CN108192913A - A kind of method for improving plant abiotic stress resistance - Google Patents
A kind of method for improving plant abiotic stress resistance Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, and in particular to a kind of method that plant abiotic stress resistance is improved to transgenic method.The invention discloses a kind of purposes for turning moss PpBURP2 gene resisting abiotic stress plants in preparation, it is characterised in that its encoding amino acid sequence is as shown in SEQ ID NO.2；Or its encoding amino acid sequence is at least with 90% homology of amino acid sequence shown in SEQ ID NO.2 and with the amino acid sequence for the resistance for improving plant.PpBURP2 genes of the present invention, there is apparent effect in high temperature resistance, drought resisting, salt resistance and preventing from heavy metal Cd stress etc., therefore it imports suitable expression vector after gene of the present invention can be combined with overexpression promoter in plant and converts plant host, improve the ability of Genes For Plant Tolerance abiotic stress.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to from the gene of bryophyte and its in raising plant
Abiotic stress resistance application.
Background technology
China is a large agricultural country, and the normal growth of crops is to ensureing that China's grain security is of great significance.But
China is the country that a natural calamity takes place frequently, and presentation occurrence frequency increases in recent years, the duration lengthens, coverage expands
The characteristics of, crops, if meeting with serious natural calamity, often result in a large amount of underproduction of crops in growth course.In order to realize
The weight that plant abiotic stress biology is agricultural cience and farming techniques research is understood in long-term grain security and sustainable development in depth
Want one of target.Plant gradually develops the mechanism for coping with various abiotic stresses in long-term evolutionary process, such as
Arid, high/low temperature, saline and alkaline, heavy metal pollution and stagnant flood etc..Representative of the bryophyte as the terrestrial plant occurred earliest, life
Period still based on gametophyte, in occupation of consequence in plant evolution history.It excavates and research moss adapts to terrestrial life
The adversity gene evolved is attempted important adversity gene carrying out genetic engineering transformation and one kind effectively to crops
The degeneration-resistant new crop varieties of acquisition breeding technique.
Scientific research finds that, to resist rugged environment variation, plant soma can experience the variation of external environment and will
Signal is transmitted into the cell, and inducing cell starts the various adversity genes of expression and resists wound of the poor environment to plant jointly
Evil.Albumen comprising BURP regions is the albuminoid found in plant, and it is that plant ABA signal transductions are ground that gene, which is considered as,
An important reference gene in studying carefully plays an important role in development of plants and degeneration-resistant reaction.The genoid is found earliest
Magazine is published in the relevant research of plant adverse circumstance《Molecular And General Genetics integrate molecular genetics》
(1993,238 (1-2):On 17-25), title of article is " The plant hormone abscisic acid mediates
the drought-induced expression but not the seed-specific expression of rd22,a
Gene responsive to dehydration stress in Arabidopsis thaliana (Abscisic Acids
The drought-induced expression of mediation but be not the arabidopsis gene rd22 of seed specific expression be desiccation stress response gene) ".This article
Expression of the arabidopsis gene RD22 under dehydration and salt stress processing is described, is the positively related of abscisic acid ABA induced expressions
Gene.Further research also confirmed RD22 gene functions (Harshavardhan et related to arabidopsis drought resistance later
al,PLoS One 2014；9(10):e110065).
On the basis of Phylogenetic Analysis, the member of BURP gene families is segmented into four major class：BNM2-like,USP-
Like, RD22-like, and PG-like subfamilies.RD22-like genes are concerned more because related to plant stress-resistance.
As rape homologous gene BnBDC1 and RD22 expression patterns in arabidopsis and protein sequence present height it is similar (Yu et al,
PhysiolPlantarum 2004；122(2):210-218).But with carrying out in a deep going way for research, find RD22-like inverse
Role in the stress of border is sufficiently complex.Come from the BgBDC3 of mangrove (Bruguieragymnorrhiza) under salt treatment,
Expression trend is just opposite with arabidopsis RD22 (Banzai et al, 2002)；From the 3 of grape (Vitis vinifera)
Response performance difference (the Matus et that a RD22 genes (VvRD22a, VvRD22b and VvRD22c) handle salt stress and ABA
al,PLoSOne 2014；9(10):e110372).It is anticipated that outer result is also found, rice (Oryza is such as derived from
Sativa the overexpression of OsBURP16) causes rice (Liu et al, Plant more sensitive to abiotic stress instead
CellEnviron 2014；37(5):1144-1158).But from the RD22 genoids GmRD22 of soybean (Glycine max)
Abiotic stress induction is responded, passes through the salt resistance of transgenic arabidopsis and rice of protoplast system identification method side light
Ability improves (Wang et al, Plant, Cell＆Environment 2012；35(11):1932-1947).In another text
Zhang Zhong has found that soybean BURP genes Sali3-2 also improves the resistance to copper ion of arabidopsis and cadmium ion stress ability (Tang et
al,PLoS One 2014；9(6):e98830).The above research illustrates that gene is played the part of in environment stress in BURP families
Role it is not consistent, the mechanism to play a role in adverse circumstance is still not apparent enough, and the genoid is in adverse circumstance in more ancient species
In effect also nobody be related to, such as from bryophyte BURP family genes assign plant stress-resistance role also not by people
Report, finds more efficient resisting abiotic Stress gene and is still of great significance applied to the most important cereal crops rice in China.
Invention content
The present invention is just to regulate and control plant drought, high temperature resistant and resistance to heavy from the PpBURP2 genes of moss based on a part
The discovery of the abiotic stress resistances such as cadmium metal.For in terms of certain, this invention provides genetically engineered plants overexpression
PpBURP2 genes amplifications are to the resistance of abiotic stress and structure genetically engineered plants method, to expand current plant biological skill
The gene that can be applied to improve plant resisting abiotic stress ability in art, obtains novel transgenosis adversity resistant plant kind.
For this purpose, the invention discloses a kind of DNA moleculars of the separation of the polynucleotides containing code nucleic acid to turn base in preparation
Because of the purposes in adversity resistant plant kind, wherein：
Its encoding amino acid sequence is as shown in SEQ ID NO.2；Or
Its polynucleotide sequence is the polynucleotides for encoding PpBURP2 albumen.
On the other hand, the invention discloses the DNA sequence dnas of the separation to be connected to composition promoter in plant, wherein, institute
PpBURP2 genes can be overexpressed by stating nucleic acid.
On the other hand, contain promoter comprising recombinant nucleic acid the invention discloses a kind of plant and be operably coupled to coding
The polynucleotides of PpBURP2, wherein, in the encoding amino acid sequence and SEQ ID NO.2 from 148-364 sequence lengths have to
Few 70%, 80%, 90% or 95% homogeneity.
On the other hand, the invention also discloses a kind of improvement plant is abiotic to arid, high temperature and heavy metal cadmium murder by poisoning etc.
The method of stress resistance, method include to plant and import nucleotide sequence, which, which contains, starts sub-operation connection
To coding PpBURP2 polynucleotides, wherein, the encoding amino acid sequence with it is long from 148-364 sequences in SEQ ID NO.2
Degree has the homogeneity of at least 70%, 80%, 90% or 95%..
In the examples below that, the nucleic acid sequence is as shown in SEQ ID NO.1.
In a preferred embodiment in accordance with this invention, the plant be selected from rice, corn, wheat, barley, broomcorn millet,
Any one in sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, citrus or cucumber.
The beneficial effects of the invention are as follows
Description of the drawings
Fig. 1 PpBURP2 are protein structure figure.
Fig. 2 is the structure schematic diagram of expression vector pCB4004-PpBURP2；
Fig. 3 is the PpBURP2 genes relative expression quantity level view in transgenic paddy rice blade.
Fig. 4 Seedling Stages turn the experiment of PpBURP2 trans-genetic hybrid rice high temperature resistants Stress treatment.
Fig. 5 Seedling Stages turn the simulation drought resisting processing experiment of PpBURP2 trans-genetic hybrid rice.
Fig. 6 Seedling Stages turn the processing experiment of PpBURP2 trans-genetic hybrid rice salt stress.
Fig. 7 Seedling Stages turn the resistance to heavy metal cadmium processing experiment of PpBURP2 trans-genetic hybrid rice.
Fig. 8 Adult plants turn the resistance to heavy metal cadmium processing experiment of PpBURP2 trans-genetic hybrid rice.
Specific embodiment
Herein, term " separation ", " purifying " DNA refer to, the DNA or segment be located under native state its two
It is separated in the sequence of side, also refers to the DNA or segment and separated, and with the component with nucleic acid under native state
With being separated in cell with its protein.
Fig. 1 PpBURP2 are protein structure figure, wherein, using 4.1 software (http of SignalP://
Www.cbs.dtu.dk/services/SignalP/ signal peptide shearing site analysis) is carried out to SEQ ID NO.2 protein sequences,
Prediction site 1-38 is signal peptide sequence, is indicated with underscore；Using BLAST softwares (http in NCBI://
Blast.ncbi.nlm.nih.gov/) to SEQ ID NO.2 sequential analysis of protein, prediction site 148-364 is
BURPdomain is indicated with double underline.
Fig. 2 is the structure schematic diagram of expression vector pCB4004-PpBURP2.
Fig. 3 is the PpBURP2 genes relative expression quantity level view in transgenic paddy rice blade, wherein, using real-time RT-
Real-time PCR methods detect PpBURP2 genes expression quantity in transgenic paddy rice blade, wherein, the code T 1-T8 of horizontal axis
Represent that difference turns PpBURP2 trans-genetic hybrid rice strains；The longitudinal axis represents：Transgenic line PpBURP2 genes and reference gene actin1
Expression quantity ratio.
Fig. 4 Seedling Stages turn the experiment of PpBURP2 trans-genetic hybrid rice high temperature resistants Stress treatment.It, will when paddy growth is to 4 leaf phase
Seedling is displaced in high temperature greenhouse, after being grown 12 days under 32 DEG C -50 DEG C of hot environment, is transplanted to the environment of 25 DEG C or so of room temperature
Lower growth carries out plant height statistics after 3 days, unit is centimetre.A, before high-temperature process；B, after high-temperature process；C, seedling after high-temperature process
It is long；D, seedling fresh weight after high-temperature process.ZH11 represents non-transgenic wild rice；S2G221-230 numbers represent to turn
PpBURP2 trans-genetic hybrid rice strains.
Fig. 5 Seedling Stages turn the simulation drought resisting processing experiment of PpBURP2 trans-genetic hybrid rice.When paddy growth is to 4 leaf phase, start
Using the Solution culture method containing 20%PEG6000, rehydration is handled using normal nutrition liquid after processing 10 days, and plant is counted after 5 days
Survival rate.ZH11 represents non-transgenic wild rice；G227, G229 number represent to turn PpBURP2 trans-genetic hybrid rice strains.
Fig. 6 Seedling Stages turn the processing experiment of PpBURP2 trans-genetic hybrid rice salt stress.When paddy growth is to 4 leaf phase, start to make
With the Solution culture method of the NaCl containing 150mM, rehydration processing is using normal nutrition liquid after processing 10 days, and statistics plant deposits after 7 days
Motility rate, wherein, ZH11 represents non-transgenic wild rice；G227, G229 number represent to turn PpBURP2 trans-genetic hybrid rice strains.
Fig. 7 Seedling Stages turn the resistance to heavy metal cadmium processing experiment of PpBURP2 trans-genetic hybrid rice.When paddy growth is to 4 leaf phase, open
Begin to use CdCl containing 75mM2Nutrient solution pour rice, processing 7 days after using normal nutrition liquid rehydration handle, counted after 3 days
Height, root long, fresh weight and dry weight of plant etc..ZH11 represents non-transgenic wild rice；S2G221-227 numbers represent to turn
PpBURP2 trans-genetic hybrid rice.
Fig. 8 Adult plants turn the resistance to heavy metal cadmium processing experiment of PpBURP2 trans-genetic hybrid rice.It is planted respectively in same nutritive cube
Transgenosis and control rice, use CdCl containing 150mM2Tap water is irrigated 2 months, is then irrigated using tap water.After 4 months
Rice maturation takes fringe to count setting percentage.ZH11 represents non-transgenic wild rice；The numbers of S2G227 and 228 represent to turn
PpBURP2 trans-genetic hybrid rice strains.* P is represented<0.05.
The invention also includes open reading frame in the SEQ ID NO.1 that can encode the albumen with PpBURP2 identical functions
The variant form of sequence.These variant forms include missing, insertion and/or the substitution of several nucleotide, usually 1-90,
Preferably 1-60, more preferably 1-20, missing, insertion and/or the substitution of the nucleotide of most preferably 1-10 and 5 '
And/or 3 ' end addition it is several, usually within 60, within preferably 30, it is most preferably 5 within 10 to be more preferably
Nucleotide within a.
In the present invention, it further includes with the variant form with the SEQ ID NO.2 sequences of PpBURP2 identical functions.This
A little variant forms include missing, insertion and/or the substitution of several amino acid, usually 1-50, preferably 1-30, more preferably
Ground 1-20, most preferably 1-10 and one or several in C-terminal and/or N-terminal addition, usually within 20, preferably
Ground is within 10, is more preferably the amino acid within 5.For example, in the art, with amino acid with similar or analogous performance
When being replaced, the function of protein is not usually changed.For another example, one or several ammonia are added in C-terminal and/or N-terminal
Base acid will not generally also change the function of protein.
The percent homology of albumen is determining by GAP (Needleman and Wunsh, 1970) analyses (GCG programs), wherein,
Parameter gap creation penalty=5, gap extension penalty=0.3.Analyzed sequence length is at least
During 15 amino acid, GAP analyses are just tested in the region for being at least 15 amino acid for participating in two sequences of test.More
Preferably, when analyzed sequence length is at least 50 amino acid, GAP analyses are just participating in two sequences of test at least
Region for 50 amino acid is tested.It is highly preferred that when analyzed sequence length is at least 100 amino acid, GAP points
Analysis is just tested in the region for being at least 100 amino acid for participating in two sequences of test.It is highly preferred that analyzed sequence
When row length is at least 250 amino acid, GAP analyses are just at least 250 amino acid for participating in two sequences of test
Region is tested.Even further preferably, when analyzed sequence length is at least 500 amino acid, GAP analyses are just participating in
The region for being at least 500 amino acid of two sequences of test is tested.
Polynucleotides (DNA or RNA), carrier, transformant and life can be detached and purified by methods known in the art
Object.
The present invention it is separated go out polynucleotides, include the nucleotide sequences of, SEQ ID NO.1 coding PpBURP2 genes；
Or the nucleotide sequence can be with the nucleotide sequence hybridization from 78-1181, nucleotide in SEQ ID NO.1；Or its work(
The subfragrnent of sequence shown in SEQ ID NO.1 can be equivalent to.
The PpBURP2 genes cloned may be used as probe, screened from cDNA and genomic library and obtain this
The gene or homologous gene of invention can also be synthesized directly using the method for gene chemical synthesis.PCR can also equally be used
(polymerase chain reaction) technology, from genome, cDNA amplification obtain the PpBURP2 genes of present aspect with
And any section of DNA or its homologous section of DNA.
Carrier for the present invention can be such as bacteriophage, plasmid, clay, minichromosome, virus or retrovirus
Carrier.The carrier of polynucleotides available for cloning and/or expressing the present invention can need to replicate and/or express polynucleotides
The carrier of polynucleotides is replicated and/or expressed in host cell.It is, in general, that carry the recombinant expression of the nucleic acid sequence of the present invention
Carrier can use Ti-plasmids, the standard biologics technical method such as plant viral vector, directly delivered DNA, microinjection, electroporation to lead
Enter plant cell (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy
Press,New York,pp.411-463；Geiserson and Corey,1998,Plant Molecular Biology
(2nd Edition)。
A variety of methods have been developed for making polynucleotides via complementary cohesive end and carrier is operable is connected.Example
Such as, complementary homopolymer sequence fragment can be added in the DNA section in carrier DNA to be inserted into.Then pass through complementary homopolymeric tail
Between hydrogen bond connection carrier and DNA section to form recombinant DNA molecules.
Synthetic linker containing one or more restriction sites provides another connection DNA section and the method for carrier.
The DNA generated with bacteriophage T4DNA polymerases or e. coli dna polymerase I processing by endonuclease restriction digestion
Section, described two kinds of its 3' of polymerase, 5 '-exonucleolytic activity removes prominent γ-single stranded end, and is lived with its polymerization
Property filling-in 3 '-female end.Therefore, combining for these activity produces flush end DNA section, then can be catalyzed blunt-ended DNA molecules company
The enzyme connect, as kept the temperature flush end section together with the linkers of molar excess in the presence of bacteriophage T4DNA ligases.Cause
This, reaction product is the DNA section that end carries polylinker sequence, then cracks these region of DNA with appropriate restriction enzyme
Section, and be connected in the expression vector for having used enzymatic lysis, the enzyme can generate the end compatible with the DNA section.From multiple
Businessman can buy the synthetic linker containing multiple restriction endonuclease sites.
Using methods of homologous recombination, will carry the polynucleotides of particular sequence connector or homologous sequence connector and carrier into
Row homologous recombination, the DNA section for being intended to be inserted into carrier DNA pass through with equally carrying the carrier of particular sequence or homologous sequence
The effect of recombinase forms recombinant DNA molecules.
Polynucleotides insert should be operably connected to compatible with the host cell of expression polynucleotides appropriate open
On mover, promoter can be strong promoter and/or inducible promoter.The example for some promoters enumerated includes bacteriophage
λ PL promoters, Escherichia coli lac, trP, phoA, tac promoter, the early and late promoters of SV40 and retrovirus
LTR promoters；Other appropriate promoters are known to the skilled in the art.Expression recombinant vector, which further contains, transcribes
Begin, termination site, and contain the ribosome bind site for being useful for translation in transcriptional domain.The coding of the transcript of recombinant vector expression
Part may include the translation initiation codon for being located at starting point and the terminator codon for being suitably positioned at the end for being translated polypeptide
(UAA, UGA or UAG).
As described above, expression vector may include at least one selected marker.The label includes encoding the resistance of antibiotic
Gene, such as：Neomycin phosphotransferase (Neomycin phosphotransferase) gene npt II, hygromycin phosphoric acid turn
Move enzyme (Hygromycin phosphotransferase) gene hpt and dihyrofolate reductase (Dihydrofolate
Reductase) gene dhfr；Another kind of is encoding herbicide resistance gene, for example, glufosinate transacetylase
(Phosphinothricin acetyltransferase) Bar gene, 5- enolpyruvyl acyl oxalic acid -3- phosphate synthases (5-
Enoylpyruvate shikimatr-3-phosphate) gene EPSPS.The representative example of appropriate host includes plasm
Body cell and plant cell, the appropriate culture medium and condition of culture of above-mentioned host cell are known in the art.
The method for transformation of target gene or polynucleotide of interest：One kind is carrier mediated method for transformation, i.e., by purpose base
Because being inserted on the carrier molecules such as the plasmid of Agrobacterium or the DNA of virus, target gene is imported with the transfer of carrier DNA
Into Plant Genome；Agriculture bacillus mediated and virus-mediated methods just belong to this method.Second class is gene direct guiding method, is
Refer to and directly imported external source target gene in the genome of plant by method physically or chemically.Physical method includes particle gun
Conversion method, Electroporation conversion, supercritical ultrasonics technology, microinjection and laser microbeam method etc.；Chemical method has PEG mediated transformation methods
With liposome method etc..Third class is germplasm systems approach, this includes pollen tube passage method, reproduction cell dip method, blastular and ovary
Injection etc..
In the present invention, the term " transformant " (transformant) that uses, the i.e. host cell with heterologous DNA molecule
Or organism.
The invention also includes containing the present invention nucleotide sequence host cell, the nucleotide sequence through this field
Heterologous control regions (such as promoter and/or enhancer) are operable is connected with one or more for the technology known.It can select adjust and insert
The expression of the gene order entered can be according to the modification of required particular form and the host strain of processed gene product.Certain
In the presence of inducer, the expression that certain promoters start can increase.
It can be identified by the cell of successful conversion by widely-known technique, i.e., containing nucleotides sequence of the present invention
The cell of the recombinant vector of row or organism.
Below in conjunction with specific embodiment, the present invention is furture elucidated.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Test method without specific conditions in the following example, usually according to conventional strip
Part or according to the normal condition proposed by manufacturer.
Embodiment 1：Separation clone's PpBURP2 genes
Open air takes eugonic moss, and moss total serum IgE is extracted using TRIzol reagents (GIBCO BRL, USA).Profit
With reverse transcriptase MLV (Tiangen, China) by its reverse transcription into cDNA.With primers F (5 '-atg
Ctctcagaattgaggctt gg-3 ') and primer R
(5 '-ttactgctgaacgggtacccaaatc-3 ') amplifies the overall length code cDNA (110bp) of gene.PCR
Reaction condition is：94 DEG C of pre-degeneration 3min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 60sec are recycled for 35 totally；72 DEG C of extensions
5min.The PCR product for expanding acquisition is connected into pGEM-T carriers (Promega, USA), screening positive clone is simultaneously sequenced, and obtains
The cDNA sequence (SEQ ID NO.1) of PpBURP2 genes.The protein sequence that PpBURP2 speculates carries out the comparison point of homologous gene
Analysis, it is the family protein for including BURP domain to find the gene coded protein, is conservative in 148-364 amino acid
BURP regions (see Fig. 1).In order to further appreciate that some other attributes of the protein sequence, using 4.1 softwares of SignalP
(http://www.cbs.dtu.dk/services/SignalP/) signal peptide shearing is carried out to SEQ ID NO.2 protein sequences
Locus Analysis in Shoots, prediction site 1-38 are signal peptide sequence (see Fig. 1).
Embodiment 2：The structure and genetic transformation of PpBURP2 gene overexpression vectors
The structure of 2.1 carriers containing destination gene expression：
According to the full length sequence (SEQ ID NO.1) of PpBURP2 genes, design amplifies drawing for complete coding reading frame
Object, and adapter-primer is added respectively on sense primer and downstream primer, so as to construction of expression vector.To be obtained in embodiment 1
Amplified production for template, after carrying out PCR amplification through high-fidelity Taq enzyme pfu enzymes (Tiangen, China), by PpBURP2 genes
CDNA clone further converts bacillus coli DH 5 alpha, before ensureing that reading frame is correct to intermediate carrier (such as pDONR207)
Identification intermediate carrier is put, then extracts plasmid, then using LR Clonase recombinases with including promoter and terminator egg
White plant expression vector carrier pCB4004 carries out recombining reaction, constitutes a complete expression unit (see Fig. 2), converts agriculture
Bacillus EHA105 finally carries out Rice Callus transformation experiment.
2.2 rice transformation
2.2.1 seed disinfection
It is put into sterile triangular flask after ripe Nipponbare rice paddy seed decladding, impregnates 1-2min with 75% alcohol, it is sterile
Water rinses 2 times；30min is sterilized with 30%NaClO again, needs often to shake therebetween, then is washed 3-4 times with sterile, uses aseptic filter paper
Extra moisture is blotted, seed is inoculated on callus inducing medium (MS+2,4-D 2.0mg/L), per about 30, ware,
In 28 DEG C of light cultures.
2.2.2 squamous subculture
By the induction in nearly January, rice grows the callus that yellow is expanded, and removes its scultellum, callus is gone to fresh
Subculture is carried out on callus inducing medium (MS+2,4-D 2.0mg/L).Subculture is primary every 2 weeks, and general subculture 2-4 times is i.e.
The bright yellow of suitable transgenosis, granular embryo callus can be obtained.After squamous subculture 2 weeks, embryo particle use is selected
In genetic transformation.
2.2.3 the culture of Agrobacterium
Picking single bacterium colony is cultivated in 1ml Agrobacterium culture mediums on conversion tablet.(contain phase in 50ml Agrobacteriums culture medium
Answer antibiotic) in add in the above-mentioned cultures of 1ml, 200rpm, 28 DEG C culture 5-6hr to OD600 be 0.6-1.0, culture terminate before
2hr adds in acetosyringone (AS, final concentration 100uM).Take above-mentioned bacterium solution at room temperature, 4000rpm, 10min abandon supernatant, add
Enter MS fluid nutrient mediums (100uM containing AS) and thalline is resuspended, cultivating 2hr under the same conditions with upper, making the OD600=of bacterium solution
0.5-1 can be used to transformed calli at this time.AS=acetosringone
2.2.4 it co-cultures
Rice embryo callus is immersed into Agrobacterium bacterium solution 20-30min, then with sterile blotting paper suck dry moisture, will be invaded
The callus of dye, which is placed in, to be co-cultured on culture medium (MS+2,4-D 2.0mg/L+AS 100uM), 28 DEG C of light cultures three days.
2.2.5 bacterium is washed
The callus of co-cultivation first uses aseptic water washing 3 times, then is immersed in the MS liquid training of the 400mg/L containing Cef/CN
In foster base after 20-30min, callus is transferred on aseptic filter paper and is blotted.
2.2.6 selection culture
The callus of suck dry moisture is inoculated in Selective agar medium (MS+2,4-D 2.0mg/L+Hyg 30mg/L+Cef
On 400mg/L).After 3 weeks, select the callus newly grown and be inoculated in Selective agar medium (MS+2,4-D 2.0mg/L+Hyg 50mg/L
+ Cef 250mg/L) on, reselection 2 weeks.
2.2.7 differentiation culture
Obtained resistant calli will be selected to be transferred to pre- differential medium (N6+KT 2.0mg/L+NAA by 2 times
0.2mg/L+6-BA 2.0mg/L+Hyg 30mg/L+Cef 200mg/L+ agar 9g/L+ sucrose 45g/L) on 10 days left sides of light culture
The right side returns again to differential medium (N6+KT 2.0mg/L+NAA 0.2mg/L+6-BA 2.0mg/L+Hyg 30mg/L+ agar
4.5g/L+ sucrose 30g/L) on illumination cultivation.
2.2.8 culture of rootage
About 1-2 months, the high seedling of 2cm or so is gone into root media (1/2MS+Hyg 15mg/L+ agar 4.5g/L
+ sucrose 20g/L) on inducing adventitious root generation.
2.2.9 the transplanting of transgenic seedling
When seedling is grown to 10cm high, seedling is taken out, the solid medium of attachment is cleaned with sterile water, moves into soil
In, just start with several days of cloche cover, removed cloche again after plant to be planted stalwartness, cultivated in greenhouse.
Embodiment 4：Expression analysis of the PpBURP2 genes in transfer-gen plant
3.1 materials prepare
It after transgenosis T1 is for rice seed germination, transplants in fluid nutrient medium, the culture medium is configured to 1/ for tap water
5MS a great number of elements.After 15 d of growth of seedling, clip blade quickly puts into Liquid nitrogen storage, for the extracting of RNA.
It is prepared by 3.2 total serum IgEs without DNA
The a small amount of extraction agent box operation instructions of leaves of plants RNA provided by Shanghai Quan Shijin Bioisystech Co., Ltd are taken out
It carries.Use Beckman CoulterTM 640 ultraviolet specrophotometers measure RNA concentration.It is remained in RNA to remove
DNA, each total serum IgE sample take 5 μ g, add in 1 μ LDNAaseI and 1 μ 10 × reaction buffers of L, supply volume to 10 μ L, often
Temperature 30 min of reaction, then often pipe adds in 1 μ L, 2 mmol L-1 EDTA and terminates reaction, and finally heating 10 min at 70 DEG C makes
DNAaseI is inactivated.
The synthesis of 3.3 first chain cDNA
Above-mentioned RNA sample is respectively taken into 2 μ L, the reagent provided by Promega companies of U.S. reverse transcription reagent box adds 4 successively
25 mmol L-1 MgCl2 of μ L, 2 μ L10 × RT buffer solutions, 2 μ L dNTP mixed liquors and 1 μ L oligo (dT) 15, add water to supply
Volume, in 70 DEG C of heat denatured 10min, quickly cools down on ice to 18.5 μ L.Then plus 0.5 μ L RNase inhibitor and
1 μ LAMVRTase heat 10 min at 42 DEG C of water-bath 60 min, 70 DEG C and terminate reaction.
3.4 quantitative PCR
According to the sequence design specific primer BF of gene PpBURP2:5 '-CAATTGTACTACTGCCACCACG-3 ',
BR:5 '-CCCACATGGTTGTATTCAAATG-3 ' are for quantitative fluorescent PCR, according to rice Actin genes (GenBank
Accession No.AY212324) cDNA sequence design specific primer AF:5 '-CTTCCTCATGCCATCCTGC-3 ',
AR:5 '-GCAAGCTTCTCCTTGATGTCC-3 ' for reference gene quantitative fluorescent PCR.PCR uses American AB I7000 quantitative PCR apparatus, each PCR settings are primary to be repeated.Reaction system includes SYBR Premix Ex TaqTM
(2 ×) 10 μ L, forward and reverse each 0.5 μ L of primer, the 1 μ L of cDNA templates of various processing adds water to supply volume to 25 μ L.Response procedures
For：Then 95 DEG C of 30s are recycled 40 times under 95 DEG C of 10s, 61 DEG C of 34s, fluorescence is read when setting 60 DEG C of 34s in each cycle
Value, is carried out at the same time the correction of ROX values, finally adds the analysis of fluorescence PCR products melt curve analysis, other operations refer to instrument operation instruction
Book.In order to detect the pollution that whether there is DNA in RNA sample, 3 samples are randomly selected, 1 μ L RNA is respectively taken to be carried out as template
PCR, method are same as above.
3.5 analysis method
Ct is to be determined as by fluorescence thresholding of the 7000system SDS Version1.2.3 softwares in PCR by manual
It is generated after 0.2, enters data into EXCEL and carry out calculating analysis.Data analysis uses method as 2-ΔΔCT, then utilize
EXCEL tables make differential expression block diagram.
3.6 analysis result
Using blank non-transgenic Nipponbare kind as reference, 8 independent transgenic line T1-T8 are had detected respectively, are removed
Outside transgenic line T8 expression quantity does not significantly increase, other transgenic lines have stronger Enhanced expressing, especially strain
T2 and T5 expression quantity is at 500 times or more, referring in particular to Fig. 3 is seen, is obtained in blade significantly after illustrating the channel genes to rice
The expression of dystopy height, can be applied to the research of further transgenic paddy rice.
Embodiment 5：Upgrowth situations of the PpBURP2 gene overexpression transgenosis T3 for family plant under the conditions of high temperature stress
It has chosen PpBURP2 genes overexpression transgenosis T3 in 5 embodiments 2 and has carried out seedling stage high temperature for family plant
Stress experiment.It is as follows：Every part of material about 20 plants or so of half plate of plantation in 96 hole PCR plates, when plant was grown to 4 leaf phases
When, seedling is displaced in high temperature greenhouse, under hot environment (32 DEG C -50 DEG C) growth 12 days after, be transplanted to (25 DEG C) lifes of room temperature
It is long, observe the upgrowth situation of transfer-gen plant.The result shows that 5 parts of transgenosis of PpBURP2 genes overexpression that the present invention clones
The growth of plant strains compares nontransgenic plants under normal operation with compareing no significant difference, but after high temperature compels processing
Growth is significantly slower than transfer-gen plant, after processing 12 days, is transferred under normal growing conditions, transfer-gen plant can be fast
Quick-recovery is grown, and nontransgenic plants growth is significantly inhibited, and just starts to grow young leaves after a week (see Fig. 4 a and figure
4b).These plant are counted into seedling length and fresh weight data (see Fig. 4 c and Fig. 4 d) respectively after processing, also illustrates and is overexpressed the gene
It can strengthen rice and resist high temperature stress, improve the high-temperature resistance of plant.
Embodiment 6：Growths of the PpBURP2 gene overexpression transgenosis T3 for family plant under the conditions of Drought stress simulation
Situation
It has chosen PpBURP2 genes overexpression transgenosis T3 in 5 embodiments 2 and has carried out seedling stage infiltration for family plant
Stress experiment.It is as follows：Every part of material about 24 plants of half plate of plantation in 96 hole PCR plates, when plant was grown to 4 leaf phase,
Rehydration is handled after seedling is displaced to the growth 10 days of (25 DEG C) of room temperature in nutrient solution containing 20%PEG6000, is converted to normal nutrition
It is cultivated 5 days in liquid, observes the upgrowth situation of transfer-gen plant.The result shows that the PpBURP2 genes overexpression that the present invention clones
Under normal operation with compareing no significant difference, but after processing is compeled in infiltration, control is non-to turn base for the growth of transfer-gen plant strain
Because plant leaf curling is significantly earlier than transfer-gen plant, after processing 10 days, it is transferred under normal growing conditions, transgenosis is planted
Strain energy survival rate is apparently higher than nontransgenic plants (see Fig. 5), illustrates that the resistance to infiltration side of body of rice can be strengthened by being overexpressed the gene
The ability of compeling improves the drought-resistant ability of plant.
Embodiment 7：Upgrowth situations of the PpBURP2 gene overexpression transgenosis T3 for family plant under condition of salt stress
It has chosen PpBURP2 genes overexpression transgenosis T3 in 5 embodiments 2 and has carried out the seedling stage salt side of body for family plant
Compel experiment.It is as follows：Every part of material about 24 plants of half plate of plantation in 96 hole PCR plates, will when plant was grown to 4 leaf phase
Seedling is displaced to rehydration processing after the growth 10 days of (25 DEG C) of room temperature in the nutrient solutions of NaCl containing 150mM, is converted in normal nutrition liquid
The upgrowth situation of transfer-gen plant is observed in culture 7 days.The result shows that the PpBURP2 genes overexpression that the present invention clones turns base
Because the growth of plant strains is under normal operation with compareing no significant difference, but after salt compels processing, nontransgenic plants are compareed
Leaf rolling after processing 10 days, is transferred under normal growing conditions significantly earlier than transfer-gen plant, and transfer-gen plant can be into
Motility rate is apparently higher than nontransgenic plants (see Fig. 6), illustrates that rice salt stress-resistant ability can be strengthened by being overexpressed the gene.
Embodiment 8：Growth shapes of the PpBURP2 gene overexpression transgenosis T3 for family plant under cadmium metal stress conditions
Condition
It has chosen PpBURP2 genes overexpression transgenosis T3 in 4 embodiments 2 and has carried out seedling stage metal for family plant
The saturating stress experiment of cadmium.It is as follows：Every part of material about 24 plants of half plate of plantation in 96 hole PCR plates, when plant was grown to 4 leaf phases
When, seedling is displaced to CdCl containing 75mM2In nutrient solution after the growth 7 days of (25 DEG C) of room temperature, be converted to and cultivated in normal nutrition liquid
3 days, observe the upgrowth situation of transfer-gen plant.In the case of heavy metal cadmium processing, control nontransgenic plants growth is apparent
It is suppressed, and the tolerance of transfer-gen plant is significantly better than control, other than root long, the height of plant is higher than compareing, and
And its fresh weight and dry weight are significantly higher than control (see Fig. 7), illustrate that the resistance to heavy metal cadmium side of body of rice can be strengthened by being overexpressed the gene
The ability of compeling.
Embodiment 8：Adult plants of the PpBURP2 gene overexpression transgenosis T3 for family plant under cadmium metal stress conditions
Setting percentage is observed
It has chosen PpBURP2 gene overexpression transgenosis T3 in 2 embodiments 2 and has carried out Adult plant weight for family plant
The saturating stress experiment of cadmium metal.It is as follows：Transfer-gen plant and non-transgenic reference plant shoots are transplanted to nutritive cube
In, 5 plants of transfer-gen plants and 5 plants of controls are planted in each alms bowl, when being reduced moisture in nutritive cube, using containing 150mM
CdCl2Tap water is irrigated, and is persistently irrigated 2 months, is irrigated later using tap water.Finally observe the solid situation of plant blossom.Make
With 150mM CdCl2Tap water is irrigated, hence it is evident that inhibits the growth of rice, florescence delay.In general, CdCl2Tap water fills
The growth of rice can be inhibited by irrigating, and reduce tiller, reduce Seed-Setting Percentage in Rice, but without significant difference between tiller number.With same nutrition
Adjoining tree ZH11 in alms bowl is compared, and is turned PpBURP2 plant setting percentages and is significantly higher than adjoining tree (see Fig. 8), illustrates to overexpress
The gene can improve the setting percentage of the rice under heavy metal cadmium stress.
The scope of the present invention is not limited by the specific embodiments described, and the embodiment is only used as illustrating of the invention each
The single example of a aspect further includes function equivalent method and component in the scope of the invention.In fact, in addition to as described herein
Outside content, those skilled in the art can easily grasp a variety of improvement to the present invention with reference to described above and attached drawing.Institute
Improvement is stated to also fall within the scope of the appended claims.Every bibliography mentioned above is all included in conduct herein in full
With reference to.
Claims (6)
1. a kind of applications of moss gene PpBURP2 in prepare transgenosis resisting abiotic stress plant, it is characterised in that：It is described
The encoding amino acid sequence of moss gene PpBURP2 is as shown in SEQ ID NO.2.
2. application as described in claim 1, which is characterized in that the moss gene PpBURP2 nucleic acid sequences such as SEQ ID
Shown in NO.1.
A kind of 3. recombinant vector containing moss gene PpBURP2 described in claim 1, which is characterized in that the recombinant vector
Moss gene PpBURP2 nucleic acid sequences as shown in SEQ ID NO.1.
A kind of 4. method for the genetically modified plants for producing stress tolerance, it is characterised in that：
This method includes the following steps：
1) moss gene, is operably connected to plant expression regulation sequence, forms plant expression vector；
2) plant expression vector obtained by step 1), is transferred to plant cell；
3), the transformed cells obtained through screening, are regenerated as plant and its offspring, and the plant includes plant cell, plant group
It knits or vegetable seeds.
5. the method described in claim 4, it is characterised in that：The plant is selected from rice, corn, wheat, barley, broomcorn millet, height
Any one in fine strain of millet, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, citrus or cucumber.
6. one kind include moss gene described in claim 1, which is characterized in that the moss gene prepare with high temperature,
Application in terms of the genetically modified plants of abiotic stress tolerances such as arid, salt and heavy metal cadmium.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722441A (en) * | 2019-01-22 | 2019-05-07 | 广东省农业科学院蔬菜研究所 | A kind of small heat shock protein Cu-sHSP gene of cucumber and its application |
| CN113913439A (en) * | 2021-11-22 | 2022-01-11 | 上海市农业生物基因中心 | Application and method of rice gene OsAL11 |
| CN114752622A (en) * | 2022-05-05 | 2022-07-15 | 安庆市长三角未来产业研究院 | Application of polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen |
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|---|---|---|---|---|
| WO2012004401A2 (en) * | 2010-07-09 | 2012-01-12 | Genoplante-Valor | Preformed defense in plants |
| CN102532287A (en) * | 2010-12-13 | 2012-07-04 | 首都师范大学 | Stress-resistant protein PpLEA3-17 of bryophyte as well as encoding gene and application thereof |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012004401A2 (en) * | 2010-07-09 | 2012-01-12 | Genoplante-Valor | Preformed defense in plants |
| CN102532287A (en) * | 2010-12-13 | 2012-07-04 | 首都师范大学 | Stress-resistant protein PpLEA3-17 of bryophyte as well as encoding gene and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722441A (en) * | 2019-01-22 | 2019-05-07 | 广东省农业科学院蔬菜研究所 | A kind of small heat shock protein Cu-sHSP gene of cucumber and its application |
| CN113913439A (en) * | 2021-11-22 | 2022-01-11 | 上海市农业生物基因中心 | Application and method of rice gene OsAL11 |
| CN113913439B (en) * | 2021-11-22 | 2023-12-01 | 上海市农业生物基因中心 | Application and method of rice gene OsAL11 |
| CN114752622A (en) * | 2022-05-05 | 2022-07-15 | 安庆市长三角未来产业研究院 | Application of polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen |
| CN114752622B (en) * | 2022-05-05 | 2023-09-01 | 安庆市长三角未来产业研究院 | Application of polypeptide receptor PSKR1 gene in improving high-temperature stress resistance of tomato plants and/or tomato pollen |
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