CN108840915A - Moso bamboo PeDi19-4 albumen and its encoding gene and application - Google Patents
Moso bamboo PeDi19-4 albumen and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of moso bamboo PeDi19-4 albumen and its encoding gene and applications.As shown in SEQIDNo.1, the nucleotide sequence of the moso bamboo PeDi19-4 albumen is as shown in SEQID No.2 for the amino acid sequence of the albumen.Carrier containing the coding gene sequence.PeDi19-4 gene provided by the invention it is different degrees of it is drought-induced under, have high expression.PeDi19-4 expression vector is transferred in wildtype Arabidopsis thaliana and arabidopsis di19 mutant by the inflorescence dip method of mediated by agriculture bacillus, the drought resistance of transgenosis wildtype Arabidopsis thaliana is significantly improved as the result is shown, and the drought-resistant ability of transgenic arabidopsis di19 mutant plants is also covered, and illustrates that PeDi19-4 gene can be improved the drought resistance of transgenic arabidopsis.
Description
Technical field
The present invention relates to field of plant molecular biology, and in particular to a kind of moso bamboo PeDi19-4 albumen and its encoding gene
With application.
Background technique
The bamboo plant in China is various in style, resourceful, cultivated area is relatively wide, yield is higher, therefore China is referred to as
" bamboo kingdom ".And moso bamboo (Phyllostachysedulis) is as the model plant in bamboo plant, the speed of growth compared with
Fastly, cultivated area is wider, there is important economy, ecology and cultureal value.Moso bamboo is suitble to produce various bamboo boards, bamboo shoots taste
It is delicious, full of nutrition containing 8 kinds of amino acid and a variety of the essential trace elements of the human bodys, it is former to belong to preferable non-wood papermaking fiber
One of material also can be processed into dried bamboo shoots, can, beverage, bamboo craftwork product and bamboo top grade ornament materials, cosmetics, health care product etc..
Di19 (Drought-induced 19) gene family, is a kind of drought induced-protein, belongs to Cys2/His2 zinc finger
One kind in protein transcription factor, is prevalent in higher plant, is primarily involved in the drought stress reaction of plant.In arabidopsis
Middle AtDi19 gene, under drought-induced, expression quantity is significantly increased.It is when carrying out Osmotic treatment for plant to T3 and wild
Type plant (WT) ratio, the overexpression plant of Di19 gene has shown higher drought-resistant ability, and mutant plants are then to arid
It is more sensitive.AtDi19 gene improve plant drought resistance, mainly by with upstream CPK11 interactions between protein, formed one it is compound
Body；AtDi19 gene can also cause the expression of downstream adverse circumstance related gene simultaneously.
The growing environment of moso bamboo is more demanding to moisture, weather and soil, is suitble to be grown in moisture abundance, warmer climate
And in subacidity or neutral loam environment.However in recent years, it with the continuous raising and the continuous deterioration of weather of Global Temperature, causes
Making plant during the growth process will receive the injury of the various unfavorable conditions from environment, and in the processing of numerous environment stresses,
Drought stress has become primary restriction factor.In the growth and development process of moso bamboo, it will receive in 1 year apparent twice dry
Drought influences, first is that the Shooting stage in spring, if the arid time is long, it will cause the unearthed slow of bamboo shoots, and yield is reduced, even if being unearthed
Also it is easy to thirst, leads to increasing for inferior bamboo；Second is that the differentiation of shoot bud period in autumn can make point of bamboo shoot bud if arid occurs
Change and reduce, and then the yield of winter current year bamboo shoots and the newborn bamboo quantity of second year can be greatly reduced.Especially young setation bamboo, it is right
The requirement of moisture is higher.Therefore, the drought-enduring moso bamboo new germ plasm of a batch is cultivated, to the yield for improving moso bamboo, promotes Foresty industry steady
Fixed development increases national income and has great importance.
The Di19 gene and its homologous gene announced at present, mostly from model plant or herbaceous plant, these plants
Object is mostly annual plant, and the function in moso bamboo about Di19 gene is not yet reported.
Summary of the invention
The purpose of the present invention is to provide a kind of good moso bamboo PeDi19-4 albumen of drought resistance and its encoding gene and applications.
In order to achieve the above objectives, present invention employs following technical proposals：A kind of moso bamboo PeDi19-4 albumen of the invention,
The amino acid sequence of the moso bamboo PeDi19-4 albumen is as shown in SEQ ID No.1.
The gene order of moso bamboo PeDi19-4 albumen described in a kind of coding of the invention, the moso bamboo PeDi19-4 egg
White nucleotide sequence is as shown in SEQID No.2.
Carrier containing coding gene sequence as claimed in claim 2 of the invention.
The construction method of carrier of the present invention, includes the following steps：It include the moso bamboo of restriction enzyme site by both ends
On PeDi19-4 protein coding gene sequence construct to pEASY T1 simple Cloning Vector carrier, it is transferred to large intestine bar
In bacterium competence cell TransT1, obtain T1-PeDi19-4 recombinant plasmid, double digestion T1-PeDi19-4 recombinant plasmid and
PCAMBIA1301a plasmid after connection, is transferred to competent escherichia coli cell, after extracting plasmid, obtains expression vector
pCAMBIA1301a-PeDi19-4。
The present invention contains the engineering bacteria of carrier as claimed in claim 3.
The present invention is used to clone the primer pair of coding gene sequence as claimed in claim 2, and the primer pair includes upper
Swim primer and downstream primer, the nucleotide sequence of the upstream primer as shown in SEQ ID No.4, the downstream primer
Nucleotide sequence is as shown in SEQ ID No.5.
PeDi19-4—F:5'—GGGGTACC ATGGAGGTGGGAAGCCTT—3'；
PeDi19-4—R:5’—GCTCTAGACTATTGTGCCTCTTTGCGG—3’
A kind of moso bamboo PeDi19-4 gene of the invention, it is characterised in that：The cDNA of the moso bamboo PeDi19-4 gene
The nucleotide sequence of overall length is as shown in SEQ ID No.3.
Application of the moso bamboo PeDi19-4 gene of the present invention in plant modification drought resisting.
Further, by the inflorescence dip method of mediated by agriculture bacillus by gene as claimed in claim 7 or the coding
Gene order is transferred in plant.
Beneficial effect：The present invention provides the relevant PeDi19-4 albumen of moso bamboo drought resisting and its encoding gene for the first time and answers
With.PeDi19-4 expression vector is transferred to wildtype Arabidopsis thaliana and arabidopsis by the inflorescence dip method of mediated by agriculture bacillus
In di19 mutant, the drought resistance for being overexpressed Arabidopsis plant as the result is shown is remarkably reinforced, and transgenic arabidopsis di19 is mutated
The drought-resistant ability of body plant is also covered, and illustrates that PeDi19-4 gene can be improved the drought resistance of transgenic arabidopsis.Benefit
The vegetable material obtained with moso bamboo PeDi19-4 expression vector has certain adaptability to extraneous drought environment, has
Good application prospect.
Detailed description of the invention
Fig. 1 is the amino acid sequence and structural domain division figure of the albumen of PeDi19-4 coded by said gene of the present invention；
Fig. 2 is the electrophoretogram of 2 Leaves of Bamboo Phyllostachys pubescens total serum IgE of the embodiment of the present invention.M：Trans2K DNA Marker；1：Moso Bamboo Leaves
Piece total serum IgE；
The plant transgene carrier pCAMBI1301a-PeDi19-4 of 4 moso bamboo PeDi19-4 gene of Fig. 3 embodiment of the present invention
Construct schematic diagram；
Fig. 4 is the phenotypic map of 5 transgenic arabidopsis of the embodiment of the present invention and wildtype Arabidopsis thaliana before and after arid；A:Turn base
Because of the phenotypic map of arabidopsis and wildtype Arabidopsis thaliana before and after arid；B：Transgenic arabidopsis and wildtype Arabidopsis thaliana are at arid
Survival rate of the reason after 10 days；C：The fresh weight of transgenic arabidopsis and wildtype Arabidopsis thaliana before and after Osmotic treatment；di19：Mutant
Arabidopsis；Col：Wildtype Arabidopsis thaliana；OE-1,OE-3：Transgenosis is overexpressed strain；OX-4,OX-7：Genetically modified mutant di19
Strain.
Specific embodiment
By explaining that the preferred embodiment of following the application, other objects and advantages of the present invention will be apparent.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair
It is bright.In addition, for the numberical range in the present invention, it is thus understood that it is also specifically disclosed that between the upper limit value and lower limit value of the range
Each median.Median and any other statement value in any statement value or stated ranges or in the range
Lesser range is also included in the present invention each of between interior median.These small range of upper limit values and lower limit value can
Independently include or exclude in range.
Reagent used in embodiment mainly includes molecular biology experiment reagent and kit etc., can pass through business
Approach obtains, and specifically includes：RNA extracts kit is purchased from Roche Biotechnology Co., Ltd；Reverse transcription reagent box (First-
Strand cDNASynthesis Kit) it is purchased from Promega company；SYBR Green Mix is purchased from American AB I company；Trizol
Purchased from the handsome Bioisystech Co., Ltd's Ago-Gel DNA QIAquick Gel Extraction Kit in Shanghai, plasmid, quickly small extraction reagent kit is purchased from
Axygen；PEASY T1 simple Cloning Vector, Taq enzyme, Trans T1 competence and relevant kit are purchased
Beijing Quan Shi King Company；Restriction enzyme KpnI and XbaI and DNAmarker is purchased from TaKaRa, and (precious bioengineering is (big
Even)) Co., Ltd；Examining order is completed by Hua Da Gene science limited liability company.Provided in the embodiment of the present invention
If method is conventional method without specified otherwise.
A kind of moso bamboo PeDi19-4 albumen of the invention, the amino acid sequence such as SEQ of the moso bamboo PeDi19-4 albumen
Shown in ID No.1.
The gene order of moso bamboo PeDi19-4 albumen described in a kind of coding of the invention, the moso bamboo PeDi19-4 egg
White nucleotide sequence is as shown in SEQID No.2.
Carrier containing coding gene sequence as claimed in claim 2 of the invention.
The construction method of carrier of the present invention, includes the following steps：It include the moso bamboo of restriction enzyme site by both ends
On PeDi19-4 protein coding gene sequence construct to pEASY T1 simple Cloning Vector carrier, it is transferred to large intestine bar
In bacterium competence cell TransT1, obtain T1-PeDi19-4 recombinant plasmid, double digestion T1-PeDi19-4 recombinant plasmid and
PCAMBIA1301a plasmid after connection, is transferred to competent escherichia coli cell, after extracting plasmid, obtains expression vector
pCAMBIA1301a-PeDi19-4。
The present invention contains the engineering bacteria of carrier as claimed in claim 3.
The present invention is used to clone the primer pair of coding gene sequence as claimed in claim 2, and the primer pair includes upper
Swim primer and downstream primer, the nucleotide sequence of the upstream primer as shown in SEQ ID No.4, the downstream primer
Nucleotide sequence is as shown in SEQ ID No.5.
PeDi19-4—F:5 ' one GGGGTACC ATGGAGGTGGGAAGCCTT -3 '；
PeDi19-4—R:5 ' one GCTCTAGACTATTGTGCCTCTTTGCGG -3 '
A kind of moso bamboo PeDi19-4 gene of the invention, it is characterised in that：The cDNA of the moso bamboo PeDi19-4 gene
The nucleotide sequence of overall length is as shown in SEQ ID No.3.
Application of the moso bamboo PeDi19-4 gene of the present invention in plant modification drought resisting.
Gene as claimed in claim 7 or the coding gene sequence are turned by the inflorescence dip method of mediated by agriculture bacillus
Enter in plant.
Embodiment 1
The protein sequence of one moso bamboo PeDi19-4 gene coding
Protein sequence translation (SEQ is carried out to the cDNA sequence overall length of moso bamboo PeDi19-4 gene using primers software
ID No.1), and according to C2H2 genetic traits, moso bamboo PeDi19-4 protein sequence carries out two zine-finger like motifs
(Fig. 1) is divided with Di19-C motif.
Embodiment 2
Moso bamboo gene related to drought tolerance PeDi19-4 inducing expression pattern analysis
1. the extraction of Leaves of Bamboo Phyllostachys pubescens totalRNA
It is 25 DEG C/18 DEG C that it is (white/black) that temperature, which will be grown in, and the photoperiod (light dark) is big for three months moons in the greenhouse of 18/6h
Moso bamboo seedling that is small, and growing fine carries out different degrees of arid (15%PEG-6000,25%PEG-6000 and 35%
PEG-6000 spray solution blade) processing, take appropriate untreated blade to compare before treatment, different time sections are distinguished after processing
Sampling (1h, 3h, 6h, 12h and for 24 hours), is put into test tube, is immediately placed in after being rapidly frozen 3h in liquid nitrogen, be put into one 80 DEG C it is ultralow
It is spare in temperature refrigerator.According to the Trizol method of improvement, the extraction of total serum IgE is carried out：
(1) blade is transferred in sterilized grinding, is slowly added to liquid nitrogen, be ground to rapidly powdered, powder is filled
Enter in 1.5mL Dof pipe；
(2) 1mL Trizol is added into pipe, shakes 20s on whirlpool instrument, cracks it completely, stands 10 points on ice
Clock；
(3) 4 DEG C, 1.2 × 104G is centrifuged 15min；
(4) supernatant for drawing 800 μ L, gets in new 2.0mL Dof pipe, adds 200 μ L chloroforms/isoamyl alcohol (24:
1) it, mixes well；
(5) 4 DEG C, 1.2 × 104G is centrifuged 10min；The supernatant for drawing 400 μ L again gets to new 2.0mL Dof pipe
In, 400 μ L isopropanols are added, makes to mix sufficiently, stands 15min；
(6) 4 DEG C, 1.2 × 104G is centrifuged 15min；Net supernatant is abandoned, the ethyl alcohol that the DEPC water of 1.0mL 75% is prepared is added；
(7) 4 DEG C, 8 × 103G is centrifuged 5 minutes, carefully discards supernatant；75% ethyl alcohol of addition 1.0mL again, 4 DEG C, 8
×103G is centrifuged 5 minutes, lightly discards supernatant, place 10min on super-clean bench, make it dry；
(8) again plus 25 μ L DEPC water, 8 × 103G is centrifuged 5min, and 3 μ L is taken to detect the concentration of RNA with spectrophotometer.
2. electrophoresis detection total serum IgE integrality
The RNA for taking 2 μ L DEPC water to dissolve, is added 1 μ L bromophenol blue and mixes, and carries out electrophoresis inspection using 1.0% Ago-Gel
It surveys, electrophoresis 3-5V/cm.After electrophoresis 20-30 minutes, gel is placed under gel imaging system and is observed, if closer from loading wells
The brightness of master tape of 28S rRNA be two times of another 18S rRNA and without other miscellaneous bands (except 5S), then show RNA
Undegraded (Fig. 3).
3. removing genomic DNA processing
In micro centrifuge tube, following reagent is sequentially added：
37 DEG C after reaction 20-30 minutes, are added the DEPC water of 50 μ L；Phenol/chloroform/isoamyl alcohol (ratio of 100 μ L is added
25：24：1) it, mixes well, 1.2 × 104G is centrifuged 5min, carefully draws in upper layer (water layer) to another microcentrifugal tube, adds
The 3M NaOAC for entering 10 μ L (1/10), adds the cold dehydrated alcohol of 250 μ L, -20 DEG C preservation 30-60 minutes；Centrifugation recycling is heavy
It forms sediment, cleans precipitating with 70% cold ethyl alcohol, after vacuum drying, dissolved and detected with suitable DEPC water, be confirmed whether to remove gene
Group DNA.
4. reverse transcription
Using Promega reverse transcription reagent box First-Strand cDNA Synthesis Kit, first chain is synthesized
CDNA, concrete operations are as follows：In micro centrifuge tube, it is separately added into following reagent in order：
After crawl mixes, 42 DEG C of 15min；95 DEG C of 5min, 5 DEG C are incubated for 5 minutes；Room temperature high speed centrifugation 5s, by all solution
Collect tube bottom, detection is placed on -20 DEG C of refrigerators and saves sample；
5. utilizing Primer3Plus software design fluorescent quantitation according to the cDNA sequence overall length of moso bamboo PeDi19-4 gene
The primer of test：
F：5'-TTTGGCCTGGGCTTCACA-3'；
R：5'-GGAATAGGAACTGGCGATTTTG-3'.
Its specificity is detected with PCR, under the premise of ensuring PCR specific amplification, can be used.With moso bamboo
TIP41 gene is the positive control of fluorescent quantitation test, and the primer of TIP41 gene is：
F：5'-AAAATCATTGTAGGCCATTGTCG-3'；
R：5-ACTAAATTAAGCCAGCGGGAGTG-3'.
Quantitative fluorescent PCR reaction system is 25 μ L, and each group is divided into 12.5 μ L of SYBR Green Mix, and template cDNA is 2 μ
L, upstream primer and downstream primer (10 μm of ol/L) each 0.5 μ L finally mends deionized water to 25 μ L.PCR response parameter is as follows：95
DEG C initial denaturation 10min；95 DEG C of 15sec, 60 DEG C of 1min, totally 40 recycle.After reaction, product is heated, is produced
The solubility curve of object.Using 2–ΔΔCTMethod is handled (Fig. 4) to the signal and data of acquisition.
Embodiment 3
The clone of moso bamboo gene related to drought tolerance PeDi19-4
According to the PeDi19-4 gene C DS sequence announced on moso bamboo genome website, drawing for the PCR amplification segment is designed
Object, upstream primer add the GGGGTACC restriction enzyme site of KpnI, and downstream primer adds the GCTCTAGA restriction enzyme site of XbaI.
Primer sequence is as follows：
PeDi19-4—F:5’—GGGGTACCATGGAGGTGGGAAGCCTT—3'；
PeDi19-4—R:5’—GCTCTAGACTATTGTGCCTCTTTGCGG—3’
Using the 1st chain cDNA of Leaves of Bamboo Phyllostachys pubescens totalRNA reverse transcription as template, PCR expansion is carried out with upstream primer and downstream primer
Increase, PCR reaction system is as shown in table 1：
Table 1
PCR reaction condition is：Initial denaturation：98℃10min；Denaturation：98℃10s；Annealing：62℃5s；Extend：72 DEG C of 30s,
33 circulations；Overall elongation：72℃10min.
After reaction, it draws PCR product and carries out 2% agarose gel electrophoresis, detect and cut in gel imaging system
It after glue, is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit, by recycling segment and pEASY T1 simple Cloning
Vector connection is transformed into the E. coli competent TransT1 cell of Quan Shijin Bioisystech Co., Ltd；PCR and enzyme
Cut detection screening positive clone；To the tentatively judicious junction fragment of testing result, sends to Shanghai Sangon Biotech Company and is sequenced,
Sequencing result is such as：SEQ ID NO in sequence table:Shown in 2 DNA sequence dna, by 747bp base composition, by itself and moso bamboo gene
The sequence alignment of group website report, it is as a result completely the same.
Embodiment 4
The plant transgene carrier pCAMBI1301a-PeDi19-4 of moso bamboo PeDi19-4 gene is constructed
To will be already connected to the PeDi19-4 segment of the small fragment on pEASY T1 simple Cloning Vector with
Agrobacterium binary vector pCAMBIA1301a carries out double digestion with KpnI and XbaI enzyme, in 37 DEG C of water-baths, 3h, digestion 20uL
System is as shown in table 2：
Table 2
It after double digestion, is detected by 1% agarose gel electrophoresis, is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit.It will
Above-mentioned size segment is connected with T4DNA ligase, 25 DEG C of 2~12h of connection, and linked system is as shown in table 3：
Table 3
Carrier pCAMBIA1301a-PeDi19-4 the plasmid of 5~10 μ L built is taken gently to be driven into 100 μ L
In EHA105 Agrobacterium competent cell, after liquid nitrogen flash freezer 1min, 37 DEG C of water-bath 5min, 200 μ L YEP liquid are added in ice bath 5min
Body culture medium, 28 DEG C, 220 4~5h of culture；1.0 × 104g is centrifuged 30s, abandons supernatant, and 100 μ L YEP fluid nutrient mediums are added,
Again suspension cell is coated on the YEP solid plate containing 100 μ g/mL Kan and 50 μ g/mL Rif, and 28 DEG C of cultures about 24~
48h；The yellowish single colonie grown on picking plate is inoculated in the YEP liquid containing 100 μ g/mL Kan and 50 μ g/mL Rif
In culture solution, 24~48h of bacterium is shaken；It to bacterium solution muddiness, is shown as orange-yellow, extracts plasmid；It is verified respectively with PCR and double digestion.
Embodiment 5
Plant transgene expression vector pCAMBIA1301a-PeDi19-4 is transferred to wildtype Arabidopsis thaliana and arabidopsis di19
In mutant
1. the plantation of arabidopsis：
(1) on the super-clean bench, full wildtype Arabidopsis thaliana seed 8min is rinsed with 12% flower king's bleaching water, then to go out
The distilled water repeated flushing of bacterium 7-8 times, rinses 3min every time.
(2) seed sterilized is drawn with 1mL liquid-transfering gun equably to blow and beat on 1/2MS solid medium, be put into 4 DEG C of ice
It case vernalization 3 days, is then transferred into greenhouse and cultivates.
(3) after a week, the arabidopsis on plate is transplanted to equipped with vermiculite and black earth (3:1) in small basin, every 4/basin.
Then preservative film covers three days.Watering in growth period 3 days is primary, keeps ground moistening in flowerpot.
2. the preparation of Agrobacterium infected liquid：
The Overexpression vector Agrobacterium bacterium solution for taking 50 μ L to build, is added in 10mLYEP Liquid Culture, adds simultaneously
Enter corresponding Kan and Rif, 28 DEG C, 230r/min, cultivates two days, OD600=08-1.0.4 DEG C, 4.5 × 103R/min, centrifugation
6min abandons supernatant.Precipitating is resuspended with conversion buffer culture medium, it is spare.
The stem of bolting is cut with scissors, breaks apical dominance, makes its mitogenetic more side shoot.To have 1/ on plant
When 3 bud, a prepared bacterium solution is drawn with rubber head dropper, drop dips in bud, and cuts off existing fruit pod with scissors, reduces kind
The false positive of son.It is protected from light dark culturing three days.After about 5 days, secondary infection is carried out with the method for sample.The seed finally harvested,
For T0 generation.
Embodiment 6
The transgenic arabidopsis of moso bamboo PeDi19-4 gene screens and identification
1. the screening of hygromycin culture medium：
It is equably laid on the MS culture medium of hygromycin, arabidopsis T0 seed such as above-mentioned steps.To 4 DEG C vernalization 3 days
Afterwards, it is transferred in greenhouse and cultivates.About one week, sprouting situation is observed, it is such as normal to sprout, then it may be transgenic positive plant.
2.PCR Molecular
First according to CTAB method into extract PeDi19-4 transgenic arabidopsis DNA,
(1) CTAB Extraction buffer is dissolved in water-bath (60 DEG C)；
(2) blade is placed in mortar, pours into liquid nitrogen, be ground to powdery.It is distributed into 2mLDof pipe, every pipe is added
1mLCTAB Extraction buffer, whirlpool shake 30s, are placed in quiet 3-5min on ice, make sufficiently to crack；
(3) Dof pipe is placed in water-bath (65 DEG C), keeps the temperature 1h, keep cell completely broken；
(4) 1mL phenol is added into Dof pipe：Chloroform：Isoamyl alcohol (25：24：1) it, and mixes well, 1.2 × 104G, centrifugation
15min takes upper strata aqueous phase into new Dof pipe, isometric chloroform isoamyl alcohol is added, mixes well, and again 1.2 × 104G,
Centrifugation 15 minutes；
(5) supernatant of 400mL is inhaled again into new Dof pipe, and the isopropanol of 400mL is added, mixes, in -20 DEG C of ice
Half an hour is stood in case, 1.2 × 104G is centrifuged 15min, removes supernatant；
(6) ethyl alcohol (70%) of 1mL is added, is centrifuged 6min, abandons supernatant；
(7) step (6) are repeated；
(8) pure water for adding 50 μ L sterilized dissolves precipitating, and carries out the purity, concentration and A of DNA260/A280The detection of value.
Using wildtype Arabidopsis thaliana DNA as control, using the primer PeDi19-4F and PeDi19-4R of vector construction, and
PCR screening is carried out to the positive plant DNA of transgenic arabidopsis.PCR amplification program：98℃10min；Denaturation：98℃10s；It moves back
Fire：62℃5s；Extend：72 DEG C of 30s, 28 circulations；Overall elongation：72℃10min.After reaction, PCR product is taken, carries out 2%
After agarose gel electrophoresis, detected in gel imaging system.
3. the phenotypic analysis of the transgenic arabidopsis of moso bamboo PeDi19-4 gene
Transgenosis wildtype Arabidopsis thaliana and genetically modified mutant arabidopsis di19 homozygote seed are planted in greenhouse,
After two weeks, it takes pictures and observes phenotype；While drought and water shortage processing is carried out, in drought process, takes pictures and observe character mutation.And to difference
Survival rate after the arabidopsis Osmotic treatment of strain counted and Osmotic treatment before and after fresh weight counted (Fig. 4).
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Claims (9)
1. a kind of moso bamboo PeDi19-4 albumen, it is characterised in that：The amino acid sequence such as SEQ of the moso bamboo PeDi19-4 albumen
Shown in ID No.1.
2. a kind of gene order for encoding moso bamboo PeDi19-4 albumen described in claim 1, it is characterised in that：The moso bamboo
The nucleotide sequence of PeDi19-4 albumen is as shown in SEQID No.2.
3. containing the carrier of coding gene sequence as claimed in claim 2.
4. the construction method of carrier as claimed in claim 3, it is characterised in that include the following steps：It include restriction enzyme site by both ends
Moso bamboo PeDi19-4 protein coding gene sequence construct to pEASY T1 simple Cloning Vector carrier on, be transferred to
In competent escherichia coli cell TransT1, T1-PeDi19-4 recombinant plasmid, double digestion T1-PeDi19-4 recombinant plasmid are obtained
With pCAMBIA1301a plasmid, after connection, it is transferred to competent escherichia coli cell, after extracting plasmid, obtains expression vector
pCAMBIA1301a-PeDi19-4。
5. containing the engineering bacteria of carrier as claimed in claim 3.
6. the primer pair for cloning coding gene sequence as claimed in claim 2, it is characterised in that：The primer pair includes
Upstream primer and downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID No.4, the downstream primer
Nucleotide sequence as shown in SEQ ID No.5.
7. a kind of moso bamboo PeDi19-4 gene, it is characterised in that：The nucleosides of the cDNA overall length of the moso bamboo PeDi19-4 gene
Acid sequence is as shown in SEQ ID No.3.
8. application of the moso bamboo PeDi19-4 gene as claimed in claim 7 in plant modification drought resisting.
9. application according to claim 8, it is characterised in that：By the inflorescence dip method of mediated by agriculture bacillus by claim
Gene described in 7 or coding gene sequence as claimed in claim 2 are transferred in plant.
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| CN110128520A (en) * | 2019-05-31 | 2019-08-16 | 安徽农业大学 | Moso bamboo PheHDZ45 albumen and its encoding gene and application |
| CN110157713A (en) * | 2019-04-08 | 2019-08-23 | 安徽农业大学 | Drought-resistant maize related gene ZmDi19-7 and its application |
| CN114478730A (en) * | 2022-02-28 | 2022-05-13 | 安徽农业大学 | Wheat TaVQ14 protein and coding gene and application thereof |
| CN116769792A (en) * | 2023-06-15 | 2023-09-19 | 安徽农业大学 | Phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157713A (en) * | 2019-04-08 | 2019-08-23 | 安徽农业大学 | Drought-resistant maize related gene ZmDi19-7 and its application |
| CN110128520A (en) * | 2019-05-31 | 2019-08-16 | 安徽农业大学 | Moso bamboo PheHDZ45 albumen and its encoding gene and application |
| CN114478730A (en) * | 2022-02-28 | 2022-05-13 | 安徽农业大学 | Wheat TaVQ14 protein and coding gene and application thereof |
| CN114478730B (en) * | 2022-02-28 | 2024-01-12 | 安徽农业大学 | Wheat TaVQ14 protein, and coding gene and application thereof |
| CN116769792A (en) * | 2023-06-15 | 2023-09-19 | 安徽农业大学 | Phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof |
| CN116769792B (en) * | 2023-06-15 | 2024-03-22 | 安徽农业大学 | Phyllostachys pubescens stem elongation related gene PheLBD12 and application thereof |
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