CN102719451B - Poncirus trifoliata basic helix-loop-helix (PtrbHLH) and application in improving cold resistance of plant - Google Patents

Abstract

The invention discloses a PtrbHLH and an application in improving cold resistance of plant. The bHLH serves as a key word to search citrus expressed sequence tag (EST) database (HarvEST: Citrusver.0.51), 17 EST sequences which are highly homologous with the bHLH, overlap sections are used for splicing, the sequences are spliced into one sequence through a splicing software DNA start, according to sequence analysis, the sequence after splicing comprises a complete open reading frame (ORF), primers are designed to amplify gene PtrbHLH on the poncirus trifoliata with the spliced sequence serving as a reference sequence, an agrobacterium mediated genetic transformation method is used for transforming tobacco and lemon, the obtained transgenic plant, after biological functional verification, proves that the cloned PtrbHLH gene has the function of improving cold resistance, obstacles of conventional breeding methods are broken; and in addition, the gene has stable resistance in various plants, important genetic resources are provided for cold resistant genetic engineering of the plants.

Description

Trifoliate orange transcription factor PtrbHLH and the application in improving plant cold resistance

Technical field

This patent belongs to plant genetic engineering field.Be specifically related to an a kind of bHLH(basic helix-loop-helix who from trifoliate orange (Poncirus trifoliata), separates, clones, the alkalescence helix-loop-helix) the transcription factor PtrbHLH of family also relates to the application of a kind of transcription factor PtrbHLH in improving plant cold resistance.

Background technology

Plant often suffers a series of environment-stress, comprises arid, high temperature, low temperature, saline and alkaline, waterflooding etc., and wherein, low temperature is to affect crop growth and one of output and the of paramount importance factor of restriction plant regional distribution.In very long evolutionary process, plant has formed the complicated mechanism of a cover and has adapted to and the abiotic stress of surviving.Recent two decades comes, and scientist is in made significant headway aspect the parsing of plant responding abiotic stress molecular mechanism (Qin et al., 2011) both at home and abroad.Be clear that now, the plant stress response is the process of very complicated a plurality of signal pathways.Extraneous stress signal activates second messenger's (such as calcium ion, active oxygen and inositolophosphate) by known or unknown sensing member identification.Afterwards, by different signal element transductions, reception and registration, amplification stress signal, finally cause a series of physiology and metabolism to change.People have obtained huge progress in identifying the Signal Transduction Components that the participation stress response is answered.

A large amount of evidences show, complicated adverse circumstance signal transduction path has comprised a large amount of stress response genes, can be divided into two classes substantially according to the function of gene product.The functional protein that the first kind is avoided damaging by direct Cell protection forms, and Equations of The Second Kind forms (Agarwal et al., 2006 by the adjusting albumen that plays regulating and controlling effect in signal transduction and genetic expression; Chinnusamy et al., 2006; Shinozaki and Yamaguchi-Shinozaki 2007; Lata and Prasad, 2011).Research to the stress response gene not only can be understood stress response mechanism, and so that people create transgenic plant (Wang et al., 2003 that resistance strengthens by transgenosis; Qin et al., 2011; Lata and Prasad, 2011).Studies show that in a large number, can significantly improve the resistance of plant by overexpression functional gene and regulatory gene.But functional gene and transcription factor are in constructive expression's the possibility of result different (Agarwal et al., 2006).Compare with functional gene, transcription factor is with better function in the resistance that improves plant.The overexpression of a transcription factor can activate a series of target gene, and they can work in the reply adverse circumstance together, rather than a functional gene that changes over to works.Therefore, the transcription factor genetic transformation is a kind of important channel and the means of plant resistance to environment stress genetic improvement.

Plant Genome has a large amount of transcription factors; For example, have in the Arabidopis thaliana more than 1500 transcription factors, the genome that accounts for it is 6%(Riechmann et al. nearly, 2000), bHLH(basic helix-loop-helix wherein, alkaline helix-loop-helix) the class transcription factor is member important in the regulated and control network, and this family protein has the bHLH territory, the HLH zone is positioned at the C end of b HLH die body, and the obvious different bHLH die bodys of two functions that are comprised of about 60 amino acid form.The formation that the HLH die body participates in homodimer or heterodimer is because the interaction of alpha-helix.The N end that the alkalescence zone is positioned at the bHLH die body contains about 15 basic aminoacidss, and it is determining specificity (Buck and Atchley, 2003 of DNA and interactions between protein; Toledo-Ortiz et al., 2003; Li et al., 2006).The bHLH transcription factor extensively distributes in eukaryote, as 147 and 167 genes (Li et al., 2006 are arranged respectively in Arabidopis thaliana and paddy rice; Toledo-Ortiz et al., 2003).In animal, bHLH family member's function is widely studied, but the research in plant is relatively less.From first plant bHLH albumen (Lc) is in the news (Ludwig et al., 1989) in corn since, in some plants, identified part bHLH transcription factor.Show, plant bHLH albumen rises in different biological procedureses and transcribes regulating and controlling effect, comprises fur or roots development (Berhardt et al., 2003; Tominaga-Wada et al., 2012; Karaset al., 2009), Development of Chloroplasts (Monte et al., 2004), flavonoid alkaloid and anthocyanidin synthesize (Nesi et al., 2000; Yamada et al., 2011).Some bHLH albumen are such as PIF3(phytochrome interaction factor 3), PIF4 and HFR1 participate in photoinduced signal transduction (Ni et al., 1998; Huq and Quail, 2002; Fairchild et al., 2000).Yet plant bHLH transcription factor is few aspect replying at environment-stress.At present, study the most clearly Stress response bHLH transcription factor be ICE1(inducer of CBF expression 1), its similar transcription factor of MYC of encoding, it improves the cold tolerance (Chinnusamy et al., 2003) of plant by the cis-acting elements in the regulation and control CBF3 promotor.Afterwards, identified that 2 have the bHLH gene of higher homology with ICE1, i.e. Arabidopis thaliana ICE2 and apple MdCibHLH1(Fursova et al., 2009; Feng et al., 2012).Other bHLH transcription factor relevant with stress response comprises AtbHLH38, AtbHLH39, FIT, and they can be induced by iron deficiency, and the overexpression of AtbHLH38, AtbHLH39 can strengthen plant to absorption (Yuan et al., 2008 of iron or cadmium; Lignam et al., 2011; Wuet al., 2012), show that they work in heavy metallic poison is replied.In addition, the bHLH gene of paddy rice also is found to participate in drought stress and replys (Seo et al., 2011).All these studies show that, the bHLH transcription factor plays an important role in the abiotic stress response, very large potentiality are arranged aspect the stress resistance of plant utilizing genetically engineered to improve.

Summary of the invention:

The object of the invention has provided a kind of bHLH class transcription factor (applicant is with its called after PtrbHLH) of separating, cloning from extremely cold-resistant trifoliate orange (Poncirus trifoliata), its nucleotides sequence is classified as shown in the SEQ ID NO.1.

Another object of the present invention has provided the application of a kind of bHLH class transcription factor PtrbHLH in improving plant cold resistance, by agriculture bacillus mediated genetic transformation it is imported tobacco and lemon, identify its cold-resistant function, for the plant stress-resistance Molecular design breeding provides new genetic resources, for enforcement green agriculture, water-saving agriculture provide new genetic resources, the development and use of this genetic resources are conducive to reduce agriculture production cost and realize environmental friendliness.In order to reach above purpose, the present invention adopts following technique means:

Take bHLH as keyword search oranges and tangerines est database (HarvEST:Citrus ver.0.51), search the est sequence of 17 and bHLH height homology, utilize its lap to splice, utilize splicing software DNAstart that it is spliced into a sequence, sequential analysis finds that spliced sequence comprises a complete ORF reading frame, splice good sequence as a reference sequences according to this, the design primer amplifies gene PtrbHLH at trifoliate orange, its nucleotides sequence is classified as shown in the SEQ IDNO:1, is the coding region of this gene at the 253-1824bp place of sequence shown in the SEQ ID NO:1; The aminoacid sequence of its coding is shown in sequence table SEQ ID NO:2, and it comprises the open reading frame of 1464bp, 487 amino acid of encoding, and iso-electric point is 5.3, the molecular weight of prediction is 53.6kD.

The applicant has designed the primer pair of the cDNA sequence of cloning above-mentioned gene PtrbHLH, and its nucleotide sequence is as follows:

Forward primer: 5 '-TTGTCGACGCAGCGAGTAAAGTATGGCCAATGG-3 ';

Reverse primer: 5 '-ATGGTACCGGATCCATGATACCATTACCCTACCAC-3 '.

Utilize agrobcterium-mediated transformation transformation of tobacco and lemon, the transfer-gen plant of acquisition shows that through biological function verification the PtrbHLH gene that the present invention clones has the cold resistant function of raising.In the embodiments of the invention part, we have set forth separation, functional verification and the application of trifoliate orange PtrbHLH transcription factor.

Compared with prior art, the present invention has the following advantages: the present invention utilizes transgenic technology to obtain the plant that winter resistance improves, and has broken through the obstacle of traditional breeding method means; In addition, gene of the present invention resistance in various plants is stable, for genetic engineering of cold-resistance in plants provides important genetic resources.

Description of drawings

Fig. 1 is a kind of techniqueflow chart of the present invention.

Fig. 2 is the expression of a kind of PtrbHLH gene under low temperature, salt stress and dehydration are compeled.

Wherein: Fig. 2 A be trifoliate orange field seedling (not transgenosis) under 4 ℃ of processing, corresponding point in time sampling adopts the relative expression quantity of Real-time PCR Analysis gene of the present invention; Fig. 2 B is trifoliate orange field seedling (not transgenosis) under 200mM sodium-chlor is processed, and corresponding point in time sampling adopts the relative expression quantity of Real-time PCR Analysis gene of the present invention; Fig. 2 C is the expression pattern of gene of the present invention under the dehydration different time points under trifoliate orange field seedling (not transgenosis) room temperature.

Fig. 3 is a kind of PtrbHLH gene Subcellular Localization schematic diagram.

Fig. 3 A, GFP gene (contrast) light field (figure left), details in a play not acted out on stage, but told through dialogues (in) lower imaging, right figure is the imaging after the two superposes; Fig. 3 B, the PtrbHLH gene light field (left side), details in a play not acted out on stage, but told through dialogues (in) under imaging, right figure is the imaging after the two stack.

Fig. 4 is that a kind of PtrbHLH genetic transcription activates the evaluation schematic diagram.

Fig. 4 A is empty carrier (pDEST ^TM32) growing state of yeast on different culture media that transforms; Fig. 4 B is the growing state of yeast on different culture media that fusion vector PtrbHLH transforms.

Fig. 5 is that a kind of PtrbHLH genophore makes up schematic diagram.

Fig. 6 is a kind of PtrbHLH gene transformation lemon process schematic diagram.

Upper left stem section for cultivating altogether, upper right for to grow resistant buds in screening culture medium, the lower-left is the clump bud on the bud elongation medium, the bottom right is the root culture seedling.

Fig. 7 is that a kind of PtrbHLH gene transformation tobacco and lemon regeneration plant PCR identify schematic diagram.

The regeneration plant PCR evaluation figure that obtains behind Fig. 7 APtrbHLH transformation of tobacco (left is NPTII gene specific primer pcr amplification figure, and the right side is 35S forward primer and PtrbHLH specific primer PCR amplification figure).Fig. 7 B is that the regeneration plant that obtains behind the PtrbHLH conversion lemon utilizes NPTII gene specific primer pcr amplification figure.Fig. 7 C is that the regeneration plant that obtains behind the PtrbHLH conversion lemon utilizes 35S forward primer and PtrbHLH specific primer PCR amplification figure.

Fig. 8 is a kind of expression analysis schematic diagram of identifying transgenosis PtrbHLH in the positive transfer-gen plant.

Fig. 8 A is the expression analysis of transgene tobacco PtrbHLH, and Fig. 8 B is the overexpression analysis of transgenosis lemon PtrbHLH.

Fig. 9 is that the PtrbHLH transgene tobacco seedling (two systems of OE8 and OE10) of a kind of 30 days sizes relatively reaches surviving rate statistics schematic diagram with the wild-type winter resistance.

Fig. 9 A(is left) be the phenotype before transgene tobacco and the contrast subzero treatment; Among the 9A() be 0 ℃ of phenotype of processing after 23 hours; 9A(is right) be 0 ℃ of processing 23 hours and the phenotype after recovering 10 days under 25 ℃.Fig. 9 B is Transgenic Tobacco system and 0 ℃ of processing of wild-type 23 hours and the surviving rate after recovering 10 days under 25 ℃.

Figure 10 is that the PtrbHLH transgene tobacco seedling (two systems of OE8 and OE10) of a kind of 60 days sizes compares schematic diagram with the wild-type winter resistance.

Left figure (on) be that PtrbHLH gene transformation tobacco and wild-type are before subzero treatment; (in) 0 ℃ of phenotype of processing after 23 hours; The phenotype of growth after 5 days recovered by (descending) 25 ℃.Right figure is that another group repeats.

Figure 11 is that 0 ℃ of PtrbHLH transgene tobacco seedling (two systems of OE8 and OE10) and the wild-type of a kind of 30 days sizes processed necrocytosis dyeing, conductance measurement schematic diagram after 23 hours.

Figure 11 A is necrocytosis dyeing; Figure 11 B is conductance measurement.

Figure 12 is that a kind of PtrbHLH transgenosis lemon (#2 and #8) is compared schematic diagram with the wild-type winter resistance.

(on) wild-type and two phenotypes that the transgenic lines subzero treatment is front; (in) be that 0 ℃ of processing changed-3 ℃ of phenotypes of processing after 2 hours in 48 hours over to; (descending) is 25 ℃ and recovers 5 days phenotype of growth.

Figure 13 is that a kind of transgenosis lemon and 0 ℃ of processing of wild-type low temperature changed-3 ℃ of conductance measurement and necrocytosis dyeing schematic diagram of processing 2 hours in 48h hour over to.

Figure 13 A conductance measurement; The dyeing of Figure 13 B necrocytosis.

Figure 14 is a kind of PtrbHLH transgene tobacco and lemon and the corresponding DAB dyeing schematic diagram of wild-type after subzero treatment.

Indication H ₂O ₂Content, bright H is more deeply felt in dyeing ₂O ₂More; Figure 14 A shows the DAB dyeing after subzero treatment of transgene tobacco (OE8 and OE10) and wild-type; Figure 14 B shows the DAB dyeing after subzero treatment of transgenosis lemon (#2 and #8) and wild-type.

Embodiment

Below in conjunction with implementation the present invention is described in detail.According to following description and these enforcements, those skilled in the art can determine essential characteristic of the present invention, and in the situation that do not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that its applicable various uses and condition.

Embodiment 1 PtrbHLH gene isolation Cloning and Expression is analyzed

Take bHLH as keyword search oranges and tangerines est database (HarvEST:Citrus ver.0.51), search the est sequence of 17 and bHLH height homology, utilize its lap to splice, utilize splicing software DNAstart that it is spliced into a sequence, sequential analysis finds that spliced sequence comprises a complete ORF reading frame, as a reference sequences, the design primer amplifies gene PtrbHLH at trifoliate orange according to this splicing sequence well.Use 4 ℃ of RNA(test kits of processing 48 hours of Trizol test kit extracting trifoliate orange blade available from Invitrogen company, extracting is according to the specification sheets operation that provides), with the total RNA sample of 1 μ g of extracting through the DNaseI(of 1U available from Fermentas company) room temperature treatment is after 30 minutes, add 1 μ l EDTA(25mM), in 65 ℃ of incubations 10 minutes.With synthetic the first chain cDNA(article No.: the K1621 of MBI reverse transcription test kit, available from Fermentas company, according to the operation of test kit specification sheets), the first chain cDNA of gained is used for amplification PtrbHLH full length gene.Comprise 200ng cDNA, 1 * damping fluid (TransStart FastPfu damping fluid), 10mM dNTP, 1U Taq polysaccharase (aforementioned damping fluid and polysaccharase are available from TRANS company) and 1.0 μ M primers in the reaction system of 50 μ l.Forward primer: 5 '-TTGTCGACGCAGCGAGTAAAGTATGGCCAATGG-3 '; Reverse primer: 5 '-ATGGTACCGGATCCATGATACCATTACCCTACCAC-3 '.

PCR reaction is at ABI 7500(Applied Biosystem) finish by following program on the amplification instrument: 95 ℃ 1 minute; 95 ℃ of 20 seconds, 58 ℃ 20 seconds, 72 ℃ extensions of annealing of sex change 60 seconds (totally 40 circulations); 72 ℃ were extended 5 minutes after circulation was finished.Produce a PCR band product, behind 1% agarose gel electrophoresis, use

Gel reclaims test kit (available from Omega company, the U.S.) and reclaims special band (extraction step is consulted and used explanation).The dna solution of recovery purifying and pMD18-T carrier are (available from precious biotechnology Dalian company limited, be TaKaRa company) carry out ligation (by specification operation), the mol ratio of PtrbHLH gene and pMD18-T carrier is 3:1 in the ligation system, the ligation cumulative volume is 10 μ l, 2 * the damping fluid (available from precious biotechnology Dalian company limited), the PCR product of 4.5 μ l purifying and the 0.5 μ l T carrier that comprise 5 μ l.16 ℃ of connections are spent the night.Get 10 μ l and connect product, the heat shock method transforms bacillus coli DH 5 alpha, screening positive clone in the LB solid plate that contains 50mgL ammonia benzyl mycin, a picking 2-4 cloning and sequencing.

For whether analyzing the PtrbHLH gene to having coerced response, adopt the expression of this gene of Real-time PCR Analysis under low temperature, salt and processed.The result shows, low temperature (seeing Fig. 2 A), salt (seeing Fig. 2 B) and processed (seeing Fig. 2 C) all can be induced this genetic expression, show that it is an induced gene in adversity.

Embodiment 2PtrbHLH gene Subcellular Localization, transcriptional activation analysis

Because the PtrbHLH gene has 1 nuclear localization signal (NLS), this research and utilization onion epidermis is studied the Subcellular Localization of PtrbHLH gene.Utilize RT-PCR to amplify the whole ORF(reading frame of PtrbHLH gene), and add respectively NcoI and two restriction enzyme sites of SpeI at its amplimer two ends.At first amplified production is contained on the pMD 18-T carrier, thereby obtains a PMD18-T _N/S-PtrbHLH recombinant vectors.Use simultaneously NcoI and SpeI double digestion PMD18-T _N/S-PtrPtrbHLH and pCAMBIA1302 reclaim product and connection, thereby obtain the pCAMBIA1302-PtrbHLH-GFP recombinant vectors, and change it over to Agrobacterium EHA105.Agrobacterium is infected onion epidermis and carries out as follows: (1) draws dull and stereotyped, chooses mono-clonal (Agrobacterium that contains recombinant plasmid) in 3 milliliters of YEB substratum (containing kantlex 40 μ g/ml, Rifampin 25mg/L), and 28 ℃ of concussions were cultivated 24 hours, to OD ₆₀₀Approximately 0.6.(2) with scalpel the onion entocuticle is divided into 1cm ²Size, tearing places upper dark the cultivation 24 hours of MS minimum medium (contain 3% sucrose, 0.75% agar does not contain hormone).(3) be the 1:1000 ratio by volume, inoculate 50 μ l Agrobacterium bacterium liquid and contain kantlex 40 μ g/ml in 50 milliliters of YEB(, Rifampin 25 μ g/ml) in, 28 ℃ shaking culture 12-24 hour.(4) 4000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant.(5) add 50 milliliters of YEB substratum and (contain 10mM MgCl ₂), onion epidermis is put into bacterium liquid soaked 30 minutes.(6) blot surperficial bacterium liquid, place upper 28 ℃ of dark the cultivations two days of MS minimum medium (contain 3% sucrose, 0.75% agar does not contain hormone).With Olympus BX61 type microscopic examination reporter gene location situation, the result shows, in the whole cell fluorescence (Fig. 3 A) is arranged all when control vector transforms, and fluorescence can only detect (Fig. 3 B) in the cell that recombinant vectors transforms in nuclear, illustrates that PtrbHLH is a nuclear locating sequence.

Adopt pcr amplification PtrbHLH gene ORF(amplimer 5 ' end and 3 ' end to add respectively EcoRI and two restriction enzyme sites of XhoI), amplified production reclaims and is subcloned into the linear pENTR with EcoR I and XhoI digestion after digesting with EcoRI and XhoI ^TM3C(is available from Invitrogen company) on the carrier, obtain fusion vector pENTR ^TM3C-PtrbHLH, the recycling recombinant technology is with pENTR ^TMThe 3C-PtrbHLH pDEST that recombinates ^TMOn 32 expression vectors, thereby obtain fusion expression vector pDEST ^TM32-PtrbHLH.Order-checking confirm sequence errorless after with fusion expression vector and empty carrier (pDEST ^TM32) difference transformed yeast bacterial strain MV203(is available from invitrogen), then cultivate at different disappearance substratum.Found that, the yeast cell that empty carrier transforms can only be in disappearance substratum SD/-Trp growth (Fig. 4 A), lack on the substratum SD/-Leu/-Trp/-His fully suppressed at the 3-AT that has added different concns, and the yeast cell that fusion vector transforms can contain at disappearance substratum SD/-Leu/-Trp/-His(the 3-AT of different concns) upper normal growth (Fig. 4 B), show that PtrbHLH has transcriptional activation activity.

Embodiment 3 Plant Transformation overexpression vectors make up

According to the restriction enzyme site analysis on the coding region sequence of the multiple clone site of pMV carrier and PtrbHLH gene, select SalI and KpnI as restriction endonuclease.At first will carry out pcr amplification take PtrbHLH gene cloning as template, and be connected on the pMD18-T carrier by the TA clone again, thereby make up a recombinant vectors pMD18-T-PtrbHLH.Double digestion system cumulative volume is 40 μ l, wherein contains pMD18-T-PtrbHLH plasmid 8 μ l, 10 * M damping fluid (available from Takana), 4 μ l, KpnI and XhoI each 2 μ l, distilled water 24 μ l.Purifying reclaims after 37 ℃ of enzymes are cut 3-4h.The double digestion system of pMV carrier is the same.PtrbHLH gene and carrier pMV are respectively 6 μ l and 2 μ l in the ligation system, and the reaction cumulative volume is 10 μ l, and 10 * T4 connects damping fluid 1 μ l, T4 ligase enzyme 1 μ l, and 16 ℃ connect 14-16 hour.Connect product and transform coli strain DH5 α, in containing the LB solid plate of 100mg/L kantlex, screen, choose after the mono-clonal PCR tests positive extracting plasmid and carry out enzyme and cut, order-checking is determined reading frame not after the sudden change, i.e. pMV-PtrbHLH construction of recombinant vector success.The carrier figure that structure is finished sees Fig. 5.Application freeze-thaw method (with reference to the Pehanorm Brooker, Huang Peitang translates, " molecular cloning experiment guide " third edition, Science Press, 2002) imports to recombinant vectors among the agrobacterium tumefaciens EHA105 and protects bacterium.

Embodiment 4 tobacco genetic transformations

Agrobacterium tumefaciens mediated tobacco genetic transformation step is as follows:

1. agrobacterium tumefaciens is cultivated: get the agrobacterium tumefaciens bacterium liquid of preserving in the Ultralow Temperature Freezer, rule at the LB flat board that has added kantlex 50mg/L, scraping line bacterial plaque adds in the liquid MS minimum medium, 28 ℃ of 180 rev/mins of shaking culture treat that bacterial concentration reaches OD ₆₀₀Contaminate during=0.3-0.8.

2. contaminate: get not genetically modified tobacco leaf, be cut into 0.5cm * 0.5cm size, then put into the agrobacterium tumefaciens bacterium liquid for preparing, soaked 8-10 minute, during constantly vibrate.

3. cultivate altogether: get the tobacco leaf after the dip-dye, aseptic filter paper blots top bacterium liquid, then is inoculated in (leaf back is downward) on the common culture medium 25 ℃ of dark cultivations 3 days.

4. screening and culturing: the tobacco leaf after cultivating altogether 3 days, wash one time with the cephamycin solution of 500mgL concentration, then aseptic water washing is 3-5 time, is transferred in the screening culture medium of having added 100mg/L kantlex and 500mgL cephamycin again.

5. root culture: the indefinite bud on the substratum to be screened is long during to the 1cm left and right sides, downcuts and changes on the root media that has added 100mg/L Km and 500mg/L Cef.

6. tobacco seedling changes earth culture over to: the transformation seedlings after taking root covers with culturing bottle, by taking out in the root media, with the substratum on the clean transformation seedlings of tap water, and plants in the Nutrition Soil of sterilization.

Transformation of tobacco seedling used medium sees Table 1.

Table 1 Transformation of tobacco seedling used medium prescription

7. positive transgene tobacco is tentatively definite

Obtain according to the method described above turning PtrbHLH tobacco resistant buds, extract DNA.Method is as follows: design primer NPTII and 35S+ gene inner primer carry out pcr amplification and identify positive seedling.Be accredited as and independently gather in the crops seed (T1 is for seed) after possible positive plant is transplanted, 4 ℃ of vernalization treatment 3 days, get 0.1g in the centrifuge tube of 1.5ml, use first 70% alcohol-pickled seed 20 seconds, then wash once with the sterilization distilled water, add again 1ml 2.5%NaClO surface sterilization 7 minutes, fully vibration, wash 3 times with the sterilization distilled water after discarding NaClO, be sowed at 50mg/l with the seed that it is good that inoculating needle will go out at last and have the upper of kantlex (Km) MS minimum medium, the result shows resistance seedling energy normal growth on resistant panel that the present invention obtains, and is green, and non-resistance seedling then yellow is die.The plantlet of transplant that survives is gathered in the crops T1 again for seed, and T2 is used for sowing and Analysis of Resistance for seed.

Embodiment 5 lemon genetic transformations

1, gets the lemon seed, NaOH with 1mol/L soaks 15 minutes afterwash, again in Bechtop 2%(volume ratio) clorox soak sterilization 15-20min, the sterilized water washing is peelled off kind of a skin 3 times again under aseptic condition, be inoculated on the MT solid medium, secretly cultivate 3-4 week again illumination cultivation 3-5d be used for to transform.

2, Agrobacterium is rule at the solid LB substratum that contains kantlex 50mg/L, 28 ℃ of dark cultivations two days; Picking list colony inoculation was secretly cultivated 2-3 days for 28 ℃ on the new LB flat board that is added with kantlex; Scrape long good Agrobacterium with scalpel, be seeded in the liquid MT substratum of added with antibiotic not, add simultaneously 20mg/L (100uM) AS, 28 ℃ of 200r/min shaking culture 2h(prepare to cut epicotyl simultaneously during this period of time); OD with spectrophotometer measurement bacterium liquid ₆₀₀Value is adjusted OD with the MT liquid nutrient medium ₆₀₀Value infects to 0.6-0.8.

3, get the seedling epicotyl, be mitered into 1-1.5cm long shoot section in Bechtop, the stem section that cuts temporarily is put in the empty triangular flask of sterilization (adding a small amount of water moisturizing), is used for transforming after the Agrobacterium bacterium solution preparation is finished.

4, the explant that cuts is immersed in the Agrobacterium bacterium liquid that has prepared, infects 20min, the centre is shaken several times.Infected the aseptic thieving paper of rear usefulness and blotted explant surface bacterium liquid, inoculated on the common substratum 21-23 ℃ of dark place and cultivated altogether 3 days.

5, cultivate altogether 3 days after, with aseptic washing 3-5 time, blot again the Agrobacterium on surface with aseptic thieving paper, forward on the screening culture medium that is added with kantlex 50mg/L and cephamycin 400mg/L, 25 ℃ dark cultivates 4 Zhou Houzai and forwards under the illumination condition and cultivate.Grow resistant buds in screening culture medium, when resistant buds〉during 0.5cm, forward again short its elongation on the elongation medium to.When treating that bud has 1.5cm long, downcut and change over to the root media root induction, the lemon conversion process is seen Fig. 6.

Culture medium prescription commonly used:

LB solid medium: peptone 10g/l+ yeast extract 5gl+NaCl 10gl+ agar 15g/l

Lemon sprouts and elongation medium: MT+BA1.0mg/l+ agar 7.5g/l+ sucrose 35gl(pH is 5.8)

The lemon root media: 1/2MT+IBA 0.1mg/l+NAA 0.5mg/l+ gac 0.5g/l+ agar 7.5g/l+ sucrose 35g/l(pH is 5.8)

Embodiment 6 transformed plant molecule and Physiological Appraisals

1 tobacco and lemon blade DNA extraction

Get an amount of blade and put into the 1.5mL centrifuge tube, add liquid nitrogen, fully grind; Then cetyltriethylammonium bromide (the being called for short CTAB) DNA extraction damping fluid (prescription: 100mM Tris-HCl that adds 65 ℃ of preheatings of 700 μ l, pH8.0,1.5M NaCl, 50mM EDTA, pH 8.0, the 1%(mass ratio) polyvinylpyrrolidone, the 2%(mass ratio) CTAB), 65 ℃ of temperature are bathed 60-90 minute (per 15 minutes taking-up centrifuge tubes are put upside down up and down mixing gently in the warm bath process); Centrifugal 10 minutes of 10000 * g; Get supernatant, add 600 μ l chloroforms, put upside down mixing and left standstill 3 minutes; Centrifugal 15 minutes of 10000 * g; Get supernatant 450 μ l, add 900 μ l precooling dehydrated alcohols, 34 μ l 5M NaCl, behind the mixing, freezing 30 minutes, centrifugal 10 minutes of 10000 * g; After abandoning supernatant, with lml 75%(volume ratio) ethanol, wash 3 times after, add an amount of distilled water and dissolve.

2 positive transfer-gen plant PCR detect

Adopt primer NPTII and 35S upstream primer and gene right side inner primer to carry out pcr amplification.Primer sequence, response procedures and system see Table respectively 2, table 3 and table 4.At first adopting NPTII primer pair tobacco regeneration plant to carry out PCR identifies, find that 6 transgenic lines all can amplify the fragment of NPTII gene, utilize 35S upstream primer and gene right side primer to carry out PCR, show that 5 transgenic lines choosing amplify the fragment (Fig. 7 A) of expection size, show their positive transgenic lines.In lemon is identified, adopting 21 regeneration systems of NPTII primer pair to carry out PCR identifies, find that 13 transgenic lines can amplify the fragment (Fig. 7 B) of NPTII gene, utilize 35S upstream primer and gene right side primer pair wherein 7 systems carry out PCR, all amplify the fragment (Fig. 7 C) of expection size, show that they all are transfer-gen plants.

Table 2, primer sequence information

Table 3, PCR response procedures

Table 4, PCR reaction system

Composition	Consumption (μ l)
		Dna profiling	1
10 * PCR damping fluid	2
		dNTP Mix（2.0mmol/L）	2
MgCl 2（25mmol/L）	1.2
		35S promoter primer (10 μ mol/L)	0.3
PtrbHLH reverse primer (10 μ mol/L)	0.3
		Taq archaeal dna polymerase (5U/ μ L)	0.2
Nuclease free water	13

[0084] Embodiment 7 sxemiquantitative RT-PCR detect the overexpression of PtrbHLH gene

The expression amount of foreign gene PtrbHLH in semi-quantitative RT-PCR analysis transgenosis lemon and the tobacco plant is adopted in this research, and transgenic line blade RNA extracts and uses the Trizol test kit, and its step is as follows: 1, mortar precooling.Mortar soaks 10min with 0.5M NaOH, washes 3-4 time with sterilization DEPC again, adds liquid nitrogen precooling (aluminium foil is put the refrigeration of precooling on ice blade); 2, weigh the 0.1g blade in the 1.5ml centrifuge tube, after the liquid nitrogen grinding, add 1ml Trizol mixing (cracking cell also protects RNA).3,4 ℃ of 10000-12000g, centrifugal 15min gets supernatant, adds 200 μ L chloroforms, firmly shakes 15s and hatches 2min, 4 ℃ of 10000-12000g, centrifugal 15min.4, get supernatant, add 500 μ L Virahols, hatch 10min.4 ℃ of 12000g, centrifugal 10min.Join 75% alcohol.5, abandon supernatant, add alcohol (joining with the sterilization DEPC water) jog of 1ml 75%.4 ℃ of 7200g, 5min is centrifugal.6, abandon supernatant, air-dry, precipitation 30-50 μ L DEPC water dissolution ,-80 ℃ of preservations.7, get concentration and the quality that 1-2 μ L detects RNA.Become its method of cDNA with embodiment 1 the RNA reverse transcription, the sxemiquantitative the primer is PtrbHLH gene specific primer (forward primer 5 '-GCACGTCAATGCTGGCTCTCTG-3 '; Reverse primer 5 '-GTGGCAGCAGTGAATGTGTCGGG-3 '), when response procedures is 94 ℃ 3 minutes, 94 ℃ of sex change 30 seconds, 61 ℃ of annealing 30 seconds, 72 ℃ were extended 28 circulations 45 seconds; 72 ℃ were extended 5 minutes after circulation was finished.With the ubiquitin gene of tobacco and oranges and tangerines actin gene respectively as reference gene, tobacco ubiquitin forward primer 5'-TACATAAACGTCACTCTCGATCAC-3', reverse primer 5'-TCCAGGACAAGGAGGGTAT-3 '; Oranges and tangerines ACTIN forward primer is 5'-CCAAGCAGCATGAAGATCAA-3'; Reverse primer is 5'-ATCTGCTGGAAGGTGCTGAG-3'.Expression level to 4 transgenic lines of tobacco, 7 transgenic lines of lemon is analyzed, the result shows, the expression level of No. 8 and No. 10 two systems obviously strengthens (Fig. 8 A in the Transgenic Tobacco plant, describe for convenient, two systems are abbreviated as OE8 and OE10), the expression level of 5 systems is arranged than the obvious enhancing of wild-type (Fig. 8 B) in the lemon, wherein two systems are used for Evaluation of Cold-Resistance, and namely E2 and C8(are abbreviated as respectively #2 and #8).

The Evaluation of Cold-Resistance of embodiment 8 transfer-gen plants

Whether can render transgenic plant winter resistance strengthen for estimating PtrbHLH, at first Transgenic Tobacco system (OE8 and OE10) and wild-type are carried out subzero treatment.Before transgenic lines and wild-type are processed on phenotype without any difference (Fig. 9 A is left).Wild-type and two of the transfer-gen plants of 30 days seedling ages are tied up to 25 ℃ of lower placements two days, then process 23 hours (among Fig. 9 A) for 0 ℃, at last in 25 ℃ of lower recoveries 10 days (Fig. 9 A is right), behind the 10d that restore normal growth, the surviving rate of OE-8 and OE-10 is respectively 80.7% and 79.65%, and contrast only has 33%(Fig. 9 B).

In order further to compare the winter resistance of PtrbHLH transfer-gen plant, with 0 ℃ of processing of 60 days big or small tobacco seedlings (wild-type and transgenosis) 23 hours, then 25 ℃ were recovered 5 days, after finding 0 ℃ of processing, contrast water stain shape degree more serious than transgenic lines, after 25 ℃ of recovery growths, transfer-gen plant recovers growth fraction contrast fast (Figure 10).

Utilize Typan blue that the tobacco leaf necrocytosis degree before and after processing is dyeed, discovery does not have difference between transgenic lines and the wild-type before processing, but the dyeing of wild-type is darker than transgenic lines after subzero treatment, shows the former necrocytosis degree more serious (Figure 11 A).In addition, the specific conductivity of transfer-gen plant is lower than wild-type, shows its film degree lighter (Figure 11 B) that is hurt.

In order to assess PtrbHLH transgenosis lemon overexpression to the resistance of low temperature, in the normal growth situation, contrast and overexpression are that form does not have obvious difference (on Figure 12 A), 0 ℃ process 48 hours after, with-3 ℃ of processing 2 hours, the discovery wild-type was wilted more serious than transgenic lines and is water stain shape (among Figure 12 A), and 25 ℃ are recovered growth 5 days, genetically modified two be recover fast and contrast can not restore normal growth become withered and yellow, and terminal bud dead (under Figure 12 A).Transgenic line and wild-type specific conductivity and necrocytosis are at low temperatures studied, shown under the low temperature, the specific conductivity of transgenic lines is lower than wild-type (Figure 13 A), and its Typan blue dye levels also is lighter than wild-type (Figure 13 B).Above-mentioned result of study shows, the cold tolerance of transgenic lines is stronger than wild-type.

H when embodiment 9 transgene tobaccos and lemon subzero treatment ₂O ₂Content analysis

H after utilizing DAB to subzero treatment in tobacco and lemon transfer-gen plant and the wild-type blade ₂O ₂Accumulating level is analyzed, and finds no matter be tobacco or lemon, and the dyeing of the blade of transfer-gen plant obviously is lighter than wild-type (Figure 14 A is tobacco, and Figure 14 B is lemon) behind the low temperature, shows the H that accumulates in the transgenic line after subzero treatment ₂O ₂Lack than wild-type.

SEQUENCE LISTING

＜110〉Hua Zhong Agriculture University

＜120〉trifoliate orange transcription factor PtrbHLH and the application in improving plant cold resistance

＜130〉trifoliate orange transcription factor PtrbHLH and the application in improving plant cold resistance

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 2179

<212> DNA

<213> Poncirus trifoliata

<400> 1

gcagcgagta aagtatggcc aatggataaa aacagaacag actcaattca tttcagtaca 60

aattttcata acataacata aggttcaaac ttggaatcaa tttctctctt gtctccattt 120

cctttcccaa acgatgacgc cctgcttctg ctgcctctgc tactaaaccc accaagcttt 180

ttacaatttt actcctctgt ggttctctct cactaacaaa aagcattaaa gaaaataaca 240

aaaaagggaa aaatggttct ggaaccaaac ggcgccgttt ggatggaagg tgaagaggag 300

cagccactct cagtttcttg gaccacagcc gccgccgcca ctgccaccac caccgcccgt 360

gccaccacgg agcctaaaga agatgaaatg cacgtcaatg ctggctctct gtcgggtttc 420

aaatcaattc tcgacactga ctggttcttg aactcaactc taaacaaccc acctcaagat 480

tttaccaaca ccaccggctt attagaaacc caccaagaac tcagagcttt caacgctttt 540

caagaaacca acctcttttt tcaaccaatt gaatcacacc ctttcacctt aaacccgaca 600

cattcactgc tgccaccaaa caacaacgac aacaacagca acagccacct cccttttgta 660

agtggctttg atttgggtgg tgaagctgct ggttttattc agccggcttc gggtttcatg 720

ggcctaacga caactcagat ttgtgccact aatgactctg attttcatgg gttcggttct 780

tcttatagca attgctttga taatttagag ggtctgtttt ttaacagtaa cagcaaggct 840

aaagtgtgtt cgcagttaca gccaactctg tttgaaaagc gagcggcttt gaggcagagt 900

tctggtaaat tagaaaactt ggagattttg ggagggaatt tgctgttgga gaacatcaag 960

tgtcggaaaa atgaggaggc tagtgttgat atttcaagtt tgaattatga gtctgatgag 1020

tataataata acaataataa taataatgct agtaatgaca ataatgtgaa tggtaaggtg 1080

gatgagagtg tcaagaattg gaatgctggt ggtagtgcga ctgttgggga taataaaggg 1140

aaaaggaaag gtttgccagc aaagaatttg atggctgaga ggaggagaag gaagaagctt 1200

aatgataggc tttacatgct taggtctgtt gtgcccaaga ttagcaaggt attttatttc 1260

attttacttt ctgttttgtt ttgtttttca attcaattga attataataa tgtgttgata 1320

ggctttacat gcttaggtct gttgtgccca agattagcaa gatggatagg gcttcaatac 1380

tgggggatgc cattgattac ctaaaggaac tcctgcagag gatcaatgat cttcacaatg 1440

aattggagtc aaccccaact ggctctttga tgcagccctc tacaagcatc caacctatga 1500

ccccaactcc acccaccctt ccgtgccgcg tcaaggaaga aataagccgg agtccaacag 1560

gcgaagctgc aagggtggaa gttaggatta gagaaggaag ggctgtgaac attcacatgt 1620

tctgcgctcg taggccgggt ctcttgctct ctactatgag ggctctggac agccttgggt 1680

tggacattca gcaggctgtc atcagctgtt tcaatgggtt tgcactggat gttttccgag 1740

cagagcaatg cagggaaggc caggatgtct tgcccaagca gatcaaatcg gtgctcttgg 1800

atacagctgg cttccatgat gtgatgtaaa atcgaatagc tgtacattca accaccttgt 1860

gagtgaagaa actaactgta gttcttctgt acctcaagta gtatgtaaat gaaattcggt 1920

tctcattttc attttgtaga ggtacgaaca agaccagact gatgcttgta ggtcatcagt 1980

catcatatgt ttggagaatt ggtagtttgg gtaagcatct ttcacagttg tgttcttagt 2040

catgcctcag aaagcacaga aactcaagtg ttgttctttt gtaatttcag ctcgtagaat 2100

aaaatctcct tttcggttac tttaggtgta tttgccatta ctggctttat tggtggtagg 2160

gtaatggtat catggatcc 2179

<210> 2

<211> 487

<212> PRT

<213> Poncirus trifoliata

<400> 2

Met Val Leu Glu Pro Asn Gly Ala Val Trp Met Glu Gly Glu Glu Glu

1 5 10 15

Gln Pro Leu Ser Val Ser Trp Thr Thr Ala Ala Ala Ala Thr Ala Thr

20 25 30

Thr Thr Ala Arg Ala Thr Thr Glu Pro Lys Glu Asp Glu Met His Val

35 40 45

Asn Ala Gly Ser Leu Ser Gly Phe Lys Ser Ile Leu Asp Thr Asp Trp

50 55 60

Phe Leu Asn Ser Thr Leu Asn Asn Pro Pro Gln Asp Phe Thr Asn Thr

65 70 75 80

Thr Gly Leu Leu Glu Thr His Gln Glu Leu Arg Ala Phe Asn Ala Phe

85 90 95

Gln Glu Thr Asn Leu Phe Phe Gln Pro Ile Glu Ser His Pro Phe Thr

100 105 110

Leu Asn Pro Thr His Ser Leu Leu Pro Pro Asn Asn Asn Asp Asn Asn

115 120 125

Ser Asn Ser His Leu Pro Phe Val Ser Gly Phe Asp Leu Gly Gly Glu

130 135 140

Ala Ala Gly Phe Ile Gln Pro Gly Ser Gly Phe Met Gly Leu Thr Thr

145 150 155 160

Thr Gln Ile Cys Ala Thr Asn Asp Ser Asp Phe His Gly Phe Gly Ser

165 170 175

Ser Tyr Ser Asn Cys Phe Asp Asn Leu Glu Gly Leu Phe Phe Asn Ser

180 185 190

Asn Ser Lys Gly Lys Val Cys Ser Gln Ser Gln Pro Thr Leu Phe Glu

195 200 205

Lys Arg Ala Ala Leu Arg Gln Ser Ser Gly Lys Leu Glu Asn Leu Asp

210 215 220

Ile Leu Gly Gly Asn Leu Leu Leu Glu Asn Ile Lys Cys Arg Lys Asn

225 230 235 240

Glu Glu Ala Ser Val Asp Ile Ser Ser Leu Asn Tyr Glu Ser Asp Glu

245 250 255

Tyr Asn Asn Asp Asn Asn Asn Asn Asn Ala Ser Asn Asp Asn Asn Val

260 265 270

Asn Gly Lys Val Asp Glu Ser Val Lys Asn Trp Asn Ala Gly Gly Ser

275 280 285

Ala Thr Val Gly Asp Asn Lys Gly Lys Arg Lys Gly Leu Pro Ala Lys

290 295 300

Asn Leu Met Ala Glu Arg Arg Arg Arg Lys Lys Leu Asn Asp Arg Leu

305 310 315 320

Tyr Met Leu Arg Ser Val Val Pro Lys Ile Ser Lys Met Asp Arg Ala

325 330 335

Ser Ile Leu Gly Asp Ala Ile Asp Tyr Leu Lys Glu Leu Leu Gln Arg

340 345 350

Ile Asn Asp Leu His Asn Glu Leu Glu Ser Thr Pro Thr Gly Ser Leu

355 360 365

Met Gln Pro Ser Thr Ser Ile Gln Pro Met Thr Pro Thr Pro Pro Thr

370 375 380

Leu Pro Cys Arg Ile Lys Glu Glu Ile Ser Arg Ser Pro Thr Gly Glu

385 390 395 400

Ala Ala Arg Val Glu Val Arg Ile Arg Glu Gly Arg Ala Val Asn Ile

405 410 415

His Met Phe Cys Ala Arg Arg Pro Gly Leu Leu Leu Ser Thr Met Arg

420 425 430

Ala Leu Asp Ser Leu Gly Leu Asp Ile Gln Gln Ala Val Ile Ser Cys

435 440 445

Phe Asn Gly Phe Ala Leu Asp Val Phe Arg Ala Glu Gln Cys Arg Glu

450 455 460

Gly Gln Asp Val Leu Pro Lys Gln Ile Lys Ser Val Leu Leu Asp Thr

465 470 475 480

Ala Gly Phe His Asp Val Met

485

Claims (3)

1. the gene of a separation, it is characterized in that: its sequence is shown in the SEQ ID NO.1.

2. the application of gene claimed in claim 1 in the raising tobacco is cold-resistant.

3. the application of gene claimed in claim 1 in the raising lemon is cold-resistant.

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