CN106906224A - A kind of corn anti contravariance related gene ZmDi19 5 and its application - Google Patents
A kind of corn anti contravariance related gene ZmDi19 5 and its application Download PDFInfo
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Abstract
The invention discloses a kind of corn anti contravariance related gene ZmDi19 5 and its application, the gene has the nucleotide sequence as shown in SEQ ID NO.1, and the encoding proteins of the gene have the amino acid sequence as shown in SEQ ID NO.2.The gene is a kind of new plant stress-resistance related gene, it is possible to increase plant stress-resistance performance, the discovery of the gene enriches the resource of anti contravariance related gene, and the seed selection to the degeneration-resistant new varieties of corn is significant.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of corn anti contravariance related gene
ZmDi19-5 and its application.
Background technology
Corn serves not only as one of three generalized grain things, also can be used as important energy and material.The breast produced from corn
Acid, after chemical reaction can generate macromolecular material (PLA), can substitute chemical plastic, be the environmental protection that can be biodegradable
Material, has important strategic importance in social and economic development.At present, China's Maize Production total amount is produced more than wheat
Amount, positioned at the second largest cereal crops of China, its importance is self-evident.China major part corn planting area be subject to arid and
The stress of salt marsh, the annual therefore underproduction, directly affects the agricultural production of drought-hit area and salt lick.Therefore, drought-resistant maize is strengthened
One of China's agricultural research major issue is turned into the ability of salt tolerant.
The new varieties method of current seed selection drought resisting and salt tolerant has a lot, and compared to traditional breeding method, transgenic technology is to realize
The method of breeding objective instant effect.The key problem in technology is to excavate the gene related to arid and salt stress, and analyzes biology
Function and degeneration-resistant mechanism.In model plant arabidopsis, being analyzed its gene function by deletion mutant body has turned into
Main flow, for the corn that compares, the method for obtaining single-gene deletion mutant is relatively difficult so that gene functional research is subject to
Certain limitation, therefore transgenic line method is obtained by heterologous transformant, weight has been played in the functional study of adversity gene
The effect wanted.
The degeneration-resistant acknowledgement mechanism of plant be unable to do without the regulation and control of transcription factor.Have been reported that display, the body of the anti-adversity of plant
It is not now the result of the independent regulation and control of some transcription factor, is by answering that multiple transcription factor coordinate expression joint effects are determined
Miscellaneous biological character.The response of the function controlling and plant stress-resistance mechanism of transcription factor, is the development of crop molecule breeding for stress tolerance
There is provided providing powerful support for, for the raising and improvement of crop synthesis anti-adversity provide theoretical foundation.
Research shows that the transcription factor of C2H2 zinc finger proteins family is primarily involved in growing and plant for plant
Resistance correlated activation.Di19 albumen is not only a member of C2H2 zinc finger proteins family, while being also drought induced-protein
Member.Di19 genes are cloned at present in the plants such as arabidopsis, cotton, soybean, wheat, paddy rice, but are had in corn
The transcription factor report for closing Di19 genes does not have also.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of corn anti contravariance related gene ZmDi19-5
And its application, to provide a kind of new gene that can be used for the degeneration-resistant genetic improvement of corn.
The present invention is achieved by the following technical solutions：
The invention provides a kind of corn anti contravariance related gene ZmDi19-5, its nucleotide sequence such as SEQ ID NO.1 institutes
Show, total length 720bp.
Present invention also offers above-mentioned corn anti contravariance related gene ZmDi19-5 in reducing or improving stress resistance of plant
Using.
Present invention also offers the encoding proteins of above-mentioned corn anti contravariance related gene ZmDi19-5, its amino acid sequence is such as
Shown in SEQ ID NO.2, with 239 amino acid.
Present invention also offers a kind of plant Overexpression vector, the plant Overexpression vector is pCAMBIA1301
Carrier, the MCS region of the pCAMBIA1301 carriers is connected with 35S promoter, ZmDi19-5 genes and end in turn
It is only sub.
Present invention also offers a kind of genetically engineered host cell, the host cell contains the plant and crosses scale
The sequence forward or backwards of the ZmDi19-5 genes of external source is integrated with up to carrier, or in its genome.
Present invention also offers a kind of method for improving stress resistance of plant, methods described is to lead the ZmDi19-5 genes
Enter in cell, tissue or the organ of plant, obtain the transfer-gen plant new varieties of strong stress resistance.
Further, the plant includes corn, paddy rice, wheat, arabidopsis.
The present invention has advantages below compared to existing technology：The invention provides a kind of corn anti contravariance related gene
ZmDi19-5 and its application, the gene are a kind of new plant stress-resistance related gene, enrich the resource of anti contravariance related gene, right
The seed selection of the degeneration-resistant new varieties of corn is significant.
Brief description of the drawings
Fig. 1 is ZmDi19-5 gene detected through gel electrophoresis result figures；
Fig. 2 is the Phylogenetic analysis result figure of ZmDi19-5 albumen and the Di19 protein family members of other crops；
Fig. 3 is ZmDi19-5 gene overexpression vector construction collection of illustrative plates；
Fig. 4 is transgenic arabidopsis and wildtype Arabidopsis thaliana root growth situation column analysis chart under salt stress；
Fig. 5 is transgenic arabidopsis and wildtype Arabidopsis thaliana plant height situation column analysis chart under salt stress；
Fig. 6 is transgenic arabidopsis and wildtype Arabidopsis thaliana mda content measurement result block diagram under salt stress；
Fig. 7 is the measurement result block diagram of transgenic arabidopsis and wildtype Arabidopsis thaliana relative conductivity under salt stress；
Fig. 8 is transgenic arabidopsis and wildtype Arabidopsis thaliana Catalase determination result block diagram under Osmotic treatment；
Fig. 9 is transgenic arabidopsis and wildtype Arabidopsis thaliana mda content measurement result block diagram under Osmotic treatment；
Figure 10 is transgenic arabidopsis and wildtype Arabidopsis thaliana leaf r elative water content measurement result column under Osmotic treatment
Figure.
Specific embodiment
Embodiment 1
1st, material
The present embodiment method therefor is known to those skilled in the art the conventional method of dawn unless otherwise instructed, used
The material such as reagent, unless otherwise instructed, be commercially available purchase product.
2nd, method
The clone of 2.1ZmDi19-5 genes
Selection corn B73 self-mating systems are experiment material, extract maize leaf tissue RNA, and reverse transcription is standby into cDNA.
The biological information of ZmDi19-5 is obtained from maize database retrieval, with ZmDi19-5 gene orders as stencil design
Specificity amplification primer, in combination with the polyclone enzyme enzyme site on cloning vector, selection Kpn I and BamHI restriction enzyme sites draw
Enter ZmDi19-5 sequences two ends, specificity amplification primer sequence is as follows：
SEQ ID NO.3：ZmDi19-5-F：5’-GGGGTACCATGGATTCCGACCTCTGGAT-3’
SEQ ID NO.4：ZmDi19-5-R：5’-CGGGATCCCTAGTCTTCAAAAATGGTGGTG-3’
With cDNA as template, ZmDi19-5-F and ZmDi19-5-R is primer, the high-fidelity produced using TAKARA companies
Enzyme PrimerSTAR Max Premix (2 ×) carries out pcr amplification reaction, and pcr amplification reaction system is as follows：
PCR amplification programs are：98 DEG C of predegenerations 10min, 98 DEG C of denaturation 10s, 62 DEG C of annealing 5s, 72 DEG C of extension 1min, altogether
After 30 circulate, the Taq enzyme of 0.5 μ L is added, PolyA tails are added with to 3 ' ends, finally, at 72 DEG C continue extension 10min complete
Into reaction, the PCR primer mass ratio of acquisition is that 1% agarose gel electrophoresis detection is taken pictures.
Result is as shown in figure 1, as can be seen that target gene band is clear in figure, and size and consistent, the explanation that predicts the outcome
PCR results are good.
Further, target gene is carried out into gel extraction with the glue reclaim kit of Axygen companies, with T1Simple
It is transformed into Escherichia coli Trans5 α competent cells after carrier connection, double digestion checking is carried out with Kpn I and BamHI, is screened
Positive clone molecule, obtains T1+ZmDi19-5 carriers, and send to the sequencing of Shanghai Sheng Gong companies.
The bioinformatic analysis of 2.2ZmDi19-5 genes
After sequencing result is through the sequence alignment in Mega4.0 softwares and database, with the nucleotides shown in SEQ ID NO.1
Sequence is consistent, illustrates that successful clone obtains ZmDi19-5 genes.By in sequence of threads PFAM to the coding egg of ZmDi19-5 genes
Domains characteristic analysis is carried out in vain, as a result shows that its protein structure domain contains zf-Di19 and (is located at the 49th to 102 amino acid
Position) and Di19_C (being located at the 123rd to 236 amino acid positions), illustrate that it is a kind of Di19 albumen.It is soft using DNAMAN
Part, as a result its amino acid sequence and the amino acid alignment of known rice Os Di19-4 shows, ZmDi19-5 genes are not
Only contain two Cys2/His2 type zinc finger protein domains, the also homology with the amino acid sequence of OsDi19-4 reaches 74%
Left and right, and it is transcription factor that OsDi19-4 genes are reported.Thus prediction ZmDi19-5 belongs to turning for drought induced-protein family
The record class of the factor one.
Using Mega4.0 softwares, the arabidopsis of function and the Di19 albumen of paddy rice will be reported, entered with corn Di19 albumen
Row Phylogenetic analysis, as a result as shown in Fig. 2 in figure, the affiliation of ZmDi19-5 and OsDi19-4 is nearest, and
OsDi19-4 genes have been proved to participate in the drought resisting mechanism of regulation and control plant, and this is found to be follow-up ZmDi19-5 gene functions
Checking lays the foundation.
The structure of 2.3ZmDi19-5 gene overexpression carriers
With pCAMBIA1301 as initial carrier, one is connected between EcoRI the and SalI restriction enzyme sites of its MCS
Individual 35S promoter, and add the preceding paragraph terminator between SphI and HindIII restriction enzyme sites, obtain the carrier of transformation
pCAMBIA1301a。
Small purpose fragment is obtained with Kpn I, BamHI double digestion T1+ZmDi19-5 carriers, while with Kpn I, BamHI
Double digestion p1301a obtains large fragment.
Coupled reaction system is：
After coupled reaction carries out 3h at 16 DEG C, in conversion to competent escherichia coli cell, choose spot, shake bacterium, upgrading grain,
And verified by double digestion, obtain ZmDi19-5 gene overexpression carriers p1301a-ZmDi19-5 as shown in Figure 3.
The arabidopsis genetic transformation of 2.4ZmDi19-5 genes
2.4.1 the cleaning of arabidopsis seed：
12ml liquor natrii hypochloritises are first taken in 88ml water, the liquor natrii hypochloritis of configuration 12% is configured to.Take to be cleaned
Seed in sterile centrifugation tube, often pipe add 1ml 12% liquor natrii hypochloritis, soaking and washing 15min, period interval shake
Shake；Rinsed with sterile water is used 5 times again, period rocks mixing, to clean the surface of the seed liquor natrii hypochloritis and impurity；By cleaning and sterilization
Colombia's wildtype Arabidopsis thaliana seed afterwards is processed 2 days in 4 DEG C of refrigerators, standby.
2.4.2 the culture of arabidopsis
Seed after cleaning is taken out, is uniformly spread on the square culture dish for being dispersed in MS solid mediums by liquid-transfering gun, intend south
Mustard culturing room cultivates；Growth, nutrition black earth and leech in the seedling mixed Nutrition Soil of immigration of 10d or so will be grown on culture medium
Stone volume ratio is 1:3, continue hot-house culture；It should be noted that the seedling just transplanted need to keep moisture, using light-passing plastic
Film cladding process, opens after 2-3d.
2.4.3 agrobacterium mediation converted arabidopsis
The over-express vector p1301a-ZmDi19-5 that will be built is imported in Agrobacterium EHA105 competent cells, is invaded
Microbiological contamination.When the most of preparation of the arabidopsis transplanted is bloomed for the first time, as arabidopsis infects best period.In order to promote to intend south
Mustard has the hyperplasia of spray, can prune the petal of Blooming；The silique having had simultaneously cuts off, and improves the sun of transgenic seed
Property rate.Method for transformation is as follows：Bacterium will be infected and be enlarged culture to OD600Be worth is 0.8.Conversion Buffer solution (place is configured simultaneously
Reason liquid)：The Buffer solution of 1L needs the MS of 2.3g, 50g sucrose, the MES of 0.5g to adjust pH value to 5.8 with NaOH.Treatment fluid
Also need to add surfactant using preceding.The bacterium block that the centrifugation of the mixing Buffer solution suspensions after stirring is obtained is taken, that is, is invaded
Dye solution.
Contaminate arabidopsis floral：When about 10 centimetres of arabidopsis bolting is high, with the wet arabidopsis floral of During Agrobacterium immersion, weight
It is multiple 2-3 times, above-ground plant parts is wrapped with preservative film, cultivated 2-3 days under dark.After one week, repeat to contaminate again.
About two months, the seed (T0 generations) of ripe arabidopsis is harvested, preserved at being placed in 4 DEG C after drying.
2.4.5 the screening of transgenic arabidopsis
By the arabidopsis seed (T0 generations) of above-mentioned results it is sterilized (with 12% colored king's bleaching water rinse 9 minutes, Ran Houyong
The distilled water flushing of sterilizing 5 times, each scavenging period about 1 minute), 4 DEG C of vernalization two days (attention lucifuge), seed is uniformly sprinkled upon and contains
On the MS culture medium flat plates of hygromycin, while using wildtype Arabidopsis thaliana seed as control.If wildtype Arabidopsis thaliana seed exists
On MS flat boards without resistance can normal germination and growth, on the flat board containing resistance can not normal germination and growth, and turn base
Because but there is the seed being capable of normal germination and growth on the flat board containing resistance in arabidopsis, then it is assumed that the positive Arabidopsis plant of screening is
Effectively.
It is purpose gene with ZmDi19-5 genes, the positive plant to screening enters performing PCR Molecular Detection, and Preliminary Identification turns base
Because whether arabidopsis converts successfully.
2.4.6 the acquisition of transgenic arabidopsis offspring
According to the method for step 2.4.5, the seed (T1 generations) of transgenic Arabidopsis plants will be initially identified as with containing damp mould
The MS culture medium flat plates of element are screened, and obtain T1 for plant, and when T1 is ripe for plant Fruit pod, results transgenosis T2 generations plant
Son, by that analogy, is finally obtained the transgenic arabidopsis seed (at least T3 generations) of stabilization heredity.
The Salt Tolerance Analysis of 2.5ZmDi19-5 transgenic arabidopsis
2.5.1ZmDi19-5 Salt Tolerance Analysis of transgenic arabidopsis Seedling Stage
After by transgenic arabidopsis L1, L2, L5 strain seed of stabilization heredity and wild type WT strain seed disinfections, uniformly
It is layered on the flat board containing MS culture mediums, 4 days or so (about 2-3 centimetres of root growth) moves it to the MS containing 0,130mM NaCl and put down
On plate, these flat boards are put in 22 DEG C of artificial greenhouses vertically, observe root growth situation.
Result is as shown in figure 4, as can be seen that under condition of salt stress, the root system of transgenic Arabidopsis plants is than open country in figure
The root system of raw type is long, illustrates that the overexpression of ZmDi19-5 genes enhances abiotic stress of the genetically modified plants to high salt.
2.5.2ZmDi19-5 Salt Tolerance Analysis of the transgenic arabidopsis into seedling stage
After by transgenic arabidopsis L1, L2, L5 strain seed of stabilization heredity and wild type WT strain seed disinfections, uniformly
It is layered on the flat board containing MS culture mediums, is placed on vernalization 2 days (being easy to synchronous germination) in 4 DEG C of refrigerators.Then, it is 22 to be placed in condition
DEG C, cultivated in the special culture greenhouse of arabidopsis of 16/8h light dark cultures.Sprout 10 days or so, choose transgenosis of the same size
It is 1 that strain and wild type seedlings move into Nutrition Soil and vermiculite volume ratio:Grown in the Nutrition Soil of 3 mixing.Use the NaCl of 220mM
The transgenic arabidopsis and wildtype Arabidopsis thaliana of solution 3 week old for the treatment of, continuous processing 4 weeks, used as test group, control group uses water generation
For the NaCl solution (normal growth) of 220mM.
2.5.2.1 phenotypic analysis
The phenotype of arabidopsis, as a result finds after observation before processing, before NaCl solution treatment, ZmDi19-5 transfer-gen plants
Growth conditions with wildtype Arabidopsis thaliana are consistent, but after being processed 3 weeks with 220mM NaCl, most of leaf of wildtype Arabidopsis thaliana
Piece then turns white withered, shows the sensitiveness to high-salt stress, and most of blade of ZmDi19-5 transgenic Arabidopsis plants
Green is remained in that, showing the expression of ZmDi19-5 genes can strengthen stress of the plant to high salt.
Further, the plant height under statistics normal growing conditions with wild type and transgenic arabidopsis under condition of salt stress,
Result as shown in figure 5, in figure as can be seen that the plant height and size of wild type and the transgenic arabidopsis of normal growth almost,
But by after salt stress treatment, wild type is substantially less than transfer-gen plant, further demonstrates that the ZmDi19-5 genes of overexpression
Enhance stress of the Arabidopsis plant to high salt.
2.5.2.2 the measure of MDA (MDA) content：
(1) tissue of experimental group and control group equivalent weight is weighed, sodium phosphate buffer is added and by means of quartz sand, is ground
Wear into tissue homogenate；
(2) take and be homogenized in centrifuge tube, 15min is centrifuged, whether observation homogenate is layered；
(3) the homogenate supernatant of layering is taken in new test tube, first adds 0.5% thiobarbituricacidα- of 3ml to mix, then
5% solution of trichloroacetic acid is added, mixing is rocked, water-bath (100 DEG C) 10min, and test tube is rinsed with fast cooling with clear water, so
After be centrifuged, be control, measurement centrifugation supernatant light absorption value A under 532nm, 600nm wavelength with deionized water532、A600。
(4) treatment of data is according to following operational formula：
In formula, A is absorbance, V1It is reaction solution total amount, V is extract solution total amount, V2It is the extraction liquid measure in reaction solution, W is
The weight of plant sample, 1.55 × 10-1It is micromole's absorptivity of MDA.
Result is as shown in fig. 6, can be seen that the mda content of transgenic line and WT lines after treatment all in figure
Having is increased, but in ascensional range, and WT lines illustrate what is accumulated in transfer-gen plant apparently higher than transfer-gen plant
Mda content is less, cell injury lesser extent.
2.5.2.3 the measure of relative conductivity (REL)
(1) experimental group after before processing and each strains of wild type WT are taken with position blade, ddH is used2O is rinsed twice, punching
10-15 disk of blade；
(2) each group blade is respectively put into test tube, while test tube is covered and fixed from gauze and breathable sealing adhesive tape,
Extract examination air in tube, 30min；
(3) test tube is put in 1h in shaking table, calibrates the conductivity gauge measurement for completing just electric conductivity value K1；
(4) 16min will be boiled in water-bath (100 DEG C) comprising sample tube again,.With originally cold rinse test tube after boiling water bath
To room temperature, after standing 10min at room temperature, its whole electric conductivity value K2 is surveyed after shaking up；
(5) with ddH2Used as control, conductance apparatus measuring value is blank electric conductivity value K0 to O；
(6) calculating of relative conductivity can be calculated according to formula below：
Result as shown in fig. 7, as can be seen that before processing each group plant relative conductivity is no significant difference in figure,
Wild type is significantly raised with the relative conductivity of transgenic arabidopsis after treatment, but the elevation amplitude of transgenic arabidopsis is notable
Less than wild type, illustrate transgenic arabidopsis than wildtype Arabidopsis thaliana leaf cell between electrolyte content it is few, film damage situations
It is lighter.
The aridity analysis of 2.6ZmDi19-5 transgenic arabidopsis
After by transgenic arabidopsis L1, L2, L5 strain seed of stabilization heredity and wild type WT strain seed disinfections, uniformly
It is layered on the flat board containing MS culture mediums, is placed on vernalization 2 days (being easy to synchronous germination) in 4 DEG C of refrigerators.Then, it is 22 to be placed in condition
DEG C, cultivated in the special culture greenhouse of arabidopsis of 16/8h light dark cultures.Sprout 10 days or so, choose transgenosis of the same size
It is 1 that strain and wild type seedlings move into Nutrition Soil and vermiculite volume ratio:Grown in the Nutrition Soil of 3 mixing.When seedling is vibrant,
Carry out water shortage Osmotic treatment 15 days, then rehydration 5 days.
2.6.1 phenotypic analysis
Observation phenotype, as a result finds, Osmotic treatment is after 15 days, wild-type Arabidopsis plants yellow leaf and seriously rolls up
Song, and transgenic arabidopsis blade is most of or green, and transgenic arabidopsis blade is bigger than wild-type leaves expansion degree；
After rehydration 5 days, wild type is all dead, and transfer-gen plant full recovery normal growth.Show the ZmDi19-5 of overexpression
Genes amplification adaptability and tolerance of the transgenic Arabidopsis plants to drought stress.
2.6.2 catalase (CAT) is determined
CAT is determined and is built up catalase (CAT) measure kit (visible ray that biological study is bought using Nanjing
Method) and total protein (TP) determine kit (Coomassie Brilliant Blue) be measured, specific method is carried out with reference to kit specification,
Specially：
Take respectively after drought stress before processing and the same leaf position of each experimental group strains of wild type WT lotus throne leaf, use ddH2O
Rinse twice, remove the soil and impurity on blade face.
(1) detection of tissue samples
A. Liquid nitrogen precooler mortar and grinding rod, add sample to be ground to powdered, weigh 1g powder, and add 9 times of volumes
Physiological saline, thus obtained tissue homogenate, and being centrifuged 10 minutes in centrifuge 2500rpm takes supernatant.
B. 5ml centrifuge tubes are taken and adds corresponding reagent by following volume, wherein, determine pipe addition tissue magma, control tube
Replaced with water：
Mix, 37 DEG C of warm bath 1min in water-bath, then according to the form below adds reagent three, the reagent four of respective volume：
C. the sample mixing of each reagent is shaken up, wavelength 405nm determines each pipe absorbance, distilled water zeroing.
(2) protein quantification test
A. a. step supernatants in (1) are taken, supernatant physiological saline is taken by 1：9 dilution proportions are homogenized into 1% concentration tissue, treat
Survey；
B. test serum is homogenized in addition 5ml centrifuge tubes, and according to the form below volume adds the reagent of respective volume.Wherein,
Coomassie brilliant blue nitrite ion needs now with the current, collocation method：Coomassie brilliant blue stock solution and distilled water are pressed 1：4 ratio is mixed
It is even.Note：0.563/L protein standard substances (ml) need to put -20 DEG C of preservations, can preserve 6 months.
Each agent formulations are mixed, room temperature places 10min, distilled water zeroing before measurement measures each cuvette sample long in harmonic
The light absorption value of 595nm.
C. experimental group sample protein concentration computing formula is such as：
In the present embodiment, standard concentration is 0.563g/L.
(3) in tissue CAT vigor calculating：
In order to accurately analyze each group of data, we complete treatment and the statistical of our department's divided data from SPSS10.0 softwares
Analysis.Significance of difference t inspections are based on P<0.01 (* *) and P<Carried out in 0.05 (*) two level.
Under adverse environmental factor, plant can produce excessive active oxygen such as hydrogen peroxide (H2O2), influence the growth hair of plant
Educate, but the catalase in plant body can eliminate hydrogen peroxide excessive in vivo, protect cells from the poison of hydrogen peroxide
Evil.The present invention hydrogen oxide enzyme activity in transgenic line and WT lines blade is measured, as a result as shown in figure 8,
It is as can be seen that after Osmotic treatment, the activity of catalase of transfer-gen plant and WT lines has declined in figure but wild
Activity of catalase pole in raw type is substantially less than the vigor in transfer-gen plant, illustrates that the cell viability of WT lines is bright
The aobvious vigor without transfer-gen plant is big.
2.6.3 mda content detection
Detection method with step 2.5.2.2, as a result as shown in figure 9, as can be seen that transfer-gen plant and wild type are planted in figure
The mda content of strain has risen, but the mda content pole of transfer-gen plant is substantially less than WT lines, illustrates open country
The cell injury degree of raw type plant is larger；
2.6.4 leaf r elative water content (RWC) is determined
(1) the forward and backward transfer-gen plant of Osmotic treatment and adjoining tree same area blade are taken respectively.
(2) ddH is used2O is rinsed twice, removes the soil and impurity on blade face, then exhausts surface moisture with filter paper, claims fresh weight
W0；
(3) the rear blade immersion 5-6h in deionized water that weighs is taken, until when water suction saturated weight is constant, claiming fresh weight W1；
(4) blade weighed in (3) is wrapped with masking foil, is put into 180 DEG C of baking oven, dry moisture, claim dry weight W2；
(5) computing formula of relative water content：
As indicated by 10, the relative water content of transfer-gen plant and WT lines has declined result figure, but transgenosis
The relative water content pole of plant is significantly higher than WT lines, illustrates that the cell of WT lines is most of dead.
2.7 conclusions
WT lines are subject to salt stress and drought stress degree to be all higher than transfer-gen plant, illustrate ZmDi19-5 gene energy
Significantly improve the resistance of genetically modified plants.
It is above a kind of detailed implementation method of the invention and specific operating process, is with technical solution of the present invention as preceding
Put and implemented, but protection scope of the present invention is not limited to the above embodiments.
SEQUENCE LISTING
<110>Agricultural University Of Anhui
<120>A kind of corn anti contravariance related gene ZmDi19-5 and its application
<130> /
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 720
<212> DNA
<213> Zea mays
<400> 1
atggattccg acctctggat ctcgcgtctc acggccgcca agcggcagtt tgcgctgcag 60
cgcgcgcagc gccagcacgc cgccccggca tcccatcacg atcgatttgg gtatgatgat 120
atcgaacctg aggatgattt gcactcagac ttcccatgcc cttactgtta tgaagatcat 180
gatgttgctt ctctctgcgc ccatttggag gacgagcacc ctttcgagtc taaaattgtg 240
tcttgcccag tttgctcagc taggatttcg aaggacttgg tggatcatat aacccttcag 300
catggctacc tatttaaatt gcagaaacat caaagagtac gcagagttac tggtaatggc 360
aaccacaatc tctcctatgc agggagagat cttcaactgc aagagaccta cttaaaggtg 420
cttcttggaa atagcagtcg aagcagcagt accaatgcat caagtacagt caccgattca 480
ctgctaccct cactagtttt aaatttatca tcaccagaag tagaagacgc atcaaagttt 540
tcagctcctg ctgtggtgga aaataattgg tttaaacgat cactgccttc aaaaacttgg 600
aaattaagaa ctgttgattc cacccttagt catgaagaga gggagcgtag gaggaggaga 660
gctgctgtga gatcagcttt tgtccagcat ctccttgtca ccaccatttt tgaagactag 720
<210> 2
<211> 239
<212> PRT
<213> Zea mays
<400> 2
Met Asp Ser Asp Leu Trp Ile Ser Arg Leu Thr Ala Ala Lys Arg Gln
1 5 10 15
Phe Ala Leu Gln Arg Ala Gln Arg Gln His Ala Ala Pro Ala Ser His
20 25 30
His Asp Arg Phe Gly Tyr Asp Asp Ile Glu Pro Glu Asp Asp Leu His
35 40 45
Ser Asp Phe Pro Cys Pro Tyr Cys Tyr Glu Asp His Asp Val Ala Ser
50 55 60
Leu Cys Ala His Leu Glu Asp Glu His Pro Phe Glu Ser Lys Ile Val
65 70 75 80
Ser Cys Pro Val Cys Ser Ala Arg Ile Ser Lys Asp Leu Val Asp His
85 90 95
Ile Thr Leu Gln His Gly Tyr Leu Phe Lys Leu Gln Lys His Gln Arg
100 105 110
Val Arg Arg Val Thr Gly Asn Gly Asn His Asn Leu Ser Tyr Ala Gly
115 120 125
Arg Asp Leu Gln Leu Gln Glu Thr Tyr Leu Lys Val Leu Leu Gly Asn
130 135 140
Ser Ser Arg Ser Ser Ser Thr Asn Ala Ser Ser Thr Val Thr Asp Ser
145 150 155 160
Leu Leu Pro Ser Leu Val Leu Asn Leu Ser Ser Pro Glu Val Glu Asp
165 170 175
Ala Ser Lys Phe Ser Ala Pro Ala Val Val Glu Asn Asn Trp Phe Lys
180 185 190
Arg Ser Leu Pro Ser Lys Thr Trp Lys Leu Arg Thr Val Asp Ser Thr
195 200 205
Leu Ser His Glu Glu Arg Glu Arg Arg Arg Arg Arg Ala Ala Val Arg
210 215 220
Ser Ala Phe Val Gln His Leu Leu Val Thr Thr Ile Phe Glu Asp
225 230 235
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<400> 3
ggggtaccat ggattccgac ctctggat 28
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
cgggatccct agtcttcaaa aatggtggtg 30
Claims (7)
1. a kind of corn anti contravariance related gene ZmDi19-5, it is characterised in that the nucleotide sequence of the gene such as SEQ ID
Shown in NO.1.
2. a kind of corn anti contravariance related gene ZmDi19-5 as claimed in claim 1 is in reducing or improving stress resistance of plant
Using.
3. a kind of encoding proteins of corn anti contravariance related gene ZmDi19-5 as claimed in claim 1, it is characterised in that described
The amino acid sequence of albumen is as shown in SEQ ID NO.2.
4. a kind of plant Overexpression vector, it is characterised in that the plant Overexpression vector is pCAMBIA1301 carriers,
The MCS region of the pCAMBIA1301 carriers is connected with 35S promoter, as claimed in claim 1 in turn
ZmDi19-5 genes and terminator.
5. a kind of genetically engineered host cell, it is characterised in that the host cell contains plant as claimed in claim 4
Be integrated with thing Overexpression vector, or its genome external source ZmDi19-5 genes as claimed in claim 1 forward direction or
Reverse sequence.
6. it is a kind of improve stress resistance of plant method, it is characterised in that methods described is to arrive the ZmDi19-5 gene integrations
In the cell of plant, tissue or organ, the transfer-gen plant new varieties of strong stress resistance are obtained.
7. it is according to claim 6 it is a kind of improve plant drought resistance method, it is characterised in that the plant include jade
Rice, paddy rice, wheat, arabidopsis.
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| CN201710307119.7A CN106906224A (en) | 2017-05-04 | 2017-05-04 | A kind of corn anti contravariance related gene ZmDi19 5 and its application |
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| CN106906224A true CN106906224A (en) | 2017-06-30 |
Family
ID=59210685
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| CN201710307119.7A Pending CN106906224A (en) | 2017-05-04 | 2017-05-04 | A kind of corn anti contravariance related gene ZmDi19 5 and its application |
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| CN110157713A (en) * | 2019-04-08 | 2019-08-23 | 安徽农业大学 | Drought-resistant maize related gene ZmDi19-7 and its application |
| CN113088526A (en) * | 2021-05-27 | 2021-07-09 | 安徽农业大学 | Heat shock related gene ZmHsf11 and application thereof in regulation and control of plant heat resistance |
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| CN114262369A (en) * | 2021-12-15 | 2022-04-01 | 中国农业大学 | ZmDi19 gene and application of ZmDi 10 target gene in cultivation of gray leaf spot resistant plants |
| CN114262369B (en) * | 2021-12-15 | 2023-05-16 | 中国农业大学 | Application of ZmDi19 gene and target gene ZmPR10 thereof in cultivation of anti-gray-spot plant |
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