CN1450165A - Method for breeding salt-resistant tomato utilizing atriplex hortensis BADH gene transfer - Google Patents
Method for breeding salt-resistant tomato utilizing atriplex hortensis BADH gene transfer Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
The method for cultivating salt-enduring tomato line by transferring betaine aldehyde dehydrogenase gene (BADH) includes the following steps: a. using Agrobacterium LBA 4404 of double-element expression vector pBin438 of gene fragment containing BADH cDNA to infect tomato euphylis blade, b. placing the infected tomato blade on the IM culture medium to make differentiation and culture, c. extracting genome DNA from teneral blade of differentiated plant to make molecular detection and d. BADH activity detection and salt-enduring property determination of transgenic plant.
Description
1. technical field
The invention belongs to plant biotechnology field.Improve by genetically engineered salt resistant gene conversion means
The salt tolerance of plant.
2. technical background:
Plant-growth often is subjected to the influence of unsuitable environmental condition in physical environment.Wherein, salt damage is a regional distribution of restriction plant and an important factor (Boyer, 1982) that influences its output and quality to a certain extent.In the evolution of long period of time process, plant has formed the mechanism that adapts to and resist salt stress.Common a kind of be exactly synthesized micromolecule organism such as amino acid derivative (proline(Pro), trimethyl-glycine etc.), carbohydrate, alcohols etc.The quaternary structure of the osmotic pressure of trimethyl-glycine by statocyte and surrounding environment, stabilizing protein and protect intracellular enzyme system to come the protective plant cell is to reduce suffered injury (Bernard etc., 1988 of plant under the salt stress; Papageorgiou etc., 1995).
In the vegetable cell, trimethyl-glycine is synthetic by two-step reaction in chloroplast(id).The first step is to form betaine aldehyde chloride by choline mono-oxygenase (CMO) catalysis choline; Second step was to generate trimethyl-glycine by the betaine aldehyde chloride oxidation under the effect of betaine-aldehyde dehydrogenase (BADH).The gene of the synthetic BADH enzyme of coding is successfully cloned in various plants, as spinach (Weretilnyk etc., 1990), prunella asiatica (Xiao Gang etc., 1995), beet (McCue etc., 1992), Chinese sorghum (Wood etc., 1996) etc.Result of study shows that many plants such as Arabidopis thaliana, tobacco and tomato belong to the salt sensitive plant but self can not synthesize and accumulate trimethyl-glycine (Weretilnyk etc., 1989; Rhodes etc., 1993; Nuccio etc., 1998).This has just caused that people are to importing trimethyl-glycine route of synthesis synthesizing betaine to improve the interest of salt sensitive plant salt tolerance.
Tomato is widely distributed in the world a kind of vegetable crop, but its Cultivar is relatively more responsive to salt.The salinization of soil and irrigation water has reduced the output of tomato to a great extent, causes very big financial loss (Foolad, 1999; Cuartero etc., 1999).Can utilize cultivated area to reduce day by day, improve the salt tolerance of cultivation tomato today that soil property worsens day by day, strengthen the adaptive faculty of tomato, not only can make full use of limited land resources, and have very big economic and social benefit bad edatope.
3. technology contents
Tomato is the conventional vegetable species of widely cultivating in the whole world, and the cultivation tomato variety is generally to the salt sensitivity.The present invention imports the cultivation tomato by agrobacterium mediation method with prunella asiatica BADH gene, has improved the salt tolerance of cultivation tomato variety.Realize that concrete technological step of the present invention is as follows:
A, the Agrobacterium LBA4404 of binary expression vector pBin438 that will contain the gene segment of BADH cDNA infects tomato true leaf blade;
B, metainfective tomato leaf place and carry out differentiation culture on the IM substratum;
C gets differentiation plant young leaflet tablet extraction genomic dna and carries out Molecular Detection;
D, the BADH of transfer-gen plant is active to be detected and the salt tolerance evaluation.
By importing salt tolerance more existing reports (Rathinasabapathi etc., 1994 of trimethyl-glycine route of synthesis raising plant; Liu Fenghua etc., 1995; Guo Yan etc., 1997; Trossat etc., 1997; Li Yinxin etc., 2000), but with vegetables as research object seldom.Tomato is the vegetables that a kind of economic worth is higher, market potential is bigger, and the cultivation tomato variety is generally to the salt sensitivity; Studies show that the endogenous BADH route of synthesis of shortage (McCue and Hanson, 1990 in the tomato plants; Weretilnyk etc., 1989), therefore,, set up tomato trimethyl-glycine route of synthesis by the BADH gene is imported tomato, can improve the salt tolerance of cultivation tomato variety.
Although the salt tolerance of plant is a complicated quantitative character by controlled by multiple genes, still there is challenge in the salt tolerance that improves plant by genetically engineered.The present technique invention proves the salt tolerance that can improve tomato by this technique means by agriculture bacillus mediated prunella asiatica BADH gene transformation tomato cultivation kind " hundred sharp spring ".
4. description of drawings:
Fig. 1, binary expression vector structure diagram.
Binary expression vector pBin438 contains the Ω fragment translational enhancer of two 35S promoters and TMV, and prunella asiatica BADH cDNA fragment is inserted BamHI and the KpnI site of pBin438.
Fig. 2 A-C, transgenic Fructus Lycopersici esculenti differentiation.
A. it is dead on that screening culture medium of card not infect blade; B. transgenic calli; C. transfer-gen plant
Fig. 3 A-C, transgenic Fructus Lycopersici esculenti Molecular Detection.
A.PCR detects; B.Southern detects; C.Northern detects
BADH activity in Fig. 4, the transgenic Fructus Lycopersici esculenti
Fig. 5, transgenic Fructus Lycopersici esculenti are in the variation of salt stress lower blade specific conductivity
Fig. 6, T1 plant PCR detect: A.T1-1; B.T1-8
Fig. 7, T1 plant Northern detect 1,2:T1-1 PCR positive plant; The negative plant of 3:T1-1 PCR; The negative plant of 4:T1-8 PCR; 5,6:T1-8 PCR positive plant
Fig. 8, salt stress are to changeing the influence of BADH gene T1 for rate of emergence
Fig. 9, commentaries on classics BADH gene T1 plant conductivity variations
5. embodiment 1) material " hundred sharp spring " tomato (Lycopersicon esculentum Mill) seed buied by the Chinese Academy of Agricultural Sciences.Seed carried out surface sterilization 1 minute and 15 minutes with 70% alcohol and 0.1% mercuric chloride respectively, then with sterile distilled water flushing three times.Be seeded in (Murashige etc., 1962) in the MS minimum medium that does not add any hormone.Culturing room's temperature remains on 25 ± 2 ℃, humidity 70%-80%, illumination every day 12 hours.
PCR reacts used Taq enzyme and primer is given birth to worker company purchase and synthetic by Shanghai.Restriction enzyme and Random Primer Labeling System Kit are bought by Takara company.Trizol is bought by GIBCO company.
Plant expression vector is provided by the Chen Shouyi researcher of Institute of Genetics, Academia Sinica.Binary expression vector pBin438 contains the Ω fragment translational enhancer of two 35S promoters and TMV, and BADH cDNA fragment is inserted BamHI and KpnI site (Fig. 1) of pBin438.2) screening of Agrobacterium-mediated Transformation and transgenic Fructus Lycopersici esculenti
After the LBA4404 Agrobacterium that will contain expression vector with liquid YEB+ kantlex 50mg/L substratum is cultured to OD560=0.5, dilute 10 times with the MS liquid nutrient medium of no hormone.With the aseptic true leaf blade of tomato in wherein infecting 5 minutes.Take out blade subsequently, blot surperficial bacterium liquid, place IM substratum (MS+IAA0.2mg/L+BA2.0mg/L+ZT0.1mg/L) to go up dark and cultivated altogether 48 hours with aseptic filter paper.Be transferred to afterwards on the IM+ kantlex 50mg/L+ Pyocianil 500mg/L substratum and carry out differentiation culture, illumination every day 12 hours.
Observations shows, without the 2 weeks back flavescence or bleach on screening culture medium of the blade of agroinfection, and blade death, the wound does not have callus and grows.Through the tomato leaf of agroinfection, 2-3 begins leaf margin and green bud point occurs after week, and 5-6 differentiates plantlet after week.Long to 2-3cm when high when these plantlets, it is transferred on the MS+ kantlex 500mg/L+ Pyocianil 500mg/L substratum takes root.These plantlets can take root smoothly and grow substantially normal (Fig. 2).3) name of transfer-gen plant
The present age, transgenic line was that TG0 adds strain system number separately, as TG0-1, and TG0-2.Each strain is that the offspring of strict selfing adds separately strain system number for TG1, as TG1-1, and TG1-2 etc.4) Molecular Detection of transfer-gen plant
The young leaflet tablet of getting 0.1-0.2g differentiation plant extracts genomic dna with the CTAB method.The PCR reaction system is by standard method.The primer is: 5 '-AGAATGGCGTTCCCAATTCCTGCTC-3 ' and 5 ' TTCAAGGAGACTTGT ACCATCCCCA-3 ', response procedures is: 95 ℃ of sex change 1 minute, and 55 ℃ of annealing 1 minute, 72 ℃ were extended 1.5 minutes, totally 35 circulations (Xiao Gang etc., 1995).
(20 μ g) cuts with HindIII and EcoRI enzyme respectively with genomic dna, 1.0% sepharose 50V electrophoresis 5 hours, and DNA is transferred to (Sambrook etc., 1989) on the Hybond-N+ nylon membrane.Probe is a template with BADH cDNA, with α-32P-dCTP, and Randon PrimerLabeling System (Takara) test kit mark.
RNA extracts and uses the Trizol single stage method, with 1.2% denaturing formaldehyde gel electrophoresis, changes film, hybridizing method and used probe and hybridizes with Southern.
PCR and Southern hybridization detected result shows that the BADH gene has been incorporated into that (Fig. 3 A, B), but from Southern result as can be seen, integration site and BADH gene copy number do not have than big-difference (Fig. 3 B) in the homophyletic system in the genome of 6 strains systems.This may influence the expression level of BADH gene in tomato to a certain extent, and one of them strain system is detected fully less than the signal (Fig. 3 C) of expressing.5) BADH of transfer-gen plant is active detects
The plantlet of transplant of different transgenic lines is in the flowerpot that soil and vermiculite (1: 1) is housed, and greenhouse experiment is growth (25 ± 2 ℃, humidity 60%-80%, illumination every day 12 hours) down.With the MS nutrition liquid irrigation.After 40 days, each transgenic line plant is divided into two groups.One group with the MS nutrition liquid irrigation; Another group is irrigated with MS+NaCl and is reached 180mM until the NaCl final concentration.The active mensuration of BADH is undertaken by the method for (1997) such as Guo Yan.An enzyme unit definition alive is the NAD of every milligram of protein depletion 1nmol of per minute in the standard reaction system.
The BADH determination of activity is the result show, no matter whether NaCl coerces existence, can detect tangible activity in the plant of 6 strain systems.But the BADH activity of transfer-gen plant under salt stress is significantly higher than (Fig. 4) under the no salt stress.6) mensuration of the relative conductivity of rotaring gene plant blade (REc)
The mensuration of relative conductivity is undertaken by the method for (1984) such as Leopold.Contrast and each transgenic line are handled with 0mM, 90mM, 180mM and 270mM NaCl respectively.
The specific conductivity of tomato leaf raises gradually along with the increase of salt stress intensity, shows injury that cell is subjected to more and more serious (Fig. 5).But as can be seen, in 5 strain systems of normal expression BADH gene, its specific conductivity all has reduction in various degree, and this shows that changeing BADH gene tomato is improving salt tolerance in varying degrees.7) change the mensuration that BADH gene tomato T1 Dai Kana resistance is separated ratio
Through the transgenic line that Molecular Identification BADH gene really has been integrated into genome and can have correctly expressed, self-fertility.Its planting seed and makes solution not have filter paper in culture dish on the filter paper that is soaked with MS+ kantlex 100mg/L solution, seals to reduce the influence that moisture evaporation causes antibiotic concentration with the Parafilm film.Add up responsive and insensitive seedling ratio after 20 days, and carry out χ2Jian Yan kantlex.
This experiment is that the seed that T0-1 and T0-8 selfing obtain has carried out that resistance evaluation of card respectively to two strains of energy normal expression BADH gene.The χ2Jian Yan result shows that these two progeny populations separation all are separation in 3: 1, meet Mendelian's segregation ratio (table 1).Can infer thus, these two transgenic lines (T0-1, T0-8) in the insertion site of BADH gene be one or on same karyomit(e), and can entail the offspring.PCR result has also proved this point (Fig. 6).Northern result shows the plant that has the BADH gene in every genome, all can detect expression signal (Fig. 7).
Table 1 changes BADH gene tomato T1 Dai Kana resistance and separates ratio
Measured value expected value colony
χ
2Significance
The responsive T1-1 147 53 150 50 0.624 0.25＜P of the responsive resistance of resistance＜0.5T1-8 145 55 150 50 0.414 0.5＜P＜0.758) transgenic Fructus Lycopersici esculenti T1 is for the plant Molecular Identification
Pcr amplification condition and Northern hybridizing method are with T0 generation.9) changeing BADH gene tomato T1 plant salt tolerance detects
The T1 seed of sterilization is placed on the MS substratum of test tube, its grain number that germinates, height of seedling, root length and fibrous root number are carried out record day by day.And use the SPSS statistical software and carry out variance analysis.Simultaneously the blade specific conductivity of 60 age in days seedling under 0mM, 180mM NaCl coerce measured.
The rate of emergence of T1-1 and T1-8 is significantly higher than contrast (Fig. 8) under 90mM and 140mM NaCl coerce.And the height of seedling of seedling, main root length and fibrous root quantity are also compared according to improving a lot (table 2).NaCl is coerced the conductance measurement of lower blade and find that the specific conductivity of transfer-gen plant is starkly lower than contrast (Fig. 9).
Table 2 T1 analyzes for plant strain growth index Ducan
Index
Long (cm) fibrous root number (bar) of height of seedling (cm) root colony
0mM 90mM 140mM 0mM 90m 140mM 0mM 90mM 140mM
MWT 6.60a 3.25c 0.60d 9.68a 4.40d 0.62e 8.4a 0.0e 0.0eT1-1 6.52a 5.04b 2.91c 9.72a 6.86b 5.84 8.3a 3.7b 1.6d
cdT1-8 6.65a 4.64b 3.10c 9.83a 6.12 6.06 8.5a 4.2b 2.8c
The expression difference of the same letter of mark is not remarkable after the bc bc numeral.
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Claims (5)
1. cultivate the method for salt tolerant tomato strain system with changeing betaine aldehyde dehydrogenase gene (BADH) for one kind, this method comprises the steps:
A, the Agrobacterium LBA4404 of binary expression vector pBin438 that will contain the gene segment of BADH cDNA infects tomato true leaf blade;
B, metainfective tomato leaf place and carry out differentiation culture on the IM substratum;
C gets differentiation plant young leaflet tablet extraction genomic dna and carries out Molecular Detection;
D, the BADH of transfer-gen plant is active to be detected and the salt tolerance evaluation.
2. according to the described method of claim 1., Agrobacterium LBA4404 bacterial concentration OD wherein
560Value equals 0.5, uses the MS liquid nutrient medium of no hormone to dilute 10 times, infects tomato true leaf blade.
3. according to the described method of claim 1., wherein metainfective tomato leaf places differentiation culture on the IM substratum, and the IM substratum is made up of MS, IAA0.2mg/L, BA0.2mg/L, ZT0.1mg/L.
4. according to the described method of claim 1., the Molecular Detection of the genomic dna of wherein said transfer-gen plant, be with α-
32The BADH cDNA template of P-dCTP mark is that probe detects.
5. according to the described method of claim 1., the active detection of the BADH of wherein said transfer-gen plant is meant that the MS substratum reaches 180mM with salt (NaCl) until the NaCl final concentration, measures the relative conductivity of BADH enzymic activity and blade.
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| CN 02105490 CN1233833C (en) | 2002-04-05 | 2002-04-05 | Method for breeding salt-resistant tomato utilizing atriplex hortensis BADH gene transfer |
| AU2003236099A AU2003236099A1 (en) | 2002-04-05 | 2003-04-03 | A method for breeding salt-tolerant tomato plants |
| PCT/CN2003/000241 WO2003097827A1 (en) | 2002-04-05 | 2003-04-03 | A method for breeding salt-tolerant tomato plants |
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| CN100465276C (en) * | 2006-12-11 | 2009-03-04 | 中国科学院植物研究所 | Method for improving stress resistance of plant |
| CN1871896B (en) * | 2005-06-01 | 2010-06-09 | 天津农学院 | High performance method for transgene tomato |
| CN101787372A (en) * | 2010-03-29 | 2010-07-28 | 天津农学院 | Gene transformation method of oil sunflower |
| WO2010127579A1 (en) * | 2009-05-08 | 2010-11-11 | 创世纪转基因技术有限公司 | Mangrove betaine aldehyde dehydrogenase gene and uses thereof |
| CN103070004A (en) * | 2013-02-17 | 2013-05-01 | 中国科学院植物研究所 | Method for irrigating transgenic tomatoes by brine |
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| DE602005009553D1 (en) | 2004-09-08 | 2008-10-16 | Univ Carmel Haifa Economic Cor | STRESS-TOLERANT MICROORGANISMS EXPRESSING MAP KINASE HOMOLOGOUS GENE |
| CN101831497B (en) * | 2010-05-11 | 2012-07-04 | 新疆农业科学院园艺作物研究所 | Molecular marking method for distinguishing salt-tolerance character of tomato |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP3144260B2 (en) * | 1995-02-01 | 2001-03-12 | トヨタ自動車株式会社 | Environmental stress-tolerant plant and production method thereof |
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2003
- 2003-04-03 AU AU2003236099A patent/AU2003236099A1/en not_active Abandoned
- 2003-04-03 WO PCT/CN2003/000241 patent/WO2003097827A1/en not_active Application Discontinuation
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1307312C (en) * | 2004-05-25 | 2007-03-28 | 山东省林业科学研究院 | Tetraploid transgenic locust and quick cultivation |
| CN1871896B (en) * | 2005-06-01 | 2010-06-09 | 天津农学院 | High performance method for transgene tomato |
| CN100465276C (en) * | 2006-12-11 | 2009-03-04 | 中国科学院植物研究所 | Method for improving stress resistance of plant |
| WO2010127579A1 (en) * | 2009-05-08 | 2010-11-11 | 创世纪转基因技术有限公司 | Mangrove betaine aldehyde dehydrogenase gene and uses thereof |
| CN101787372A (en) * | 2010-03-29 | 2010-07-28 | 天津农学院 | Gene transformation method of oil sunflower |
| CN103070004A (en) * | 2013-02-17 | 2013-05-01 | 中国科学院植物研究所 | Method for irrigating transgenic tomatoes by brine |
| CN103070004B (en) * | 2013-02-17 | 2014-12-03 | 中国科学院植物研究所 | Method for irrigating transgenic tomatoes by brine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1233833C (en) | 2005-12-28 |
| AU2003236099A1 (en) | 2003-12-02 |
| WO2003097827A1 (en) | 2003-11-27 |
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