CN105624180B - HbCS3 gene is improving prokaryotic expression bacterium growth rate, the anti-environment stress of research rubber tree and is producing the application in glue ability - Google Patents
HbCS3 gene is improving prokaryotic expression bacterium growth rate, the anti-environment stress of research rubber tree and is producing the application in glue ability Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Abstract
The present invention provides HbCS3 genes to improve prokaryotic expression bacterium growth rate, the anti-environment stress of research rubber tree and produce the application in glue ability and the recombinant and recombination bacillus coli of carrying HbCS3 gene.After HbCS3 gene is transferred to prokaryotic expression bacterial strain, growth rate of the bacterial strain under normal and heavy metal stress under condition of culture can be significantly improved, is had a good application prospect in the genetic engineering of microorganism.The high expression in male flower, bark and seed of HbCS3 gene, it is expressed simultaneously by ethephon (CEPHA),2-(chloroethyl) phosphonic acid, rubber tapping, high temperature and low temperature stress inducible up regulation, it is closely related with rubber tree yield traits and anti-environment stress, the important target gene that can be used as rubber tree transgenic breeding is expected to the expression by regulating and controlling the gene rationally to excavate the production glue of rubber tree and degeneration-resistant potentiality.In addition, the gene can be used as important genetic resources, it is also possible to be applied in the genetic engineering of the other plant other than rubber tree.
Description
Technical field
The invention belongs to biological fields, and in particular to HbCS3 gene improve prokaryotic expression bacterium growth rate in application,
HbCS3 gene in the research anti-environment stress of rubber tree and produces the application in glue ability as target gene, further relates to a kind of carrying
The recombinant and recombination bacillus coli of HbCS3 gene.
Background technique
Citrate synthase (citrate synthase, CS, EC4.1.3.7) is almost present in all organisms, is
The intracellular crucial rate-limiting enzyme of multiple metabolic pathways and marker enzyme (Ge Yadong, Pan Wei, Wang's Jie, Zhu state duckweed lemon of metabolic alterations
Progress on Molecular Biology biology magazine .2010,27 (3): 59-62. of acid synthase).CS has the substrate specificity of height
Property, it is only catalyzed acetyl coenzyme A and oxaloacetic acid condensation generates citric acid and coacetylase.CS has a variety of isodynamic enzymes, according to its presence
In different subcellular structures, CS points are mitochondria CS (mCS) and peroxisome CS (gCS) (Schnarrenberger
C,Martin William.Evolution of the enzymes of the citric acid cycle and the
glyoxylate cycle of higher plants.A case study of endosymbiotic gene
transfer.European Journal of Biochemistry.2002,269(3):868-883.).MCS is in mitochondria
The key enzyme of tricarboxylic acid cycle (TCA) starting participates in intracellular main energetic supersession；PCS is the key that in peroxidase precursor
One of enzyme participates in the oxidation and cell detoxification process of intracellular fatty acid.The activity change of CS directly results in intracellular acetyl
The variation in coacetylase pond, and acetyl coenzyme A is the primary raw material source of fatty acid synthesis, the variation in acetyl coenzyme A pond is by direct shadow
Ring the synthesis of fatty acid.In recent years, research find CS participate in various physiological processes, as mitochondrial, seed sprout and
Degeneration-resistant etc. (Schmidtmann E,AC,Orwat A,Leister D,Hartl M,Finkemeier I.Redox
regulation of Arabidopsis mitochondrial citrate synthase.Mol Plant.2014,7(1):
156-69.Pracharoenwattana I,Cornah JE,Smith SM.Arabidopsis peroxisomal citrate
synthase is required for fatty acid respiration and seed germination.Plant
Cell.2005,17(7):2037-48.Koyama H,Kawamura A,Kihara T,Hara T,Takita E,Shibata
D.Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana
improved growth on a phosphorus-limited soil.Plant Cell Physiol.2000,41(9):
1030-7. child Shanxi, Zhan Gaomiao, Wang Xinfa, Liu Guihua, Hua Wei, the clone of Wang Hanzhong rape citrate synthase gene and in adverse circumstance
Under expression Acta Agronomica Sinica .2009,35 (1): 33-40.).
Para rubber tree is the main source of one of four big raw materials of industry natural rubber, and natural rubber (rubber hydrocarbon), which has, to be closed
At the synthesis merit that rubber is incomparable, there is high economic value and application prospect.Acetyl coenzyme A is that rubber hydrocarbon closes
At precursor substance, directly participate in rubber tree natural rubber biosynthesis, it is auxiliary that the activity change of CS directly affects acetyl intracellular
The variation in the pond enzyme A, and then influence rubber tree and produce the glue ability (biology of rubber in Zou Zhi, Yang Lifu, Wang Zhenhui, Yuan Kun rubber tree
Synthesis and regulation Plant Physiology Communications .2009:45 (12): the good fortune cities 1231-1238., old to keep ability Para rubber tree natural
Key enzyme and Advances of Genes tropical agricultural science, 2006,26 (1): 42-46. in rubber biosynthesis).In addition,
Rubber tree produces glue ability and the extraneous poor environment factor ability of resistance is closely related, and such as cold damage, windburn and disease are directly affected
Rubber tree yield.Therefore, rubber tree CS rubber tree growth and development (rubber hydrocarbon biosynthesis) and resist biology with non-life
Play a significant role in object stress, analysis CS will be helpful to further appreciate that the regulatory mechanism in acetyl coenzyme A pond intracellular and in rubber
Important function in gum metabolism.Rubber tree citrate synthase (CS) gene and enzyme activity characteristic have not been reported at present.
Summary of the invention
It is an object of the invention to overcome deficiency in the prior art, to rubber tree citrate synthase gene HbCS3 gene
Gene expression analysis and functional study have been carried out, it is found that it is transferred to the transgenic strain obtained after prokaryotic expression bacterial strain to the gene
Growth rate significantly improves under condition of culture under normal and heavy metal stress, and related to elastomer gum milk production, can answer
For improving rate of bacterial growth, it can be used as target gene and apply in the research that rubber tree produces glue and anti-environment stress ability.
The first aspect of the invention is to provide a kind of genetic recombinants, described for the recombinant for carrying HbCS3 gene
The sequence of HbCS3 gene is as shown in seqid no:1.
Wherein, the carrier of the recombinant is expression plasmid or viral vectors.
Wherein, the viral vectors is selected from adenovirus vector, herpes simplex virus vector, retrovirus vector, gland companion
With one of viral vectors, slow virus carrier, preferably adenovirus vector.
The second aspect of the invention is to provide a kind of recombination bacillus coli of fast-growth, and the recombination bacillus coli contains
There are any one genetic recombinants described in first aspect of the present invention.
The third aspect of the invention is to provide recombination bacillus coli described in the second aspect of the present invention in genetic engineering
Application in bacterium.
The fourth aspect of the invention is to provide HbCS3 gene and is improving the application in prokaryotic expression bacterium growth rate.
Further, HbCS3 gene is applied to improve growth rate of the prokaryotic expression bacterium under heavy metal stress.
Wherein, the heavy metal be copper, and/or lead, and/or zinc, and/or tin, and/or nickel, and/or cobalt, and/or antimony,
And/or mercury, and/or cadmium, and/or bismuth.
Further, the prokaryotic expression bacterium is Escherichia coli.
The fifth aspect of the invention is to provide HbCS3 gene as target gene in the research anti-environment stress ability of rubber tree
In application.
The sixth aspect of the invention be to provide HbCS3 gene as target gene research rubber tree production glue ability in
Using.
Beneficial effects of the present invention:
After HbCS3 gene is transferred to prokaryotic expression bacterial strain, bacterial strain can be significantly improved and cultivated under normal and heavy metal stress
Under the conditions of growth rate, by carry HbCS3 gene prokaryotic expression bacterial strain be applied to engineering bacteria, the engineering bacteria speed of growth is fast,
Efficiency can be significantly improved, cost etc. is reduced, is had a good application prospect in the genetic engineering of microorganism.In addition, HbCS3 base
Because of expression high in male flower, bark and seed, while by ethephon (CEPHA),2-(chloroethyl) phosphonic acid and rubber tapping inducible up regulation expression, it is in elastomer gum milk production
It is positively correlated, which is also expressed by high temperature and low temperature stress inducible up regulation simultaneously, therefore the gene may be yield with rubber tree
Shape and anti-environment stress are closely related, can be used as the important target gene of rubber tree transgenic breeding, are expected to by regulating and controlling the gene
Expression come rationally excavate rubber tree production glue and degeneration-resistant potentiality.In addition, the gene can be used as important genetic resources, it is also possible to
It is applied in the genetic engineering of the other plant other than rubber tree.
Detailed description of the invention
The prokaryotic expression that Fig. 1 is HbCS3 is analyzed；
Fig. 2 is HbCS3 recombinant protein Enzyme activity assay；
Fig. 3 is the growth rate measurment of transgenic strain under normal operation；
Fig. 4 is growth rate measurment of the transgenic strain under heavy metal copper ionic stress；
Fig. 5 is that the tissue specific expression of HbCS3 gene is analyzed；
Fig. 6 is influence of the rubber tapping to HbCS3 gene expression；
Fig. 7 is influence of the ethephon (CEPHA),2-(chloroethyl) phosphonic acid to HbCS3 gene expression；
Fig. 8 is influence of the high temperature stress to HbCS3 gene expression；
Fig. 9 is influence of the low temperature stress to HbCS3 gene expression.
Specific embodiment
With reference to the accompanying drawings, the present invention is further described in conjunction with specific embodiments, to more fully understand this hair
It is bright.
1. the acquisition of rubber tree citrate synthase encoding gene (HbCS)
Analysis in the nucleotide sequence of the citrate synthase (CS) of NCBI arabidopsis, rice, poplar and the castor-oil plant logged in,
By searching for the latex of panama rubber tree est sequence database of our foundation, the rubber tree lemon for screening and splicing a 1900bp or so
Sequence (contig) after the assembling of lemon synthase genes, design a pair of of special primer expand to obtain the cDNA comprising complete reading frame it is complete
Long sequence.
The specific method is as follows for cDNA clone:
Specific primer design is as follows:
F (5 ' end): 5 '-AATCGACTATCGGTTTCGCTT-3 '；
R (3 ' end): 5'-GCCACGAATTGCAAAATAGTAGAT-3'.
Grinding 7-33-97 with Para rubber tree heat, (Rubber Institute, Chinese Academy of Agricultural Science cultivates, Tropical China agricultural
There is seedling sale in rubber research institute of the academy of sciences for a long time) leaf cDNA is template (being obtained with random primed reverse transcription), F and R are to draw
Object, final concentration of 0.4 μm of ol/L carry out PCR amplification in 20 μ l reaction systems.Amplification program are as follows: 94 DEG C of initial denaturation 4min；
94 DEG C of denaturation 45S, 68 DEG C of annealing 3min, totally 30 times circulation；72 DEG C of extension 10min.
The PCR segment obtained is connected to pMD18-T carrier (TaKaRa) to be sequenced, sequencing shows above-mentioned acquisition
Segment is citrate synthase of the invention (CS) gene, which has the nucleotide sequence of SEQ ID NO:1, sequence
In list SEQIDNO:1 overall length be 1779 nucleotide, comprising a length be 1536 nucleotide open reading frame (ORF,
Hold 45-1580 nucleotide sequences from the 5 ' of sequence 2), 5 '-UTR of 44 nucleotide are (from 5 ' 1-44, ends of sequence 2
Nucleotide sequence) and 219 nucleotide 3 '-UTR (holding 1581-1799 nucleotide sequences from the 5 ' of sequence 2), encode one
The albumen that a length is 511 amino acid (SEQ ID NO:2), molecular weight is about 56kDa, as citrate synthase
(CS), which is named as HbCS3.The pMD18-T of the above-mentioned nucleotide containing SEQIDNO:1 is recombinated and is carried
Body is named as pMD18-HbCS3.In addition to this, online using subcellular localization forecasting software (ProtComp Version 9.0)
The albumen is positioned in peroxidase precursor by the amino acid sequence for analyzing the albumen, therefore HbCS3 belongs to rubber tree peroxide
Figure citrate synthase.
2. the functional verification of prokaryotic expression and HbCS3 gene
Using pMAL-c5E expression vector, (pMAL-c5E (plasmid) expression vector is public purchased from New England Biolabs
Department) construct HbCS3 gene prokaryotic expression carrier (it should be understood that expression vector is only for example in the present embodiment, this hair
It is bright to use other expression plasmids and viral vectors etc.), while utilizing E. coli expression strains E.coli BL21
(DE3) (bacterial strain is purchased from TransGen Biotech company) induction recombinant protein, measurement recombinant protein are active and its raw to BL21
The influence of long rate, the specific method is as follows:
<1>acquisition of the recombinant vector of the gene coding region containing HbCS3
Design the gene coding region HbCS3 primer (F:5'-CGG GGT ACC GAT GTC AGA ATC TGA TTC TTC
CTT ATC CTC CG-3', R:5'-CCG GAA TTC CTA AAT CCC AGA TCC AGC AAG TCG TCG-3', should
Kpn I and EcoR I restriction enzyme site is respectively provided with to primer), using pMD18-HbCS3 as template, carry out PCR amplification, amplification program
Are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 45s, 68 DEG C of annealing 2min, totally 30 times circulation；72 DEG C of extension 5min.By amplified production
Kpn I and EcoR I double digestion is carried out with pMAL-c5E expression vector and is connect, and is obtained recombinant vector, is utilized gene specific primer
(gene coding region HbCS3 primer) carries out PCR identification, it is ensured that citrate synthase coding segment is cloned into expression vector.Recombination carries
Body carries out sequencing identification, and identification is shown that correctly 45-1580 nucleotide sequences, frames containing SEQIDNO:1 are accurate
Recombinant expression carrier be named as pMAL-c5E-HbCS3.
<2>prokaryotic expression of HbCS3 gene
The recombinant vector pMAL-c5E-HbCS3 of acquisition and control vector pMAL-c5E-Empty (empty carrier) is imported
In E.coli BL21 (DE3) (competence is purchased from Tiangeng biochemical technology Co., Ltd), recombinant expression bacterium is obtained, identification is correct
Recombinant bacterium in the LB culture medium containing 50 μ g/mL ampicillins and 25 μ g/mL chloramphenicol culture to OD600=0.4~
0.6, add IPTG (isopropyl-beta D-thio galactopyranoside) extremely final concentration of 1mM, Fiber differentiation 4h at 30 DEG C, centrifugation
Thallus is collected, mycoprotein carries out 12%SDS-PAGE electrophoresis detection, as a result as shown in Figure 1.Fig. 1 shows in the case where IPTG is induced
HbCS3 gene realizes the efficient heterogenous expression in cytoplasm, and recombinant protein contains HbCS3 and cytomixis albumen
MBP (maltose-binding protein) molecular weight is about at 100kDa (HbCS3 about 56kDa, MBP approximately 45 kda).
<3>HbCS3 recombinant protein Enzyme activity assay
Thallus is collected according to " prokaryotic expression of<2>HbCS3 gene " above method, low temperature ultrasonic is broken, is collected by centrifugation
Clear measurement citrate synthase is active (citrate synthase kit purchases Suzhou Ke Ming Bioisystech Co., Ltd).Measurement result is such as
Shown in Fig. 2: enzyme activity is the results show that the activity containing pMAL-c5E-HbCS3 bacterial strain citrate synthase is apparently higher than containing pMAL-
C5E-Empty control strain (5 times), this illustrates that the albumen of HbCS3 gene coding has the activity of citrate synthase really.
<4>influence of the HbCS3 recombinant protein to strain growth rate
According to " prokaryotic expression of<2>HbCS3 gene " above method activated strains, and will containing pMAL-c5E-HbCS3 and
The strain culturing of pMAL-c5E-Empty adds IPTG (isopropyl-beta D-thio galactopyranose to identical OD600=0.5
Glycosides) to final concentration of 1mM, it measures its OD (600nm) value at 30 DEG C after Fiber differentiation 0.5h, 1h and 2h respectively, calculates growth speed
Rate.As a result as shown in Figure 3: the growth rate containing pMAL-c5E-HbCS3 bacterial strain is higher than pMAL-c5E-Empty control strain,
And become apparent in induction 1h, illustrate that the growth rate of bacterial strain under normal operation can be improved in the albumen of HbCS3 gene coding.
In addition to this, it determines containing pMAL-c5E-HbCS3 and pMAL-c5E-Empty bacterial strain in heavy metal copper ion
(Cu2+, 600 μm) under growth rate, induction time is respectively 0h, 1h, 2h, 3h, 4h and 5h, measures its OD (600nm) respectively
Value calculates growth rate.As a result as shown in Figure 4: the growth rate containing pMAL-c5E-HbCS3 bacterial strain is higher than pMAL-c5E-
Empty control strain, and become apparent in induction 2h and 3h, illustrate that bacterial strain can be improved in weight in the albumen of HbCS3 gene coding
Growth rate under metal copper ion stress.It is coerced using other heavy metals such as lead, zinc, tin, nickel, cobalt, antimony, mercury cadmium, bismuths
As a result test is tested with heavy metal copper ionic stress, the growth rate of bacterial strain can be improved in the albumen of HbCS3 gene coding.
Other prokaryotic expression bacterium of HbCS3 channel genes are subjected to above-mentioned test, as a result same Escherichia coli, HbCS3 gene is compiled
Code albumen can be improved other protokaryon bacterial strains under normal operation with the growth rate under the conditions of heavy metal stress.
3.HbCS3 Gene Expression Profile Analysis
<1>HbCS3 Tissue-specific expression
Using rubber tree 7-33-97 blade, female flower, male flower, latex, seed and the cDNA of the random reverse transcription of bark RNA as mould
Plate, with HbCS3 gene specific primer (F:5'-CAA AGC CTC TGA TTT CAA AAA GAT-3'；R:5'-CAC TTC
CAA AAA GGT GCT ACT-3') real-time fluorescence quantitative PCR is carried out, as a result (relative expression quantity is with the table of latex as shown in Figure 5
It is control up to amount), HbCS3 gene is high in male flower, bark and seed to express, and expresses in female flower, blade and latex relatively low.
<2>tapping rubber influences the expression of HbCS3 gene
Using do not open cut Para rubber tree heat grind 7-33-97 difference knife time the random reverse transcription of latex RNA cDNA as template
(three days knives, five knife of continuous sampling), with HbCS3 gene specific primer (F:5'-CAA AGC CTC TGA TTT CAA AAA
GAT-3'；R:5'-CAC TTC CAA AAA GGT GCT ACT-3') real-time fluorescence quantitative PCR is carried out, as a result as shown in Figure 6
(relative expression quantity is control with the expression quantity of the first knife latex).The result shows that rubber tapping influences the expression of HbCS3 gene, preceding two knife
The expression of middle HbCS3 gene upregulation, is declined slightly in third and the 4th knife, and stable gene expression abundance is kept in four subsequent knives not
Become.
<3>ethephon (CEPHA),2-(chloroethyl) phosphonic acid influences the expression of HbCS3 gene
Ethephon (CEPHA),2-(chloroethyl) phosphonic acid ET is applied to above rubber tree secant and secant at 1cm face stimulate rubber tree (0h, 12h, for 24 hours and
48h), using the cDNA of the random reverse transcription of latex RNA as template, with HbCS3 gene specific primer (F:5'-CAA AGC CTC TGA
TTT CAA AAA GAT-3'；R:5'-CAC TTC CAA AAA GGT GCT ACT-3') carry out real-time fluorescence quantitative PCR, knot
Fruit is as shown in Figure 7 (relative expression quantity is to induce the expression quantity of 0h for control).The result shows that: under ethephon (CEPHA),2-(chloroethyl) phosphonic acid ET stimulation, HbCS3
Gene upregulation expression, and in 48h to reach to peak value.
<4>high temperature stress influences the expression of HbCS3 gene
Rubber tree seedling is carried out high temperature (45 DEG C) Stress treatment (0,3h, 6h, 9h, 12h and for 24 hours), it is random with blade RNA
The cDNA of reverse transcription is template, with HbCS3 gene specific primer (F:5'-CAA AGC CTC TGA TTT CAA AAA GAT-
3'；R:5'-CAC TTC CAA AAA GGT GCT ACT-3') real time fluorescent quantitative PC is carried out, it is as a result (opposite as shown in Figure 8
Expression quantity is to induce the expression quantity of 0h for control).The result shows that: under high temperature stress, up-regulated expression in the HbCS3 gene short time
Restore again afterwards to Initial abundance, reaches peak value in 3h.
<5>low temperature stress influences the expression of HbCS3 gene
Rubber tree seedling is carried out low temperature (4 DEG C) Stress treatment (0,3h, 6h, 9h, 12h and for 24 hours), it is random with blade RNA
The cDNA of reverse transcription is template, with HbCS3 gene specific primer (F:5'-CAA AGC CTC TGA TTT CAA AAA GAT-
3'；R:5'-CAC TTC CAA AAA GGT GCT ACT-3') real-time fluorescence quantitative PCR is carried out, it is as a result (opposite as shown in Figure 9
Expression quantity is to induce the expression quantity of 12h for control).The result shows that: under low temperature stress, upper mileometer adjustment in the HbCS3 gene short time
Up to rear and recovery to Initial abundance, reaches peak value in 3h, minimized in 12h.
The present invention has cloned the full-length cDNA (peroxide figure) of rubber tree citrate synthase HbCS3 gene for the first time, warp
Prokaryotic expression simultaneously measures recombinant protein activity and shows that the albumen of gene coding has the function of citrate synthase, the gene turn
Entering the transgenic strain that obtains after prokaryotic expression bacterial strain, growth rate is obvious under condition of culture under normal and heavy metal stress
It improves.Gene expression analysis shows the high expression in male flower, bark and seed of HbCS3 gene, while being lured by ethephon (CEPHA),2-(chloroethyl) phosphonic acid and rubber tapping
Up-regulated expression is led, is positively correlated with elastomer gum milk production, in addition, the gene is simultaneously by high temperature and low temperature stress inducible up regulation table
It reaches, therefore the gene and rubber tree yield traits and anti-environment stress are closely related, can be used as the weight of rubber tree transgenic breeding
Target gene is wanted, is expected to the expression by regulating and controlling the gene rationally to excavate the production glue of rubber tree and degeneration-resistant potentiality.In addition, the gene
It can be used as important genetic resources, it is also possible to be answered in the genetic engineering of other plant or microorganism other than rubber tree
With.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (10)
1. a kind of genetic recombinants, which is characterized in that for the recombinant for carrying HbCS3 gene, the sequence of the HbCS3 gene is such as
Shown in SEQ ID NO:1.
2. genetic recombinants according to claim 1, which is characterized in that the carrier of the recombinant is expression plasmid or disease
Poisonous carrier, wherein it is adjoint that the viral vectors is selected from adenovirus vector, herpes simplex virus vector, retrovirus vector, gland
One of viral vectors, slow virus carrier.
3. a kind of recombination bacillus coli of fast-growth, which is characterized in that the recombination bacillus coli contains described in claim 2
Genetic recombinants, and the carrier of the recombinant be expression plasmid.
4. application of the recombination bacillus coli as claimed in claim 3 in genetic engineering bacterium.
5.HbCS3 gene is improving the application in prokaryotic expression bacterium growth rate, the sequence of the HbCS3 gene such as SEQ ID
Shown in NO:1.
6. application according to claim 5, which is characterized in that HbCS3 gene is applied to improve prokaryotic expression bacterium in a huge sum of money
Belong to the growth rate under stress.
7. application according to claim 6, which is characterized in that the heavy metal be copper, and/or lead, and/or zinc, and/or
Tin, and/or nickel, and/or cobalt, and/or antimony, and/or mercury, and/or cadmium, and/or bismuth.
8. the application according to any one of claim 5-7, which is characterized in that the prokaryotic expression bacterium is large intestine bar
Bacterium.
Application of the 9.HbCS3 gene as target gene in the research anti-environment stress ability of rubber tree, the sequence of the HbCS3 gene
Column are as shown in SEQ ID NO:1.
The application that produces in glue ability of the 10.HbCS3 gene as target gene in research rubber tree, the sequence of the HbCS3 gene
As shown in SEQ ID NO:1.
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