CN107602679A - TabHLH44 albumen and its encoding gene and application - Google Patents
TabHLH44 albumen and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses TabHLH44 albumen and its encoding gene and application.The invention provides albumen, is named as TabHLH44, be it is following 1) or 2)：1) protein in sequence table shown in sequence 2；2) amino acid sequence shown in sequence in sequence table 2 by the substitution of one or several amino acid residues and/or missing and/or addition and had into identical function protein as derived from sequence 2.The experiment proves that, the present invention passes through to the processing of wheat environment stress, filter out the transcription factor TabHLH44 genes of response environment stress, it is induced by salt, PEG, cold stress and ABA, it is conducted into arabidopsis, obtains turning TabHLH44 arabidopsis, carried out Function Identification, show that TabHLH44 improves the drought resistance, salt-resistance and frost resistance of transgenic arabidopsis, basis is provided to cultivate resistance plant.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of TabHLH44 albumen and its encoding gene and application.
Background technology
Severe growing environment (extreme temperature, saline and alkaline, arid etc.) has a strong impact on growing, reducing work for plant
The yield of thing.Plant can not avoid environment stress by mobile, therefore in order to eliminate or reduce environment stress to certainly
Harm caused by body, plant form the regulated and control network of complexity so that plant is facing adverse circumstance during evolution
Can be by cellular level during stress, the regulation of molecular water equality adapts to unfavorable growing environment.It is unfavorable in order to reduce
Growing environment caused by crops huge loss, ensure grain security, how to improve the resistance of crops, by
The concern of countries in the world government is arrived.Improvement of genes is carried out to crops and improves the resistance of itself, is a kind of effective
Method.
Transcription factor is a kind of regulatory gene, is molecule important in regulated and control network, it is by signal transduction and to adverse circumstance
The regulation and control of responsive genes, all life processes of organism almost being participated in, wherein bHLH is a major class transcription factor family,
And wide participation plant response biotic and abiotic stress, play key among plant response stress procedure.
But the functional study of the TabHLH transcription factors in wheat moderate resistance inversely related is relatively fewer.
The content of the invention
It is an object of the present invention to provide TabHLH44 albumen and its encoding gene.
Albumen provided by the invention, is named as TabHLH44, be it is following 1) or 2)：
1) protein in sequence table shown in sequence 2；
2) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or
Lack and/or add and there is identical function protein as derived from sequence 2.
In order that the protein in (1) is easy to purify, amino acid sequence that can be in as sequence table shown in sequence 2
Arrange the amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of composition.
The sequence of the label of table 1
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (2) can be artificial synthesized, also can first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of protein in above-mentioned (2) can be by will lack one in the DNA sequence dna shown in sequence in sequence table 1
Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 '
The coded sequence that end and/or 3 ' ends connect the label shown in table 1 obtains.
It is also the scope of protection of the invention to encode above-mentioned protein DNA molecule.
Above-mentioned DNA molecular is following 1) -4) in any DNA molecular：
1) code area is the DNA molecular shown in sequence 1 in sequence table；
2) code area is the DNA molecular shown in the 271-1458 positions of sequence 1 in sequence table；
1) or 2) 3) under strict conditions with the DNA sequence dna hybridization limited and coding with identical function protein
DNA molecular；
4) 1) or 2) with the DNA sequence dna that limits at least with 70%, at least with 75%, at least with 80%, extremely
Less with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with
98% or at least with 99% homology and coding with identical function protein DNA molecule.
Above-mentioned stringent condition can be 6 × SSC, 0.5%SDS solution in, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned DNA molecular are also protection of the present invention
Scope.
Recombinant vector is by two attB sites of the carriers of 1 271-1458 positions nucleotides inserted pEarleyGate of sequence 100
Between obtained carrier, to be overexpressed TabHLH44 genophores, be named as pEarleyGate 100-TabHLH44.
The primer pair for expanding above-mentioned DNA molecular total length or its any fragment is also the scope of protection of the invention.
Above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are being adjusted
The application controlled in stress resistance of plant is also the scope of protection of the invention；
Or above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell or recombinant bacterium are being trained
It is also the scope of protection of the invention to educate the application that resistance is improved in genetically modified plants.
In above-mentioned application, the resistance is salt-resistance, drought-resistant and/or low-temperature resistance；
The plant is monocotyledon or dicotyledon.
Another object of the present invention is to provide a kind of method cultivated resistance and improve genetically modified plants.
Method provided by the invention, for the DNA molecular for encoding above-mentioned albumen is imported into purpose plant, obtain transgenosis and plant
Thing,
The resistance of the genetically modified plants is higher than the purpose plant；
In the above method, the resistance is salt-resistance, drought-resistant and/or low-temperature resistance；
In the above method, the drought-resistant of above-mentioned genetically modified plants is higher than the purpose families of plant described under drought stress
The survival rate of genetically modified plants is higher than the purpose plant；
The salt-resistance of above-mentioned genetically modified plants is higher than purpose families of plant genetically modified plants under salt stress
Survival rate or root long are above the purpose plant；
The low-temperature resistance of above-mentioned genetically modified plants is higher than purpose families of plant genetically modified plants under low temperature stress
Survival rate be higher than the purpose plant.
Drought stress is not water；Salt stress is 150mM NaCl；Low temperature stress is -10 DEG C；
In the above method, the plant is monocotyledon or dicotyledon.
The low temperature is specially -10 DEG C；
The dicotyledon is specially arabidopsis.
It is the experiment proves that of the invention by the processing of wheat environment stress, filtering out response environment stress
Transcription factor TabHLH44 genes, it is induced by salt, PEG, cold stress and ABA, is conducted into arabidopsis, obtains
To TabHLH44 arabidopsis is turned, Function Identification is carried out, shows that TabHLH44 improves transgenic arabidopsis
Drought resistance, salt-resistance and frost resistance, basis is provided to cultivate resistance plant.
Brief description of the drawings
Fig. 1 is expression patterns of the TabHLH44 under Different stress processing.
Fig. 2 is the Identification of Drought for turning TabHLH44 arabidopsis.
Fig. 3 handles survival rate to turn the drought resisting of TabHLH44 arabidopsis.
Fig. 4 identifies to turn TabHLH44 arabidopsis arabidopsis salt-resistance.
Fig. 5 handles root long to turn the salt resistance of TabHLH44 arabidopsis.
Fig. 6 identifies to turn TabHLH44 arabidopsis frost resistance.
Fig. 7 is to turn the freeze proof processing survival rate of TabHLH44 arabidopsis.
In above-mentioned each figure, WT：Wild type；L1,L2,L3:Turn TabHLH44 Arabidopsis plants.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The discovery of embodiment 1, TabHLH44 genes
First, the discovery of TabHLH44 genes
The work of laboratory early stage constructs several Wheat Full-length cDNA libraries, by the sequencing to cDNA plasmids,
BHLH transcription factors are predicted according to transcription factor design feature, have obtained the full-length cDNA of TabHLH44 genes
Sequence.
The nucleotides sequence of TabHLH44 genes in wheat is classified as sequence 1, and ORFs is the 271-1458 of sequence 1
Position nucleotides；The albumen TabHLH44 of coding, its amino acid sequence are sequence 2.
2nd, expression of the TabHLH44 genes under adverse environmental factor
By the China spring for Stress treatment, Drought resistant Wheat drought selects No. 10, salt resistance wheat tea form sediment it is red, respectively 25 DEG C,
Cultivated 10 days under 16h illumination/8h dark photoperiods, then carry out different Stress treatments.China spring is cold for 4 DEG C
Processing and ABA processing, leaf tissue, ABA processing are taken when 0,3,7,12,24 and 48h during cold treatment
When take root tissue when 0,1,3,7,12 and 24h.Drought resistant Wheat drought selects No. 10, for 16.1%PEG6000
Processing, root tissue is taken when 0,1,3,7,12 and 24h.Red, the salt for 250mM in salt resistance wheat tea shallow lake
Processing, root tissue is taken when 0,1,3,7,12 and 24h.The RNA of samples taken is extracted, reverse transcription obtains cDNA
As template, with specific primer (F primers, the ATGACAGGTTCGCGTTCGTC of gene；R primers
TCTTGTTCCCATTCGTTGGG) expanded, with Tubulin genes (F primers, ACCGTGGTGATGTTGTGC；R draws
Thing, TGGTGGCTGGTAGTTGATA) it is internal reference.
As a result it is as shown in Figure 1, it can be seen that under NaCl treatment conditions, to handle in 3h, with processing time
The expression quantity for increasing TabHLH44 gradually rises, and then expression declines, and is substantially increased to expression quantity when handling 12h,
TabHLH44 genes still keep high expression status when 24h.Under PEG processing, TabHLH44 expression
Amount slowly rises, and reaches maximum to expression quantity when handling 7h, then expression quantity starts to reduce, table when handling 24h
Begun to ramp up again up to amount.Under conditions of cold treatment, when handling 3h, TabHLH44 expression quantity raises rapidly, 7h
Begin to decline, 24h expression quantity is raised again, and high expression level is still maintained when handling 48h.In ABA processing
Under the conditions of, TabHLH44 expression quantity has just raised in processing 3h, and expression quantity declines afterwards.From result,
TabHLH44 expression is coerced by NaCl, PEG stress, cold stress and ABA stress are induced, you can response
A variety of environment stresses, therefore further Function Identification is carried out to TabHLH44 arabidopsis thaliana transformations.
Embodiment 2, TabHLH44 genes resistance application
First, the preparation of TabHLH44 arabidopsis is turned
1st, the over-express vector of transformation of Arabidopsis thaliana is built using Gateway methods
BP plasmids are that the TabHLH44 genes shown in the 271-1458 positions of sequence 1 are inserted into entry vector
pDONRTMThe plasmid that/Zeo (Invitrogen, 12535035) is obtained.
LR reacts：Above-mentioned BP plasmids are entered with the over-express vectors of pEarleyGate 100 by the method for Gateway technologies
Row LR reacts, and obtains recombinant vector.LR reaction systems such as table 2 below：
Table 2 reacts for LR
Recombinant vector is by two attB sites of the carriers of 1 271-1458 positions nucleotides inserted pEarleyGate of sequence 100
Between obtained carrier, to be overexpressed TabHLH44 genophores, be named as pEarleyGate 100-TabHLH44.
2nd, the conversion of Agrobacterium
Recombinant vector pEarleyGate 100-TabHLH44 are transferred into Agrobacterium GV3101, and (GV3101 Agrobacterium competence is thin
Born of the same parents, Shanghai Chao Yan bio tech ltd, CC3201) in, obtain recombinant bacterium GV3101/pEarleyGate 100-
TabHLH44 (extraction plasmid, sequencing are correct).
3rd, Agrobacterium infects the screening of arabidopsis thaliana transformation and transfer-gen plant
A) the culture of arabidopsis
1) aseptically, with 10 ℅ liquor natrii hypochloritis by wildtype Arabidopsis thaliana clo-0 (Nitschke S,
Cortleven A,Iven T,Feussner I,Havaux M,Riefler M,Schmülling T.2016.
Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic
Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants.Plant Cell.
[Epub ahead of print]) seed disinfection 15min, it is clean with sterile water wash, then seed is laid on
Cultivated on MS culture mediums.
2) after MS culture dishes being put in into 4 DEG C of vernalization 2 days, transfer in 22 DEG C of greenhouses and cultivate, stay on MS culture mediums
After cultivating 1 week, seedling is moved into soil and continues to cultivate, when being transferred in soil, needs moisturizing 2-3 days.
3) when Arabidopsis plant grows to most of bud and will bloomed, proceed by Agrobacterium and infect conversion.
B) Agrobacterium infects arabidopsis thaliana transformation
1) picked with sterile toothpick and identify correct agriculture through GV3101/pEarleyGate 100-TabHLH44 bacterium solutions PCR
Bacillus bacterium solution, (contain antibiotic Rif in YEB solid mediums:30μg/mL,Gen:30μg/mL,Kan:
50 μ g/mL) line, it is inverted in 28 DEG C of incubators, light culture 2-4 days.
2) after monoclonal is grown, (antibiotic Rif is contained to YEB fluid nutrient mediums:30μg/mL,Gen:30μg/mL,
Kan:50 μ g/mL) in, single bacterium colony is inoculated with, is put in 28 DEG C of shaking tables, 210rpm overnight incubations.
3) prepare 200ml YEB fluid nutrient mediums and (contain antibiotic Rif:30μg/mL,Gen:30μg/mL,Kan:
50 μ g/mL), the Agrobacterium bacterium solution that 1mL is incubated overnight is added thereto.In 28 DEG C of shaking tables, 210rpm mistakes
Night cultivates, untill bacterium solution color is rendered as crocus.
4) above-mentioned cultured 200mL Agrobacteriums bacterium solution is poured into sterile centrifuge tube, thalline is collected in room temperature,
5000rpm, centrifuge 15min.
5) with the μ lsilwet ratios of 1/2MS+5% sucrose+20, in the conversion penetrating fluid for preparing 100mL, by the agriculture of collection
Bacillus thalline is suspended in conversion penetrating fluid.
6) the conversion penetrating fluid of Agrobacterium is loaded onto with big culture dish, the whole inflorescence of arabidopsis is invaded into 30s in penetrating fluid,
Arabidopsis after infecting is lain against in pallet, and culture 24h, the plan that then will be infected are placed under dark condition
Southern mustard is placed under regular culture conditions and cultivated.According to the growth conditions of arabidopsis, may be selected again after one week
Arabidopsis is infected, to improve transformation efficiency, harvest obtains the seed for turning TabHLH44 arabidopsis in T1 generations.
4th, the identification of TabHLH44 arabidopsis is turned
In extraction T1 generations, turn the DNA of TabHLH44 Arabidopsis leafs, use gene-specific primer：F primers,
ATGACAGGTTCGCGTTCGTC；R primers TCTTGTTCCCATTCGTTGGG enters performing PCR amplification,
Obtain 240bp turns TabHLH44 arabidopsis for the positive.
The RNA that the above-mentioned positive turns TabHLH44 Arabidopsis leafs is extracted, it is template that reverse transcription, which obtains cDNA, is used
State gene-specific primer carry out RT-PCR amplifications, using AtActin genes as internal reference (F primers,
GTCTGGATTGGAGGGTC；R primers, TGAGAAATGGTCGGAAA), obtain 240bp is
In positive T1 generations, turn TabHLH44 arabidopsis.
In positive T1 generation, turns TabHLH44 arabidopsis by expanding numerous subculture, obtains T2 generations and turns TabHLH44 arabidopsis, as pure
Conjunction turns TabHLH44 arabidopsis strains.
Using same method, empty carrier pEarleyGate 100 is transferred in wildtype Arabidopsis thaliana, turned
The arabidopsis of pEarleyGate 100.
2nd, the degeneration-resistant Journal of Sex Research of TabHLH44 arabidopsis is turned
Selection homozygosis turns TabHLH44 arabidopsis and wildtype Arabidopsis thaliana is handled as follows, each 10 plants of strain, real
Test and be repeated 3 times, results averaged：
1) drought-resistant processing
Wildtype Arabidopsis thaliana, homozygosis turn TabHLH44 arabidopsis strain L1, L2, L3 and turn pEarleyGate 100
Arabidopsis thaliana Seedlings are transferred in soil respectively after being cultivated on MS culture mediums 1 week, under the conditions of 22 DEG C of 9h short-day after
Continuous culture.Early stage Arabidopsis plant be grown on regular culture conditions, treat growth of seedling 3 weeks, when more healthy and strong, stop
Only water, observe the growth conditions of seedling at any time, when plant to be planted serious wilting occurs, dried up, rehydration is carried out to plant,
Rehydration observes the growth conditions of Arabidopsis plant again after 5 days.It is control with normal culture always.
As a result as shown in fig. 2, it can be seen that after Osmotic treatment, homozygosis turn TabHLH44 arabidopsis strains L1, L2,
L3 growths are better than wildtype Arabidopsis thaliana, show that homozygosis turns the raising of TabHLH44 arabidopsis drought resistance.
After rehydration 5 days, most of WT lines are substantially dead, and some transfer-gen plants can restore normal growth
State, the respectively homozygous survival for turning TabHLH44 arabidopsis strain L1, L2, L3 strain and wildtype Arabidopsis thaliana of statistics
Rate=(surviving plant number/total plant number) * 100%.
As a result as shown in figure 3, after Osmotic treatment, homozygosis turns TabHLH44 arabidopsis strain L1, L2, L3 strains
Survival rate is significantly higher than WT strain.
Turn the arabidopsis of pEarleyGate 100 and wildtype Arabidopsis thaliana result without significant difference.
2) salt resistance is handled
In 22 DEG C of 16h illumination cultivations greenhouses, homozygosis turns TabHLH44 arabidopsis strains L1, L2, L3, wild type and intended
Southern mustard and turn after the Arabidopsis thaliana Seedlings of pEarleyGate 100 grow 5 days in MS culture mediums respectively, respectively by transgenosis
Strain and wild type seedlings are transferred to the MS cultures for being not added with NaCl (0mM NaCl, CK) and adding 150mM NaCl
In base, and MS culture mediums are disposed vertically, observe the change of the growth conditions and root long degree of Arabidopsis thaliana Seedlings daily.
As a result it is as shown in Figure 4, it can be seen that in 150mMNacl culture mediums, homozygosis turns TabHLH44 arabidopsis strains
It is that L1, L2, L3 growth are better than wildtype Arabidopsis thaliana, shows that homozygosis turns the raising of TabHLH44 arabidopsis salt-resistance.
Measurement homozygosis turns the root long of TabHLH44 arabidopsis strain L1, L2, L3 and WT strain respectively, as a result such as
Shown in Fig. 5, in the NaCl containing 150mM, homozygosis turn the root longs of TabHLH44 arabidopsis strains apparently higher than
Wild type.
Turn the arabidopsis of pEarleyGate 100 and wildtype Arabidopsis thaliana result without significant difference.
3) low-temperature resistance is handled
22 DEG C of week old positive homozygosis of 16h illumination cultivations 3 turn TabHLH44 arabidopsis strains L1, L2, L3, wild type
Arabidopsis (WT) and turn the arabidopsis of pEarleyGate 100, grow 3h at -10 DEG C.With it is normal culture for control (CK,
- 10 DEG C of low-temperature treatments are not carried out).
As a result it is as shown in Figure 6, it can be seen that under the conditions of -10 DEG C, homozygosis turn TabHLH44 arabidopsis strains L1,
L2, L3 growth are better than wildtype Arabidopsis thaliana, show that homozygosis turns the raising of TabHLH44 arabidopsis low temperature resistivity.
After jelly processing, most of WT lines are substantially dead, and some transfer-gen plants can restore normal growth shape
State, respectively statistics homozygosis turn the survival rate of TabHLH44 arabidopsis strain L1, L2, L3 and wild type=(survive plant
Strain number/total plant number) * 100%.
As a result as shown in fig. 7, the homozygous survival rate for turning TabHLH44 arabidopsis strains L1, L2, L3 is significantly higher than open country
Raw type strain.
Turn the arabidopsis of pEarleyGate 100 and wildtype Arabidopsis thaliana result without significant difference.
In the above results, the arabidopsis for being overexpressed TabHLH44 has resistance, shows that TabHLH44 has resistance.
Claims (10)
1. a kind of albumen, be it is following 1) or 2)：
1) protein in sequence table shown in sequence 2；
2) amino acid sequence shown in sequence in sequence table 2 by the substitution of one or several amino acid residues and/or missing and/or addition and had into identical function protein as derived from sequence 2.
2. encode protein DNA molecule described in claim 1.
3. DNA molecular as claimed in claim 2, it is characterised in that：The DNA molecular is following 1) -4) in any DNA molecular：
1) code area is the DNA molecular shown in sequence 1 in sequence table；
2) code area is the DNA molecular shown in the 271-1458 positions of sequence 1 in sequence table；
1) or 2) 3) there is identical function protein DNA molecule with the DNA sequence dna hybridization limited and coding under strict conditions；
1) or 2) 4) at least have 70% with the DNA sequence dna limited, at least with 75%, at least with 80%, at least with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least with 99% homology and encode with identical function protein DNA molecule.
4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing DNA molecular described in Claims 2 or 3.
5. expand the primer pair of DNA molecular total length or its any fragment described in Claims 2 or 3.
6. albumen described in claim 1, DNA molecular described in Claims 2 or 3 or the application of recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in stress resistance of plant is regulated and controled described in claim 4；
Or the application of albumen described in claim 1, DNA molecular described in Claims 2 or 3 or recombinant vector, expression cassette, transgenic cell line or recombinant bacterium described in claim 4 in cultivating resistance and improving genetically modified plants.
7. application according to claim 6, it is characterised in that：
The resistance is salt-resistance, drought-resistant and/or low-temperature resistance；
The plant is monocotyledon or dicotyledon.
8. a kind of method cultivated resistance and improve genetically modified plants, for the DNA molecular for encoding albumen described in claim 1 is imported into purpose plant, genetically modified plants are obtained,
The resistance of the genetically modified plants is higher than the purpose plant.
9. according to the method for claim 8, it is characterised in that：
The resistance is salt-resistance, drought-resistant and/or low-temperature resistance.
10. method according to claim 8 or claim 9, it is characterised in that：
The plant is monocotyledon or dicotyledon.
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| CN110428865A (en) * | 2019-08-14 | 2019-11-08 | 信阳师范学院 | A kind of method of high-throughput prediction Antifreeze protein |
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| CN104531751A (en) * | 2008-08-20 | 2015-04-22 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
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| MATSUMOTO,T.等: ""AK357521.1"", 《GENBANK》 * |
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