CN105132436B - A kind of growth hormone receptor gene of poplar adjusted and controlled indefinite root development and its application - Google Patents
A kind of growth hormone receptor gene of poplar adjusted and controlled indefinite root development and its application Download PDFInfo
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- CN105132436B CN105132436B CN201510659032.7A CN201510659032A CN105132436B CN 105132436 B CN105132436 B CN 105132436B CN 201510659032 A CN201510659032 A CN 201510659032A CN 105132436 B CN105132436 B CN 105132436B
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Abstract
Growth hormone receptor gene and its application the present invention relates to a kind of poplar adjusted and controlled indefinite root development, belong to plant genetic engineering and biological technical field.The nucleotide sequence of PtrFBL1 is as shown in the sequence 1 in sequence table；The amino acid sequence of the expressing protein of PtrFBL1 is as shown in sequence 2 in sequence table.The present invention by PtrFBL1 genes by being transferred to 84k poplars, the transgenic poplar of overexpression PtrFBL1 is compared with wild type, it is apparent to take root ahead of time, and adventitious root quantity showed increased, adventitious root overall length and the adventitious root gross area significantly increase, it is the key regulatory genes of poplar adjusted and controlled indefinite root development to illustrate PtrFBL1 genes, has significant application value in Forest-tree Gene Engineering field and Developing Clonal Forestry field.
Description
Technical field
The present invention relates to a kind of growth plain gene of indefinite root development of regulation and control and its application more particularly to one kind are poplar adjusted and controlled
The growth hormone receptor gene PtrFBL1 of indefinite root development and its application, belong to plant genetic engineering and biological technical field.
Background technology
With the completion of the development of Protocols in Molecular Biology and the work of willow gene order-checking in recent years, willow has become
It is a kind of to study the wood formation of perennial plant, growth and development, Seasonal fluctuation, Sex Determination, bloom and biological interaction
Model plant.Willow is a kind of economic tree in world's distribution in extensive range in itself, is not only important wood raw material,
A kind of important energy-source plant is also considered as, has and is distributed wide, adaptable, early stage fast-growing, easy crossbreeding and improvement and breeding etc.
Feature, thus it is widely used in fast growing wood cultivation.Poplar Cultivation mostly carries out vegetative propagation by cuttage, and many excellent at present
Good poplar clones especially Properties of Populus Clones seeds cuttage root-taking is extremely difficult.Therefore, strengthen xylophyta Rooting Mechanism of Cutting to grind
Study carefully, improving willow cuttage radication capability using molecular biology and technique for gene engineering has most important theories meaning and practical application
Value.
Adventitious root refers to the root formed on the stem, leaf or hypocotyl of aboveground vegetation part, and its growth is developed by external ring
Border and the synergistic effect of endogenous hormones.Plant makes it possess the ability of the more root systems of formation by the generation type of adventitious root,
Plant or cell is allowed to be provided with power of regeneration, its this action mode is in the vegetative propagations such as plant tissue cuttage and tissue cultures
It is middle to play increasingly huger effect.In hormone regulating and controlling, auxin is the endogenous hormones of most critical, other hormones mainly lead to
Cross with auxin interaction to adjust the development of adventitious root, auxin not only can directly act on intracellular component and to influence cell anti-
Should, the expression of growth and development related gene can be regulated and controled with indirect mode.Therefore, the molecule of the indefinite root development of xylophyta is studied
Formation mechenism must be related to the specific expression study of related gene of Auxin Signal Tranducation, this process core content
It is the identification of auxin signal and the expression of downstream related gene.Separate the relevant crucial base of auxin regulation and control adventitious root development
Cause identifies its biological function, by genetic improvement plant with difficult rooting is promoted to take root, is in forest molecular breeding important research
Hold, not only there is most important theories meaning to fine-root Developmental Biology research, but also in the production of high quality tree species clonal reproduction
With potential application value.Auxin is tied with growth hormone receptor (TRANSPORT INHIBITOR RESPONSE 1, TIR1)
After conjunction, pass through ubiquitination pathway degradation Aux/IAA transcription inhibitory factors, activation auxin response factor (Auxin response
Factor, ARF) albumen, and then regulate and control the expression of auxin responsive genes.TIR1 is that downstream growth is uniquely adjusted in nucleus
The growth hormone receptor of plain responsive genes transcription, therefore, TIR1 is the key gene of Auxin Signal Tranducation system, is initially formed
SCFTIR1- Aux/IAA complexs, then combined with the cis-acting elements AuxRE of Aux/IAA promoter regions, start Aux/IAA
The proteolysis process of proteins ubiquitin mediation；Aux/IAA protein degradations make ARF start or inhibit auxin downstream responses gene
Transcription, generate auxin effect, complete auxin to the adjusting process of gene expression.In recent years, people plant in a variety of draft
The growth and development for finding different TIR1 genes by regulating and controlling different Gene Expression root systems is studied in object, illustrates TIR1 genes
Plant root system development is played an important role.
At present in xylophyta, the mechanism that growth hormone receptor regulates and controls indefinite root development is rarely reported.In addition, xylophyta
It experienced 2 genome duplication events during evolution, portion gene family is expanded or lost, some gene functions
Also broken up, such as TIR1 gene families, there are 6 members in arabidopsis, there are 8 members (PtrFBL1s) in willow,
Its gene family is expanded, and the expression pattern of homeologous gene breaks up.Therefore, it is research pair using xylophyta
As binding molecule biology and technique for gene engineering parse the molecule developmental mechanism of the poplar adjusted and controlled adventitious root of growth hormone receptor, right
Forest difficult to take root is improved in the molecular basis for understanding xylophyta root system development and by molecular breeding, accelerates forest breeding
It is of great significance.
The content of the invention
For the deficiencies in the prior art, the main object of the present invention is to provide a kind of poplar adjusted and controlled indefinite root system
Growth hormone receptor gene PtrFBL1, it is a further object of the present invention to provide a kind of growth hormone receptors of poplar adjusted and controlled indefinite root system
The application of gene PtrFBL1.
To achieve the above object, the present invention takes following technical scheme：
A kind of growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled indefinite root development, in nucleotide sequence such as sequence table
Sequence 1 shown in.
A kind of expressing protein of the growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled indefinite root development, amino acid sequence
As shown in sequence 2 in sequence table.
A kind of carrier PMDC32- of the growth hormone receptor gene PtrFBL1 containing poplar adjusted and controlled indefinite root development
PtrFBL1, the carrier is in 5 ' end assembling composing type strongly expressed promoter P35S of PtrFBL1 genes；In PtrFBL1 genes
3 ' ends are assembled with strong terminator NOS.
A kind of HPT genes assembled by above-mentioned carrier can be turned as the selection markers of transgenic poplar with hygromycin
The screening of gene willow.The carrier over-assemble has LB sequences and RB sequences, and LB sequences and RB sequences can promote to be assembled in it
Between PtrFBL1 gene integrations into willow recipient cell chromosome.
Applications of the growth of poplar element acceptor gene PtrFBL1 in poplar adjusted and controlled growth and development process.
Advantages of the present invention：The present invention has cloned PtrFBL1 genes using 84k silver gland poplars as material；Meanwhile it builds excessive
Expression vector PMDC32-PtrFBL1, which is located at after promoter P35S, under the driving of promoter P35S, PtrFBL1
Can in willow body high efficient expression, so as to poplar adjusted and controlled adventitious root development.Wherein, PtrFBL1 genes are poplar adjusted and controlled indefinite
The key gene of root development.
Compared with prior art, it is of the invention by the way that PtrFBL1 genes are transferred to 84k silver gland poplars, overexpression PtrFBL1's
Transgenic poplar is compared with wild type, hence it is evident that takes root ahead of time, and adventitious root quantity showed increased, adventitious root overall length and adventitious root are total
Area significantly increases, and it is the key regulatory genes of poplar adjusted and controlled indefinite root development to illustrate PtrFBL1 genes, in Forest-tree Gene Engineering
There is significant application value in field and Developing Clonal Forestry field.
Description of the drawings
Fig. 1 is the structure diagram of plant expression vector PMDC32-PtrFBL1.
Fig. 2-1 to Fig. 2-4 be overexpression PtrFBL1 non-transgenic poplar compared with transgenic poplar root system figure；Figure
2-1 and Fig. 2-2 is 15 days strain root systems after cuttage, and Fig. 2-3 and Fig. 2-4 are strain root system after cuttage 5 months.
Fig. 3 is the quantitative detection figure of transcriptional level of the transgenic poplar of overexpression PtrFBL1.
Fig. 4 is that the 15th day root system occurs for the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, adventitious root not
Determine total root long and total root area after radical mesh compares figure and plants 2 months and compare figure.
Fig. 5-1 and Fig. 5-2 is that the non-transgenic poplar of overexpression PtrFBL1 and transgenic poplar adventitious root occur respectively
6th born chromosome.
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 are to handle wild type (84k) and the root growth figure of transgenic poplar (#B, #D) using auxin
And rooting rate statistical chart.
Fig. 8 A to Fig. 8 H are using root growth figure of the BA processing wild types (84k) with transgenic poplar (#B, #D) and life
Root rate statistical chart.
Fig. 9 A to Fig. 9 D are using the wild type (84k) of PEG6000 processing and the root growth of transgenic poplar (#B, #D)
Figure and rooting rate statistical chart.
Specific embodiment
With reference to specific embodiment, the present invention is described further, is not described in detail in following embodiment
Operation can refer to molecular cloning, and the operation of related kit operation instruction is realized.
Embodiment 1 clones PtrFBL1 genes
Using the silver-colored gland poplars of 84K (P.alba X P.glandulosa) as material, RNeasy Plant Mini kits are used
It is total with the 84K tissue-cultured seedling of RNase-free DNase I kits (Qiagen, Hilden, Germany) extraction cuttage 10 days
RNA.Each sample takes about 3.0 μ g RNA by using SuperScript III first-strand synthesis
System (Life Technologies, Carlsbad, CA, USA) synthesizes the first chains of cDNA.With reference to the comospore Yankee delivered
Because of a group sequence, using 5 software Design primers of Primer (amplicon includes initiation codon and terminator codon), gene is carried out
Overall length amplification (introduces GATEWAY connectors) in primer.
Wherein, PtrFBL1ORF forward primers are (sequence 3 in such as sequence table)：
GGGGACAACTTTGTACAAAAAAGTTGGAATGTTGAGAAAGGCGAATTC,
PtrFBL1ORF reverse primers are (sequence 4 in such as sequence table)：
GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATCAAGAAAACCTTGACACAGAATC；
High fidelity PCR reaction system is as follows：12 μ l of TaKaRa high-fidelity amplification enzymes PrimeSTAR, forward primer (10 μM)
0.8 μ l, 0.8 μ l of reverse primer (10 μM), template (84K poplar cDNA) 0.8 μ l, sterile ddH2O complements to 20 μ l.Response procedures：
98 DEG C of pre-degeneration, 5min；98 DEG C, 30s；56 DEG C, 30s；72 DEG C, 3min, 10 Xun Huans；98 DEG C, 30s；60 DEG C, 30s；72 DEG C,
3min, 25 Xun Huans；72℃10min.
The final full length gene cDNA sequence that obtains is 1755bp, is named as PtrFBL1 genes, sequence in sequence such as sequence table
Shown in 1, expressing protein sequence is compiled as shown in sequence 2 in sequence table.
2 PtrFBL1 gene plant expression vector establishments of embodiment
Using clone technology structure PtrFBL1 genes Overexpression vector, using specific PCR primers (embodiment 1
PtrFBL1ORF primers), using 84K cDNA as template, PCR amplification is carried out, PtrFBL1 genes ORF is building up to entry vector.
Entry vector is PDNOR222.1, and sequence is as shown in sequence 5 in sequence table.Reaction system is Fresh PCR product
80ng；PDNOR222.1 vector 0.4μl；BP ClonaseⅡenzyme mix 0.6μl；Sterile ddH2O complements to 5 μ l.
Response procedures are：25 DEG C of reactions are more than 5h.
Picking positive colony carries out PCR detections and sequence verification from sifting motion cultivation plate, by entering with PtrFBL1 genes
After door carrier is linearized by Mlu I restriction enzymes, with plant expression vector PMDC32, sequence 6 in sequence such as sequence table
It is shown, carry out LR reactions.Reaction system is：Linearized entry clone 50ng；purified destination
vector 75ng；LR ClonaseⅡenzyme mix 0.6μl；TE buffer (pH 8.0) supply 5 μ l.Reaction condition：25
DEG C reaction be more than 5h.After LR reactions, in PtrFBL1 gene transfered plant expression vectors PMDC32, at 5 ' ends of PtrFBL1 genes
Composing type strongly expressed promoter P35S is assembled, PtrFBL1 genes high efficient expression in willow body can be made；In PtrFBL1 genes
3 ' ends are assembled with strong terminator NOS, can effectively terminate the transcription of PtrFBL1 genes, be as shown in Figure 1 plant expression vector
The structure of PMDC32-PtrFBL1.
In carrier over-assemble hygromix phosphotransferase HPT, as the selection markers of transgenic poplar, hygromycin can be used
Carry out the screening of transgenic poplar.In carrier over-assemble LB and RB sequence, promote to assemble PtrFBL1 gene expression frames therebetween
Frame and riddled basins HPT are integrated into willow recipient chromosome.By PCR detections and sequence verification, overexpression is confirmed
Vector construction success, is named as PMDC32-PtrFBL1.The gene is located at after promoter P35S, in the driving of promoter P35S
Under, PtrFBL1 genes can in willow body high efficient expression.
The genetic transformation of embodiment 3PtrFBL1 genes
Constructed PMDC32-PtrFBL1 Overexpression vectors are transferred in Agrobacterium GV3101 by electric shocking method, are passed through
Agriculture bacillus mediated that PtrFBL1 genes are transferred to willow, step of converting is as follows：84K tissue cultures are cloned for the Hybrid Poplar of genetic transformation
Seedling cultivation temperature is 23-25 DEG C, illumination is 16/8h (day/night), intensity of illumination be 50 μM of m-2s-1Under conditions of train
It supports.Agrobacterium containing PMDC32-PtrFBL1 expression vectors infects 84K leaf dishes in OD600=0.6~0.8.After infecting
Leaf dish is in adventitious bud induction culture base (SIM, Murashige-Skoog (MS) minimal medium addition 0.5mg/l 6-benzyl
Aminopurine (6-BA) and 0.05mg/l naphthaleneacetic acid (NAA)) on, it is 23 ± 2 DEG C in temperature
It is co-cultured 3 days under dark condition.Leaf dish after co-cultivation is transferred to containing 3mg/L hygromycin B and 200mg/L
On the SIM of Timentin, cultivation temperature is 23-25 DEG C, illumination is 16/8h (day/night), intensity of illumination be 50 μM of m- 2s-1Under conditions of induction and screening resistance adventitious bud.By the Fiber differentiation of about 30 days, by resistance adventitious bud be transferred to containing
(RIM, 1/2MS minimal medium add in the root media of 3mg/L hygromycin B and 200mg/LTimentin
0.05mg/L IBA and 0.02mg/L NAA), until inducing adventitious root.Extract plant leaf DNA, PCR verification of having taken root.
4 PtrFBL1 genes of embodiment promote Adventitious root initiation by enhancing auxin signal
Using the mode of the transgenic line blade of auxin processing PtrFBL1, verification PtrFBL1 ectopic expressions promote not
Determine the formation of root.It takes wild type (84k) and turns the poplar leaf of PtrFBL1 genes, remove petiole, leave and take the leaf of about 2/3 size
In piece insertion culture medium, (0mg/L IAA, 1mg/L IAA) carries out experiment of taking root in two kinds of culture mediums respectively, is opened from the 11st day
Begin to observe situation of taking root.
As shown in Fig. 2-1 to Fig. 9 D, Fig. 2-1 to Fig. 2-4 is the wild type of overexpression PtrFBL1 and turns base experimental result
Because willow root is compared, wherein, Fig. 2-1 and Fig. 2-2 are respectively the non-transgenic poplar (84k) of overexpression PtrFBL1 and turn
15 days cuttage strains of gene willow (PtrFBL1) root system, Fig. 2-3 and Fig. 2-4 are respectively the non-transgenosis of overexpression PtrFBL1
Willow (84k) and transgenic poplar (PtrFBL1) root (PtrFBL1) are that cuttage is colonized 5 months strains.
Fig. 3 is the quantitative PCR detection of the transgenic poplar of overexpression PtrFBL1, and column diagram shows PtrFBL1 bases in figure
Because of the expression quantity respectively in wild type (84k) and transgenic poplar (#B, #D, #F), #B, #D and #F transgenic poplar are difference
It is different transgenic lines that batch Agrobacterium, which infects wild type (84k) conversion to obtain adventitious bud,.
Fig. 4 is that the 15th day root system occurs for the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, adventitious root not
Determine total root long and total root area after radical mesh compares figure and plants 2 months and compare figure, every group is respectively from left to right transgenosis poplar
Set #B, #D and #F and wild type willow (84k).
Fig. 5-1 and Fig. 5-2 is respectively that the wild type (84k) of overexpression PtrFBL1 and transgenosis (PtrFBL1) willow give birth to
Root compares (adventitious root occurs the 6th day) sooner or later.
Fig. 6 is that the transgenic poplar of overexpression PtrFBL1 and non-transgenic poplar, different time adventitious root are taken root
Rate (roated cuttings, %) compares figure, and column diagram shows root induction wild type (84k) and transgenic poplar (#B, #
D rooting rate), every group is respectively from left to right transgenic poplar #B, #D and wild type willow (84k)；Wild type and transgenosis
The different clones of plant at least count 30 plants, are repeated 3 times.
Fig. 7 A to Fig. 7 H verify PtrFBL1 dystopys by the way of the transgenic line blade of auxin processing PtrFBL1
It is to rely on auxin approach that expression, which promotes the formation of adventitious root, and Fig. 7 A to Fig. 7 C are respectively the in 0mg/LIAA processing procedures
The root growth figure of 12 days wild types (84k) and transgenic poplar (#B, #D), Fig. 7 D to Fig. 7 F are respectively at 1mg/L IAA
The root growth figure of 12nd day wild type (84k) and transgenic poplar (#B, #D) during reason；Fig. 7 G and Fig. 7 H are wilder
Type and the transgenic line of PtrFBL1 (#B, #D), respectively in 0mg/L IAA, 1mg/L IAA processing procedures rooting rate system
It counts (ARs frequency, %).
Fig. 8 A to Fig. 8 H are that PtrFBL1 promotes the rooting rate of adventitious root to be subject to the inhibition of 6-BA (6- benzamido groups purine).Figure
8A to Fig. 8 C is respectively the root of the 12nd day wild type (84k) and transgenic poplar (#B, #D) in 0.05mg/L BA processing procedures
Portion's growth figure, Fig. 8 D to Fig. 8 F are respectively the 12nd day wild type (84k) and transgenic poplar in 0.1mg/L BA processing procedures
The root growth figure of (#B, #D)；Fig. 8 G and Fig. 8 H are to compare wild type and the transgenic line (#B, #D) of PtrFBL1, are existed respectively
Rooting rate statistics (ARs frequency, %) in 0.05mg/L BA, 0.1mg/L BA processing procedures.
Fig. 9 A to Fig. 9 D are that PtrFBL1 promotes the rooting rate of adventitious root to be inhibited be subject to drought stress.A to Fig. 9 C points of Fig. 9
Not Wei in 5%PEG6000 processing procedures the 10th day wild type (84k) with the root growth figure of transgenic poplar (#B, #D), figure
9D is wild type (84k) and the rooting rate statistics (ARs of transgenic poplar (#B, #D) in 5%PEG6000 processing procedures
Frequency, %).
From the results, it was seen that the present invention, by the way that PtrFBL1 genes are transferred to willow, overexpression PtrFBL1's turns base
Because willow is compared with wild type, hence it is evident that it takes root ahead of time, and adventitious root quantity showed increased, adventitious root overall length and the adventitious root gross area
It significantly increases, it is the key regulatory genes of poplar adjusted and controlled indefinite root development to illustrate PtrFBL1 genes, in Forest-tree Gene Engineering field
There is significant application value with Developing Clonal Forestry field.
Claims (1)
1. a kind of growth hormone receptor gene PtrFBL1 of poplar adjusted and controlled indefinite root development is in poplar adjusted and controlled adventitious root growth course
Application, the nucleotide sequence of the growth hormone receptor gene PtrFBL1 is as shown in the sequence 1 in sequence table.
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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 |
| CN115211321B (en) * | 2022-08-22 | 2024-02-20 | 中国林业科学研究院林业研究所 | Early identification method for male and female sex of 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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