CN104962563A - BpMyB106 gene in Betula platyphylla and amino acid sequence and application thereof - Google Patents

Abstract

The invention relates to and aims at providing a BpMyB106 gene in Betula platyphylla and an amino acid sequence and application thereof. A nucleotide sequence of the BpMyB106 gene in Betula platyphylla is show in a sequence list SEQ ID NO.1; the amino acid sequence thereof is shown in a sequence list SEQ ID NO.2. The BpMyB106 gene helps improve expressive abundance of photosynthesis related genes. The yeast one-hybrid technique shows that a BpMyB106 transcription factor is capable of directly controlling photosynthetic genes to increase the photosynthetic speed and growth speed of Betula platyphylla by the combination with photosynthesis related cis-acting elements; a new variety of Betula platyphylla with the trans-BpMYB106 gene, which is high in luminous effect and growth speed, can be cultivated with the BpMyB106 gene; the BpMyB106 gene is applicable to the field of molecular breeding of trees.

Description

White birch BpMYB106 gene, its aminoacid sequence and application

Technical field

The present invention relates to a kind of white birch gene, its aminoacid sequence and application.

Background technology

Along with the continuous consumption of socio-economic development and the energy, the energy that the earth stores is day by day exhausted, and the lignum of trees constitutes 20% of Global land carbon storage, and these carbon amounts then derive from photosynthetic product.Therefore, the application efficiency increasing the carbon solidification effect of xylophyta, biomaterial output and biofuel is the important channel solving renewable energy source problem, and the photosynthesis improving forest then becomes effective means.

Photosynthesis of plant ubiquity inefficiencies, in photosynthesis, light energy conversion is only 2% ~ 4% in order to the efficiency of plant-growth by plant.Photosynthetic efficiency in reality and potential photosynthetic efficiency also exist very large gap, and plant photosynthesis efficiency has the space that can improve.To the raising in the photosynthetic research of forest and efficiency, be not only the one side of basic theory Study on Problems in life science, the solution of the problems such as the energy dilemma that it has also faced with people, crisis of resource and ecocrisis has close relationship.

Traditional breeding technique is difficult to the demand realizing height of stand light efficiency character improvement in a short time.The rise of the development of biotechnology in recent years and molecular breeding research, improveing forest for utilizing genetic engineering means, accelerating the seed selection of Forest Tree New Varieties and lay the foundation.By transgenic approach, in plant materials, import the transcription factor that a controllable downstream photosynthesis genes is expressed, effectively can improve plant photosynthetic rate.Therefore the transcription factor studying forest regulation and control photosynthesis genes is very necessary.

The indirect adjustments and controls factor that what current research was more is to photosynthesis genes, generally make response under Adversity-stressed Plant, this type of transcription factor mainly comprises: CBF/DREB transcription factor, main participation low temperature stress, NAC (NAM, ATAF and CUC) and ZF-HD (zinc finger protein homeodomain) transcription factor, main participation arid and high-salt stress, AREB/ABF (ABA response element associated proteins/ABA binding factor), it is the arid response transcription factor of important dependence ABA, MYC/MYB transcription factor, the arid response and the biotic response factors that rely on ABA.These four kinds of transcription factors respectively can under plant abiotic or biotic stress conditions, and indirect shadow plant rings photosynthesis.In numerous transcription factor family, the regulation and control of myb transcription factor multiple vital process in plant materials, HvMCB1and HvMCB2 in Arabidopis thaliana CCA1 (CIRCADIAN CLOCKASSOCIATED 1) and barley belongs to the transcription factor of R1/2MYB family, all direct regulation and control can participate in the genes involved of photosynthesis, but plant R2R3-MYB class transcription factor rarely has report in direct regulation and control photosynthesis gene.

White birch (Betulaplatyphylla Suk.) is the important reproducting tree species in China northeast and Inner Mongolia, is the excellent wealthy commerical tree species of short period.Carry out genetic improvement by transgenic approach to it, object is the white birch new variety of cultivating fast-growing, high-quality, specular removal, accelerates forest breed improvement simultaneously also have greater significance for genetic engineering breeding means.

Summary of the invention

The invention provides a kind of white birch gene, its aminoacid sequence and application.

The nucleotide sequence of white birch BpMYB106 gene of the present invention is as shown in SEQ ID NO:1 in sequence table.

The present invention encodes the aminoacid sequence of white birch BpMYB106 gene as shown in SEQ ID NO:2 in sequence table.

The application of white birch BpMYB106 gene of the present invention refers to and is increasing the application on Photosynthesis Related Genes gene expression abundance.

The invention discloses encoding gene and the application of the R2R3-MYB transcription factor BpMYB106 in white birch, the present invention utilizes agrobacterium-mediated transformation this gene of process LAN in white birch, and the birch transgenic strain of acquisition has the feature that the increase of trichome density, Net Photosynthetic Rate, transpiration rate and growth velocity all improve.Numeral expression spectral technology is utilized to carry out differential gene analysis to wild-type and transgenic white birch, find that some gene upregulation directly related with photosynthesis in transgenosis white birch are expressed, mix technical identification BpMYB106 transcription factor through yeast list can in conjunction with the relevant cis-acting elements of photosynthesis, and then direct regulation and control photosynthesis genes, thus the photosynthetic rate of white birch and growth velocity are improved.The white birch new variety turning BpMYB106 gene of specular removal, fast-growing can be cultivated by the present invention, to the rearing new variety aspect of forest molecular breeding, there is important theory and practical significance.

Accompanying drawing explanation

Fig. 1 is that real-time fluorescence quantitative PCR analyzes the expression of BpMYB106 gene in white birch is respectively organized;

Fig. 2 is the electrophoresis detection figure of BpMYB106 gene cDNA fragment amplified production, M is DL2000DNA Marker, and 1 is BpMYB106 gene;

Fig. 3 is that intestinal bacteria (pROK II-BpMYB106) bacterium liquid PCR detects figure, and M is DL2000DNA Marker, and 1 is BpMYB106 gene;

Fig. 4 is intestinal bacteria (pROK II-BpMYB106) plasmid double digestion (BamH I/Sac I) product electrophoresis detection figure, M is DL2000DNA Marker, and 1 is BpMYB106 gene;

Fig. 5 is that Agrobacterium (pROK II-BpMYB106) plasmid PCR detects figure, and M is DL2000DNA Marker, and 1 is BpMYB106 gene;

Fig. 6 is white birch leaf explant, and arrow points is the position that the blade Kan resistant calli transforming BpMYB106 gene occurs;

Fig. 7 is white birch stem explants, and arrow points is the position that the stem section Kan resistant calli transforming BpMYB106 gene occurs;

Fig. 8 is the blade Kan resistant calli transforming BpMYB106 gene;

Fig. 9 is the stem section Kan resistant calli transforming BpMYB106 gene;

Figure 10 is the Kan resistance Multiple Buds transforming BpMYB106 gene;

Figure 11 is that the white birch Kan resistance transforming BpMYB106 gene is taken root seedling;

Figure 12 is the DNA level Molecular Detection of transgenosis white birch; M is DL2000DNA Marker, and 1 is wild-type white birch, and 2 is pROK II-BpMYB106 recombinant plasmid, and 3 ~ 18 is 16 transgenosis white birch strains;

Figure 13 is that the transcriptional level RT-PCR of transgenosis white birch detects; Wherein WT is wild-type white birch, and OE1-11 is transgenosis white birch;

Figure 14 is that the transcriptional level qRT-PCR of transgenosis white birch detects, * P<0.05, * * P<0.01; Wherein WT is wild-type white birch, and OE1-11 is transgenosis white birch;

Figure 15 is the blade upper epidermis trichome Density Detection of transgenosis white birch, and wherein WT is wild-type white birch, and OE1, OE3, OE8, OE9 and OE11 are transgenosis white birch;

Figure 16 is the blade lower epidermis trichome Density Detection of transgenosis white birch, and wherein WT is wild-type white birch, and OE1, OE3, OE8, OE9 and OE11 are transgenosis white birch;

Figure 17 is the mensuration of the plant height of transgenosis white birch, * P<0.05, * * P<0.01; Wherein WT is wild-type white birch, and OE1, OE3, OE8, OE9 and OE11 are transgenosis white birch, and a is WT, b is OE1; C is OE3, d be OE8, e be OE9, g is OE11;

Figure 18 is that growth 8 months white birch plant heights compare, and wherein WT is wild-type white birch, and OE1, OE3, OE8 and OE9 are transgenosis white birch;

Figure 19 is that the morphology of stomata of transgenosis white birch is observed, and observe pore under stereomicroscope, scale length is 100 μm, and wherein WT is wild-type white birch, and OE1, OE3, OE8 and OE9 are transgenosis white birch;

Figure 20 is that qRT-PCR verifies differential gene expression amount, and wherein is DEG order-checking, and ■ is qRT-PCR;

Figure 21 is that yeast one-hybrid effector plasmid (pGADT7-Rec2-BpMYB106) bacterium liquid PCR detects.M is DL2000DNA Marker, and 1 is pGADT7-Rec2-BpMYB106 carrier bacterium liquid PCR ,+be pROK II-BpMYB106 plasmid PCR;

Figure 22 is that the bacterium liquid PCR of yeast one-hybrid report carrier pHIS2-component carrier detects, and M is DL2000DNA Marker ,+be pHIS2 empty carrier plasmid PCR, all the other electrophoretic bands are respectively the recombinant vectors connecting corresponding element sequences;

Figure 23 is the yeast one-hybrid analysis that BpMYB106 identifies MYB and light response element;

To be that tobacco is instantaneous infect experimental verification yeast one-hybrid result to Figure 24.

Embodiment

Embodiment one: the nucleotide sequence of present embodiment white birch BpMYB106 gene is as shown in SEQ ID NO:1 in sequence table.

Present embodiment discloses encoding gene and the application of the R2R3-MYB transcription factor BpMYB106 in white birch, present embodiment utilizes agrobacterium-mediated transformation this gene of process LAN in white birch, and the birch transgenic strain of acquisition has the feature that the increase of trichome density, Net Photosynthetic Rate, transpiration rate and growth velocity all improve.Numeral expression spectral technology is utilized to carry out differential gene analysis to wild-type and transgenic white birch, find that some gene upregulation directly related with photosynthesis in transgenosis white birch are expressed, mix technical identification BpMYB106 transcription factor through yeast list can in conjunction with the relevant cis-acting elements of photosynthesis, and then direct regulation and control photosynthesis genes, thus the photosynthetic rate of white birch and growth velocity are improved.The white birch new variety turning BpMYB106 gene of specular removal, fast-growing can be cultivated by the present invention, to the rearing new variety aspect of forest molecular breeding, there is important theory and practical significance.

Embodiment two: the aminoacid sequence of present embodiment coding white birch BpMYB106 gene is as shown in SEQ ID NO:2 in sequence table.

Embodiment three: the application of present embodiment white birch BpMYB106 gene refers to and increasing the application on Photosynthesis Related Genes gene expression abundance.

The preparation method of embodiment one, white birch BpMYB106 gene is as follows:

The clone of 1.1 goal gene

1. utilize CTAB method to extract white birch total serum IgE, after reverse transcription, with this cDNA for template, LA Taq enzyme system is used to carry out the clone of goal gene, response procedures is that 95 DEG C of 5min → (95 DEG C of 30s → 60 DEG C 30s → 72 DEG C 70s) 30 circulates → 72 DEG C of 7min, and 1.2% agarose gel electrophoresis detects.

PCR system is:

2. pair object fragment products reclaims, and recovery product is connected to carrier T, and intestinal bacteria carry out blue hickie screening after transforming, and positive bacteria are dropped into row enlarged culturing, checks order after extracting plasmid.

Sequencing result shows that white birch BpMYB106 gene has the nucleotide sequence of SEQ ID NO:1 in sequence table, SEQ ID NO:1 in sequence table is by 1206 based compositions, its encoding sequence is that coding has the protein of the aminoacid sequence of SEQ ID NO:2 in sequence table from 5 ' end the 1 to 1206 bit base.

1.2 white birch BpMYB106 gene expression characteristics analyze (qRT-PCR)

1. design BpMYB106 gene primer MYBS, MYBA and 18S internal reference primer 18s-S, 18s-A, as table 1.

Table 1 primer sequence

2. with the reverse transcription product of white birch root, stem, leaf, stem-tip tissue for template, carry out real-time fluorescence quantitative PCR reaction, system is as follows:

Increase by two-step approach response procedures: denaturation 95 DEG C of 30s; 95 DEG C of 5s → 64 DEG C 30s, 45 circulations, are progressively warmed up to 99 DEG C from 55 DEG C, collect 1 fluorescence every 0.5 DEG C.The data obtained inputted in excel, analytical data also uses 2 ^{-Δ Δ Ct}method carries out data calculating, is figure (Fig. 1).

Result shows that the expression amount of this gene in blade is the highest, and be secondly stem apex, the relative expression quantity in stem and root is very low.

Embodiment two, transgenic approach analyze BpMYB106 gene function

2.1 design primers

The primer with restriction enzyme site of design construction over-express vector and carrier sense primer, as following table.

Table 2 primer sequence

2.2 method

2.2.1pROK II-BpMYB106 over-express vector builds

BpMYB106 gene is with ML-S and ML-A for after primer amplification, and product is building up on expression vector pROK II by BamH I, Sac I multiple clone site.After PCR rear electrophoresis, double digestion and order-checking detect, determine vector construction success.

2.2.2 agriculture bacillus mediated white birch genetic transformation

1. be transformed in EHA105 agrobatcerium cell by the plant expression vector containing BpMYB106 gene be cloned into, transformed bacteria is at 50mgL ^-1kan and 50mgL ^-1on the solid LB media of Rif, cultivate 2 days for 28 DEG C.

2. picking mono-clonal Agrobacterium, 28 DEG C are shaken bacterium to bacterium liquid OD 600 is 0.8 ~ 1.0, and bacterium liquid is diluted 20 times, 28 DEG C are continued to cultivate until bacterium liquid OD 600 is 0.6 ~ 0.8, the resuspended thalline of WPM liquid nutrient medium, and to be diluted to OD 600 be 0.1, as infecting engineering bacteria liquid.

3. choose the white birch that leaf look bud green, leaf area is larger to take root seedling leaf, near petiole base 3mm namely total vein crotch carry out cut mechanically, explant puts into engineering bacteria liquid, infects 3 ~ 5min, takes out blade aseptic filter paper and sucks unnecessary bacterium liquid.

4. explant and Agrobacterium are at substratum WPM+6-BA 0.8mgL ^-1+ NAA 0.02mgL ^-1+ GA ₃0.5mgL ^-1upper Dual culture 3 ~ 4 days.

5. Dual culture explant is put into 200mgL ^-1wash bacterium in the sterilized water of cephamycin, suck excessive moisture with thieving paper, blade is put in (with Dual culture substratum) on Selective agar medium and cultivates, cefotaxime na concn is 400mgL ^-1, Kan concentration is 30mgL ^-1.

6. select cultivation 15 days to start, can see successively has callus to expand in wound, grows after resistant buds, then break up succeeding transfer culture until callus.

7. succeeding transfer culture uses substratum WPM+6-BA 0.5mgL ^-1+ KT 0.5mgL ^-1+ Kan 30mgL ^-1, when resistant buds Growth and Differentiation is to 2-4cm, individual plant is gone in root media and cultivates (WPM+Kan 30mgL ^-1).

8. treat that resistance seedling of taking root grows to a certain degree, stem section is cut, move to (the WPM+6-BA0.5mgL on division culture medium that puts forth of resistant buds screening ^-1+ KT 0.5mgL ^-1+ Kan 30mgL ^-1), carry out second selecting cultivation, screening is expanded numerous.

2.2.3 the expansion of transfer-gen plant numerous and transplant

After a large amount of transgenosis white birch tissue cultured seedling to be obtained, select root length about 1 ~ 3cm, the transgenosis of the high 5 ~ 10cm of stem and non-transgenic seedlings, carefully wash away the substratum being attached to root, be transplanted to soddy soil with the vermiculite matrix that 1:1 mixes in mass ratio, close in order to keep humidity with preservative film, within about 7 ~ 10 days, survive.

2..2.4 the PCR of transgenosis white birch detects

Respectively with non-transgenic and each transgenosis white birch blade for material, CTAB method extracts white birch genomic dna, and method is with reference to embodiment one.With recombinant plasmid (pROK II-BpMYB106) for positive control, with non-transformed white birch STb gene for negative control, PCR detection is carried out to the transformed plant of postsearch screening.

2..2.5 the RT-PCR of transgenosis white birch and fluorescence quantitative PCR detection

Respectively with non-transgenic and each transgenosis white birch blade for material, extract total serum IgE.Use the gene of quantitative PCR in embodiment one and internal reference primer to carry out RT-PCR, response procedures is that 95 DEG C of 5min → (95 DEG C of 30s → 58 DEG C 30s → 72 DEG C 30s) 35 circulates → 72 DEG C of 7min, obtains PCR primer through 3% agarose gel electrophoresis analysis.Adjustment template concentrations, repeats one to take turns PCR, until internal reference to be the product brightness of primer identical, add the PCR that identical template amount carries out BpMYB106 gene.The method in embodiment one and BpMYB106 gene, internal reference primer is adopted to carry out quantitative fluorescent PCR.

2.2.6 the trichome distribution of transgenosis white birch detects

With scalper, blade is cut into 0.5 × 0.5cm ²size, carries out leaf upper epidermis under scanning electron microscope and lower epidermis trichome is observed and takes pictures.Each strain gets 3 strain earth culture seedlings, and every young plant gets the blade of same position formed objects, and each blade is got 3 orders and taken pictures.

2.2.7 the Stoma of Leaves of transgenosis white birch distributes and quantity detection

To tear gently blade lower epidermis, about 0.5 × 0.5cm with tweezers ²be placed on slide glass, drip 20% glycerine and carry out observation under an optical microscope and take pictures.Same strain gets the blade of the same position of 3 plant, and same blade is got 3 orders respectively and taken pictures, and the pore quantity in comparison film is carried out counting and carried out variance analysis.

2.2.8 transgenosis white birch plant height measures

Each strain white birch tissue cultured seedling is transplanted after taking root 1 month, 25 DEG C of hot-house cultures, and the plant that a Zhou Houyi survives is measured once for every 10 days, totally 60 days.Each strain measures 10 ~ 15 strain earth culture seedlings, carries out variance analysis and data analysis.

2.2.9 the Photosynthetic Index of transgenosis white birch detects

The photosynthetic instrument of LI6400 is utilized to measure the photosynthetic parameters index of wild-type white birch and transgenosis white birch, setting CO ₂concentration is 400 μm of olmol ^-1, intensity of illumination is 1400 μm of olm ^-2s ^-1, measure at room temperature is 25 DEG C, from the top of each plant, the functional leaf of several 4th or the 5th is as determination object downwards, and each strain arranges 6 strains and repeats, and repeat to arrange 3 readings, mensuration process all completes during 10:00 ~ 11:30 at every turn.Derived data is to Net Photosynthetic Rate (A), stomatal conductance (Gs), intercellular CO ₂concentration (Ci) transpiration rate (E), leaf water utilization ratio (A/E) carry out data statistic analysis and mapping process.

2.3 results and analysis

The present embodiment application Microsoft Excel software carries out one-way analysis of variance to data.

2.3.1BpMYB106 the acquisition of gene overexpression carrier

The BpMYB106 gene being template amplification with white birch cDNA is as Fig. 2, and bacterium liquid PCR detection (Fig. 3) electrophoresis result of recombinant vectors shows this length and mrna length meets.Carry out again plasmid double digestion simultaneously and detected (Fig. 4).The sample choosing more than 3 is with ML-S and ML-A for primer checks order, and the sequence that order-checking produces is mated completely with object fragment.Three kinds of different detection methods all show that object fragment is connected in carrier, vector construction success.PROK II-BpMYB106 the vector plasmid successfully constructed is proceeded in agrobacterium tumefaciens EHA105 by electric method for transformation, is detected by plasmid PCR, electrophorogram display result errorless (Fig. 5).

2.3.2 the acquisition of transgenosis white birch

The Agrobacterium plant expression vector pROK II-BpMYB106 built is infected white birch blade and stem section by agriculture bacillus mediated, resistant calli produces (Fig. 6 ~ 11) in white birch blade vein wound and stem section wound, screened by Kan, obtain 16 transgenosiss and to take root seedling.By breaking up, taking root, break up again, obtain a large amount of transgenosis white birch seedling.

2.3.3 the Molecular Detection of transgenosis white birch

2.3.3.1 the PCR qualification of transgenosis white birch

Extract the transgenosis white birch genomic dna of wild-type and each strain, with recombinant plasmid (pROK II-BpMYB106) for positive control, wild-type white birch DNA is negative control, pcr amplification is carried out with Auele Specific Primer pROK-1 and pROK-2 (according to carrier sequence design), result as shown in figure 12, have in 16 Kan resistance strains 11 consistent with positive control, be the specific band of 1206bp, wild-type white birch is without band.Result can preliminary proof BpMYB106 gene integration in the white birch genome of 11 positive PCR results.

2.3.3.2 the real-time fluorescence quantitative PCR qualification of transgenosis white birch

Carry out RT-PCR and qRT-PCR to 11 transgenic lines and wild-type white birch to analyze, result is as Figure 13 and 14, the BpMYB106 gene expression amount that result all shows transgenic line raises, and it is 2 ~ 12 times that qRT-PCR result shows positive strain BpMYB106 gene expression amount rising multiple.This illustrates that OE1 ~ OE11 transgenosis white birch is BpMYB106 process LAN strain, and this gene all has expression in various degree in these 11 strains.

2.3.4 ciliary distribution

Preliminary genetic function prediction due to BpMYB106 gene is grown relevant with trichome, and therefore white birch wild-type and 6 transgenic strains observe ciliary distribution situation under scanning electron microscope.Figure 15 and Figure 16 display be upper epidermis and lower epidermis trichome distribution, result show, all apparently higher than WT strain in the trichome density of transgenic line.But find no difference in the trichome of white birch stem is observed.This result illustrates the process LAN of BpMYB106 gene, improves white birch blade trichome density.It should be noted that the lower epidermis trichome density of OE10 and OE11 two strains is as seen from the figure higher than other transgenic lines.

2.3.5 the mensuration of white birch plant height and analysis

In order to determine ciliaryly increase and lengthen whether have impact to white birch plant height, by wild-type and 6 transgenosis white birch hardening, transplantings at one time, measure plant height, as Figure 17, plant bury in time be designated as the first day of measurement, the tissue cultured seedling that what choose is plant height, upgrowth situation is identical is transplanted.In originally three weeks, the not too big-difference of the plant height between each strain, but from the 21st day, OE1, OE3, OE8, OE11 were all significantly higher than wild-type (P<0.05).The decreased growth of OE10 and OE11 after 31 days, plant height is concordant with wild-type gradually, a little less than wild-type white birch when even arriving 61d.Plant height no significant difference between other transgenic lines.This result shows, the process LAN of BpMYB106 gene have impact on the growth of white birch.And it should be noted that, two strain OE10 that this gene expression amount is the highest and OE11 trichome density high, but plant height is relatively short, and the trichome density of all the other process LAN strains is higher than wild-type, lower than OE10 and OE11, but plant height is significantly higher than OE10, OE11 and wild-type white birch.

White birch earth culture 8 months on the occasion of autumn time plant height mensuration is carried out to wild-type, OE1, OE3, OE8 and OE9, the average 100cm of wild-type white birch, the average 130cm of transgenic, as shown in figure 18.

The mensuration of the plant height of white birch is shown, the process LAN of BpMYB106 gene adds the ciliary density of white birch blade, have impact on the growth of white birch, but simultaneously also there is difference between strain, so have selected 4 transgenic lines OE1, OE3, OE8 and OE9 that plant height is significantly higher than wild-type white birch to carry out follow-up study.

2.3.6 the photosynthesis index of transgenosis white birch

Get the mensuration that above-mentioned four transgenic lines and WT carry out Photosynthetic Index.The Net Photosynthetic Rate (A) that table 3 shows 4 transgenic lines is all significantly higher than WT, and transpiration rate (E) and the stomatal conductance (Gs) of OE1 and OE3 are significantly higher than WT, but the E of OE8 and OE9 and Gs and WT is without significant difference.In addition, in the data evaluating Photosynthetic Index, intercellular CO ₂concentration (Ci) no less important, this value of 4 transgenic lines is all remarkable in WT, and the CO of transgenic line is described ₂utilization ratio is higher, and efficiency of water application value display transgenic line takes advantage.These 5 Photosynthetic Indexes comprehensive, the photosynthetic level of entirety of transgenic line is better than WT.

The mensuration of table 3 Photosynthetic Index

* P<0.05, * * P<0.01 and * * * P<0.001.

2.3.7 pore analysis

Stomatal frequency is also one of important indicator of research plant growth state, observe the gas cell distribution situation of each strain blade lower epidermis under an optical microscope, as Figure 19, what show in figure is the gas cell distribution of each strain under 20 times of opticmicroscopes, in the form of pore and indifference, at pore quantitatively, statistical analysis, without significant difference (table 4, P>0.05), illustrate that the process LAN of BpMYB106 gene there is no impact to the Stoma of Leaves form of white birch and quantity.

The component analysis of table 4 stomatal number

The numeral expression spectrum analysis of embodiment three, transgenosis white birch

3.1 plants and sequencing analysis material

Transgenosis white birch (OE1) and non-transgenic reference white birch (WT), choose respectively from stem apex downwards several 4th or the 5th leaf as this strain express spectra order-checking material, in triplicate.

3.2 design primers

Following primer is used for express spectra result verification.

Table 5 primer sequence

3.3 method

3.3.1 RNA extracts

Get the blade of white birch process LAN transgenosis earth culture seedling (35S::BpMYB106) and wild-type earth culture seedling as material, extract RNA by CTAB method respectively.Remain a part of RNA and carry out reverse transcription, for the template of express spectra result verification.

3.3.2 the acquisition of express spectra data

Acquisition and the statistics of cDNA library structure, order-checking, express spectra data complete by Hua Da gene.Sequencing result and the white birch genome in library are compared ( http:// birch.genomics.cn/page/species/index.jsp).

3.3.3 real-time fluorescence quantitative PCR checking

The reverse transcription product mentioned with step 3.3 is template, choose up-regulated expression gene, comprise Photosynthesis Related Genes, oxidative phosphorylation genes involved and BpMYB106 gene (BP015308.1), verify expression modal data, reaction system is with reference to the real time fluorescent quantitative step of embodiment one.

3.3.4 downstream gene and promotor functional element thereof are analyzed

Utilize in pathway and the approach at some the differential gene places in express spectra is analyzed, and utilize UltraEdit software, in white birch genome database, search the promoter sequence of portion gene.Utilize PLACE database ( http:// www.dna.affrc.go.jp/PLACE/) functional element analysis is carried out to promoter sequence.

3.7 experimental result

3.7.1 Pathway and related gene promoter sequential analysis

Adopt KEGG to carry out pathway analysis, obtain the gene participating in photosynthesis and oxidative phosphorylation, as table 6.

The genes involved of table 6 photosynthesis and oxidative phosphorylation

The analysis of promotor functional element is carried out to photosynthesis and oxidative phosphorylation relevant difference gene, in the upstream promoter sequence of these differential genes, as shown in table 7 containing a large amount of photoresponse functional element.Same containing a large amount of MYB binding site in above-mentioned differential gene promoter sequence in addition, such as MYB1AT, MYB2AT, MYBCORE, MYB2CONSENSUS, MYBST1.

The analysis of cis-acting elements in differential gene promoter sequence that table 7 photosynthesis is correlated with

3.7.2 real-time quantitative PCR checking differential gene expression amount

Figure 20 shows that the relative expression quantity trend of all genes all comes to the same thing with express spectra, and the result demonstrating express spectra is accuracy.This result also demonstrates photosynthesis and oxidative phosphorylation genes involved up-regulated expression in white birch process LAN strain simultaneously, shows that the process LAN of BpMYB106 gene causes the expression amount of these genes to raise.

Embodiment four, BpMYB106 are to the identification of downstream gene functional element

4.1 design of primers

Table 8 primer sequence

4.2 experimental technique

4.2.1 the structure of effector plasmid pGADT7-Rec2-BpMYB106

1. use ADM-F/R primer (with pGADT7-Rec2 carrier restriction enzyme site SmaI and both sides homologous sequence thereof), take cDNA as template, LA Taq enzyme system is used to carry out pcr amplification, response procedures: 95 DEG C of 5min → (95 DEG C of 30s → 65 DEG C 30s → 72 DEG C 80s) 35 circulate → 72 DEG C of 7min, obtain PCR primer through 1.5% agarose gel electrophoresis analysis.The glue of object fragment PCR products reclaims.

2. extract pGADT7-Rec2 plasmid, and with Sma I for restriction enzyme, carry out single endonuclease digestion to plasmid, product, after 1.2% agarose gel electrophoresis is analyzed, reclaims the glue of digestion products.

3., after measuring pGADT7-Rec2 plasmid (Sma I enzyme cuts purifying) and BpMYB106 (purifying) concentration respectively, connect according to following reaction system:

Mix above reaction solution, 37 DEG C of insulation 30min, 50 DEG C of insulation 15min.

4. pair connection product carries out intestinal bacteria conversion, and microbiotic is 50mgmL ^-1amp.With AD-F, AD-R for primer, after PCR rear electrophoresis and order-checking detect, determine vector construction success.

4.2.2 the structure of report carrier pHIS2-element

1. (oligonucleotide fragment of often pair of primer all containing respective element is concatenated into 3 tumor-necrosis factor glycoproteinss to each pHIS2 element primer in use table 8, and according to pHIS2 multiple clone site, endonuclease EcoR I, Sac I restriction enzyme site is introduced respectively at 5 ' end of the upstream and downstream primer of element), carry out renaturation according to following system:

10×LA Taq PCR Buffer 2μL

Upstream primer 9 μ L

Downstream primer 9 μ L

Response procedures: DEG C 2min → 25, DEG C 2min → 37,95 DEG C of 30s → 72 DEG C 2min, the product dilution 100 obtained is doubly in order to connect.

2.pHIS2 plasmid extraction, utilizes EcoR I, Sac I restriction enzyme carries out plasmid double digestion, and product, after 1.2% agarose gel electrophoresis is analyzed, carries out glue recovery to digestion products.

3. pair each double-stranded DNA short-movie section and carrier connect respectively, and reaction system is as follows:

16 DEG C of connections of spending the night.

4. pair connection product carries out intestinal bacteria conversion, and microbiotic is 50mgmL ^-1kan.With HIS-F, HIS-R for primer, after PCR rear electrophoresis and order-checking detect, determine vector construction success.

4.2.3 yeast one-hybrid

1. prepare competent yeast.

2. the cotransformation of yeast adds each reagent according to table 9:

Table 9 yeast cotransformation reagent

3. reaction solution is 30 DEG C of insulations 30min (softly mixing once every 10min); In each reaction solution, add 25 μ L DMSO respectively, mix rear 42 DEG C of insulations 15min (softly mixing once every 5min), rapid ice bath 1 ~ 2min; 12000rpmmin ^-1centrifugal 15s, sedimented yeast cell; Abandon supernatant, add the resuspended yeast cell of 1mL YPD Plus Medium, 30min is cultivated in 30 DEG C of shaking table concussions; 12000rpmmin ^-1centrifugal 15s, sedimented yeast cell, abandons supernatant, adds 1mL 0.9%NaCl Solution (sterilizing also can replace with aseptic deionized water), resuspended yeast cell;

4. the yeast liquid of cotransformation is coated DDO, TDO and TDO/3-AT screening culture medium respectively, each flat board coating 100 μ L, are inverted cultivation 3 ~ 5 days for 30 DEG C.

5. the single bacterium colony on picking TDO/30mM 3-AT or DDO flat board, with TDO liquid nutrient medium 200rpmmin at 30 DEG C ^-1activated yeast 1 ~ 2d.Adjust the yeast liquid activated consistent to concentration, dilute 10 times, 100 times, 1000 times successively, get bacterium liquid 2 μ L that is undiluted and dilution respectively, put in DDO and TDO/30mM3-AT dull and stereotyped, be inverted cultivation 3 days for 30 DEG C, observe growing state.

4.2.4 instantaneous conversion infects tobacco altogether

1. the cultivation of tobacco aseptic seedling: the alcohol-pickled seed 2min of 95%, quick sucking-off alcohol, the clorox of 50% soaks seed 5min, fast by liquid sucking-off, sterile water wash 5 ~ 6 times.The seed of disinfecting is tiled on the flat board of 1/2MS solid medium, cultivates in tissue culture room.About 7 ~ 10d seed germination, transfers to seedling in the tissue culture flasks of 1/2MS solid medium and cultivates, about about 10 ~ 15d, can be used as the instantaneous vegetable material infected when Nicotiana tabacum leaves is about 2cm2.

2. Agrobacterium cotransformation tobacco: the Agrobacterium of effector plasmid and report carrier is carried out actication of culture.

3. Agrobacterium is instantaneous infects tobacco: use effector plasmid pROK II-BpMYB106, report carrier p1301-MYB1AT, p1301-MYB2AT, p1301-MYBCORE, p1301-MYBST1, p1301-MYBPZM, p1301-RBCS, p1301-Box II and p1301-SORLIP2.With the resuspended thalline of 1/2MS liquid nutrient medium, transfer in same sterilized triangular flask respectively by the resuspended bacterium liquid of effector plasmid and different report carriers, final volume 100ml (AS containing 150 μMs), as cotransformation bacterium liquid.Get in the corotation bacterium liquid that tobacco seedling is put into above, in 22 ~ 25 DEG C of shaking tables, 120rpm shakes 2 days slowly, the 1/2MS liquid nutrient medium (AS containing 150 μMs) more renewed every day.

4. clean the bacterium liquid on tobacco surface, GUS dyes.

4.3 experimental result

4.3.1 the structure of effector plasmid

Result, as Figure 21, has obvious band at about 1200bp, checks order to this band, and result shows vector construction success.

4.3.2 the structure of report carrier

Result is as Figure 22, and the report carrier that each element builds all is chosen 2 ~ 3 resistant clones and carried out PCR detection, and in Figure 22, the PCR electrophoretic band of pHIS2 empty carrier is more smaller than positive colony, and choosing PCR result is that positive clone is sent to the order-checking of rich bodyguard biology.Sequencing result shows that the report carrier of each element successfully constructs.

4.3.3 the yeast one-hybrid of pGADT7-Rec2-BpMYB106 and pHIS2-element

Have chosen typical MYB element and light response element, finishing screen is selected 5 MYB elements and 3 light response elements and can be combined by the identification of BpMYB106 transcription factor, and as Figure 23, all cotransformation list bacterium colonies all grow on DDO substratum, illustrate that thalline is normal.On TDO/30mM 3-AT, negative control pGADT7-Rec2-BpMYB106/p53HIS2 yeast conversion list bacterium colony does not grow, and all the other bacterium colonies all grow.Yeast liquid dilution is the bacterium liquid of different multiples, 5 MYB elements and 3 light response elements all have colony growth, show that BpMYB106 can identify and in conjunction with MYB1AT, MYB2AT, MYBCORE, MYBST1, MYBPZM, RBCS, Box II and SORLIP2 element.

4.3.4 BpMYB106 and the mutual of element are verified

Utilize the instantaneous infestation method of tobacco, confirmatory experiment is carried out to above 8 functional elements.Effector plasmid pROK II empty carrier report carrier of recombinating with 8 is respectively done mutually, as a control group, effector plasmid pROK II-BpMYB106 report carrier of recombinating with 8 is respectively done mutually, as experimental group, as Figure 24, control group uniform dyeing result is feminine gender, and except MYBST1 element, the dyeing of all the other experimental group is the positive.Result shows, BpMYB106 can identify MYB1AT, MYB2AT, MYBCORE, MYBPZM, RBCS, Box II and SORLIP2 element, and combines with it.

Utilize yeast one-hybrid and the instantaneous infestation method of tobacco, demonstrate BpMYB106 to combine with 4 MYB and 3 photosynthesis elements of the Photosynthesis Related Genes promotor of 11 differential expressions, perhaps this be that BpMYB106 transcription factor can the immediate cause of direct regulation and control Photosynthesis Related Genes differential expression.

In implementation column 1 ~ 3, medicine used is all commercially available prod.From embodiment, the present embodiment utilizes agrobacterium-mediated transformation this gene of process LAN in white birch, and the birch transgenic strain of acquisition has the feature that the increase of trichome density, Net Photosynthetic Rate, transpiration rate and growth velocity all improve.Numeral expression spectral technology is utilized to carry out differential gene analysis to wild-type and transgenic white birch, find that some gene upregulation directly related with photosynthesis in transgenosis white birch are expressed, mix technical identification BpMYB106 transcription factor through yeast list can in conjunction with the relevant cis-acting elements of photosynthesis, and then direct regulation and control photosynthesis genes, thus the photosynthetic rate of white birch and growth velocity are improved.The white birch new variety turning BpMYB106 gene of specular removal, fast-growing can be cultivated by the present embodiment, to the rearing new variety aspect of forest molecular breeding, there is important theory and practical significance.

Claims (3)

1. white birch BpMYB106 gene, is characterized in that the nucleotide sequence of this gene is as shown in SEQ ID NO:1 in sequence table.

2. the aminoacid sequence of coding white birch BpMYB106 gene according to claim 1, is characterized in that the aminoacid sequence of this gene is as shown in SEQ ID NO:2 in sequence table.

3. the application of white birch BpMYB106 gene according to claim 1, is characterized in that it is increasing the application on Photosynthesis Related Genes gene expression abundance.