CN103540607B - The new opplication of Arabidopis thaliana At4g09820 gene in plant anti-salt - Google Patents
The new opplication of Arabidopis thaliana At4g09820 gene in plant anti-salt Download PDFInfo
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Abstract
The invention belongs to genetically engineered applied technical field, be specifically related to Arabidopis thaliana At4g09820 gene and cultivating the application in salt-tolerant plant new variety.Arabidopis thaliana At4g09820 gene, also known as <i>TT8</iGreatT. GreaT.GT gene, its CDS length is 1557bp, to encode 518 amino acid whose albumen, do you belong to bHLH (basic? helix-loop-helix) type transcription factor, regulates the pycnogenols of flavonoid route of synthesis and the biosynthesizing of anthocyanidin.Contriver is by the research in high salinity adverse circumstance to At4g09820 gene, find the Ion toxicity reaction <b> that the regulation and control NaCl of this gene specific causes, </b> shows good salt resistance characteristic, therefore there is the application of outbalance in plant anti-salt, by to the further research of this gene and conversion, excellent salt-tolerant plant kind can be cultivated.
Description
Technical field
The invention belongs to genetically engineered applied technical field, be specifically related to Arabidopis thaliana At4g09820 gene and cultivating the application in salt-tolerant plant new variety.
Background technology
Current, global water resources shortage aggravates increasingly, and the mankind use chemical fertilizer to make the soil salinization day by day serious in a large number, constrains continuous production and the development of agricultural.Along with going deep into of molecular biology and genetically engineered research, a lot of scientific worker is also striving to find the gene relevant to plant anti-salt in the world, intend the salt resistance by genetic engineering technique Crop Improvement, but current available achievement is very few.
Arabidopis thaliana (
arabidopsisthaliana) belong to Cruciferae mouse ear mustard, have another name called mouse ear mustard, Arabic mustard, Arabian cron, be plant genetics, one of model plant that molecular biology is conventional, be also usually used in resistant gene research under plant adverse environmental factor.
Existing study general thinks that the color of seed coat is except as except form instruction shape, and associated by also normal and plant quality has, the research that therefore Land use models plant Arabidopsis thaliana carries out seed coat chromogenesis Regulation Mechanism also has more achievement.Pigment mainly flavonoid in Arabidopis thaliana seed coat is specifically the pycnogenols in anthocyanidin and the flavonol in quercetin.
tT8gene (arabidopsis gene group coding is At4g09820) is one of gene of regulation and control Arabidopis thaliana seed coat pigment, belong to bHLH (basichelix-loop-helix) type transcription factor, 518 amino acid whose albumen of its coding, are mainly used in regulating the pycnogenols of flavonoid route of synthesis and the biosynthesizing of anthocyanidin.Other purposes about this gene there is no about research and report.
Summary of the invention
The object of the invention be to provide Arabidopis thaliana At4g09820 gene (
tT8gene) new opplication in plant salt resistance characteristic, thus provide new possibility for cultivating salt-resistant plant kind.
The technical solution adopted in the present invention is as follows:
The application of Arabidopis thaliana At4g09820 gene in plant anti-salt.
The application of described Arabidopis thaliana At4g09820 gene in plant anti-salt, this gene is used for anti-NaCl growing environment, and namely the existence of this gene can make plant materials as Arabidopis thaliana normal growth in certain density NaCl environment.
The application of described Arabidopis thaliana At4g09820 gene in plant anti-salt, this gene enables Arabidopis thaliana resist 100mMNaCl growing environment.
The application of described Arabidopis thaliana At4g09820 gene in plant anti-salt, transform the plant materials of this gene for anti-NaCl environment, namely, after this gene transformation being entered other plant body by genetic engineering means, this plant materials normal growth under certain density NaCl environment can be made.
The application of described Arabidopis thaliana At4g09820 gene in plant anti-salt, transform this gene and the plant materials of overexpression for anti-NaCl environment, namely after this gene transformation being entered other plant body by genetic engineering means, this gene is overexpression in plant materials in post-conversion, can make this plant materials normal growth under certain density NaCl environment.
The application of described Arabidopis thaliana At4g09820 gene in plant anti-salt, transforms the Arabidopis thaliana of this gene and overexpression
tt8-1mutant is used for anti-100mMNaCl environment.
Now there are some researches show, Arabidopis thaliana At4g09820 gene, also known as
tT8gene, its CDS length is 1557bp, and 518 amino acid whose albumen of encoding, belong to bHLH (basichelix-loop-helix) type transcription factor, regulate the pycnogenols of flavonoid route of synthesis and the biosynthesizing of anthocyanidin, grow with TTG1 and TT2 co-controlling seed coat.And the present invention is by the research in high salinity adverse circumstance to At4g09820 gene, find the Ion toxicity reaction that the regulation and control NaCl of this gene specific causes, show salt resistance characteristic, therefore by further research and the conversion of this gene, excellent salt-tolerant plant kind can be cultivated.
Accompanying drawing explanation
Fig. 1 be germination and growth under 100mMNaCl and 200mM treatment with mannitol 7 days WT and
tt8-1seedling;
Fig. 2 is that germination and growth is at 100mMNaCl, KCl or NaNO
3process lower 7 days WT and
tt8-1seedling;
Fig. 3 is germination and growth under 0.1,0.5 and 1 μM of ABA process WT and the tt8-1 seedling of 7 days;
Fig. 4 be germination and growth under 100mMNaCl process 7 days WT and
tt8-1function covering seedling.
Embodiment
Introduce embodiment below the present invention will be further explained explanation.
embodiment 1
Relevant to plant anti-salt effect for proving At4g09820 gene, inventors performed comparative analysis experiment.
Following
tt8-1mutant is At4g09820 gene T-DNA insertion mutation body (CS111) obtained from Arabidopis thaliana resource center, the At4g09820 gene inactivation of this mutant, can not correct coding related protein, checking can find that At4g09820 gene is relevant to plant anti-salt function further.
Specific experiment proof procedure is as follows:
(1) the Ion toxicity reaction that At4g09820 gene specific regulates NaCl to cause is verified
By Arabidopis thaliana wild-type (WT) and
tt8-1mutant seeds is seeded in the MS substratum of 0.6% agar and contains on the MS substratum of 100mMNaCl or 200mM N.F,USP MANNITOL.Growing environment is: light/dark cycle is 8/16h, temperature 22 DEG C, dark temperature 18 DEG C, relative humidity 70%, intensity of illumination 90 μm of olm
-2s
-1., specifically to observe after growing 1 week until Seed Germination of Arabidopsis Pumila.
Experimental result as shown in Figure 1, can find on the substratum containing 100mMNaCl,
tt8-1mutant seeds is only sprouted, and growth is subject to serious suppression, and wild-type (WT) seed can normally be sprouted and grow, namely under condition of salt stress
tt8-1mutant seeds is sprouted and energy for growth is significantly smaller than wild-type (WT); And under normal or 200mM N.F,USP MANNITOL (with the infiltration such as 100mMNaCl) treatment condition, the two upgrowth situation without any difference, under namely waiting the treatment with mannitol situation of infiltration, wild-type (WT) and
tt8-1mutant does not have notable difference in sprouting and growth perfonnance.
This result shows, At4g09820 gene (
tT8gene) specific regulating NaCl cause Ion toxicity reaction, and do not participate in osmotic stress regulate.
(2) verify that At4g09820 gene specific regulates Na
+
the Stress responses caused
For prove further At4g09820 gene (
tT8gene) Ion toxicity that regulates NaCl to cause is Na
+or Cl
-, experiment uses 100mMKCl and KNO respectively
3process wild-type (WT) and
tt8-1the seed of mutant.Process and culture condition are: Arabidopis thaliana wild-type (WT) and tt8-1 mutant seeds are seeded in the MS substratum of 0.6% agar and contain respectively on the MS substratum of 100mMNaCl, 100mMKCl or 100mMNaNO3.Growing environment is: light/dark cycle is 8/16h, temperature 22 DEG C, dark temperature 18 DEG C, relative humidity 70%, intensity of illumination 90 μm of olm
-2s
-1., specifically to observe after growing 1 week until Seed Germination of Arabidopsis Pumila.
As shown in Figure 2, what can find that 100mMNaNO3 process can obtain 100mMNaCl process coerces phenotype to experimental result, and 100mMKCl process, and wild-type and mutant do not have phenotypic difference, thus can confirm At4g09820 gene (
tT8gene) specific regulating Na
+the Stress responses caused.
(3) verify that At4g09820 gene specific regulates Na
+
whether the Stress responses caused relies on ABA signal transduction
For prove further At4g09820 gene (
tT8gene) regulate Na
+whether the Stress responses caused, depend on ABA signal transduction pathway, contriver with ABA process wild-type (WT) and
tt8-1mutant, concrete cultivation and treatment condition are: Arabidopis thaliana wild-type (WT) and mutant (tt8-1) planting seed are in the MS substratum of 0.6% agar with respectively containing on the MS substratum of 0.1,0.5 and 1 μM of ABA.Growing environment is: light/dark cycle is 8/16h, temperature 22 DEG C, dark temperature 18 DEG C, relative humidity 70%, intensity of illumination 90 μm of olm
-2s
-1., specifically to observe after growing 1 week until Seed Germination of Arabidopsis Pumila.
Experimental result as shown in Figure 3, result display there is no phenotypic difference, show At4g09820 gene (
tT8gene) regulate Na
+coercing of causing does not rely on ABA.
(4) examine
tt8-1mutant is to Na
+
sensitivity response be that At4g09820 transgenation causes
For examining further
tt8-1mutant is to Na
+sensitivity response, be really At4g09820 gene (
tT8gene) sudden change causes, the present invention construct At4g09820 gene (
tT8gene) overexpression vector, transform
tt8-1mutant, concrete overexpression and conversion process as follows:
(1) the CDS sequence of At4g09820 gene is obtained from Arabidopis thaliana website (http://www.arabidopsis.org), according to overexpression vector super-1300(Caulimovirus 35S promoter, energy effective expression foreign gene, containing hygromycin gene) restriction enzyme site, utilize primer5.0 software design Auele Specific Primer, with arabidopsis gene group cDNA for template, pcr amplification goes out CDS full length sequence, through enzyme cut link object fragment is linked on super-1300 carrier, through sequence verification with
at4g09820CDS sequence is consistent, namely obtains the overexpression vector containing goal gene.
(2) by the above-mentioned overexpression vector recombinant plasmid transformed Agrobacterium GV3101 competent cell containing goal gene.
(3) at Arabidopis thaliana
tt8-1when mutant seedlings inflorescence grows to 5-10cm height, carry out Agrobacterium-medialed transformation, be immersed in bacterium liquid by Arabidopis thaliana inflorescence, every strain leaching flower 30s, lying in pallet by soaking the seedling basin spent, covering keep humidity with plastic film, lucifuge is taken out after placing 16-24h, normally cultivates; After 5-7 days, superinfection once.
(4) seed of the Arabidopis thaliana results infected by above-mentioned Agrobacterium, sows seeds on the MS culture medium flat plate containing 25mg/L Totomycin, 4 DEG C of vernalization 3d, move on to usable material chamber to cultivate, positive plant has resistance to Totomycin, can normal growth, negative plant is responsive to Totomycin, can not normal growth finally dead.
(5) by the positive seedling replanting that filters out in Nutrition Soil, sowing after ripe, gained seed continuation hygromycin selection also does further Molecular Identification, and by further for positive seedling expanding propagation, obtains the homozygote of goal gene stably express.
Experimental result as shown in Figure 4, can find through transforming
tt8-1mutant has recovered wild type phenotype.
In sum, through some row confirmatory experiments, inventors have demonstrated that At4g09820 gene has huge potential function in plant anti-salt, by this gene overexpression is transformed into plant materials, can obtain and stand the relevant transgenic plant of Salt Stress, realize the anti-salt functional of plant, the present invention is that the genetically modified crops new variety of cultivating anti-salt have established theoretical and production practice basis.
Claims (2)
1. the application of Arabidopis thaliana At4g09820 gene in plant anti-salt, is characterized in that, described plant is Arabidopis thaliana, and this gene is used for anti-Na
+coerce growing environment.
2. the application of Arabidopis thaliana At4g09820 gene in plant anti-salt as claimed in claim 1, is characterized in that, to Arabidopis thaliana
tt8-1mutant transform and overexpression At4g09820 gene after, for anti-100mMNaCl environment, described Arabidopis thaliana
tt8-1mutant is At4g09820 gene T-DNA insertion mutation body, the At4g09820 gene inactivation of this mutant.
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| CN104263735A (en) * | 2014-07-31 | 2015-01-07 | 河南大学 | Novel application of arabidopsis TTG1 genes to plant salinity-resisting and drought-resisting aspects |
| CN105018522B (en) * | 2015-07-17 | 2018-02-09 | 山东省水稻研究所 | Gene OsPIL16 improves the application in the arid and salt stress patience of rice at the same time |
| CN105002212B (en) * | 2015-07-28 | 2018-01-05 | 山东省水稻研究所 | Applications of the gene OsPIL13 in Under Salt Stress in Rice patience is improved |
| CN110241124A (en) * | 2019-07-09 | 2019-09-17 | 中国农业科学院北京畜牧兽医研究所 | Arabidopsis At4g36920 gene is in regulation plant proanthocyanidin biosynthesis and the application of the anti-hoove of ruminant |
| CN111690662B (en) * | 2020-06-03 | 2021-11-12 | 吉林大学 | Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis |
| CN111733166B (en) * | 2020-06-18 | 2021-12-14 | 中国农业科学院郑州果树研究所 | Vitis davidii anthocyanin synthetic gene VdbHLH037 and application thereof |
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