CN113736805A

CN113736805A - Application of HbACLA-1 gene in improving growth rate of prokaryotic expression bacteria and researching rubber production capacity of rubber tree

Info

Publication number: CN113736805A
Application number: CN202111039512.5A
Authority: CN
Inventors: 龙翔宇; 胡斌; 房平昌; 方永军; 秦云霞; 阳江华; 肖小虎
Original assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Current assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-12-03
Anticipated expiration: 2041-09-06
Also published as: CN113736805B

Abstract

The invention provides a rubber tree citrate lyase coding gene shown as SEQ ID NO. 2, and a coding protein and application thereof. The invention discovers that after the gene is transferred into the prokaryotic expression strain, the growth rate and the sodium and aluminum stress resistance of the strain under normal conditions can be obviously improved, and the prokaryotic expression strain is applied to engineering bacteria, so that the growth speed is high, the efficiency can be obviously improved, the cost is reduced and the like; the gene is highly expressed in rubber tree reservoir tissues (latex), is up-regulated by gene and protein expression influenced by a normal tapping system, is positively correlated with the yield of rubber tree latex, and can be used as a target gene to be applied to the research of rubber production of rubber trees; the expression of the gene is reduced by ethephon stimulation, which indicates that the gene can participate in external stress reaction and can be used as a target gene to be applied to the research of the adversity stress resistance of the rubber tree; the gene can be used as an important gene resource and can also be applied to the genetic engineering of other plants except the rubber tree.

Description

Application of HbACLA-1 gene in improving growth rate of prokaryotic expression bacteria and researching rubber production capacity of rubber tree

Technical Field

The invention belongs to the field of biology, and particularly relates to application of HbACLA-1 gene in improving the growth rate of prokaryotic expression bacteria and researching the rubber production capacity of rubber trees.

Background

In the organism, the assimilation of carbon dioxide reduction occurs during the carboxylic acid cycle, i.e., four molecules of CO2 are assimilated to produce one molecule of oxaloacetate. In each cycle, acetyl-coa as a precursor reacts with oxaloacetate to form citrate to progress through the cycle of the organism. As a point of entry into this cycle, acetyl-coa is an essential building block for the biosynthesis of fatty acids, cholesterol, isopentenyl phosphate and certain amino acids, and is also an essential intermediate for the production of phytochemicals. Acetyl-coa cannot be produced directly from plastids, mitochondria and peroxisomes because the membrane boundaries of these three intracellular sites are impermeable to derivatives of coa. Instead, acetyl-coa in the mitochondria needs to be converted to citrate by citrate synthase and then transported to the cytoplasm to be cleaved again to acetyl-coa by citrate lyase. In addition, there is no evidence that acetyl-CoA synthase exists in the cytoplasm and that it is produced directly by cytoplasmic glycolysis. Thus, Citrate Lyase is the major source of Cytosolic Coenzyme A, a metabolic process That is the key pathway for the initial synthesis of fatty acid chains within the plastid, the production of isoprenoid compounds and certain phytochemicals in the cytosol (Fatland BL, Ke J, Anderson MD (2002) Molecular Characterization of a heterotomeric ATP-Citrate lyases at genes cytotoxic Acetyl-Coenzyme A in Arabidopsis plant Physiology 130(2): Liu 740-.

Citrate lyase is a member of the acyl-coa synthetase superfamily, which is derived from succinyl-coa synthetases that catalyze the biosynthesis of succinyl-coa. Similar to succinyl-coa synthetase, citrate lyase has a phosphorylated histidine group, which forms an unstable intermediate of citrayl phosphate, and thus oxaloacetate and acetyl-coa are formed under nucleophilic attack by coa. The X-ray research of the enzyme crystal shows that the enzyme crystal can be divided into an amino-terminal acyl-CoA synthetase homologous structure domain and a carbon-terminal citrate synthase homologous structure domain, has a citrate binding functional site and catalyzes the generation of acetyl-CoA and oxaloacetate from the citrate-CoA. Biochemical studies in light and bacteria have shown that Citrate Lyase consists of two Subunits of size and different Catalytic activities, which are reconfigurable in vitro into the Citrate Lyase holoenzyme structure (Wonduck Kim and F. Robert Tabita (2006) Both subunit of ATP-Citrate Lyase from Chrobium tepidum control to Catalytic activity. Journal of Bacteriology 188(18): 6544-.

Citrate lyase is widely present in animals, plants, bacteria and fungi. Unlike lytic enzymes in bacteria, citrate lyase enzymes catalyze the decomposition of citric acid in animals and plants, requiring ATP and coenzyme a as indispensable substrates. In animals on high carbohydrate diets, the cytosolic form of citrate lyase plays a key role in the formation of fatty acids, which requires the transport of citrate from mitochondria as a substrate. In developing soybean cotyledons, the substrate source of acetyl-coa in fatty acid synthesis is the decomposition of citric acid by citrate lyase. In mature mangoes, an increase in cucurbitacin content is accompanied by an increase in Citrate lyase activity (A.K. Matto and V.V.Modi (1970) Citrate clearance enzyme in biochemical and Biophysical Research Communications 39(5): 895-19; T.M. Kaeth and T.ap Rees (1985) Intracellular location of ATP Citrate clearance enzymes of Pisum sativum L.plant 163: 290-294).

Natural rubber (cis-1, 4-polyisoprene) is an important industrial raw material, which is extracted from the cytoplasm (i.e., latex) of highly specialized milk duct cells. In the hevea brasiliensis tree, sucrose is the main precursor substance for latex biosynthesis. Sucrose needs to be cleaved into glucose and fructose by medium-alkaline invertases before entering the glycolytic pathway, pentose phosphate pathway and tricarboxylic acid cycle, forming three basic units for latex biosynthesis: acetyl-coa, NADPH and ATP. During tapping, a large amount of sugar, enzymes, organic acids and isoprene compounds are co-flowed out with the latex, and for the sustainability of latex yield, these substances need to be fully regenerated before the next tapping. In rubber trees, acetyl-coa is one of the important limiting factors for rubber biosynthesis as a precursor of rubber hydrocarbons. ACL catalyzes the cleavage of citric acid into acetyl-coa and oxaloacetate in the cytoplasm, thereby enabling the transport of mitochondrial acetyl-coa and cytosolic acetyl-coa, which is the major source of cytosolic acetyl-coa. ACL is used as a key enzyme of a cell citric acid metabolic pathway, determines carbon flow distribution by regulating and controlling the synthesis of acetyl coenzyme A, and is directly involved in the biosynthesis of rubber. In addition, the rubber production capacity of the rubber tree is closely related to the capacity of resisting external adverse factors, so that the analysis of the effects of ACL rubber biosynthesis and abiotic stress resistance, such as salt stress and metal ion stress, is helpful for further understanding of the action mechanism of ACL. At present, no report is found in the ACL gene correlation analysis of the rubber tree.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and performs gene expression analysis and function research on the citrate lyase gene HbACLA-1 of the rubber tree, and finds that a transgenic strain obtained after the gene is transferred into a prokaryotic expression strain grows under normal conditionsLong speed rate is obviously increased, and the sodium (NaCl) and aluminum (AlCl) resistance is improved₃) The stress capability is obviously improved, the gene can be applied to improving the growth rate and the stress resistance of bacteria, is related to the latex yield of the rubber trees, and can be applied to the research of the rubber tree latex production and the stress resistance as a target gene.

The first aspect of the invention provides a rubber tree citrate lyase coding gene which is named as HbACLA-1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

The second aspect of the invention provides a protein, which is the protein encoded by the rubber tree citrate lyase encoding gene in the first aspect of the invention, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

In a third aspect of the present invention, there is provided a recombinant expression vector comprising an original vector and the gene encoding hevea brasiliensis citrate lyase or its open reading frame according to the first aspect of the present invention.

As the original vector, there can be used a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. Wherein, the virus vector can be selected from one of adenovirus vector, herpes simplex virus vector, retrovirus vector, adeno-associated virus vector and lentivirus vector, and is preferably adenovirus vector. In one embodiment of the present invention, the primary vector is a pMAL-c5E vector plasmid or a pMD18-T vector plasmid, but it is understood that other plasmids, viruses, or the like may be used in the present invention.

Preferably, the original vector is a pMAL-c5E vector plasmid, and the gene encoding the rubber tree citrate lyase of the first aspect of the invention is ligated to the pMAL-c5E vector plasmid by double cleavage with Kpn I and EcoR I.

In a fourth aspect, the invention provides the use of a gene encoding a citrate lyase in rubber trees according to the first aspect of the invention, or a protein according to the second aspect of the invention, or a recombinant expression vector according to the third aspect of the invention, for increasing the growth rate of a prokaryotic expression strain.

The prokaryotic expression strain may be a prokaryotic expression strain commonly used in the field of gene recombination, but the invention is not limited thereto. In a particular embodiment of the invention, the prokaryotic expression strain is e.coli BL21(DE 3).

Preferably, the nuclear expression strain is escherichia coli, more preferably escherichia coli e.coli BL21(DE 3).

In a fifth aspect of the present invention, there is provided a gene encoding a citrate lyase for hevea brasiliensis according to the first aspect of the present invention, or a protein according to the second aspect of the present invention, or a recombinant expression vector according to the third aspect of the present invention for improving the sodium (NaCl) and/or aluminum (AlCl) resistance of a prokaryotic expression strain₃) Application in stress capacity.

In a sixth aspect, the present invention provides a recombinant E.coli comprising the recombinant expression vector of the third aspect of the present invention. The growth rate and sodium (NaCl) and aluminum (AlCl) resistance of the recombinant escherichia coli₃) The stress capability is obviously better than that of the Escherichia coli before recombination.

The seventh aspect of the invention provides the use of the recombinant Escherichia coli of the sixth aspect of the invention in genetically engineered bacteria.

The eighth aspect of the invention provides an application of the HbACLA-1 gene as a target gene in researching the stress resistance capability of the rubber tree and/or the rubber production capability of the rubber tree.

The invention has the beneficial effects that:

(1) the invention carries out gene expression analysis and function research on the rubber tree citrate lyase gene HbACLA-1, and finds that the conversion of the rubber tree citrate lyase gene HbACLA-1 into a prokaryotic expression strain can obviously improve the growth of the strain under normal conditionsLong rate and sodium (NaCl), aluminium (AlCl) resistance₃) The stress capability, the prokaryotic expression strain carrying the HbACLA-1 gene is applied to engineering bacteria, the growth speed of the engineering bacteria is high, the efficiency can be obviously improved, the cost is reduced, and the like, and the prokaryotic expression strain has good application prospect in the genetic engineering of microorganisms;

(2) the HbACLA-1 gene is highly expressed in rubber tree library tissues (latex), is up-regulated by gene and protein expression influenced by a normal tapping system, is positively correlated with the yield of rubber tree latex, can provide theoretical basis for reasonably formulating the tapping system, and can be applied to the research of rubber tree latex production as a target gene;

(3) the HbACLA-1 gene is expressed and reduced by ethephon stimulation, which indicates that the gene can participate in external stress reaction and can be used as a target gene to be applied to the research of the adversity stress resistance of rubber trees;

(4) the HbACLA-1 gene can be used as an important gene resource and can also be applied to genetic engineering of other plants except the rubber tree.

Drawings

FIG. 1 shows the results of tissue-specific expression analysis of HbACLA-1 gene (latex, flower, stem, bark, seed, leaf).

FIG. 2 shows the results of gene expression analysis of HbACL family.

FIG. 3 is a graph of the effect of normal tapping on the activity of latex HbACL protein.

FIG. 4 is a graph of the effect of normal tapping on HbACLA-1 protein expression.

FIG. 5 is a graph showing the effect of normal tapping on HbACLA-1 gene expression.

FIG. 6 is a graph showing the effect of ethephon on HbACLA-1 gene expression.

FIG. 7 shows the result of SDS-PAGE analysis of HbACLA-1 prokaryotic expressed protein.

FIG. 8 shows the results of the activity detection of HbACLA-1 prokaryotic expressed protein.

FIG. 9 shows the results of measuring the optimal temperature of HbACLA-1 prokaryotic expression protein.

FIG. 10 shows the results of measuring the optimum pH of HbACLA-1 prokaryotic expression protein.

FIG. 11 shows the results of determining potassium citrate Michaelis constant of prokaryotic HbACLA-1 expression protein.

FIG. 12 shows the result of measuring the ATP michaelis constant of the prokaryotic expression protein HbACLA-1.

FIG. 13 shows the expression of Na under normal conditions of genetically modified engineering bacteria⁺And Al⁺Growth rate under stress assay results.

Detailed Description

The invention will be better understood from the following description of specific embodiments with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

1. Obtaining of rubber tree citrate lyase coding gene (HbACL)

Analyzing the nucleotide sequence of citric acid lyase (ACL) of arabidopsis thaliana, rice and jatropha curcas which are logged in at NCBI, screening and splicing a rubber tree citric acid lyase gene assembled sequence (contig) of about 1700bp by searching a rubber tree latex EST sequence database established by us, and designing a pair of specific primers to amplify to obtain a cDNA full-length sequence containing a complete reading frame.

The specific method for cDNA cloning is as follows:

specific primers were designed as follows:

f (5' end): 5'-GAACCCACGCAAGGTAAACGAATCGAT-3'

R (3' -end): 5'-AGA GAA CCC AGA TGG ATA TCA GGA GTT GGA T-3'

The Brazilian rubber tree hot grinding 7-33-97 (cultivated by rubber institute of Chinese tropical agrology institute, sold by rubber institute of Chinese tropical agrology institute for long term seedling) latex cDNA is used as a template (obtained by random primer reverse transcription), F and R are upstream and downstream primers, the final concentration is 0.4 mu mol/L, and PCR amplification is carried out in a 20 mu L reaction system. The amplification procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45S, annealing at 68 ℃ for 3min, and 34 cycles; extension at 72 ℃ for 10 min.

Connecting the fragment obtained by the PCR to a pMD18-T vector (TaKaRa) for sequencing, wherein the sequencing shows that the fragment obtained is the citrate lyase gene of the invention, the fragment has a nucleotide sequence of a sequence 1 in a sequence table, the full length of the sequence 1 in the sequence table is 1686 nucleotides, the fragment comprises an open reading frame (ORF, nucleotide sequence 382 and 1653 from the 5' end of the sequence 2), a 5' -UTR (nucleotide sequence 1 to 381 from the 5' end of the sequence 2) of 381 nucleotides and a 3' -UTR (nucleotide sequence 1654 and 1686 from the 5' end of the sequence 2) of 33 nucleotides, the fragment encodes an amino acid (sequence 1 in the sequence table) with the length of 423 (amino acid not encoded by a stop codon), and a protein with the molecular weight of about 46kDa, namely the citrate lyase, this gene was named HbACLA-1. The pMD18-T recombinant vector containing the nucleotide of the sequence 2 in the sequence table is named as pMD 18-HbACLA-1. In addition, the amino acid sequence of the protein is analyzed by using subcellular localization online software, the protein is found to be localized in cytoplasm, and therefore HbACLA-1 may belong to the hevea brasiliensis cytoplasmic type protein.

2. Prokaryotic expression and functional verification of HbACLA-1 gene

Prokaryotic expression vectors of HbACLA-1 gene are constructed by utilizing pMAL-c5E expression vectors (pMAL-c5E (plasmid) expression vectors are purchased from New England Biolabs company) (the expression vectors in the embodiment are only examples, other expression plasmids, virus vectors and the like can be adopted in the invention), meanwhile, Escherichia coli expression strains E.coli BL21(DE3) (the strains are purchased from TransGen Biotech company) are utilized to induce recombinant protein, and the activity of the recombinant protein and the influence of the recombinant protein on the growth rate of BL21 are determined, and the specific method is as follows:

<1> obtaining of recombinant vector containing HbACLA-1 gene coding region

The HbACLA-1 gene coding region primer (F: 5'-GACGATGACAAGGTACCG CATATGGCACGCAAGAAGATCAG-3', R: 5'-ATTACCTGCAGGGAATTC GGATCCTTAATGGTGATGGTGATGATGTGCAGCCGCGGAG-3' the flow of the air in the air conditioner, performing PCR amplification by taking pMD18-HbACLA-1 as a template, wherein the amplification procedure is as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 68 ℃ for 2min, and 34 cycles; extending for 5min at 72 ℃, carrying out Kpn I and EcoR I double enzyme digestion and connection on the amplification product and a pMAL-c5E expression vector to obtain a recombinant vector, carrying out PCR identification by using a gene specific primer (a gene coding region primer of HbACLA-1) to ensure that a citrate lyase coding fragment is cloned to the expression vector, carrying out sequencing identification on the recombinant vector, and naming the recombinant expression vector which contains the correct 382-channel 1653 nucleotide sequence containing the sequence 2 in the sequence table and has accurate reading frame as pMAL-c 5E-HbACLA-1.

<2> prokaryotic expression of HbACLA-1 Gene

The recombinant vector pMAL-c5E-HbACLA-1 and the control vector pMAL-c5E-Empty vector obtained were introduced into E.coli BL21(DE3) (competence purchased from Gentiangen Biochemical technology Co., Ltd.) to obtain recombinant expression bacteria, the correctly identified recombinant bacteria were cultured in LB medium containing 100. mu.g/mL carbenicillin until OD600 became 0.4-0.6, IPTG (isopropyl-. beta. -D-thiogalactopyranoside) was added to a final concentration of 1mM, induction culture was carried out at 16 ℃ for 8-12h, the bacteria were collected by centrifugation, and the mycoprotein was subjected to 12% SDS-PAGE electrophoresis, with the results shown in FIG. 7. The results show that the HbACLA-1 gene realizes high-efficiency heterologous expression in cytoplasm under IPTG induction, and the recombinant protein comprises HbACLA-1 protein and cytoplasmic fusion protein MBP (maltose binding protein), and the total molecular weight is about 95kDa (HbACLA-1 protein is about 50kDa, and MBP is about 45 kDa).

<3> activity detection of HbACLA-1 recombinant protein after purification

According to "<2>Prokaryotic expression of HbACLA-1 Gene "the above-mentioned method collects the bacterial cells, sonicates them at low temperature, centrifugates them to collect the supernatant, and measures the citrate lyase activity after purification. The activity assay was performed under normal conditions (30 ℃, pH 7.0) and the assay system was as follows (working concentrations are as follows): 100mM Tris-HCl,5mM MgCl₂10mM potassium citrate, 0.2mM coenzyme A,10mM ATP,40U malate dehydrogenase, 1mM DTT, 10. mu.l ACLA-1 (added based on actual protein concentration), supplemented with ddH₂O to 200. mu.l. And continuously measuring the change of absorbance value within 30min at the wavelength of 340nm by using a microplate reader, and detecting the reduction amount of NADH. The measurement results are shown in fig. 8: the change in absorbance values of the strain containing pMAL-c5E-HbACLA-1 was significantly higher than that of the control strain containing pMAL-c5E-Empty, indicating that the protein encoded by the HbACLA-1 gene indeed has the catalytic activity of citrate lyase.

<4> determination of Mie's constant of HbACLA-1 recombinant protein

The determination of the Michaelis constant is carried out at the optimum temperature (30 ℃) and the optimum pH. The optimum temperatures were measured at 25,27.5,30,35,40 and 45 ℃ respectively, and as shown in FIG. 9, the optimum temperature of the HbACLA-1 recombinant protein was determined to be 35 ℃. The pH optimum measurements were performed at 6.5,6.75,7.0,7.5,8.0,8.5, respectively, and as shown in FIG. 10, the pH optimum of the recombinant HbACLA-1 protein was determined to be 7.0. According to "<3>HbACLA-1 recombinant protease activity assay "the change in the Michaelis constant of HbACLA-1 was determined by changing the loading amounts of the substrates potassium citrate and ATP (working concentrations were 10,20,40,80,200,500,1000, 2000. mu.M, respectively) as described above, and the results are shown in FIGS. 11 and 12. K of recombinant protein HbACLA-1 when potassium citrate is used as substrate_m22.95. mu.M, V_max45.76(nmol/mg pro/min) (numerically the same activity) (FIG. 11); k of recombinant protein HbACLA-1 when ATP is used as a substrate_mIs 14.56. mu.M, V_maxIt was 45.94(nmol/mg pro/min) (same activity as in the case of FIG. 12).

<5>HbACLA-1 transgenic engineering bacteria in Na⁺And Al⁺Growth augmentation under stress

According to "<2>Prokaryotic expression of HbACLA-1 Gene "the strain was activated as described above, and the strain containing pMAL-c5E-HbACLA-1 and pMAL-c5E-Empty was cultured to the same OD600 of 0.4, IPTG (isopropyl-. beta. -D-thiogalactopyranoside) was added to a final concentration of 1mM, OD600 was cultured at 37 ℃ to 0.8-1.0, a part of the bacterial liquid was aspirated, supplemented with LB medium to 100ml (initial OD value as in Table 1), and IPTG was added to a final concentration of 1 mM. Under no ion stress, 100mM NaCl stress, 300mM NaCl stress and 50mM AlCl₃OD600 values were determined after 0h, 4h and 8h incubation at 37 ℃ under stress (see Table 1, FIG. 13), and growth increases were calculated for 4h and 8h versus the 0h time point, respectively, and comparisons of pMAL-c5E-HbACLA-1 and pMAL-c5E-Empty strains were made (see Table 2). Wherein the growth increase of 4h is (OD 600)_4h-OD600_0h)/OD600_0hX is 100%; growth was increased by (OD 600) for 8h_8h-OD600_0h)/OD600_8hX 100%. In the absence of ionic stress (FIG. 13A), the growth of the strain containing pMAL-c5E-HbACLA-1 was significantly increased over the pMAL-c5E-Empty control strain by 97.932% increase at 4 hours and by 97.932% increase at 8 hours169.095%, indicating that the protein encoded by the HbACLA-1 gene can enhance the growth of the strain under normal conditions. At 100mM Na⁺(FIG. 13B) strains containing pMAL-c5E-HbACLA-1 showed a 4 hour rise of 66.562% and an 8 hour rise of 108.473% over pMAL-c5E-Empty control strain under stress; at 300mM Na⁺(FIG. 13C) strains containing pMAL-C5E-HbACLA-1 showed a 4 hour rise of 41.743% and an 8 hour rise of 84.404% over pMAL-C5E-Empty control strain under stress; at 50mM Al⁺(FIG. 13D) strains containing pMAL-c5E-HbACLA-1 showed a 4 hour rise of 8.338% and an 8 hour rise of 13.354% over pMAL-c5E-Empty control strain under stress; the above experimental results show that the protein encoded by the HbACLA-1 gene can promote the growth of prokaryotic expression bacteria and increase the expression level of Na⁺、Al⁺Growth under stress.

TABLE 1 Strain of pMAL-c5E-HbACLA-1 (HbACLA-1) and pMAL-c5E-Empty control Strain (Empty) without treatment, with 100mM NaCl treatment, with 300mM NaCl treatment, and with 50mM AlCL₃Growth OD600 Change under treatment

TABLE 2 Strain of pMAL-c5E-HbACLA-1 (HbACLA-1) and pMAL-c5E-Empty control Strain (Empty) without treatment, with 100mM NaCl treatment, with 300mM NaCl treatment, and with 50mM AlCL₃Growth under treatment for 4h and 8h

"HbACLA-1: Empty" means the increase in growth gain of the strain containing pMAL-c5E-HbACLA-1 relative to the pMAL-c5E-Empty control strain

HbACLA-1 expression Pattern analysis

<1> tissue-specific expression of HbACLA-1 Gene

The results of real-time fluorescence quantitative PCR using RNA random reverse transcription cDNA of six tissues such as 7-33-97 latex, flower, stem, bark, seed and leaf of Hevea brasiliensis as templates and HbACLA-1 gene specific primers (F:5'-CGA ATC GAT GGT GAT AGG CAT C-3'; R:5'-ACA ATC TCT TGG AGT CGT ACT CT-3') are shown in FIG. 1 (relative expression is compared with the expression of seed), the HbACLA-1 gene is highly expressed in latex, seed and bark, and the expression of flower, stem and leaf is relatively low. Further analysis of the latex ACL family gene transcriptome data revealed that HbACLA-1 was the highest expressed gene, as shown in figure 2.

<2> Effect of tapping on latex ACL Activity and HbACLA-1 protein expression

Hevea brasiliensis Regrinding 7-33-97 after tapping, the latex was collected and subjected to ACL activity analysis of C-whey in the latex. As shown in fig. 3, the activity of ACL was not significantly changed in the first three knives, while the ACL activity was significantly increased in the fifth, seventh and ninth knives. As shown in FIG. 4, the expression of HbACLA-1 protein was not significantly changed in the first and third cuts, but was significantly increased in the fifth and seventh cuts. This suggests that HbACLA-1 protein may be associated with total ACL activity.

<3> Effect of tapping on HbACLA-1 Gene expression

The cDNA randomly reverse-transcribed by latex RNA of 7-33-97 times of different tapping knives of the hot grinding of the uncut Brazilian rubber tree is taken as a template (one blade in three days and five blades continuously sampled), and real-time fluorescence quantitative PCR is carried out by using HbACLA-1 gene specific primers (F:5'-CGA ATC GAT GGT GAT AGG CAT C-3'; R:5'-ACA ATC TCT TGG AGT CGT ACT CT-3'). As shown in FIG. 5, the results indicated that tapping affected the expression of HbACLA-1 gene, and that the expression of HbACLA-1 gene was continuously up-regulated from the second cut.

<4> Effect of ethephon HbACLA-1 Gene expression

Selecting a cut rubber tree, coating ethephon on the cut line of the rubber tree and a cut surface 1cm above the cut line, and performing stimulation treatment, wherein the stimulation is divided into three time periods, namely 12h, 24h and 48 h. Real-time fluorescent quantitative PCR is carried out by taking cDNA randomly reverse-transcribed by latex RNA as a template and using HbACLA-1 gene specific primers (F:5'-CGA ATC GAT GGT GAT AGG CAT C-3'; R:5'-ACA ATC TCT TGG AGT CGT ACT CT-3'). As shown in FIG. 6, the expression level of HbACLA-1 gene was significantly lower after 12h, 24h, and 48h of the ethephon stimulation than that of the untreated gene, and as the treatment duration was increased, the expression level of HbACLA-1 was continuously reduced, and the expression was most significantly reduced at 48 h.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

<110> rubber institute of tropical agricultural academy of sciences of China

Application of HbACLA-1 gene in improving growth rate of prokaryotic expression bacteria and researching rubber production capacity of hevea brasiliensis

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tgatgccgaa cccacgcaag gtaaacgaat cgatggtgat aggcatcacc atagcaacga 300

gtagaaagta ctaaatcatt ttccaccaga aaaattacga agagatcagc ggttgaaacg 360

ttggtcctgg atggtaaaca aatggcacgc aagaagatca gagagtacga ctccaagaga 420

ttgttgaagg atcatttcaa gaggctttct ggctatgaat tgcccatcaa atccgcacaa 480

gttacagaat caactgattt caatgagcta gcagagaagg aaccctggct tttgtcagga 540

aaactggttg tgaagcctga catgctgttt ggtaagcgtg ggaagagtgg tctagttgct 600

ttaaatctag atttggctga agctgctgtt tttgtgaaag aacgccttgg aaaagaggtt 660

gagatgagtg gatgtaaagg acctataaca acattcattg ttgaaccttt catcccccac 720

aatgaggagt tttaccttaa tattgtctcg gagcgacttg ggtgcagcat aagcttttct 780

gattgtggag gaattgaaat tgaagagaat tgggataagg ttaagactat atatgttcca 840

acagggtcat catttacatc agaaacatgc gctccacttg ttgcaaccct tccattggag 900

ataaaacgag aaattgagga gtttattaaa tcaatttttg ctctatttca agatcttgac 960

ttcactttcc tggagatgaa tcctttcact ttggttaatg gaaagcctta tcccttggat 1020

atgagaggcg agctggatga cactgctgct ttcaagaatt tcaagaagtg gggcaacatt 1080

ggatttccaa tgccatttgg tagagttatg agctccacag agagctttat tcatggacta 1140

gatgaaaaga caagtgcatc tttgaaattc acagtcctga atccaaaggg gcgaatttgg 1200

actatggtgg ctggaggagg tgcaagtgtc atctatgcag atacagttgg agatcttggt 1260

tatgcttctg agcttgggaa ttatgcagaa tatagtggag cccccaatga agaggaagta 1320

ttgcagtatg ccagagttgt aattgattgt gcaacttctg atcctgatgg ccgtaagaga 1380

gcccttgtaa ttggaggagg gattgctaac ttcactgatg tagctgctac atttaatggc 1440

ataattcgag ccttgaagga aaaggaatct aagcttaaag cagcaaggat gcacatgtat 1500

gtgaggagag gaggtcctaa ttaccagaaa ggccttgtaa aaatgaggtc acttggagaa 1560

gaaattggac ttccaataga ggtttacggg cctgaagcaa caatgactag tatatgcaag 1620

caggcgattg agtgcatctc cgcggctgca taagtatcca actcctgata tccatctggg 1680

ttctct 1686

<210> 3

<211> 27

<212> DNA

<213> Artificial

<400> 3

gaacccacgc aaggtaaacg aatcgat 27

<210> 4

<211> 31

<212> DNA

<213> Artificial

<400> 4

agagaaccca gatggatatc aggagttgga t 31

<210> 5

<211> 41

<212> DNA

<213> Artificial

<400> 5

gacgatgaca aggtaccgca tatggcacgc aagaagatca g 41

<210> 6

<211> 58

<212> DNA

<213> Artificial

<400> 6

attacctgca gggaattcgg atccttaatg gtgatggtga tgatgtgcag ccgcggag 58

<210> 7

<211> 22

<212> DNA

<213> Artificial

<400> 7

cgaatcgatg gtgataggca tc 22

<210> 8

<211> 23

<212> DNA

<213> Artificial

<400> 8

acaatctctt ggagtcgtac tct 23

Claims

1. A coding gene of rubber tree citrate lyase is characterized in that the coding gene is named HbACLA-1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

2. A protein encoded by the gene encoding hevea brasiliensis citrate lyase of claim 1, wherein the amino acid sequence of the protein is represented by SEQ ID NO. 1.

3. A recombinant expression vector comprising a primary vector and the hevea brasiliensis citrate lyase encoding gene or open reading frame thereof of claim 1.

4. The recombinant expression vector of claim 3, wherein the original vector is a pMAL-c5E vector plasmid, and the rubber tree citrate lyase-encoding gene of claim 1 is ligated to the pMAL-c5E vector plasmid by double cleavage with Kpn I and EcoR I.

5. Use of a gene encoding a citrate lyase from Hevea brasiliensis as claimed in claim 1, or a protein as claimed in claim 2, or a recombinant expression vector as claimed in claim 3 or 4 for increasing the growth rate of a prokaryotic expression strain.

6. Use of the gene encoding citrate lyase from Hevea brasiliensis as claimed in claim 1, or the protein as claimed in claim 2, or the recombinant expression vector as claimed in claim 3 or 4 for improving the sodium and/or aluminum stress resistance of a prokaryotic expression strain.

7. Use according to claim 5 or 6, characterized in that the prokaryotic expression strain is E.coli BL21(DE 3).

8. A recombinant Escherichia coli comprising the recombinant expression vector of claim 3 or 4.

9. The recombinant Escherichia coli of claim 8, which is used in a genetically engineered bacterium.

The application of the HbACLA-1 gene as a target gene in researching the stress resistance capability of the rubber tree and/or the rubber production capability of the rubber tree.