encoding sox also can be used to cultivate the transgenic plant of tool systemic acquired resistance.

Experiment of the present invention proves; Compare with commentaries on classics empty carrier plant with wild-type, change the SeNHX1 genetic tobacco and be enhanced except salt resistance, its disease resistance also is improved; And activities of antioxidant enzymes is higher, and the expression amount of the marker gene of its systemic acquired resistance (PR gene) is more.

Description of drawings

Fig. 1 coerces seed germination, over-ground part dry weight and K for NaCl ⁺/ Na ⁺Influence figure.

Fig. 2 is H ₂O ₂The dead figure that detects of evoking tobacco blade cell.

Fig. 3 is for changeing the resistance analysis of SeNHX1 genetic tobacco to wildfire.

Fig. 4 is for changeing the resistance analysis of SeNHX1 genetic tobacco to balck shank.

Fig. 5 is measure transgenic TS11 strain system and empty carrier plant after connecing

bacterium

0,12,24 and 36 hour anti-oxidant response diagram.

Fig. 6 carried out the expression pattern analysis in 0,6,12,18,24 and 36 hours for PR1a (a) in transgenic TS11 strain system and empty carrier strain being and Gns1 (b) gene after connecing bacterium.

Fig. 7 is that transgenic, wild-type and empty carrier tobacco sprout under the different concns Whitfield's ointment.

Wherein, among Fig. 1-Fig. 7 on the standard error line identical letter representation statistical study under P≤0.05 situation do not have significant difference.

Embodiment

Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.

Among the following embodiment,, be ordinary method like no specified otherwise.

Among the following embodiment, said percentage composition is the quality percentage composition like no specified otherwise.

The resistance experiment of embodiment 1. transgene tobaccos

One, the acquisition of vegetable material

1, obtains wild-type tobacco, empty carrier tobacco and transgene tobacco

Vegetable material:

The wild-type tobacco kind ' represent with WT by winconsin 38 ' (Nicotiana tabacum cv ' Wisconsin 38 ') (Tobacco Institute, Chinese Academy of Agricultural Science's tobacco kind money resources bank is bought).

Transgene tobacco passes through Agrobacterium leaf dish method with Na ⁺/ H ⁺The encoding sox SeNHX1 of reverse transport protein transforms wild-type tobacco, and ' winconsin 38 ' (" W38 ") obtains (Zhou SF; Chen XY; Zhang XG, Li YX (2008) Improved salt tolerance in tobacco plants by co-transformation ofa betaine synthesis gene BADH and a vacuolar Na ⁺/ H ⁺Antiporter gene SeNHX1.Biotechnol Lett 30:369-376).Wherein, The SeNHX1 gene is to be primer with following

primer

1 and 2; With the RNA synthetic first chain cDNA of salicornia europaeal as template; Pcr amplification goes out nucleotide sequence such as the Genbank number encoding sox for the 301-2200 position of AY131235, and this encoding sox encoding amino acid sequence is the albumen shown in the AAN08157 as Genbank number.

Primer 1:5 '-TCTAGATGGAGGGAATTTGGAGGAGC-3 ';

Primer 2: 5 '-GGATCCTGCCTAACTGCCTCGGATT-3 '.

The MCS that the SeNHX1 gene that amplification obtains inserts the pBI121 that contains 35S promoter has made up the pBI121-35S::SeNHX1 expression vector, after the order-checking affirmation is errorless, carrier is successfully transformed Agrobacterium LBA4404 bacterial strain.Subsequently, utilize agriculture bacillus mediated leaf disc transformation method, the leaf dish tissue of " W38 " is carried out bacterium liquid infect conversion, the leaf dish after infecting is positioned on the division culture medium that contains kantlex to be cultivated.Through the screening of kantlex, the tobacco regrowth that will have resistance is transferred to hot-house culture, and regrowth is carried out PCR detect, and finally obtains 11 strains and transforms positive seedling, from the positive seedling of 11 strains, chooses 5 strains at random then, carries out Southern and Northern and analyzes; And through results of hybridization, selected T0 for the SeNHX1 gene can transcriptional expression, and single copy insert genomic T1, T11 and T15 strain system.When carrying out T1 for the transgene tobacco Function Identification, we use TS1, TS11 and TS15 numbering to distinguish T0 for tobacco to these three strain systems.

The empty carrier plant is to change the T1 of pBI121 empty expression vector for tobacco plant, representes with pBI.

2, the cultivation of vegetable material

Be sowed on the MS substratum after the wild type seeds sterilization, and SeNHX1 transgenic and empty carrier plant seed are broadcast and are being contained 150mg ml ^-1The enterprising row filter of MS substratum of kantlex.After two weeks, the kalamycin resistance transgenic of great-hearted wild-type and survival is sprouted seedling transfer to the greenhouse, be planted in and contained vermiculite, peat and soil ulmin (1: 1: 1; Volume ratio) in the plastic tub.Greenhouse experiment is controlled to be: temperature daytime is 23-27 ℃, and be 18-22 ℃ night; Illumination every day 16 hours; Relative humidity 50 ± 10%.Water 1/2 Hoagland nutritive medium seedling stage weekly once.

Two, the resistance of tobacco experiment

1, salt resistance experiment

The seed of being tied for transgenic and empty carrier plant with wild-type, by T0 is sowed on the MS substratum that contains 200mmol/L (mM) NaCl to be sprouted.Simultaneously, with the seed germination on the MS substratum that does not contain NaCl as experiment contrast.

Plant culturing as stated.Transplanted seedlings back 30 days, each week waters with the 1/2 Hoagland nutritive medium that contains 200mM NaCl.After six weeks, select the complete unfolded blade in top to measure Na ⁺And K ⁺Content.In addition, following formula is selected in the calculating of living weight decrement for use: decrement (%)=[1-(NaCl handle back over-ground part dry weight/the over-ground part dry weight is untreated)] * 100%.According to (Alian A; Altman A; Heuer B (2000) Genotypic difference in salinity and water stress tolerance of fresh markettomato cultivars.Plant Sci 152:59-65) method is utilized flame spectrophotometer (CorningLtd; Essex England) carries out blade Na ⁺And K ⁺Assay.

The salt resistance experimental result is seen Fig. 1, and wherein (a) is the result of seed germination under 200mM NaCl; (b) 200mM NaCl handles the over-ground part living weight is carried out in 6 all backs to transgenic, wild-type and empty carrier plant mensuration; (c) after 200mM NaCl handled for 6 weeks, transgenic, wild-type and empty carrier plant are carried out K ⁺/ Na ⁺Than analyzing.

Experiment repetition 3 times, sprouting test are sprouted the result shown in Fig. 1 a during 2 weeks, and the comparison of the seed of transgenic line is according to showing higher germination rate.Further experiment shows that tobacco seedling was coerced for 6 weeks under 200mM NaCl, wild-type and empty carrier plant have significantly minimizing (Fig. 1 b) than the dry weight of transgenic line.Simultaneously, receiving under the salt stress situation, transgenic line shows higher K ⁺/ Na ⁺Ratio, for example: wild-type and empty carrier plant K ⁺/ Na ⁺Than being about 0.7, and the K of TS1 strain system ⁺/ Na ⁺Than being 0.96, TS11 is 1.01, and TS15 is 0.97 (Fig. 1 c).All these results show that changeing the SeNHX1 genetic tobacco shows higher NaCl tolerance than wild-type and empty carrier plant.

2, H ₂O ₂Induce experiment

This is tested, and used strain is identical in used plant and the experiment of above-mentioned salt resistance, chooses from the top that down fully extended the 3rd leaf carries out H ₂O ₂The inductive necrocytosis is measured.Blade is cut into 1cm * 1cm size, and first vacuum infiltration 10 minutes is then respectively at the H of different concns ₂O ₂Soaked 4 hours and 8 hours in (0,1,10 and 100mM).In addition, choose near the zone of each leaf spot lesion and carry out the necrocytosis analysis that disease causes.Necrocytosis is measured (Harding SA, Roberts DM (1998) Incompatible pathogeninfection results in enhanced reactive oxygen and cell death responses in transgenictobacco expressing a hyperactive mutant calmodulin.Planta 206:253-258) with the azovan blue method.According to weighing with light absorption ratio A600/A680 described in the document, dead multiple statistics is compared with 0 hour light absorption ratio by the light absorption ratio in each period and is obtained.

The result sees Fig. 2, and wherein (a) is the H of leaf tissue piece at different concns ₂O ₂Soak 4 hours figure as a result in (0,1,10 and 100mM); (b) be the H of leaf tissue piece at different concns ₂O ₂Soak 8 hours figure as a result in (0,1,10 and 100mM).Experiment repetition 5 times is at 10mM and 100mM H ₂O ₂Handle after 4 hours, transfer-gen plant shows still less blade cell than wild-type and empty carrier plant, and dead (Fig. 2 a).Similar, at 1mM, 10mM and 100mM H ₂O ₂Middle immersion treatment is after 8 hours, and wild-type and empty carrier plant show more multiple-blade necrocytosis (Fig. 2 b) than transfer-gen plant.These presentation of results change the anti-oxidant ability of coercing of SeNHX1 genetic tobacco and have improved.

3, wildfire infection process

With reference to (Guo ZJ; Chen XJ; Wu XL; Ling JQ; Xu P (2004) Overexpression of theAP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance intobacco.Plant Mol Biol 55:607-618) method that provides will contain the 10mMMgCl of wildfire germ (Pseudomonas syringae pv tabaci) (American Type Culture Collecti's (American TypeCulture Collection (ATCC) claims that again the US mode bacterial classification collects the center) preserving number 11527) through plastic injector without a head ₂(the germ final concentration is 10 to suspension-s ⁷CFU/ml) be injected into the excised leaf of transgenic, wild-type and empty carrier plant.Wherein, with injection 10mM MgCl ₂The leaf tissue of the aqueous solution is as contrast.After infecting 6 days, get near the leaf tissue that infects the position and carry out the bacterial growth flow measurement.Use MgCl ₂Leaf dish tissue is ground and dilutes, and coated plate is in KB substratum (peptone 20g l ^-1, Magnesium Chloride Anhydrous 1.4g l ^-1, anhydrous potassium sulfate 10g l ^-1, agar 15g l ^-1, glycerine 10ml l ^-1) on.Bacterial plaque clone number is through (Bertoni G; Mills D (1987) A simplemethod to monitor growth of bacterial-populations in leaf tissue.Phytopathology 77:832-835) method is measured, and the result uses log ₁₀(CFU cm ^-2) represent.

Experiment repetition 3 times, the result is as shown in Figure 3, at injection MgCl ₂Control group in, transgenic, wild-type and empty carrier plant all have similar bacterial plaque number; But in the experimental group of injection germ, we can be clear that wild-type and empty carrier plant demonstrate more bacterial plaque number than transgenic line.Presentation of results, commentaries on classics SeNHX1 genetic tobacco has strengthened the resistance of wildfire.

4, balck shank infection process and analysis

1) balck shank infection process

Choose tobacco black shank bacterium (Phytophthora parasitica var.nicotianae) No. 0 physiological strain (ATCC preserving number 13611), bacterial classification is with PDA solid medium (potato 200g l ^-1Sucrose 20g l ^-1Agar 15g l ^-1PH 6.5) cultivate and preserve.Black shank bacterium infects blade method such as document (Guo ZJ; ChenXJ; Wu XL, Ling JQ, Xu P (2004) Overexpression of the AP2/EREBP transcriptionfactor OPBP1 enhances disease resistance and salt tolerance in tobacco.Plant Mol Biol55:607-618) shown in; When germ is covered with culture medium flat plate, get the bacterium piece that the hole device gets homalographic from flat board with plastics and be used to connect bacterium.Select tobacco top the 3rd leaf down to carry out the in vitro inoculation test, and each prick 2 holes with the toothpick symmetry in the vein both sides.The bacterial plaque piece is inoculated in two wounds in vein right side, and the vein of not inoculating left side is as experiment contrast.The inoculation blade is cultivated in cushioning the flat board of moistening filter paper, is put in 28 ± 2 ℃ of temperature, and illumination in 16 hours is in 8 hours dark culturing room.

2) the susceptible situation of plant

0,24,36 and 48 hours blade is taken pictures behind the butt joint bacterium, measures the scab diameter then, and each numerical value all is to record through 20 repeated experiments, and according to H in the step 2 ₂O ₂The method of inducing experiment to provide is carried out the necrocytosis analysis, experiment repetition 5 times.

Shown in Fig. 4 a (hpi representes the postvaccinal hours of germ), in all times of infection, the contrast position of blade (the vein left side does not have the wound of inoculation) all do not have the sign of morbidity.Shown in Fig. 4 a and Fig. 4 b, at the position of inoculation balck shank, wild-type and empty carrier plant scab all occurred connecing bacterium after 24 hours; The scab diffusion rapidly after 36 hours; During by 48 hours, the large-area outburst of germ presents large-area tissue necrosis.Yet tangible scab does not appear in transgene tobacco preceding 24 hours after connecing bacterium; Simultaneously, after connecing

bacterium

36 and 48 hours, transfer-gen plant showed slower course of disease development than wild-type and empty carrier plant.

The result of necrocytosis multiple is connecing in the bacterium 36 hours shown in Fig. 4 c, and transgene tobacco shows necrocytosis still less than wild-type and empty carrier plant.

These results explain that transgene tobacco has strengthened the resistance of balck shank.

3) balck shank inductive oxidative damage detects

For the more effect of in-depth explanation SeNHX1 in plant disease-resistant, choose TS11 transgenic line and empty carrier plant, investigate commentaries on classics SeNHX1 genetic tobacco and whether can alleviate the oxidative damage that causes by the balck shank infection process.

DAB dyeing:

At first transgenic TS11 strain system and empty carrier strain are tied up to the different vaccination time point and carry out DAB dyeing; Method is with reference to Hernandez JA, Rubio M, Olmos E; Ros-Barcelo A; The method of Martinez-Gomez P (2004) Oxidative stress induced by long-term plum pox virus infection in peach (Prunuspersica) .Physiol Plantarum 122:486-495 is carried out, and the dyeing picture is seen Fig. 5 a (hpi representes the postvaccinal hours of germ), carries out the diameter measurement of DAB stain then; DAB stain diameter multiple (Fig. 5 b) is through obtaining experiment repetition 5 times than scab diameter before the dyeing.

Find that from Fig. 5 a and Fig. 5 b after connecing

bacterium

24 and 36 hours, the empty carrier plant demonstrated more large-area dyeing foxiness than transgenic line near the infection process position, explain behind infection process, the empty carrier plant has produced more H ₂O ₂

CAT and POD enzyme are lived and are analyzed:

To transgenic TS11 and empty carrier strain is that CAT and the work of POD enzyme are analyzed, and enzyme liquid extracts institute and all under 4 ℃ of conditions, accomplishes in steps.The enzymic activity of px (POD) and katalase (CAT) is according to (RaoMV; Paliyath G; Ormrod DP (1996) Ultraviolet-B-and ozone-induced biochemicalchanges in antioxidant enzymes of Arabidopsis thaliana.Plant Physiol 110:125-136 and Wang YS; Tian SP; Xu Y; Qin GZ, 20 ℃ of .Postharvest Biol of Yao HJ (2004) Changes in the activities of pro-andanti-oxidant enzymes in peach fruit inoculated with Cryptococcus laurentii orPenicillium expansum at 0 or Tec 34:21-28) method is measured.Enzyme ratio alive obtains through relatively 0 hour enzyme work.Experiment repetition 5 times.

The result is shown in Fig. 5 c, and transgene tobacco CAT activity is all lived high than the enzyme of empty carrier plant in the time that all infect; Equally, Fig. 5 d demonstrates, and is compared to the CAT activity, and the POD activity of transfer-gen plant was lived than control enzyme after connecing bacterium and also is significantly increased in 12 hours.These results show: after receiving infection process, the oxidative damage ability that transfer-gen plant removing disease causes has strengthened.

4) the expression amount analysis of the marker gene of systemic acquired resistance

Many researchs generally believe that PR-1 (PR1a) and PR-2 (Gns1) are the marker gene (Yasuda M (2007) Regulation mechanisms of systemic acquired resistanceinduced by plant activators.J Pestic Sci 32:281-282) of systemic acquired resistance (SAR).PR1a and Gns1 (the GenBank sequence number is X06361 and EU867448) encode respectively pathogenesis-related proteins PR1a and beta-1,3-glucanase.

The present invention also connects the investigation of PR gene expression amount behind the bacterium to empty carrier and TS11 strain system.After the inoculation balck

shank

0,6,12,18,24 and 36 hours is respectively to select 5 blades to transgenic TS11 strain system and empty carrier strain, carries out the RT-PCR analysis.Tissue block to around the scab is carried out the extraction of RNA, and mixes and form a RNA pond.MLV ThermoScript II and cycle and taking corresponding operation with Promega are carried out reverse transcription.The PR1a and Gns1 special primer (the PR1a upper reaches: 5 ' TTG CCT TCA TTT CTT CTT GTC TC 3 ', downstream: 5 ' CCT CCA TTG TTA CAC TGA ACC CT 3 ' are used in the PCR reaction; The Gns1 upper reaches: 5 ' GCC CTG TCA CTGGCA CAT CTT ACC T 3 ', downstream: 5 ' GTT GTT CTC ATC AAA CAT GGC AAA T 3 '), and carry out 24,28 with the amplification repetition of 32 3 different cycle numbers.The expression amount of Actin gene in tobacco is used for trim as confidential reference items.

Experiment repetition 3 times, the result is as shown in Figure 6, and the relative expression quantity among the figure is through relatively obtaining with 0 hour expression amount; Hpi, the postvaccinal hours of expression germ.After connecing bacterium the 24th hour, transgene tobacco had higher PR1a genetic expression than empty carrier plant; Similarly, before the 36th hour, the comparison of Gns1 expression of gene amount all is significantly increased according to gene expression amount in the transgenic line after connecing bacterium.These presentation of results, transfer-gen plant PR gene (PR1a and Gns1) presents higher expression amount after plant receives infection process, hinted that SeNHX1 possibly regulate PR genetic expression through participating in the SAR approach.

5) mechanism research

Transgenic, wild-type and empty carrier T0 are sowed on the MS flat board of the SA (Whitfield's ointment) that contains different concns statistics seed germination situation for seed.

Experiment repetition 3 times, the result is as shown in Figure 7, (a) is to sprout photo, (b) is the statistical graph of germination rate.On the MS flat board that does not contain SA or lower concentration SA (0.01mM), transgenic all shows similar germination rate (Fig. 7 a and Fig. 7 b) with contrast.0.1 with 0.5mM SA concentration under, most of wild-type and empty carrier plant seed all can not normally be sprouted, and the TS11 strain is and shows 90% and 80% germination rate (Fig. 7 b).Similar, shown in Fig. 7 b, 0.1 with 0.5mM SA concentration under, TS1 is also to show higher seed germination rate than wild-type and empty carrier plant with the TS15 strain.These presentation of results, transgene tobacco has higher seed germination rate under the acid of high density bigcatkin willow.

Existing research shows, H ₂O ₂Participate in inducing PR expression of gene and initial SAR reaction (Kvaratskhelia M as the secondary signal molecule; George SJ, Thorneley RN (1997) Salicylic acid is a reducingsubstrate and notan effective inhibitor of ascorbate peroxidase.J Biol Chem 272:20998-21001).Simultaneously; Many evidences also show; The raising of activities of antioxidant enzymes (for eliminating H2O2) strengthens with PR genetic expression; Occur in simultaneously in the SA inductive SAR reaction (Ananieva EA, Christov KN, Popova LP (2004) Exogenous treatment with Salicylic acid leads to increased antioxidantcapacity in leaves of barley plants exposed to Paraquat.J Plant Physiol 161:319-328).Show that like Fig. 6 transfer-gen plant is after receiving infection process, two SAR marker gene are all compared according to higher expression amount is arranged.Now find that also transfer-gen plant and wild-type and empty carrier plant have response (Fig. 7) in various degree to Whitfield's ointment.In addition, this paper result of study shows, transfer-gen plant receives CAT and the active increase of POD behind the infection process, H ₂O ₂The minimizing of concentration and the coupling of PR genetic expression wild phase; This and consistent (the Chan Z of SAR reaction result of forefathers report; Wang Q; Xu X; Meng X, Qin G, B Li; S Tian (2008) Functions ofdefense-related proteins and dehydrogenases in resistance response induced by salicylicacid in sweet cherry fruits at different maturity stages.Proteomics 8:4791-4807 and Xu XB, Tian SP (2008) Salicylic acid alleviated pathogen-induced oxidative stress in harvestedsweet cherry fruit.Postharvest Biol Tec 49:379-385).These conclusions show that SeNHX1 possibly participate in the SAR reaction path, thereby show its disease-resistant function in plant.

Claims

1.Na ⁺/ H ⁺The application in cultivating anti-wildfire transgene tobacco of reverse transport protein or its encoding sox; Said Na ⁺/ H ⁺The aminoacid sequence of reverse transport protein is shown in Genbank AAN08157; The nucleotide sequence of said encoding sox is shown in Genbank AY131235.