CN106868018B - White birch BpSPL9 gene and its coding albumen and application - Google Patents

White birch BpSPL9 gene and its coding albumen and application Download PDF

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CN106868018B
CN106868018B CN201710154656.2A CN201710154656A CN106868018B CN 106868018 B CN106868018 B CN 106868018B CN 201710154656 A CN201710154656 A CN 201710154656A CN 106868018 B CN106868018 B CN 106868018B
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gene
bpspl9
white birch
transgenic
strain
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CN106868018A (en
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李慧玉
宁坤
董京祥
姜静
黄海娇
陈肃
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Northeast Forestry University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Abstract

White birch BpSPL9 gene and its coding albumen and application, it is related to a kind of white birch BpSPL9 gene and its coding albumen and application.The purpose is to provide a kind of white birch BpSPL9 gene and its coding albumen and application.The nucleotide sequence of white birch BpSPL9 gene is as shown in SEQ ID NO:1 in sequence table；Amino acid sequence is as shown in SEQ ID NO:2 in sequence table.The anti-environment stress ability of plant can be enhanced in the gene.The leaf disc transformation method mediated using agrobacterium tumefaciens, carries out the genetic transformation of white birch, and carried out salt tolerance and Drought Resistance Analysis to the transgenosis white birch strain of acquisition, the results show that white birch BpSPL9 gene can be improved the salt resistance of white birch, drought-resistance ability.The white birch new varieties for turning BpSPL9 gene of salt resistance, drought resisting can be cultivated through the invention, and the present invention is applied to forest field of molecular breeding.

Description

White birch BpSPL9 gene and its coding albumen and application
Technical field
The present invention relates to white birch BpSPL9 gene and its coding albumen and applications.
Background technique
Transcription factor (transcription factor, TF) is also referred to as trans-acting factor, refers to those with same eukaryon Cis-acting elements is specifically bound in gene promoter region, or combines the egg of known dna binding domain structure feature White matter molecule has the function of activating or inhibiting genetic transcription effect.Transcription factor quantity in higher plant is big, type is more and Transcriptional control is complex, and the research of transcription factor is increasingly subject to the extensive concern of people.Divide from higher plant in succession at present The relevant transcription factors such as arid a large amount of regulations, with high salt, low temperature, hormone, cause of disease reaction and growth and development are separated out.
Typical plant transcription factor has generally comprised 4 functional areas, i.e., DNA binding domain (DNA-bindingdomain), Transcriptional regulatory domain (transcription regulation domain) includes activating and inhibiting domain, oligomerisation site (oligomerization site) and nuclear localization signal (nuclear localization signal).But different turns The record factor may lack a certain functional areas, such as special DNA binding domain or transcriptional regulatory domain, and transcription factor passes through these functional areas Domain enters in nucleus in the specific time with promoter cis-acting elements or mutual with the functional area of other transcription factors Effect carrys out the transcriptional expression of controlling gene.
The development of biotechnology in recent years and the rise of molecular breeding research, to improve woods using genetic engineering means Wood accelerates the breeding of Forest Tree New Varieties to lay the foundation.White birch is China northeast and the important reproducting tree species in Inner Mongolia, is excellent The wealthy commerical tree species of good short cycle, traditional breeding technique are difficult to realize the demand of forest character improvement in a short time, by turning Gene means carry out genetic improvement to it with great significance.
Summary of the invention
The present invention provides a kind of white birch gene and its coding albumen and application.
The nucleotide sequence of white birch BpSPL9 gene of the present invention is as shown in SEQ ID NO:1 in sequence table.
The present invention encodes the amino acid sequence of the albumen of white birch BpSPL9 gene as shown in SEQ ID NO:2 in sequence table.
The application of white birch BpSPL9 gene of the present invention refers to the application in the anti-environment stress ability of enhancing plant.
The invention discloses white birch BpSPL9 genes and its coding albumen and application, the present invention to construct one kind and contain BpSPL9 The plant expression vector of gene, which has used cauliflower mosaic virus (CaMV35S) promoter, and joined selective mark Remember kanamycins.The leaf disc transformation method mediated using agrobacterium tumefaciens, carries out the genetic transformation of white birch, and to the transgenosis of acquisition White birch strain has carried out salt tolerance and Drought Resistance Analysis, the results show that transgenic line is tied up to by 1.0%NaCl and 15%PEG Coerce 12h, for 24 hours after, the activity of both enzymes shows that white birch BpSPL9 gene can be positive obviously higher than non-transgenic control Regulate and control the intracorporal SOD activity of plant and POD activity, to enhance the anti-environment stress ability of plant, the H of measurement2O2In content, H in Stress treatment 12h, the transgenic line measured afterwards for 24 hours2O2Content is than non-transgenic strain H2O2Content is reduced, and illustrates white birch BpSPL9 gene can be improved the salt resistance of white birch, drought-resistance ability.
Detailed description of the invention
Fig. 1 is BpSPL9 gene PCR amplified production electrophorogram；
Fig. 2 is bacterium solution PCR detection electrophorogram after BpSPL9 gene TOPO reaction；
Fig. 3 is bacterium solution PCR detection electrophorogram after BpSPL9 gene LR reaction；
Fig. 4 is bacterium solution PCR detection electrophorogram after the electroporated Agrobacterium of BpSPL9 gene；
Fig. 5 is primer combinations of pairs PCR detection electrophorogram after BpSPL9 gene electric shock Agrobacterium；
Fig. 6 is the PCR detection electrophorogram for turning BpSPL9 gene plant；
Fig. 7 is the expression analysis figure for turning BpSPL9 gene in white birch difference strain；
Fig. 8 is DAB the and NBT coloration result of 1.0%NaCl with 15%PEG Stress treatment different time difference strain；Its Middle a is 1.0%NaCl Stress treatment part, and b is 15%PEG Stress treatment part；
Fig. 9 is the SOD determination of activity figure of birch transgenic strain under 1.0%NaCl Stress treatment；Wherein a is NT, and b is Ox4, c Ox15；
Figure 10 is the POD determination of activity figure of birch transgenic strain under 1.0%NaCl Stress treatment；Wherein a is NT, and b is Ox4, c Ox15；
Figure 11 is the H of birch transgenic strain under 1.0%NaCl Stress treatment2O2Assay figure；Wherein a is NT, and b is Ox4, c Ox15；
Figure 12 is the SOD determination of activity figure of birch transgenic strain under 15%PEG Stress treatment；Wherein a is NT, and b is Ox4, c Ox15；
Figure 13 is the POD determination of activity figure of birch transgenic strain under 15%PEG Stress treatment；Wherein a is NT, and b is Ox4, c Ox15；
Figure 14 is the H of birch transgenic strain under 15%PEG Stress treatment2O2Assay figure；Wherein a is NT, and b is Ox4, c Ox15.
Specific embodiment
Specific embodiment 1: SEQ in the nucleotide sequence of present embodiment white birch BpSPL9 gene such as sequence table Shown in IDNO:1.
Present embodiment discloses white birch BpSPL9 gene and its coding albumen and application, present embodiment construct one kind and contain There is the plant expression vector of BpSPL9 gene, which has used cauliflower mosaic virus (CaMV35S) promoter, and joined Selected marker kanamycins.The leaf disc transformation method mediated using agrobacterium tumefaciens, carries out the genetic transformation of white birch, and to acquisition Transgenosis white birch strain carried out salt tolerance and Drought Resistance Analysis, the results show that transgenic line tie up to by 1.0%NaCl and 15%PEG coerce 12h, for 24 hours after, both enzymes activity obviously higher than non-transgenic control, show white birch BpSPL9 gene Can the intracorporal SOD activity of positive regulation plant and POD activity, to enhance the anti-environment stress ability of plant, the H of measurement2O2 In content, H in Stress treatment 12h, the transgenic line measured afterwards for 24 hours2O2Content is than non-transgenic strain H2O2Content is reduced, Illustrate that white birch BpSPL9 gene can be improved the salt resistance of white birch, drought-resistance ability.
Specific embodiment 2: the amino acid sequence such as sequence table of the albumen of present embodiment coding white birch BpSPL9 gene Shown in middle SEQ ID NO:2.
It is coerced specific embodiment 3: the application of present embodiment white birch BpSPL9 gene refers in the degeneration-resistant border of enhancing plant Application in the ability of compeling.
Embodiment 1, the acquisition of white birch BpSPL9 gene and the building of pGWB5-BpSPL9 recombinant expression carrier
The acquisition of 1.1 objective gene sequences
1.1.1 the clone of gene and sequencing
Using 10 years wild white birches as material, its male flower is taken, extracts RNA, the extraction of RNA is according to generic plant Total RNAs extraction The method of kit (centrifugal column type) carries out.Use the ReverTra of TOYOBOqPCR RT Master Mix WithgDNA Remover kit is by RNA reverse transcription at cDNA.It is expanded using white birch cDNA as template, the production expanded Object carries out 1% agarose gel electrophoresis detection, electrophoresis detection the result is shown in Figure 1.TIANGEN gel reclaims kit recovery purifying expands Increase production object, be sequenced after carrying out TA clone, sequencing result shows that white birch BpSPL9 gene has the core of SEQ IDNO:1 in sequence table Nucleotide sequence, for the SEQ ID NO:1 in sequence table by 1140 base compositions, coded sequence is from 5 ' the 1st to 1140, ends Base encodes the protein with the amino acid sequence of SEQ ID NO:2 in sequence table.PCR amplification system is as shown in table 1.
1 PCR amplification system of table
1.1.2 target fragment purifying and the connection with pBackZero-T Vector carrier:
By PCR amplification obtain BpSPL9 target fragment carry out gel-purified recycling, carry out target fragment with PBackZero-T Vector connection, it is shown that reaction system such as table 2, and 16 DEG C of reactions are overnight.
2 target fragment of table and pBackZero-T Vector coupled reaction system
1.1.3 the identification of Escherichia coli and positive colony is converted:
5 μ L of connection product is added in 50 μ L competent escherichia coli cells and is converted, the LB solid containing ammonia benzyl mycin Positive single colonie is screened in culture medium, carries out the identification of positive colony.
The analysis of 1.2 white birch BpSPL9 gene expression characteristics
Real-time PCR analyzes white birch BpSPL9 in the expression quantity of different parts different times
With wild ripe age white birch June 5, June 20, July 5, July 20, August 5 days, August 20 days, September 5 days and 9 Leaf, bud, stem, male flower and the overwintering cDNA of April 5, April 10 and male flower on April 15 and female flower afterwards of experience on the moon 20 are mould Plate carries out real-time quantitative PCR amplification, detects the expression of white birch BpSPL9 gene, selects 18SrRNA as reference gene, All samples carry out independent biology three times and repeat, and the relative quantitative assay of gene is carried out using-Δ Δ Ct method.According to BpSPL9 distinguished sequence design upstream and downstream quantifies primer, and the reference gene and its primer sequence that this research is selected are shown in Table 3.
3 white birch BpSPL9 quantification PCR primer sequence of table
Real-time quantitative PCR kit isPremixExTaqTMII (Perfect Real Time), reaction system Are as follows:PremixExTaqTMII (2 ×) 10 μ L, each 0.8 μ L of primer (10 μm of ol/L), 6 μ L of water, 2 μ L, Rox Dye of template II 0.4 μ L, circular response parameter are 95 DEG C of initial denaturation 30s, 95 DEG C of denaturation 5s, and 60 DEG C of annealing extend 34s, circulation 40 times, drafting Solubility curve, temperature is by 95 DEG C of 15s, 60 DEG C of 1min, until 95 DEG C of 15s stop.The above reaction is in ABI7500 fluorescent quantitations It is completed in PCR instrument.
In blade, BpSPL9 lowers 2 in the significant downward of the expression of June 20 and September 5th5-29Times, wherein September table on the 5th Reach most ebb up to level, other times variation is not significant；In terminal bud, BpSPL9 was July 5 and August 20 days to September 20th Significant up-regulated expression, up-regulation 25-27Times, July 20 and August downward on the 5th are expressed；In stem, BpSPL9 integrally shows expression and lowers Trend, July 5 to August 5 days and September 5 days, expression in September 20th is significant lowers, and lowers 23-29Times, the wherein table on July 20 Reach most ebb up to amount；In male inflorescence, BpSPL9 is in June 20 to the average daily significant up-regulation of September 5, up-regulation 23-26Times, and second The developmental stage expression in April in year is relatively low, is to lower expression；In female inflorescence, BpSPL9 is in April 10 and April 15 average daily up-regulated expressions.BpSPL9 gene is most of in selected tissue site expression, in entire growth phase, The trend gradually risen is presented in the expression of BpSPL9, and highest level is reached before flower_bud formation.
1.3 the building of plant expression vector pGWB5-BpSPL9
1.3.1TOPO reaction
It is reacted according to the specification operation of TOPO kit.Then reactant is placed on ice, carries out conversion large intestine Bacillus.In the selective LB solid medium tablets of 50mg/L kanamycins, picking positive monoclonal is in LB liquid medium In 37 DEG C be incubated overnight.PCR detection is carried out by template of the bacterium solution after cracking, reaction system is 20 μ L, 94 DEG C of initial denaturation 3min Afterwards, carry out PCR cycle: 94 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extensions 1min, 35 recycle after continuation in 72 DEG C of extensions 7min.Electrophoresis is carried out in 1.0% Ago-Gel, electrophoresis detection result is shown in Fig. 2, identifies positive monoclonal.
1.3.2LR reaction
According toLR ClonaseTMThe specification of II Enzyme Mix carries out LR reaction, and 5 μ L LR is taken to react Product is added in DH5 α competent cell and is converted.
After PCR identifies that positive colony, reaction system are 20 μ L, 94 DEG C of initial denaturation 3min, PCR cycle: 94 DEG C of denaturation is carried out 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min continue in 72 DEG C of extension 7min after 35 circulations.In 1.0% Ago-Gel into Row electrophoresis, electrophoresis detection result are shown in Fig. 3, are initially identified as positive monoclonal for subsequent experimental.
1.3.3 electroporated Agrobacterium
Recombinant plasmid pGWB5-BpSPL9 is imported into EHA105 Agrobacterium competent cell by electroporated method.? It is screened on solid LB (containing 50mg/L kanamycins and 50mg/L rifampin) culture medium, obtain with recombinant plasmid and there is card The agrobacterium tumefaciens of that chloramphenicol resistance and rifampicin resistance, PCR identification is carried out to it, and reaction system is 20 μ L, 94 DEG C of initial denaturations After 3min, carry out PCR cycle: 94 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 recycle after continuation at 72 DEG C Extend 7min.PCR product is detected through 1.0% agarose gel electrophoresis, and qualification result is the list of test positive shown in Fig. 4 It clones and is saved as the engineering bacteria for infecting plant.
It is matched using gene upstream and downstream primer and the combination of pGWB5 carrier upstream and downstream primer, is obtained after detection electric shock simultaneously Positive monoclonal carries out PCR detection using the bacterium solution after cracking as template, and electricity is carried out in the Ago-Gel that concentration is 1% Swimming detection.Electrophoresis detection result is as shown in Figure 5.
Embodiment 2, pGWB5-BpSPL9 recombinant expression carrier are to the genetic transformation of white birch
The genetic transformation of white birch is carried out using the leaf disk method of mediated by agriculture bacillus.The engineering bacteria kept is activated first, is added Enter in containing kanamycin and hygromycin LB liquid medium, 28 DEG C of overnight incubations, next day takes the bacterium solution shaken, according to 2- The LB liquid medium of fresh antibiotic-free is added in 5% ratio, and 28 DEG C are shaken bacterium 4-6h, OD600It controls between 0.2-0.4, As the engineering bacterium solution infected.
Health is chosen, the good explant of growth conditions takes its top or less 3-4 piece leaf, away from petiole base 0.3cm nearby (i.e. main lobe arteries and veins crotch) is cut.The time of infection general control of white birch is between 3-5min, by cutting Blade is immersed in bacterium solution, during which shaken several times, comes into full contact with blade with bacterium solution, is then drawn off being placed on aseptic paper In, absorb extra bacterium solution.
It is placed on containing 0.8mg/L 6-BA, 0.02mg/L NAA and 0.5mg/LGA3WPM culture medium in carry out total training It supports, 2-3d is cultivated in dark place, during which replaces a subculture.Then continue at the WPM selection culture of the kanamycins containing 50mg/L It is cultivated in base, the Selective agar medium for carrying out de- bacterium depending on Agrobacterium growing state and more renewing, after selection culture 15-20d, The wound of cutting is observed that the callus expanded is grown, and grows to a certain size to callus, i.e., available tweezers or blade will It is removed, then is broken up squamous subculture.
The Molecular Identification of BpSPL9 gene in embodiment 3, transgenosis white birch
The PCR detection of 3.1 turns of BpSPL9 gene plants
18 transgenic lines of acquisition are extracted into DNA, are detected through PCR, PCR reaction system is 20 μ L, 94 DEG C of initial denaturations After 5min, carry out PCR cycle: 94 DEG C of denaturation 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 recycle after continuation at 72 DEG C Extend 7min.Confirm that BpSPL9 gene has successfully been integrated on the genome of white birch.PCR product is solidifying in 1% agarose Glue carries out electrophoresis, and as the result is shown: target gene BpSPL9 can detected in this 18 transformants, while non-transgenic It is not detected in control (NT), and the position consistency of purpose band position and positive control, turns to prove to have had successfully been obtained Gene plant, testing result are as shown in Figure 6.
The expression analysis of BpSPL9 gene in 3.2 transgenosis white birches
In order to verify expression of the BpSPL9 gene in transgenosis white birch, to its 18 transgenic line (Ox1- Ox18) and 1 non-transgenic strain (NT) has carried out Total RNAs extraction, according to the ReverTra of TOYOBOqPCR RT The specification of Master Mix with gDNA Remover kit carries out reverse transcription.Choose non-transgenic strain (NT) conduct The expression of 18 different transgenic lines is divided into 3 groups by control, it may be assumed that height expression, middle expression and low expression, according to-Δ Δ Ct calculates the relative expression quantity of gene, and relative expression quantity is 26-29Between division height expression group, 24-25Between be divided into middle expression Group, 21-23Between be then classified as low expression group, as seen from Figure 7, Ox1, Ox2, Ox4, Ox6, Ox7, Ox8, Ox9 and Ox17 be high table Up to group, Ox10-14 is middle expression group, and Ox3, Ox5, Ox15, Ox16 and Ox18 are low expression group, it follows that same gene The differential expression of gene is significant in different transformant strains, and the maximum value of most highly expressed Ox4 transformant strain is 29, minimum The Ox15 transformant strain of expression only has 21, highest expression quantity is the 2 of the minimum strain of expression quantity8Times.
Embodiment 4, the resistance analysis for turning BpSPL9 gene white birch
The Stress treatment of 4.1 transgenosis white birch excised leafs
In order to detect the effect that white birch BpSPL9 gene may have under abiotic stress, c4 plant leaf discs punch is utilized Method is sampled, and chooses the blade of health status, vane thickness and solid colour, is avoided main lobe arteries and veins and is chosen two sides blade, beats and take Diameter is the small leaf dish of 10mm.The small leaf dish of selection is carried out to the PEG Stress treatment of 1.0% NaCl and 15%, each processing Three repeated experiments are carried out, processing blade is placed in the WPM fluid nutrient medium containing NaCl and PEG, is cultivated in culturing room, The state of different time points excised leaf is observed in timing, final to select to find out the biggish duration of processing variation as the processing time Take 0h, 12h and for 24 hours three time points.
The staining analysis of 4.2 transgenosis white birch excised leafs
4.2.1DAB dyeing
The DAB dye liquor for preparing 1mg/mL adds salt acid for adjusting pH to 3.8, adjusts pH to 5.8 with sodium hydroxide when use, existing With existing tune.The different leaves of processing are respectively placed in the Erlenmeyer flask of 50mL, the DAB dye liquor of every bottle of same volume of sucking, Guarantee that each small leaf dish can be adequately exposed to dye liquor, and be not overlapped between each other, room temperature is protected from light overnight incubation.Siphon away DAB 95% ethyl alcohol is added in dye liquor, and 10min is boiled in water-bath, until leaf green is sloughed completely, during which replaces destainer twice, to protect Card decoloration thoroughly, the leaf dish of de- lechery is placed in dehydrated alcohol, scanning result.
4.2.2NBT dyeing
The NBT dye liquor of 0.5mg/mL is prepared, the different leaves of processing are placed in and are placed with same volume dye liquor by matching while using In 50mL Erlenmeyer flask, make each small leaf dish that can be adequately exposed to dye liquor, and be not overlapped between each other, 28 DEG C are protected from light dye Color 3h, sucks dye liquor, and 80% ethyl alcohol is added, and water-bath is boiled to leaf green sloughs completely, during which replaces destainer twice, with Guarantee that decoloration thoroughly, the leaf dish of de- lechery is placed in dehydrated alcohol, scanning result.
The NaCl of the 1.0% concentration and PEG of 15% concentration difference Stress treatment 0h, 12h, small leaf dish for 24 hours carry out DAB and NBT dyeing, each processing carry out 3 experiments and repeat, and coloration result is as shown in Figure 8.
The Determination of Physiological And Biochemical Indices of transgenosis white birch excised leaf under 4.3 abiotic stress
Plant tissue copper Zu-Superoxide dismutase (Cu2+Zn2+- SOD) assay kit, peroxidase (POD) survey Determine kit and hydrogen peroxide (H2O2) assay kit carries out relevant operation to specifications.
4.3.1NaCl the measurement of lower birch transgenic strain physiological and biochemical index is coerced
4.3.1.1 superoxide dismutase (SOD) active measurement
Under 1.0%NaCl Stress treatment, there is significant differences for the SOD activity of three kinds of strains of 0h measurement, wherein The SOD activity highest of No. 15 transgenic lines, up to 906U/g humidity strip, minimum non-transgenic control strain only has 740U/g wet Piece, it can be seen that the active originally activity of SOD is different between different strains, this may be by caused by after transgene 's；In the result measured after 12h stress, the SOD activity difference of No. 4 and No. 15 transgenic lines is not significant, the activity of the two SOD Higher than non-transgenic strain, and significant difference；As stress time extends to for 24 hours, there is significant difference again between three kinds of strains, As can be seen that the SOD activity of transgenic line will be higher than non-transgenic strain always from 3 observed time points, Its intracorporal SOD activity is higher, illustrates that white birch BpSPL9 gene can improve the resistance of plant to a certain extent.As a result such as Shown in Fig. 9.
4.3.1.2 peroxidase (POD) active measurement
Under 1.0%NaCl Stress treatment, in the POD activity of three kinds of strains of 0h measurement, control strain and No. 4 transgenosis Difference is not significant between strain, the significant difference between No. 15 transgenic lines, it can be seen that POD is active between different strains originally lives Property is different；In the result measured after 12h stress, the POD activity difference of No. 4 and No. 15 transgenic lines is significant, the two POD Activity be higher than non-transgenic strain, and significant difference；Occur again as stress time extends to for 24 hours, between three kinds of strains aobvious Write difference.It can be seen that the POD activity of the transgenic line after stress from observed 12h and on two time points for 24 hours always Non-transgenic strain will be higher than, intracorporal POD activity is higher, and it is intracorporal to illustrate that white birch BpSPL9 gene can regulate and control plant POD activity, degeneration-resistant for plant play an important role.The results are shown in Figure 10.
4.3.1.3 hydrogen peroxide (H2O2) content measurement
Under 1.0%NaCl Stress treatment, the H of three kinds of strains of 0h measurement2O2In content, control strain turns base with No. 15 Because difference is not significant between strain, and the H of No. 4 transgenic lines2O2Content is minimum, it can be seen that H between different strains2O2Content Originally content is different；In the result measured after 12h stress, the H of No. 4 and No. 15 transgenic lines2O2Content difference is not Significantly, the two H2O2Content is below non-transgenic strain, and significant difference；As stress time extends to for 24 hours, No. 4 and No. 15 The H of transgenic line2O2Content difference is not significant, the two H2O2Content is significantly lower than non-transgenic strain, and significant difference. It can be seen that the H of the transgenic line after stress from observed 12h and on two time points for 24 hours2O2Content always will be low In non-transgenic strain, intracorporal H2O2Content is lower, illustrate white birch BpSPL9 gene can negative regulation plant it is intracorporal H2O2Content, to be played an important role to the degeneration-resistant of plant.As a result as shown in figure 11.
4.4PEG coerces the measurement of lower birch transgenic strain physiological and biochemical index
4.4.1 superoxide dismutase (SOD) active measurement
Under 15%PEG Stress treatment, there is significant differences for the SOD activity of three kinds of strains of 0h measurement, wherein No. 4 turn The SOD activity highest of gene strain, up to 788U/g humidity strip, minimum non-transgenic control strain only has 680U/g humidity strip, no It is different with the active originally activity of SOD between strain；In the result measured after 12h stress, the SOD activity of No. 4 transgenic lines is bright It is aobvious to be higher than non-transgenic strain, and significant difference；For 24 hours after Stress treatment, difference is smaller between two kinds of transgenic lines, and non-turns base It is upper it can also be seen that transgenosis from 3 observed time points because the SOD activity of strain is significantly lower than two transgenic lines The SOD activity of strain will be higher than non-transgenic strain always, and intracorporal SOD activity is higher, illustrates white birch BpSPL9 base Because that can regulate and control the intracorporal SOD activity of plant, degeneration-resistant for plant is played an important role.As a result as shown in figure 12.
4.4.2 peroxidase (POD) active measurement
Under 15%PEG Stress treatment, the POD activity difference of three kinds of strains of 0h measurement is not significant, shows between different strains The active originally activity of POD is identical；In the result measured after 12h stress, the POD activity of No. 4 and No. 15 transgenic lines It is apparently higher than non-transgenic strain, and significant difference；For 24 hours after Stress treatment, the POD activity of No. 15 transgenic lines is higher than No. 4 Transgenic line is higher than non-transgenic strain, and significant difference.It can be seen that from observed 12h and on two time points for 24 hours The POD activity of transgenic line will be higher than non-transgenic strain always, and the intracorporal POD activity of plant is high, illustrate white birch BpSPL9 gene can regulate and control the intracorporal POD activity of plant, and degeneration-resistant for plant plays an important role.As a result such as Figure 13 institute Show.
4.4.3 hydrogen peroxide (H2O2) content measurement
Under 15%PEG Stress treatment, the H of three kinds of strains of 0h measurement2O2In content, control strain and No. 15 transgenic lines Difference is not significant between system, and the H of No. 4 transgenic lines2O2Content is slightly higher compared with the two, illustrates H between different strains2O2The original of content First content is different；In the result measured after 12h stress, the H of non-transgenic strain and No. 15 transgenic lines2O2Contain It is not significant to measure difference, the two H2O2Content is above No. 4 transgenic lines, and significant difference；As stress time extends to for 24 hours, The H of No. 4 and No. 15 transgenic lines2O2Content difference is not significant, the two H2O2Content is significantly lower than non-transgenic strain, and Significant difference.It can be seen that the H of the transgenic line after stress from observed 12h and on two time points for 24 hours2O2Content begins Non-transgenic strain, intracorporal H will be lower than eventually2O2Content is lower, illustrates that white birch BpSPL9 gene can regulate and control in plant H2O2Content, to be played an important role to the degeneration-resistant of plant.As a result as shown in figure 14.
Used kit and reagent are all commercial product in implementation column 1-4.
It is dyed in conclusion carrying out DAB and NBT to the blade after Stress treatment, it is living to measure its intracorporal SOD activity, POD Property and H2O2Content, the results showed that with the extension of stress time, the dye levels of two kinds of dye liquors are aggravated between different strains, with Non-transgenic control strain is compared, and the dyeing of two BpSPL9 gene overexpression strains is shallower；SOD activity and the active survey of POD It is fixed the results show that transgenic line tie up to by 1.0%NaCl and 15%PEG stress 12h, for 24 hours after, the activity of both enzymes is bright It is aobvious to be higher than non-transgenic control, show white birch BpSPL9 gene can the intracorporal SOD activity of positive regulation plant and POD activity, To enhance the anti-environment stress ability of plant, the H of measurement2O2In content, Stress treatment 12h, the transgenic line measured afterwards for 24 hours H in system2O2Content is than non-transgenic strain H2O2Content is reduced, and illustrates that white birch BpSPL9 gene can be improved the salt resistance, resistance to of white birch Non-irrigated ability.
Sequence table
<110>Northeast Forestry University
<120>white birch BpSPL9 gene and its coding albumen and application
<160> 8
<210> 1
<211> 1140
<212> DNA
<213>white birch (Betula platyphylla Suk.)
<400> 1
atgaaaatgg gttcgggttc tctggccgag tcaggagggt cctcttcttc gtccccgccc 60
atctcgtcct ccgagtcgct caacggcttg aaatttgggc ggaaaatcta ctttgaggat 120
gtgggtaccg gggctccgga caaaccgggt ggtgggtccg ggtcctcgtc ttctgtgtcc 180
accccgccga agaagacgag gggtggtggt gtggtgcagg ggggtcagcc acctcggtgt 240
caggttgagg ggtgtaagct agatctgagt gatgccaagg cttactattc caggcacaaa 300
gtctgtggca tgcattccaa gtcacctaag gtcattgttg ctggccttga gcagaggttt 360
tgtcaacagt gcagcagatt tcatcagctt ccagaatttg accaaggaaa acgaagttgt 420
cgtaggcgtc ttgctggtca taatgaacga aggaggaagc caccacctgg atcattattg 480
tcctcacgct atggccgatt ctcttcatct atctttgaaa acagcaccag aggagggttt 540
ctggtggact tttctacata cccaaggctt actgggaggg atgcatggcc atctacaaga 600
tcttctgagc aggtatctgg aaaccaaact accactgcgg cggcaaagta tcttccacat 660
ccatggcaaa gcaacactga aaatcctcca tctgaccttt atctgcaagg gtcagcaagt 720
gggactggtt atcccggtcc tggaattcct ccgggagaat gcttcacagg agtcattgac 780
tcaacttgtg ctctctctct tctgtcaaat caaacatggg gctccagaaa ccaagcatca 840
ggtcttgggg tgaagaacgt gatgaatgct gaaagggctc ctatggttca accaacagct 900
cctcatggtg cagttgttac tgaatttcca agcggctctt gggttttgaa gggcaatgag 960
gctggtagca gttcacatga cctgcccccc gatctgggtt tgggtcaatt ttctcagcct 1020
ctaaccagtc agttttctgg tgagcttggg ctgtctcaac agggtaggag gcaatacatg 1080
gaacttgggc actccagggc ttatgactcc tcccctcagc agatgcactg gtcactttga 1140
<210> 2
<211> 379
<212> PRT
<213>white birch (Betula platyphylla Suk.)
<400> 2
Met Lys Met Gly Ser Gly Ser Leu Ala Glu Ser Gly Gly Ser Ser
1 5 10 15
Ser Ser Ser Pro Pro Ile Ser Ser Ser Glu Ser Leu Asn Gly Leu
20 25 30
Lys Phe Gly Arg Lys Ile Tyr Phe Glu Asp Val Gly Thr Gly Ala
35 40 45
Pro Asp Lys Pro Gly Gly Gly Ser Gly Ser Ser Ser Ser Val Ser
50 55 60
Thr Pro Pro Lys Lys Thr Arg Gly Gly Gly Val Val Gln Gly Gly
65 70 75
Gln Pro Pro Arg Cys Gln Val Glu Gly Cys Lys Leu Asp Leu Ser
80 85 90
Asp Ala Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Gly Met His
95 100 105
Ser Lys Ser Pro Lys Val Ile Val Ala Gly Leu Glu Gln Arg Phe
110 115 120
Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro Glu Phe Asp Gln
125 130 135
Gly Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg
140 145 150
Arg Arg Lys Pro Pro Pro Gly Ser Leu Leu Ser Ser Arg Tyr Gly
155 160 165
Arg Phe Ser Ser Ser Ile Phe Glu Asn Ser Thr Arg Gly Gly Phe
170 175 180
Leu Val Asp Phe Ser Thr Tyr Pro Arg Leu Thr Gly Arg Asp Ala
185 190 195
Trp Pro Ser Thr Arg Ser Ser Glu Gln Val Ser Gly Asn Gln Thr
200 205 210
Thr Thr Ala Ala Ala Lys Tyr Leu Pro His Pro Trp Gln Ser Asn
215 220 225
Thr Glu Asn Pro Pro Ser Asp Leu Tyr Leu Gln Gly Ser Ala Ser
230 235 240
Gly Thr Gly Tyr Pro Gly Pro Gly Ile Pro Pro Gly Glu Cys Phe
245 250 255
Thr Gly Val Ile Asp Ser Thr Cys Ala Leu Ser Leu Leu Ser Asn
260 265 270
Gln Thr Trp Gly Ser Arg Asn Gln Ala Ser Gly Leu Gly Val Lys
275 280 285
Asn Val Met Asn Ala Glu Arg Ala Pro Met Val Gln Pro Thr Ala
290 295 300
Pro His Gly Ala Val Val Thr Glu Phe Pro Ser Gly Ser Trp Val
305 310 315
Leu Lys Gly Asn Glu Ala Gly Ser Ser Ser His Asp Leu Pro Pro
320 325 330
Asp Leu Gly Leu Gly Gln Phe Ser Gln Pro Leu Thr Ser Gln Phe
335 340 345
Ser Gly Glu Leu Gly Leu Ser Gln Gln Gly Arg Arg Gln Tyr Met
350 355 360
Glu Leu Gly His Ser Arg Ala Tyr Asp Ser Ser Pro Gln Gln Met
365 370 375
His Trp Ser Leu
379
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer BpSPL9-F.
<400> 3
caccatgaaa atgggttcgg gttc 24
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer BpSPL9-R.
<400> 4
aagtgaccag tgcatctgc 19
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer qSPL-1.
<400> 5
ggtcctcgtc ttctgtgtcc 20
<210> 6
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer qSPL-2.
<400> 6
cgttcattat gaccagcaag acg 23
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer 18s-S.
<400> 7
atcttgggtt gggcagatcg 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>nucleotide sequence of PCR primer 18s-A.
<400> 8
cattactccg atcccgaagg 20

Claims (1)

1. a kind of application of white birch BpSPL9 gene, which is characterized in that the application is the salt resistance drought-resistant ability in enhancing plant On application, the nucleotide sequence of the white birch BpSPL9 gene is as shown in SEQ ID NO:1 in sequence table.
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