CN104086635B - A new anti-drought gene CkDHN1 in Caragana korshinskii - Google Patents
A new anti-drought gene CkDHN1 in Caragana korshinskii Download PDFInfo
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- CN104086635B CN104086635B CN201410152592.9A CN201410152592A CN104086635B CN 104086635 B CN104086635 B CN 104086635B CN 201410152592 A CN201410152592 A CN 201410152592A CN 104086635 B CN104086635 B CN 104086635B
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- 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
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Abstract
The invention discloses Caragana korshinskii (Caragana korshinskii Kom.) Dehydrins and its an encoding gene CkDHN1.The Dehydrins are made up of 134 amino acid residues, have a K conserved sequence " QKKGIMDKIKDKLPG ", therefore the albumen belongs to Kn type Dehydrins.Full length gene 745bp, it is a 402bp ORFs from 5 ' end 108bp to 510bp, in plant by strong expression under drought stress, mitigates plant cell aggrieved, improve drought-resistant ability, be a key gene of drought tolerance in plants mechanism.Amino acid sequence (CBE44592) similarity highest of the gene and robur dehydrin gene, only 40%, it is a new gene.Imported by building the plant expression vector of the gene, and by this Drought-tolerant gene in all kinds of plants, the drought resistance of plant can be improved.
Description
Technical field
The present invention relates to plant genetic engineering field, in particular to from Caragana korshinskii (Caragana
Korshinskii Kom.) in a new anti-drought gene CkDHN1, the gene codified plant dehydration element, its function be improve
The drought resistance of plant, belongs to plant genetic engineering field.
Background technology
Abiotic stress is the main reason for causing the world food underproduction, and per food yield can be caused to reduce every year
More than 50%.In various abiotic stress, arid is to influence the most important factor of grain yield.Arid be it is a kind of it is main from
Right disaster, there is very big harm to agricultural, water resource and natural environment, it is huge on grain yield influence, to economic and society
It is stable that there is the influence for being difficult to estimate.China is because the crisis that water shortage is brought is particularly acute, in recent years, Droughts occurrence frequency
The high, duration is lengthened, coverage constantly expands, and significant damage is brought to the grain security of China.Research plant resistant is done
The mechanism of drought stress, screens and cultivates the variety of crops of resistance to adverse circumstance, and it is to lower nature calamity to tackle Droughts by genetic engineering
One of important channel that evil influences.
Research shows, under drought stress, a series of change, including infiltration occur for the physiological and biochemical index on cellular level
It is mutual between the reduction of pressure, the change of the mobility of film and composition, the change of solution concentration, protein-protein and albumen-lipid
Effect etc..Dehydrin gene plays an important role in plant drought, salt resistance and the various abiotic stress such as low temperature resistant.It is de-
Water element (DHN) is one kind of LEA protein, can largely be accumulated in the dehydrated tissue of plant, for example, during seed maturity or at plant
When the environment-stress such as arid, low temperature and high salt, the accumulation of Dehydrins is the important means that plant adapts to adverse environment.Dehydrins
Plant cell can be protected to be escaped injury in adverse circumstance, strengthen the anti-adversity ability of plant.Experiment in vitro research shows Dehydrins
Major function is：Associated proteins and lipid；Remove free radical；With reference to metal；Low-temperature protection.It is separated in different plants more next
More dehydrin genes, research of the substantial amounts of transgenic technology to these genes are shown：Dehydrin gene is in plant drought, anti-
Played an important role in salt and the various abiotic stress such as low temperature resistant.
Caragana korshinskii (Caragana korshinskii Kom.) is distributed in desert, Semi-desert Area, growing environment bar
Part is severe, and very long evolutionary process makes it possess stronger resistance.Caragana korshinskii drought resisting, salt resistance alkali, impoverishment tolerant, adaptation
Extreme temperature, it is the ideal material for studying plant stress-resistance mechanism.Crops are carried out using Caragana korshinskii gene related to drought tolerance to change
Good, cultivating degeneration-resistant new varieties has important application value.
The content of the invention
It is an object of the invention to provide one in Caragana korshinskii (Caragana korshinskii Kom.) with
Resistance related Dehydrins and its encoding gene.
A kind of encoding gene from Caragana korshinskii of present invention offer, its entitled CkDHN1, full length gene 748bp,
ORFs is 405bp, possesses SEQ ID NO：Full length DNA sequence and SEQ ID NO shown in 2：Complete reading code shown in 3
Frame.
The CkDHN1 genes are following a)-d) in any described gene：
A) its coded sequence is SEQ ID NO in sequence table：2 from the 1-748 positions of 5 ' ends；
B) its nucleotide sequence is the SEQ ID NO in sequence table：3；
C) and the gene of the Dehydrins is encoded with gene recombination a) or b) under strict conditions；
D) gene and a) or b) has more than 90% homology, and encodes the gene of the Dehydrins.
Above-mentioned stringent condition can be miscellaneous in DNA or RNA with 0.1 × SSPE (or 0.1 × SCC), 0.1 × SDS solution
Hand over and hybridize in experiment at 65 DEG C and wash film.
The present invention also provides the plant dehydration fibroin encoded by CkDHN1, is LEA protein family from Caragana korshinskii
In one, be following protein 1) or 2) or 3)：
1) by SEQ ID NO in sequence table：The protein of amino acid sequence composition shown in 1；
2) amino acid sequence of the protein 1) limited by the substitution of one or several amino acid residues and/or is lacked
Lose and/or add, and the protein as derived from 1) related to Dehydrins.
SEQ ID NO in sequence table：1 protein being made up of 134 amino acid residues, molecular weight about 13.82KD, etc.
Electricity point 9.13.
Applications of the plant dehydration element encoding gene CkDHN1 in drought-resistant plant is cultivated falls within the protection of the present invention
Scope.
The gene is cloned in Caragana korshinskii first, and the expression of the gene is by arid, cold induction, in cold-resistant and drought resisting
There is regulating and controlling effect in reaction.
Present invention also offers a kind of application of polynucleotides of separation in Studies of Gene Engineering on Plant Drought-resistance, wherein described more
The sequence of nucleotides such as SEQ ID NO：2 or SEQ ID NO：Shown in 3.Wherein, plant is preferably Caragana korshinskii.
Brief description of the drawings
Fig. 1：The drought-induced expression of CkDHN1
Fig. 2：CkDHN1 nucleotide sequences and amino acid sequence
Embodiment
The experimental procedure of the present invention is described in detail below in conjunction with embodiment：
Embodiment 1. twists the structure in SSH libraries under bar blade drought stress
One month seedling age seedling of Caragana korshinskii is taken to be used to test.Caragana microphylla seedling, distilled water flushing are carefully taken out from culturing pot
Clean institute's band soil, avoids causing to damage as far as possible.Blotting paper blots water purification, is placed in former growing environment 3 hours and carries out arid
Processing, untreated caragana microphylla seedling is as control.The blade of clip control and processing caragana microphylla seedling is placed in 50mL centrifuge tubes respectively, liquid
Nitrogen is quick-frozen, and it is standby as structure library sample to be stored in -80 DEG C of refrigerators.TRIzole methods extract sample total serum IgE, are purified through mRNA
Kit handles to obtain mRNA.Using the caragana microphylla sample of Osmotic treatment as tester, untreated caragana microphylla sample as driver,
Suppression Subtractive Hybridization method (Suppression subtractive hybridization, SSH) is according to PCR-SelectTM
CDNA Subtract ion Ki t (Clontech) specification carries out library construction.By the PCR primer and pMD19T after abatement
Vector (TaKaRa) carrier connects, and is transformed into E. coli competent TOP10 (Tiangeng) cell, screening positive clone.
The random EST sequencings in embodiment 2.SSH libraries
1359 monoclonals are randomly selected from library and carry out 5 ' end sequencings, splicing, est sequence will be obtained and carry out BLAST
And annotate.
Select to annotate the est sequence for Dehydrins encoding gene after BLAST, design RACE primers, utilize RACE technologies
(concrete operation step is with reference to the precious biological TaKaRa RACE kit specifications in Dalian) obtains cDNA sequence total length.
3 ' RACE the primers
DHN1-3’-OU：GGAGGAAGCACAGGAACAGGT
DHN1-3’-IN：TGATGCGTATGGTGCTGGACA
5 ' RACE the primers
DHN1-5’-OU：TGTTGTCCAGCACCATACGC
DHN1-5’-IN：CATAACCTGTTCCTGTGCTTCCT
The cDNA sequence for splicing to obtain according to RACE designs following specific primer and enters performing PCR checking, with Caragana korshinskii
CDNA is template, high-fidelity enzyme PrimeSTAR (TaKaRa companies) amplification gene total length, is cloned into pEASY-Blunt-T
Hua Da gene Co., Ltd is sequenced carrier (Quan Shi King Companies) afterwards, obtains Caragana korshinskii CkDNA1 full length genes cDNA.
F-DHN1：CGTTTTCCTCCTTTGTGC
R-DHN1：AATTATTGAAAAGTTAAAAAAGAGAC
PCR amplification conditions
The drought-induced expression of example 3.CkDHN1 genes
The korshinskii Kom of Osmotic treatment is taken to extract RNA, while the blade extraction RNA for extracting normal growth is control, is inverted
CDNA is recorded into, according to the SEQ ID NO of the present invention：2 design fluorescent quantitative PCR primers, the primer expand 134bp nucleosides
Acid sequence length.
F-DHN1-RT：GAAGAAAGGGATCATGGATAAGATCA
R-DHN1-RT：GAAAAGTTAAAAAAGAGACACCAGTACG
According to EF1 α (KC679842) the sequences Design reference gene primer announced in GeneBank databases.
F-EF1α-RT：TGGGTGGGACATTCTCTGATT
R-EF1α-RT：GCACGGTTCACTTCTTCTTAGC
UseGreen I fluorescent dye determinations, it is glimmering in real time in LightCycler480 (Roche Diagnostics)
The transcriptional expression level for carrying out gene on Fluorescent Quantitative PCR instrument is analyzed, 2- Δ Δ Ct method analyze datas.As a result find that arid lures
The expression for leading down CkDHN1 genes involved in the present invention dramatically increases than control, is reached at drought-induced 12 hours and 24 hours
To 600-800 times (Fig. 1) of control, it was demonstrated that it can play a significant role in drought resisting.
The present invention has cloned a new dehydrin gene from Caragana korshinskii, by GeneBank databases
BLAST, the transcription factor and the amino acid sequence similarity highest of robur Dehydrins, only 40%, the albumen, which contains 1 K, to be protected
Domain (Fig. 2) is kept, is a new dehydrin gene.
It is described above, only presently preferred embodiments of the present invention, not the present invention is made it is any in form and substantial limit
System, all those skilled in the art, without departing from the scope of the present invention, when using disclosed above skill
Art content, and the equivalent variations for a little variation, modification and evolution made, it is the equivalent embodiment of the present invention；Meanwhile it is all according to
The variation, modification and evolution for any equivalent variations made according to the substantial technological of the present invention to above example, still fall within this
In the range of the technical scheme of invention.
Claims (1)
- A kind of 1. application of polynucleotides of separation in Studies of Gene Engineering on Plant Drought-resistance, wherein the sequence of the polynucleotides is such as SEQ ID No：2 or SEQ ID No：Shown in 3, the plant is Caragana korshinskii.
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| CN201410152592.9A CN104086635B (en) | 2014-04-10 | 2014-04-10 | A new anti-drought gene CkDHN1 in Caragana korshinskii |
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Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111041034B (en) * | 2018-10-11 | 2022-09-13 | 内蒙古农业大学 | Middle caragana Senescence _ reg-1 type protein coding gene CiDUF584-1 and application thereof |
| CN110184253B (en) * | 2019-06-06 | 2022-06-07 | 内蒙古农业大学 | Application of CiCPK32 gene of caragana intermedia in regulation and control of plant stress resistance |
| CN110760524B (en) * | 2019-10-10 | 2020-08-07 | 中国农业科学院生物技术研究所 | Specific DNA fragment com58276 and application thereof in regulating and controlling plant stress resistance |
| CN114621960B (en) * | 2022-02-23 | 2024-05-10 | 内蒙古农业大学 | Caragana microphylla drought-tolerant gene Chr8.227 and application thereof in preparation of drought-tolerant transgenic plants |
| CN114645057B (en) * | 2022-02-23 | 2023-06-09 | 兰州大学 | Caragana microphylla drought-tolerant gene Chr8.229 and application thereof in preparation of drought-tolerant transgenic plants |
| CN114645058B (en) * | 2022-02-23 | 2023-05-02 | 兰州大学 | Caragana microphylla drought-tolerant gene Chr8.231 and application thereof in preparation of drought-tolerant transgenic plants |
| CN114645056B (en) * | 2022-02-23 | 2024-05-21 | 内蒙古农业大学 | Caragana microphylla drought-tolerant gene Chr8.225 and application thereof in preparation of drought-tolerant transgenic plants |
| CN114645059B (en) * | 2022-02-23 | 2024-05-21 | 内蒙古农业大学 | Caragana microphylla drought-tolerant gene Chr8.226 and application thereof in preparation of drought-tolerant transgenic plants |
| CN114645060B (en) * | 2022-02-23 | 2023-05-23 | 兰州大学 | Caragana microphylla drought-tolerant gene Chr8.228 and application thereof in preparation of drought-tolerant transgenic plants |
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