CN113736805B

CN113736805B - Application of HbACLA-1 gene in improving growth rate of prokaryotic expression bacteria and researching rubber-producing capability of rubber tree

Info

Publication number: CN113736805B
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: 2023-07-18
Anticipated expiration: 2041-09-06
Also published as: CN113736805A

Abstract

The invention provides a coding gene of a rubber tree citrate lyase shown as SEQ ID NO. 2, and a coded protein and application thereof. The invention discovers that after the gene is transferred into a prokaryotic expression strain, the growth rate and the sodium stress resistance 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 rate is high, the efficiency can be obviously improved, the cost is reduced and the like; the gene is highly expressed in a rubber tree stock tissue (latex), is up-regulated by the influence of a normal rubber cutting system on gene and protein expression, is positively related to the yield of rubber latex, and can be used as a target gene in the research of rubber tree gum production; the expression of the gene is down-regulated by ethephon stimulation, which indicates that the gene can participate in external stress reaction, and can be used as a target gene in the research of the stress resistance of rubber trees; the gene can be used as an important gene resource and can be applied to genetic engineering of other plants except rubber trees.

Description

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

Technical Field

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

Background

In organisms, carbon dioxide reduction assimilation occurs during carboxylic acid cycle, that is, assimilation of four molecules of CO2 to produce one molecule of oxaloacetate. In each cycle, acetyl-coa reacts as a precursor with oxaloacetate to form citric acid to perform the entire cycle of the organism. As an entry point into this cycle, acetyl-CoA is an essential component of biosynthesis of fatty acids, cholesterol, isopentenyl phosphate and certain amino acids, and is also an essential intermediate for phytochemical production. 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 citric acid by a citrate synthase and then transported into the cytoplasm to be cleaved again to acetyl-coa by a citrate lyase. In addition, there is no basis for demonstrating that acetyl-CoA synthase is present in the cytoplasm and that it is produced directly by cytoplasmic glycolysis. Thus, citrate lyase is a major source of cytoplasmic CoA, a metabolic process that is the initial synthesis of the fatty acid chain in the plastid, a critical pathway for isoprenoid compounds and the production of certain phytochemicals in the cytoplasm (Fatland BL, ke J, anderson MD (2002) Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coendezyme A in Arabidopsis. Plant Physiology 130 (2): 740-756; liu Guyi, li Yangrui, yang Litao (2014). ATP-citrate lyase research evolution. Southern agricultural journal 045 (002), P.204-208).

Citrate lyase is a member of the acyl-coa synthase superfamily, which is derived from succinyl-coa synthase that catalyzes the biosynthesis of succinyl-coa. Similar to succinyl-CoA synthetases, citrate lyase has phosphorylated histidine groups, which form an unstable citric acid phosphate intermediate, which in turn generates oxaloacetate and acetyl-CoA under nucleophilic attack by CoA. X-ray research of the enzyme crystallization shows that the enzyme can be divided into an amino-terminal acyl-CoA synthetase homologous domain and a carbon-terminal citrate synthase homologous domain, and has a citrate binding functional site and can catalyze the formation of acetyl-CoA and oxaloacetate from the citrate-CoA. Biochemical studies in light and bacteria have shown that citrate lyase is composed of two subunits of size and different catalytic activity, which can be reconstituted in vitro into the citrate lyase holoenzyme structure (Wonduck Kim and F.Robert Tabita (2006) Both Subunits of ATP-Citrate Lyase from Chlorobium tepidum Contribute to Catalytic Activity. Journal of Bacteriology 188 (18): 6544-6552; sun et al (2010) Identification of the Citrate-binding Site of Human TP-Citrate Lyase Using X-ray crystal technology. The Journal of Biological Chemistry 285 (35): 27418-27428).

Citrate lyase is widely present in animals, plants, bacteria and fungi. Unlike the lyase in bacteria, citrate lyase catalytically breaks down citric acid in animals and plants, requiring ATP and coa as indispensable substrates. In high carbohydrate fed animals, cytoplasmic citrate lyase plays a key role in fatty acid formation, requiring citric acid transport from mitochondria as a substrate. In developing soybean cotyledons, the source of substrate for acetyl-coa in fatty acid synthesis is citrate lyase to break down citric acid. 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 cleavage enzyme in mango frit. Biochemical and Biophysical Research Communications (5): 895-904;T.M.Kaethner and T.ap Rees (1985) Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.planta 163:290-294).

Natural rubber (cis-1, 4-polyisoprene) is an important industrial feedstock that is extracted from the cytoplasm (i.e., latex) of highly specialized milk tube cells. In rubber trees, sucrose is the primary precursor for latex biosynthesis. Sucrose needs to be cleaved into glucose and fructose by a medium alkaline invertase 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, enzyme, organic acid and isoprene compounds flow out together with the latex, and these substances need to be sufficiently regenerated before the next tapping for the sustainability of the latex yield. In rubber trees, acetyl-coa is one of the important limiting factors for rubber biosynthesis as a precursor substance for rubber hydrocarbons. ACL catalyzes the cleavage of citric acid to acetyl-coa and oxaloacetate in the cytoplasm, thereby effecting transport of mitochondrial acetyl-coa and cytosolic acetyl-coa, which are the major sources of cytosolic acetyl-coa. ACL is used as a key enzyme of the cell citrate metabolic pathway, determines carbon flow distribution by regulating and controlling synthesis of acetyl-coa, and directly participates in biosynthesis of rubber. In addition, the rubber production capacity of the rubber tree is closely related to the capacity of resisting external bad factors, so that analysis of ACL rubber biosynthesis and the role of resisting abiotic stress, such as salt stress and metal ion stress, is helpful for further understanding of the action mechanism of ACL. At present, the related analysis of the ACL genes of the rubber trees is not reported.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, carries out gene expression analysis and functional research on the citrate lyase gene HbACLA-1 of the rubber tree, discovers that the growth rate of a transgenic strain obtained after the gene is transferred into a prokaryotic expression strain is obviously improved under normal conditions, and resists sodium (NaCl) and aluminum (AlCl) ₃ ) The stress capability is obviously improved, the gene can be applied to improving the bacterial growth rate and stress resistance, is related to the yield of rubber latex, and can be used as a target gene in the research of rubber tree gum production and stress resistance.

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

In a second aspect, the invention provides a protein which is encoded by the gene encoding the citrate lyase of the rubber tree according to 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, the present invention provides a recombinant expression vector comprising a primary vector and a gene encoding a hevea brasiliensis citrate lyase or an open reading frame thereof according to the first aspect of the present invention.

Among them, the original vector may be 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 viral vector may be selected from one of adenovirus vector, herpes simplex virus vector, retrovirus vector, adeno-associated virus vector, and lentivirus vector, preferably adenovirus vector. In one embodiment of the invention, the primary vector is a pMAL-c5E vector plasmid or a pMD18-T vector plasmid, but it is to be understood that the invention may be used with other plasmids, or viruses, etc.

Preferably, the original vector is a pMAL-c5E vector plasmid, and the coding gene of the rubber tree citrate lyase of the first aspect of the invention is connected with the pMAL-c5E vector plasmid through Kpn I and EcoRI double cleavage.

In a fourth aspect, the invention provides the use of a gene encoding a rubber tree citrate lyase 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, and the invention is not limited thereto. In a specific embodiment of the invention, the prokaryotic expression strain is E.coli BL21 (DE 3).

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

A fifth aspect of the present invention provides a method for increasing sodium (NaCl) resistance and/or aluminum (AlCl) resistance of a prokaryotic expression strain by using a gene encoding a rubber tree citrate lyase 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 ₃ ) Application in stress capability.

In a sixth aspect, the present invention provides a recombinant E.coli comprising the recombinant expression vector according to the third aspect of the present invention. The growth rate of the recombinant escherichia coli and the resistance to sodium (NaCl) and aluminum (AlCl) ₃ ) The stress capability is obviously better than that of the E.coli before recombination.

The seventh aspect of the invention provides an application of the recombinant escherichia coli in genetically engineered bacteria.

An eighth aspect of the present invention is to provide an application of HbACLA-1 gene as a target gene in researching stress resistance of rubber trees and/or researching gum-producing ability of rubber trees.

The invention has the beneficial effects that:

(1) The invention carries out gene expression analysis and functional research on the citrate lyase gene HbACLA-1 of the rubber tree, discovers that after the citrate lyase gene HbACLA-1 is transferred into a prokaryotic expression strain, the growth rate of the strain under normal conditions and the resistance to sodium (NaCl) and aluminum (AlCl) can be obviously improved ₃ ) The stress capability, the prokaryotic expression strain carrying HbACLA-1 gene is applied to engineering bacteria, the engineering bacteria grow fast, the efficiency can be obviously improved, the cost is reduced, and the like, and the method has good application prospect in the genetic engineering of microorganisms;

(2) HbACLA-1 gene is highly expressed in rubber tree warehouse tissue (latex), is up-regulated by the influence of a normal rubber cutting system on gene and protein expression, is positively related to the yield of rubber latex, can provide theoretical basis for reasonably formulating the rubber cutting system, and can be used as a target gene in the research of rubber tree rubber production;

(3) HbACLA-1 gene is subjected to the expression down regulation by ethephon stimulation, which shows that the gene can participate in external stress reaction and can be used as a target gene in the research of the stress resistance of rubber trees;

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

Drawings

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

FIG. 2 shows HbACL family gene expression analysis results.

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

FIG. 4 shows the effect of normal tapping on HbACLA-1 protein expression.

FIG. 5 shows the effect of normal tapping on HbACLA-1 gene expression.

FIG. 6 shows the effect of ethephon on HbACLA-1 gene expression.

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

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

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

FIG. 10 shows the results of measurement of the optimal pH of HbACLA-1 prokaryotic expression protein.

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

FIG. 12 shows the measurement result of ATP Miq constant of HbACLA-1 prokaryotic expression protein.

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

Detailed Description

The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

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

Analyzing nucleotide sequences of citric acid lyase (ACL) of Arabidopsis, rice and jatropha which are logged in NCBI, screening and splicing a sequence (contig) after assembling a rubber tree citric acid lyase gene of about 1700bp by searching a rubber tree latex EST sequence database established by us, and designing a pair of specific primers for amplification to obtain cDNA full-length sequence containing 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 PCR amplification was performed in a 20. Mu.l reaction system using Brazil rubber tree hot-ground 7-33-97 (cultivated by the national academy of Tropical agriculture rubber institute, which is sold for a long time by the national academy of Tropical agriculture rubber institute) latex cDNA as a template (obtained by reverse transcription with random primers), F and R as upstream and downstream primers at a final concentration of 0.4. Mu. Mol/L. The amplification procedure was: pre-denaturation at 94℃for 4min; denaturation at 94℃45S, annealing at 68℃for 3min for 34 cycles total; extending at 72℃for 10min.

The PCR obtained fragment is connected to a pMD18-T vector (TaKaRa) for sequencing, and the sequencing shows that the obtained fragment is the citrate lyase gene of the invention, the fragment has the nucleotide sequence of sequence 1 in a sequence table, the total length of the sequence 1 in the sequence table is 1686 nucleotides, the fragment comprises an open reading frame (ORF, the nucleotide sequence 382-1653 from the 5' end of sequence 2), the 5' -UTR of 381 nucleotides (the nucleotide sequence 1-381 from the 5' end of sequence 2) and the 3' -UTR of 33 nucleotides (the nucleotide sequence 1654-1686 from the 5' end of sequence 2) with the length of 423 (the amino acid coded by a stop codon is not contained in the sequence 1 in the sequence table), and the fragment has the molecular weight of about 46kDa, and the fragment is the citrate lyase, and the gene is named HbACLA-1. The pMD18-T recombinant vector containing the nucleotide of the sequence 2 in the sequence table is named pMD18-HbACLA-1. In addition, the amino acid sequence of the protein was analyzed using subcellular localization on-line software, and it was found that the protein was localized in the cytoplasm, so HbACLA-1 may belong to the rubber tree cytoplasmic protein.

2. Functional verification of prokaryotic expression and HbACLA-1 gene

Prokaryotic expression vectors of HbACLA-1 gene were constructed by using pMAL-c5E expression vectors (pMAL-c 5E (plasmid) expression vectors are purchased from New England Biolabs company) (the expression vectors in this example are only examples, other expression plasmids and virus vectors can be used in the present invention), and recombinant proteins were induced by using E.coli expression strain E.coli BL21 (DE 3) (strain is purchased from TransGen Biotech company), and the activity of the recombinant proteins and their effect on BL21 growth rate were determined as follows:

<1> production of recombinant vector containing HbACLA-1 Gene coding region

Designing HbACLA-1 gene coding region primer (F: 5'-GACGATGACAAGGTACCG CATATGGCACGCAAGAAGATCAG-3', R:5'-ATTACCTGCAGGGAATTC GGATCCTTAATGGTGATGGTGATGATGTGCAGCCGCGGAG-3'), carrying out PCR amplification by taking pMD18-HbACLA-1 as a template, carrying out pre-denaturation at 95 ℃ for 4min, denaturation at 94 ℃ for 45s, annealing at 68 ℃ for 2min, carrying out 34 cycles altogether, and extension at 72 ℃ for 5min, carrying out Kpn I and EcoR I double digestion and connection on the amplified product and a pMAL-c5E expression vector to obtain a recombinant vector, carrying out PCR identification by utilizing a gene specific primer (HbACLA-1 gene coding region primer), and ensuring that a citrate lyase coding fragment is cloned into the expression vector.

<2> prokaryotic expression of HbACLA-1 Gene

The recombinant vector pMAL-c5E-HbACLA-1 and the control vector pMAL-c5E-Empty (Empty vector) were introduced into E.coli BL21 (DE 3) (competence purchased from Tiangen Biotechnology Co., ltd.) to obtain recombinant expression strain, the recombinant strain identified correctly was cultured in LB medium containing 100. Mu.g/mL of carbenicillin to OD 600=0.4 to 0.6, IPTG (isopropyl-. Beta. -D-thiopyran galactoside) was added to a final concentration of 1mM, and the strain was collected by centrifugation and subjected to 12% SDS-PAGE electrophoresis detection at 16℃to obtain the results shown in FIG. 7. The results show that the HbACLA-1 gene achieves a high heterologous expression in the cytoplasm under IPTG induction and that the recombinant protein comprises HbACLA-1 protein and cytoplasmic fusion protein MBP (maltose binding protein) with a total molecular weight of about 95kDa (HbACLA-1 protein about 50kDa, MBP about 45 kDa).

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

According to'<2>Prokaryotic expression of HbACLA-1 Gene "thallus is collected by the above method, broken by low temperature ultrasonic, supernatant is collected by centrifugation, and the citrate lyase activity is measured after purification. Activity measurements were carried out under normal conditions (30 ℃, pH 7.0) and the measurement system was as follows (working concentrations): 100mM Tris-HCl,5mM MgCl ₂ 10mM potassium citrate, 0.2mM CoA, 10mM ATP,40U malate dehydrogenase, 1mM DTT, 10. Mu.l ACLA-1 (added according to the actual protein concentration), and ddH was supplemented ₂ O to 200. Mu.l. The decrease in NADH was detected by continuously measuring the change in absorbance at 340nm for 30min using a microplate reader. The measurement results are shown in fig. 8: the absorbance of the strain containing pMAL-c5E-HbACLA-1 was significantly changed over the absorbance of the strain containing pMAL-c5E-Empty control strain, which indicated that the protein encoded by HbACLA-1 gene did have the catalytic activity of citrate lyase.

<4> measurement of HbACLA-1 recombinant protein Mitsubishi constant

The Michaelis constant was determined at the optimum temperature (30 ℃) and at the optimum pH. The optimum temperatures were determined at 25,27.5,30,35,40,45 ℃and the HbACLA-1 recombinant protein was determined to have an optimum temperature of 35℃as shown in FIG. 9. The optimum pH was measured at 6.5,6.75,7.0,7.5,8.0,8.5, and as shown in FIG. 10, the optimum pH of HbACLA-1 recombinant protein was determined to be 7.0. According to'<3>HbACLA-1 recombinant protease Activity detection "the above method changes the loading amounts of potassium citrate and ATP (working concentrations were 10,20,40,80,200,500,1000,2000. Mu.M, respectively) of the substrates and the Michaelis constant change of HbACLA-1 was measured, and the results are shown in FIGS. 11 and 12. When potassium citrate is used as a substrate, recombinant protein HbACLA-1K _m 22.95. Mu.M, V _max 45.76 (nmol/mg pro/min) (numerically homoactive) (FIG. 11); when ATP is used as a substrate, K of recombinant protein HbACLA-1 _m 14.56. Mu.M, V _max 45.94 (nmol/mg pro/min) (numerically homoactive) (FIG. 12).

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

According to'<2>Hbacla-1 radicalProkaryotic expression "the strains were activated by the above method, and the strains containing pMAL-c5E-HbACLA-1 and pMAL-c5E-Empty were cultured to the same OD 600=0.4, IPTG (isopropyl-. Beta. -D-thiogalactopyranoside) was added to the final concentration of 1mM, OD600 was cultured at 37℃to 0.8-1.0, part of the bacterial liquid was aspirated, and the culture medium was supplemented to 100ml (initial OD values are shown in Table 1), and IPTG was added to the final concentration of 1mM. In the absence of ionic stress, 100mM NaCl stress, 300mM NaCl stress and 50mM AlCl stress ₃ OD600 values were measured after incubation at 37℃for 0h, 4h and 8h under stress (see Table 1, FIG. 13), respectively, growth amplification at time points of 4h and 8h relative to 0h was calculated, and a comparison of pMAL-c5E-HbACLA-1 and pMAL-c5E-Empty strains was performed (see Table 2). Wherein the growth gain for 4h was (OD 600 _4h -OD600 _0h )/OD600 _0h X 100%; growth amplification for 8h was (OD 600 _8h -OD600 _0h )/OD600 _8h X 100%. In the absence of ionic stress (FIG. 13A), the growth gain of the strain containing pMAL-c5E-HbACLA-1 was significantly higher than that of the pMAL-c5E-Empty control strain, with a 4-hour increase of 97.932% and an 8-hour increase of 169.095%, indicating that the protein encoded by the HbACLA-1 gene was able to increase the growth of the strain under normal conditions. At 100mM Na ⁺ (FIG. 13B) upon stress, the strain containing pMAL-c5E-HbACLA-1 had an increase of 66.562% by 4 hours and 108.473% by 8 hours over the pMAL-c5E-Empty control strain; in 300mM Na ⁺ (FIG. 13C) upon stress, the strain containing pMAL-C5E-HbACLA-1 had an increase of 41.743% by 4 hours and 84.404% by 8 hours over the pMAL-C5E-Empty control strain; at 50mM Al ⁺ (FIG. 13D) upon stress, the strain containing pMAL-c5E-HbACLA-1 had an increase of 8.338% by 4 hours and 13.354% by 8 hours over the pMAL-c5E-Empty control strain; the experimental result shows that the protein coded by HbACLA-1 gene can promote the growth of prokaryotic expression bacteria and can be used in Na ⁺ 、Al ⁺ Growth under stress.

TABLE 1 pMAL-c5E-HbACLA-1 Strain (HbACLA-1) and pMAL-c5E-Empty control strain (Empty) in no treatment, 100mM NaCl treatment, 300mM NaCl treatment, and 50mM AlCl ₃ Under-treatment growth OD600 change

TABLE 2 pMAL-c5E-HbACLA-1 Strain (HbACLA-1) and pMAL-c5E-Empty control strain (Empty) in no treatment, 100mM NaCl treatment, 300mM NaCl treatment, and 50mM AlCl ₃ Growth under treatment 4h, 8h amplification

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

HbACLA-1 expression Pattern analysis

<1> HbACLA-1 Gene tissue-specific expression

The real-time fluorescence quantitative PCR was performed using six tissues, such as rubber tree 7-33-97 latex, flowers, stems, barks, seeds and leaves, as templates, 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'), and as shown in FIG. 1 (relative expression amount was compared with the expression amount of seeds), hbACLA-1 gene was highly expressed in latex, seeds and barks, and expression in flowers, stems and leaves was relatively low. Further analysis of the latex ACL family gene transcriptome data found that HbACLA-1 was the highest expressed gene as shown in fig. 2.

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

The hot-ground Brazilian rubber without cutting 7-33-97 was used to collect latex after tapping, and ACL activity analysis of C-whey in the latex was performed. As shown in fig. 3, the ACL activity 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 protein expression of the first, third, fifth and seventh lanes shows that there is no obvious change in the expression of HbACLA-1 protein in the first, third lanes, and the protein expression in the fifth and seventh lanes is obviously increased. This suggests that HbACLA-1 protein may be involved in total ACL activity.

<3> Effect of rubber cutting on HbACLA-1 Gene expression

Real-time fluorescent quantitative PCR was performed with 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') using random reverse transcribed cDNA of latex RNA of different tapping strokes of hot-ground 7-33-97 of untreamed Brazil rubber tree as a template (three days one stroke, five strokes were sampled consecutively). As shown in FIG. 5, the results indicate that the rubber tapping affects the expression of HbACLA-1 gene, and that HbACLA-1 gene expression is continuously up-regulated from the second knife.

<4> Effect of ethephon on HbACLA-1 Gene expression

The cut rubber tree is selected, ethylene is coated on the cut line of the rubber tree and the cut surface 1cm above the cut line for stimulation treatment, and the stimulation is divided into three time periods of 12 hours, 24 hours and 48 hours. Real-time fluorescent quantitative PCR was performed using cDNA from random reverse transcription of latex RNA as a template 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'). As shown in FIG. 6, the expression level of HbACLA-1 gene was significantly lower than that of the untreated gene after ethephon stimulation for 12h,24h and 48h, and the expression level of HbACLA-1 was continuously decreased with the increase of the treatment time, and the decrease of HbACLA-1 expression was most significant at 48h.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Sequence listing

<110> rubber institute of Tropical agricultural academy of sciences in China

Application of <120> HbACLA-1 gene in improving growth rate of prokaryotic expression bacteria and researching rubber tree rubber production capacity

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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. Application of coding gene of rubber tree citrate lyase with nucleotide sequence shown as SEQ ID NO. 2, or protein with amino acid sequence shown as SEQ ID NO. 1, or recombinant expression vector in improving growth rate of prokaryotic expression strain; the recombinant expression vector comprises an original vector and the coding gene of the citrate lyase of the rubber tree or an open reading frame thereof.

2. Application of a coding gene of the citrate lyase of the rubber tree with a nucleotide sequence shown as SEQ ID NO. 2, or a protein with an amino acid sequence shown as SEQ ID NO. 1, or a recombinant expression vector in improving the sodium stress resistance and/or aluminum stress resistance of a prokaryotic expression strain; the recombinant expression vector comprises an original vector and the coding gene of the citrate lyase of the rubber tree or an open reading frame thereof.

3. The use according to claim 1 or 2, wherein the original vector is a pMAL-c5E vector plasmid, and the hevea brasiliensis citrate lyase encoding gene is linked to the pMAL-c5E vector plasmid by double cleavage with Kpn I and EcoR I.

4. The use according to claim 1 or 2, characterized in that the prokaryotic expression strain is escherichia coli e.coli BL21 (DE 3).

Application of HbACLA-1 gene as target gene in research of stress resistance of rubber tree and/or research of gum production capacity of rubber tree, wherein the nucleotide sequence of HbACLA-1 gene is shown as SEQ ID NO. 2.