CN105132436A - Auxin receptor gene for regulating and controlling development of adventitious roots of poplar and application of auxin receptor gene - Google Patents
Auxin receptor gene for regulating and controlling development of adventitious roots of poplar and application of auxin receptor gene Download PDFInfo
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Abstract
The invention relates to an auxin receptor gene for regulating and controlling development of adventitious roots of a poplar and application of the auxin receptor gene and belongs to the technical field of plant genetic engineering and biology. A nucleotide sequence of PtrFBL1 is shown as the sequence 1 in a sequence table, and an amino acid sequence of expression protein of PtrFBL1 is shown as the sequence 2 in the sequence table. According to the auxin receptor gene for regulating and controlling development of the adventitious roots of the poplar and the application of the auxin receptor gene, the PtrFBL1 gene is transferred into the 84 k poplar, compared with a wild type poplar, the transgenetic poplar with over-expressing PtrFBL1 obviously grows roots early, the number of adventitious roots is obviously increased, the total length of the adventitious roots and the total area of the adventitious roots are obvious enlarged, so that it means that the PtrFBL1 gene is the key regulating and controlling gene for regulating and controlling the development of the adventitious roots of the poplar, and the auxin receptor gene and the application of the auxin receptor gene have important application value in the forest gene engineering field and the clone forest field.
Description
Technical field
The present invention relates to a kind of regulate and control the adventive root growth hormone gene of growing and application thereof, particularly relate to growth hormone receptor gene PtrFBL1 and application thereof that a kind of poplar adjusted and controlled adventive root grows, belong to plant genetic engineering and biological technical field.
Background technology
Along with the development of Protocols in Molecular Biology in recent years and completing of willow gene order-checking work, willow become a kind of study perennial plant wood formation, grow, Seasonal fluctuation, sex determination, to bloom and the biological model plant done mutually.Willow itself is a kind of economic tree of the distribution in extensive range in the world, be not only important wood raw material, also be considered to a kind of important energy-source plant, there is distribute wide, strong adaptability, early stage fast-growing, the easy feature such as crossbreeding and improvement and breeding, be thus widely used in fast growing wood cultivation.Poplar Cultivation is many carries out vegetative propagation by cuttage, and current many excellent poplar clones especially Properties of Populus Clones seeds cuttage root-taking is very difficult.Therefore, strengthen the research of xylophyta Rooting Mechanism of Cutting, utilize molecular biology and genetic engineering technique to improve willow cuttage radication capability and there is most important theories meaning and actual application value.
Adventive root refers to the root that the stem of aboveground vegetation part, leaf or hypocotyl are formed, its synergy by external environment and endogenous hormones of growing.Plant has made it possess by the generation type of adventive root and has formed the ability of more root systems, and allow plant or cell be provided with regenerative power, its this action mode plays more and more huger effect in the vegetative propagations such as plant tissue cuttage and tissue culture.In hormone regulating and controlling, growth hormone is the endogenous hormones of most critical, other hormones are mainly through doing with growth hormone the growth regulating adventive root mutually, and growth hormone not only can directly act on component in born of the same parents and affect cell response, can also indirect mode regulation and control to grow the expression of genes involved.Therefore, the molecule formation mechenism that research xylophyta adventive root is grown, must relate to the expression study that the genes involved of Auxin Signal Tranducation is concrete, this process core content is the identification of growth hormone signal and the expression of downstream genes involved.Be separated growth hormone regulation and control adventive root and grow relevant key gene, identify its biological function, promote that plant with difficult rooting is taken root by genetic improvement, it is forest molecular breeding important research content, not only there is most important theories meaning to the research of fine-root developmental biology, and in high quality tree species clonal reproduction is produced, there is potential using value.Growth hormone and growth hormone receptor (TRANSPORTINHIBITORRESPONSE1, TIR1) after combining, by ubiquitination pathway degraded Aux/IAA transcription inhibitory factor, activate growth hormone response factor (Auxinresponsefactor, ARF) albumen, and then the expression of regulation and control growth hormone responsive genes.TIR1 is the growth hormone receptor uniquely regulating downstream growth hormone responsive genes to transcribe in nucleus, and therefore, TIR1 is the key gene of Auxin Signal Tranducation system, and it first forms SCF
tIR1-Aux/IAA complex body, then be combined with the cis-acting elements AuxRE of Aux/IAA promoter region, start the proteolysis process of Aux/IAA proteins ubiquitin mediation; Aux/IAA proteolytic degradation, makes ARF start or Developing restraint element the transcribing of downstream responses gene, produces growth hormone effect, complete the regulate process of growth hormone to genetic expression.In recent years, people study and find that different TIR1 gene is by regulating and controlling growing of different Gene Expression root systems, illustrates that TIR1 gene pairs plant root system development has important effect in multiple herbaceous plant.
At present in xylophyta, the mechanism that growth hormone receptor regulation and control adventive root is grown rarely has report.In addition, xylophyta experienced by 2 genome duplication events during evolution, portion gene family there occurs expansion or loses, some gene functions also there occurs differentiation, as TIR1 gene family, in Arabidopis thaliana, there are 6 members, in willow, have 8 members (PtrFBL1s), its gene family is expanded, and the expression pattern of portion homologous gene breaks up.Therefore, utilize xylophyta for research object, binding molecule biology and genetic engineering technique, resolve the molecule developmental mechanism of the poplar adjusted and controlled adventive root of growth hormone receptor, for the molecular basis understanding xylophyta root system development, and to be taken root forest by molecular breeding improvement difficulty, accelerate forest and breed significant.
Summary of the invention
For the deficiencies in the prior art, main purpose of the present invention is to provide a kind of growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled indefinite root system, and another object of the present invention is to provide the application of a kind of growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled indefinite root system.
For achieving the above object, the present invention takes following technical scheme:
The growth hormone receptor gene PtrFBL1 that poplar adjusted and controlled adventive root is grown, its nucleotide sequence is as shown in the sequence 1 in sequence table.
The expressing protein of the growth hormone receptor gene PtrFBL1 that poplar adjusted and controlled adventive root is grown, its aminoacid sequence is as shown in sequence in sequence table 2.
A carrier PMDC32-PtrFBL1 containing the growth hormone receptor gene PtrFBL1 that poplar adjusted and controlled adventive root is grown, described carrier is at 5 ' end assembling composing type strongly expressed promotor P35S of PtrFBL1 gene; Strong terminator NOS is assembled with at 3 ' end of PtrFBL1 gene.
The HPT gene assembled by above-mentioned carrier can be used as a selection markers for transgenic poplar, carries out the screening of transgenic poplar with Totomycin.Described carrier is assembled with LB sequence and RB sequence, LB sequence and RB sequence can impel the PtrFBL1 gene integration be assembled in therebetween in willow recipient cell karyomit(e).
The described application of growth of poplar element acceptor gene PtrFBL1 in poplar adjusted and controlled growth and development process.
Advantage of the present invention: the present invention for material, has cloned PtrFBL1 gene with 84k silver gland poplar; Meanwhile, build Overexpression vector PMDC32-PtrFBL1, after this gene is positioned at promotor P35S, under the driving of promotor P35S, PtrFBL1 can in willow body high expression, thus the growth of poplar adjusted and controlled adventive root.Wherein, PtrFBL1 gene is the key gene that poplar adjusted and controlled adventive root is grown.
Compared with prior art, the present invention is by proceeding to 84k silver gland poplar by PtrFBL1 gene, the transgenic poplar of overexpression PtrFBL1 compares with wild-type, obviously take root ahead of time, and adventive root quantity showed increased, adventive root overall length and the adventive root total area enlarge markedly, and illustrate that PtrFBL1 gene is the key regulatory genes that poplar adjusted and controlled adventive root is grown, have significant application value in Forest-tree Gene Engineering field and Developing Clonal Forestry field.
Accompanying drawing explanation
Fig. 1 is the structural representation of plant expression vector PMDC32-PtrFBL1.
Fig. 2-1 to Fig. 2-4 is non-transgenic poplar and the transgenic poplar root system comparison diagram of overexpression PtrFBL1; Fig. 2-1 and Fig. 2-2 is 15 days strain root systems after cuttage, and Fig. 2-3 and Fig. 2-4 is cuttage strain root system after 5 months.
Fig. 3 is the transcriptional level detection by quantitative figure of the transgenic poplar of overexpression PtrFBL1.
Fig. 4 is the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, and adventive root the 15th day root system adventive root number comparison diagram occurs and plants total root length and total root Area comparison figure after 2 months.
Fig. 5-1 and Fig. 5-2 is that the 6th born chromosome occurs for the non-transgenic poplar of overexpression PtrFBL1 and transgenic poplar adventive root respectively.
Fig. 6 is the rooting rate of root induction wild-type (84k) and transgenic poplar (#B, #D, #F) different time.
Fig. 7 A to Fig. 7 H is the root growth figure and the rooting rate statistical graph that adopt growth hormone process wild-type (84k) and transgenic poplar (#B, #D).
Fig. 8 A to Fig. 8 H is the root growth figure and the rooting rate statistical graph that adopt BA process wild-type (84k) and transgenic poplar (#B, #D).
Fig. 9 A to Fig. 9 D adopts the wild-type (84k) of PEG6000 process and the root growth figure of transgenic poplar (#B, #D) and rooting rate statistical graph.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, and the operation be not described in detail in following examples can refer to molecular cloning, and the operation of related kit operation instruction realizes.
Embodiment 1 clones PtrFBL1 gene
With the silver-colored gland poplar of 84K (P.albaXP.glandulosa) for material, RNeasyPlantMini test kit and RNase-freeDNaseI test kit (Qiagen, Hilden, Germany) is used to extract the cuttage 84K tissue cultured seedling total serum IgE of 10 days.Each sample is got about 3.0 μ gRNA and is synthesized cDNA first chain by using SuperScriptIIIfirst-strandsynthesissystem (LifeTechnologies, Carlsbad, CA, USA).With reference to the comospore poplar genome sequence delivered, use Primer5 software design primer (amplicon comprises initiator codon and terminator codon), carry out full length gene amplification (introducing GATEWAY joint in primer).
Wherein, PtrFBL1ORF forward primer is (as sequence in sequence table 3):
GGGGACAACTTTGTACAAAAAAGTTGGAATGTTGAGAAAGGCGAATTC，
PtrFBL1ORF reverse primer is (as sequence in sequence table 4):
GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATCAAGAAAACCTTGACACAGAATC；
High fidelity PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR12 μ l, forward primer (10 μMs) 0.8 μ l, reverse primer (10 μMs) 0.8 μ l, template (84K poplar cDNA) 0.8 μ l, aseptic ddH
2o complements to 20 μ l.Response procedures: denaturation 98 DEG C, 5min; 98 DEG C, 30s; 56 DEG C, 30s; 72 DEG C, 3min, 10 circulations; 98 DEG C, 30s; 60 DEG C, 30s; 72 DEG C, 3min, 25 circulations; 72 DEG C of 10min.
Final acquisition full length gene cDNA sequence is 1755bp, called after PtrFBL1 gene, and sequence is as shown in sequence in sequence table 1, and it compiles expresses protein sequence as shown in sequence in sequence table 2.
Embodiment 2PtrFBL1 gene plant expression vector establishment
Utilize clone technology to build the Overexpression vector of PtrFBL1 gene, use specific PCR primers (the PtrFBL1ORF primer of embodiment 1), take 84KcDNA as template, carry out pcr amplification, PtrFBL1 gene ORF is building up to entry vector.Entry vector is PDNOR222.1, and sequence is as shown in sequence in sequence table 5.Reaction system is FreshPCRproduct80ng; PDNOR222.1vector0.4 μ l; BPClonase II enzymemix0.6 μ l; Aseptic ddH
2o complements to 5 μ l.Response procedures is: 25 DEG C of reactions are greater than 5h.
From sifting motion cultivation plate, picking positive colony carries out PCR detection and sequence verification, by the entry vector with PtrFBL1 gene by after the linearizing of MluI restriction enzyme, with plant expression vector PMDC32, sequence, as shown in sequence in sequence table 6, carries out LR reaction.Reaction system is: Linearizedentryclone50ng; Purifieddestinationvector75ng; LRClonase II enzymemix0.6 μ l; TEbuffer (pH8.0) supplies 5 μ l.Reaction conditions: 25 DEG C of reactions are greater than 5h.After LR reaction, in PtrFBL1 gene transfered plant expression vector PMDC32, at 5 ' end assembling composing type strongly expressed promotor P35S of PtrFBL1 gene, PtrFBL1 gene high expression in willow body can be made; Be assembled with strong terminator NOS at 3 ' end of PtrFBL1 gene, can effectively stop transcribing of PtrFBL1 gene, be illustrated in figure 1 the structure of plant expression vector PMDC32-PtrFBL1.
Carrier assembles hygromix phosphotransferase HPT, as the selection markers of transgenic poplar, the screening of transgenic poplar can be carried out with Totomycin.Carrier is assembled LB and RB sequence, impels the PtrFBL1 gene expression construct that is assembled in therebetween and riddled basins HPT to be integrated in willow recipient chromosome.Detected and sequence verification by PCR, confirm the success of overexpression vector construction, called after PMDC32-PtrFBL1.After this gene is positioned at promotor P35S, under the driving of promotor P35S, PtrFBL1 gene can in willow body high expression.
The genetic transformation of embodiment 3PtrFBL1 gene
By electric shocking method, constructed PMDC32-PtrFBL1 Overexpression vector is proceeded in Agrobacterium GV3101, by agriculture bacillus mediated, PtrFBL1 gene is proceeded to willow, step of converting is as follows: for genetic transformation Hybrid Poplar clone 84K tissue cultured seedling culture temperature be 23-25 DEG C, illumination is 16/8h (daytime/night), intensity of illumination is 50 μMs of m
-2s
-1condition under cultivate.Agrobacterium containing PMDC32-PtrFBL1 expression vector infects 84K leaf dish when OD600=0.6 ~ 0.8.Leaf dish after infecting is at adventitious bud induction culture base (SIM, Murashige-Skoog (MS) minimum medium adds 0.5mg/l6-benzylaminopurine (6-BA) and 0.05mg/lnaphthaleneaceticacid (NAA)) on, be Dual culture 3 days under the dark condition of 23 ± 2 DEG C in temperature.Leaf dish after Dual culture is transferred on the SIM containing 3mg/LhygromycinB and 200mg/LTimentin, culture temperature be 23-25 DEG C, illumination is 16/8h (daytime/night), intensity of illumination is 50 μMs of m
-2s
-1condition under induction and screening resistance indefinite bud.Through the inducing culture of about 30 days, resistance indefinite bud is transferred to (RIM in the root media containing 3mg/LhygromycinB and 200mg/LTimentin, 1/2MS minimum medium adds 0.05mg/LIBA and 0.02mg/LNAA), until induce adventive root.Extract plant leaf DNA, PCR checking of having taken root.
Embodiment 4PtrFBL1 gene promotes Adventitious root initiation by strengthening growth hormone signal
Utilize the mode of the transgenic line blade of growth hormone process PtrFBL1, checking PtrFBL1 ectopic expression promotes the formation of adventive root.Get wild-type (84k) and turn the poplar leaf of PtrFBL1 gene, remove petiole, the blade leaving and taking about 2/3 size inserts in substratum, (0mg/LIAA in two kinds of substratum respectively, 1mg/LIAA) carry out experiment of taking root, from the 11st day, observation was taken root situation.
Experimental result is as shown in Fig. 2-1 to Fig. 9 D, Fig. 2-1 to Fig. 2-4 is that the wild-type of overexpression PtrFBL1 compares with transgenic poplar root, wherein, Fig. 2-1 and Fig. 2-2 is respectively non-transgenic poplar (84k) and transgenic poplar (PtrFBL1) root system 15 days cuttage strains of overexpression PtrFBL1, and Fig. 2-3 and Fig. 2-4 is respectively the non-transgenic poplar (84k) of overexpression PtrFBL1 and transgenic poplar (PtrFBL1) root (PtrFBL1) is cuttage field planting 5 months strains.
Fig. 3 is the quantitative PCR detection of the transgenic poplar of overexpression PtrFBL1, the column diagram display expression amount of PtrFBL1 gene respectively in wild-type (84k) and transgenic poplar (#B, #D, #F) in figure, #B, #D and #F transgenic poplar is that different batches Agrobacterium is infected wild-type (84k) and transforms acquisition indefinite bud and be different transgenic line.
Fig. 4 is the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, there is the 15th day root system adventive root number comparison diagram and plant total root length and total root Area comparison figure after 2 months in adventive root, often organizes and be from left to right respectively transgenic poplar #B, #D and #F and wild-type willow (84k).
The wild-type (84k) that Fig. 5-1 and Fig. 5-2 is respectively overexpression PtrFBL1 and transgenosis (PtrFBL1) willow take root and sooner or later compare (adventive root occurs the 6th day).
Fig. 6 is the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, different time adventive root generation rooting rate (roatedcuttings, %) comparison diagram, column diagram display root induction wild-type (84k) and the rooting rate of transgenic poplar (#B, #D), often organize and be from left to right respectively transgenic poplar #B, #D and wild-type willow (84k); Wild-type clone different from transfer-gen plant at least adds up 30 strains, repeats 3 times.
Fig. 7 A to Fig. 7 H adopts the mode of the transgenic line blade of growth hormone process PtrFBL1, checking PtrFBL1 ectopic expression promotes that the formation of adventive root relies on growth hormone approach, Fig. 7 A to Fig. 7 C is respectively the root growth figure of the 12nd day wild-type (84k) and transgenic poplar (#B, #D) in 0mg/LIAA treating processes, and Fig. 7 D to Fig. 7 F is respectively the root growth figure of the 12nd day wild-type (84k) and transgenic poplar (#B, #D) in 1mg/LIAA treating processes; Fig. 7 G and Fig. 7 H is the transgenic line (#B, #D) comparing wild-type and PtrFBL1, rooting rate statistics (ARsfrequency, %) respectively in 0mg/LIAA, 1mg/LIAA treating processes.
Fig. 8 A to Fig. 8 H is that PtrFBL1 promotes that the rooting rate of adventive root is subject to the suppression of 6-BA (6-benzamido group purine).Fig. 8 A to Fig. 8 C is respectively the root growth figure of the 12nd day wild-type (84k) and transgenic poplar (#B, #D) in 0.05mg/LBA treating processes, and Fig. 8 D to Fig. 8 F is respectively the root growth figure of the 12nd day wild-type (84k) and transgenic poplar (#B, #D) in 0.1mg/LBA treating processes; Fig. 8 G and Fig. 8 H is the transgenic line (#B, #D) comparing wild-type and PtrFBL1, rooting rate statistics (ARsfrequency, %) respectively in 0.05mg/LBA, 0.1mg/LBA treating processes.
Fig. 9 A to Fig. 9 D is that PtrFBL1 promotes that the rooting rate of adventive root is subject to the suppression of drought stress.Fig. 9 A to Fig. 9 C is respectively the root growth figure of the 10th day wild-type (84k) and transgenic poplar (#B, #D) in 5%PEG6000 treating processes, Fig. 9 D is that wild-type (84k) adds up (ARsfrequency, %) with the rooting rate of transgenic poplar (#B, #D) in 5%PEG6000 treating processes.
As can be seen from the results, the present invention is by proceeding to willow by PtrFBL1 gene, the transgenic poplar of overexpression PtrFBL1 compares with wild-type, obviously take root ahead of time, and adventive root quantity showed increased, adventive root overall length and the adventive root total area enlarge markedly, and illustrate that PtrFBL1 gene is the key regulatory genes that poplar adjusted and controlled adventive root is grown, have significant application value in Forest-tree Gene Engineering field and Developing Clonal Forestry field.
Claims (3)
1. a growth hormone receptor gene PtrFBL1 for poplar adjusted and controlled adventive root growth, is characterized in that: its nucleotide sequence is as shown in the sequence 1 in sequence table.
2. an expressing protein of the growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled adventive root growth, is characterized in that: its aminoacid sequence is as shown in sequence in sequence table 2.
3. the growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled adventive root growth according to claim 1, the application in poplar adjusted and controlled growth and development process.
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| CN115211321A (en) * | 2022-08-22 | 2022-10-21 | 中国林业科学研究院林业研究所 | Early identification method for sex of male and female poplar hybrid progeny |
| CN116064593A (en) * | 2023-02-09 | 2023-05-05 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
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Cited By (5)
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| CN106916828A (en) * | 2017-05-03 | 2017-07-04 | 中国林业科学研究院林业研究所 | A kind of growth regulator gene of poplar adjusted and controlled leaf development and its application |
| CN115211321A (en) * | 2022-08-22 | 2022-10-21 | 中国林业科学研究院林业研究所 | Early identification method for sex of male and female poplar hybrid progeny |
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| CN116064593A (en) * | 2023-02-09 | 2023-05-05 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
| CN116064593B (en) * | 2023-02-09 | 2024-05-14 | 四川大学 | PGAG gene of populus tomentosa and application thereof |
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