CN104195151B - A kind of method combining bioinformatics specificity screening rhizopus chinensis lipase gene based on full-length genome - Google Patents
A kind of method combining bioinformatics specificity screening rhizopus chinensis lipase gene based on full-length genome Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of method combining bioinformatics specificity screening rhizopus chinensis lipase gene based on full-length genome, belong to genetic engineering field.The present invention is directed to the aimed aliphatic enzyme with specific catalytic performance and purposes, excavated by full-length genome lipase gene, classification, catalysis characteristics prediction obtain candidate gene；By candidate gene respectively at expression in escherichia coli, screen by detecting soluble protein and inclusion body activity respectively, it is thus achieved that there is the rhizopus chinensis lipase gene of specific catalytic performance, and in recombination bacillus coli, carry out activity expression prepare.It is finally obtained the gene that coding only shows the lipase of Lipase absobed activity and methanol solution activity under inclusion body state.The inventive method has, by significantly increasing, the efficiency that particular characteristics novel lipase gene obtains, and screening and preparation for other enzymes also provide valuable reference.
Description
Technical field
The present invention relates to a kind of combine bioinformatics specificity screening rhizopus chinensis lipase gene based on full-length genome
Method, especially one combine bioinformatics specificity screening rhizopus chinensis lipase gene based on full-length genome and are recombinating greatly
Method recombinant expressed in enterobacteria, belongs to genetic engineering field.
Background technology
Lipase, also known as triacylglycerol hydrolytic enzyme (EC3.1.1.3), is a kind of wide variety of hydrolase, it is possible to
Catalyzing hydrolysis substrate ester bond in aqueous phase, some lipase can also in nonaqueous phase catalytic esterification and ester exchange reaction etc..
Microbial lipase has been widely used in a lot of industry, such as oil prodution industry, pharmaceuticals industry, food industry and feed industry etc..
At present existing hundreds of microbial lipases report, and many commercializations, but, extensive many due to lipase kind
Sample, the catalysis characteristics of different lipases, such as catalysis activity (Lipase absobed activity, hydrolysing activity, methanol solution activity), substrate specificity
Property, regioselectivity, stereo selectivity etc. have significant difference.For meeting the different industries need to particular characteristics lipase
Ask, excavate further microbial lipase resource, develop and prepare the lipase of given activity still there is important reality meaning
Justice.The present invention, from the microorganism with specific gene resource, is intended to use and combines bioinformatics and specificity screening technology,
Lipase gene based on microorganism full-length genome excavates, on the basis of classification, catalysis characteristics prediction, by detecting large intestine respectively
The triage techniques of the different catalysis characteristicses of bacillus heterogenous expression soluble protein and inclusion body, it is thus achieved that there is the novel China of particular characteristics
Rizolipase gene, and in recombination bacillus coli, carry out activity expression prepare.
Due to microbiologic properties and the restriction of screening technique, obtain particular characteristics fat by traditional microbiological screening technique
Enzyme, and then the efficiency of exploitation novel lipase genetic resources is the highest.In recent years, modern genetic omics technology is deep development
Microbial gene resource provides possibility, by the genome sequencing to specified microorganisms, excavates the side of wherein related gene
Method the most studied person used.Rhizopus chinensis (Rhizopus chinensis) CCTCC No:M201021 is from China's micro-life of tradition
A strain filamentous fungi of isolated in thing carrier Daqu (massive raw stater for alcholic liquor), for liquor production, have significant catalytic esterification synthesis and
Esterolytic ability, shows that it contains abundant lipase, and one of which lipase is separated and clonal expression.But further
Research finds, the rhizopus chinensis lipase of a kind of heterogenous expression does not have wild rhizopus chinensis film bound fat enzyme catalysis Lipase absobed
Ability, shows to there may be in rhizopus chinensis the lipase of other activities present's forms.This bacterial strain genome sequencing is at present
Complete (Genome Announcements, 2013,1 (2): e00195-12), for excavating rhizopus chinensis lipase gene money further
Source provides possibility.Modern biotechnology informatics technology and related software are also that the excavation of lipase gene resource provides effectively
Means.
Additionally, the catalysis characteristics of lipase is final or is determined by determination of activity.And the activity determination method of lipase
More, conventional method have olive oil indicator titration method, p-nitrophenyl fatty acid ester (p-NP) method, organic facies Lipase absobed method,
Methanol-water solution etc..These methods reflect the different catalytic performances of lipase respectively, and between the activity that records of distinct methods
Not there is obvious corresponding relation.Only by a kind of determination of activity screening technique, it is intended to obtain the novel fat of different catalysis characteristics
Fat enzyme is typically relatively difficult.Therefore, for specific objective, it is necessary to select suitable activity determination method specific to screen
The lipase of catalysis characteristics.
It is generally of the enzyme of activity, including wild enzyme and heterogenous expression enzyme, is soluble protein；And insoluble protein,
Such as the inclusion body of escherichia coli heterogenous expression, the most do not show the activity of enzyme, be only possible to after needing renaturation show corresponding activity.
The present invention is based on rhizopus chinensis genome resource, mainly for lipase-catalyzed Lipase absobed and the different performance of hydrolysis, respectively
Candidate's lipase gene escherichia coli heterogenous expression soluble protein and inclusion body are carried out different activities detection and screening, to obtain
There is the novel rhizopus chinensis lipase gene of particular characteristics, and then in recombination bacillus coli, carry out activity expression prepare.This
Bright method has the efficiency that particular characteristics novel lipase gene obtains, for the sieve of other characteristic lipases by significantly increasing
Choosing and preparation also provide valuable technical scheme.
Summary of the invention
It is an object of the invention to the multiformity for lipase and catalysis characteristics thereof, overcome existing lipase screening technique pin
The problem the strongest, inefficient to property, it is provided that one combines bioinformatics specificity screening rhizopus chinensis fat based on full-length genome
The method of fat enzyme gene, combines bioinformatics based on rhizopus chinensis whole genome sequence and excavates potential candidate's lipase gene,
By candidate gene respectively at expression in escherichia coli, sieved by the activity detecting soluble protein and inclusion body part respectively
Choosing, it is thus achieved that there is the rhizopus chinensis lipase gene of specific catalytic performance.
Technical scheme mainly comprises the steps that
(1) obtain potential lipase and esterase gene according to rhizopus chinensis full-length genome gene annotation, and to lipase and
Esterase is classified, and has the lipase gene of specific catalysis characteristics known to selection, potential with gained rhizopus chinensis genome
Lipase and esterase gene carry out sequence analysis, select lipase or esterase gene that homology is higher, it was predicted that its catalysis
Characteristic, as candidate gene；
(2) candidate gene is set up lipase gene library, at expression in escherichia coli, detection soluble protein and bag respectively
Containing the different catalysis characteristicses of body, screening obtains the rhizopus chinensis lipase gene with specific catalysis characteristics.
Technical solution of the present invention can also include that the rhizopus chinensis lipase gene with particular characteristics by screening obtains exists
Recombination bacillus coli carries out activity expression, prepares corresponding enzyme preparation.Specifically, it is the protein active according to escherichia coli expression
Form, processes soluble protein or soluble albumen (inclusion body) respectively, prepares corresponding enzyme preparation.
There is described in step (1) rhizopus chinensis of specific function, be to separate from China's traditional microbiological carrier Daqu (massive raw stater for alcholic liquor)
Strain filamentous fungi rhizopus chinensis (Rhizopus chinensis) the CCTCC No:M201021 obtained.This bacterial strain is raw for Chinese liquor
Produce, there is the synthesis of significant catalytic esterification and esterolytic ability, multiple lipase can be produced.Its full-length genome has checked order,
See document Genome Announcements, 2013,1 (2): e00195-12, gene annotation seeshttp:// genome.jgi.doe.gov/Rhich1/Rhich1.home.html。
Step (1) described employing bioinformatics technique carries out lipase gene screening and catalysis characteristics prediction, preferably root
Obtain all potential lipases and esterase gene according to gene annotation, and utilize bioinformatics tools to carry out lipase and esterase
Classification；There is the gene of the lipase of specific catalysis characteristics, the lipase potential with rhizopus chinensis genome known to selection
Carry out sequence analysis with esterase gene, select to determine the lipase or esterase gene that homology is higher, it was predicted that its catalysis characteristics,
Set up lipase candidate gene storehouse.
The lipase known to described with specific catalysis characteristics could be for the Rhizopus oryzae fat of biodiesel synthesis
Enzyme (Rhizopus oryzae Lipase, ROL) and/or the candida antarctica lipase B for nonaqueous phase organic synthesis
(Candida antarctica Lipase B, CALB) and/or the Fusarium oxysporum lipase for food processing
(Fusarium oxysporum Lipase, FOL, FOL1) etc..
Step (2) preferably with e. coli bl21 (DE3) as host, with pET-22b as expression vector.
Step (2) described detection soluble protein and the different catalysis characteristicses of inclusion body respectively, different according to lipase
Catalysis characteristics and purposes, use different lipase activity determination methods, as olive oil indicator titration method measures lipase water
Solve activity (with reference to national standard GB/T 23535-2009 lipase preparation), organic facies Lipase absobed method measures lipase synthesis activity
(with reference to Bioprocess and Biosystems Engineering, the method described in 2007,30 (3): 147-155), first
Alcohol Hydrolyze method mensuration lipase methanol solution activity (with reference to Journal of Bioscience and Bioengineering,
Method described in 2006,101 (4): 328-333).Detection object includes the solvable and soluble albumen of recombination bacillus coli simultaneously
(inclusion body) expression product.
Second technical problem that the invention solves the problems that is to provide two should screen the rhizopus chinensis fat obtained in aforementioned manners
Fat enzyme gene, nucleotide sequence is respectively as shown in SEQ ID NO.1 or SEQ ID NO.2.
The lipase that described nucleotide sequence gene (A06672) as shown in SEQ ID NO.1 encodes, with escherichia coli
BL21 (DE3) is host, during with pET-22b for expression vector, expresses the soluble protein fraction obtained and has lipase hydrolysis
Activity (62U/mg) and relatively low synthesizing activity (0.029U/mg), inclusion body part has lipase hydrolysis activity the most simultaneously
(108U/mg), synthesizing activity (0.42U/mg) and methanol solution activity (201.7U/mg).
Further, the preparation method of described inclusion body is preferably by pET-22b Transformed E .coli with genes of interest
BL21 (DE3), accesses in the test tube of the 5mlLB fluid medium containing Amp resistance, in 37 DEG C by positive transformant list bacterium colony
200rpm cultivates 3-4 hour as seed liquor；3 test tube seed liquor, 37 DEG C of 200rpm trainings are added in 1LLB fluid medium
Supporting 4-5 hour, add IPTG and induce to final concentration 0.4mmol/L, 28 DEG C of 180rpm induce 3-5 hour, are centrifuged and collect
Thalline, ultrasonication in suitable buffer, centrifuged deposit is inclusion body protein.Gained inclusion body can be directly as urging
The lipase changing nonaqueous phase Lipase absobed or alcoholysis reaction uses.
The lipase that described nucleotide sequence gene (A07506) as shown in SEQ ID NO.2 encodes, with escherichia coli
BL21 (DE3) is host, during with pET-22b for expression vector, expresses the soluble protein fraction obtained and has lipase hydrolysis
Activity (201U/mg) and relatively low synthesizing activity (0.015U/mg), inclusion body part the most also has lipase hydrolysis activity
(78U/mg) with relatively low synthesizing activity (0.042U/mg) and methanol solution activity (19.0U/mg).
Further, the pET-22b with genes of interest is preferably converted by the preparation method of described soluble protein
E.coli BL21 (DE3), accesses in the test tube of the 5ml LB fluid medium containing Amp resistance by positive transformant list bacterium colony, in
37 DEG C of 200rpm cultivate 3-4 hour as seed liquor, 1L LB fluid medium adds 3 test tube seed liquor, 30 DEG C of 200rpm
Cultivating 1 hour, add IPTG and induce to final concentration 0.4mmol/L, 16 DEG C of 180rpm induce 2 hours, centrifugal collection thalline,
Ultrasonication in suitable buffer, the supernatant obtained after being centrifuged is lipase solution.
The present invention compared with prior art has the advantage that
(1) the present invention is directed to the aimed aliphatic enzyme with specific catalysis, utilize microorganism full-length genome data and life
Thing Bioinformatic tool carries out the Preliminary screening of lipase gene；Different catalysiss according to lipase and purposes, be respectively adopted
Different lipase actives is analyzed method and is screened the lipase with specific catalysis further.Can effectively alleviate experiment
Workload, with strong points, screening efficiency is higher.
(2) under normal circumstances, activated enzyme includes wild enzyme and heterogenous expression enzyme, is soluble protein；And it is insoluble
Property albumen, such as the inclusion body of escherichia coli heterogenous expression, the most do not show enzyme and live.Owing to lipase can be catalyzed aqueous phase, profit circle
Reaction in face and organic facies (nonaqueous phase) medium, therefore, insoluble in the lipase in reaction system, such as inclusion body, also
Likely show some catalytic capability.The present invention is according to the catalysis of lipase, for can during escherichia coli heterogenous expression
The different protein form that can occur, including soluble protein and soluble albumen (inclusion body), all carry out activity screening assay, effectively
Improve acquisition genes of interest and the probability of albumen.When can avoid only analyzing soluble protein activity, miss specific base
Because the enzyme of coding has the situation of multiple different catalysis.Such as shown in SEQ ID NO.1 or SEQ ID NO.2 sequential coding
Enzyme, in screening process, if abandon analyze inclusion body different catalytically active, even if gene function analysis predicts that it has
Methanol solution activity, in the case of not detecting concrete activity, then still cannot excavate, determine the methanol solution activity of described enzyme.And
The present invention by inclusion body is carried out activity analysis, finally determine described enzyme also have methanol solution activity, the most solvable with
Under soluble state, the vigor of different catalysis characteristicses is different.
The inventive method has the efficiency that particular characteristics novel lipase gene obtains, for other enzymes by significantly increasing
Screening and preparation also provide valuable reference.
Detailed description of the invention
Embodiment 1 lipase gene based on rhizopus chinensis full-length genome excavates
Based on rhizopus chinensis (Rhizopus chinensis) CCTCC No:M201021 full-length genome data, (GenBank compiles
Number ANKS00000000), by Augustus software with Genemark software tool respectively to whole genome sequence number after order-checking
According to carrying out predictive genes, by predicted gene sequence and each data base NR, PFAM, CDD, KEGG, COG, SwissProt and TrEMBL
Compare, it is thus achieved that corresponding functional annotation information (http://genome.jgi.doe.gov/Rhich1/ Rhich1.home.html).119 esterase genes and 123 lipase genes, wherein part bases are found altogether from gene annotation
Because not only belonging to esterase but also belong to lipase.
Embodiment 2 prediction has the determination of the lipase candidate gene of purpose enzyme function
Select Rhizopus oryzae lipase (Rhizopus oryzae Lipase, ROL, the ammonia for biodiesel synthesis respectively
Base acid sequence is as shown in GeneBank:BAG16821.1), for the candida antarctica lipase B of nonaqueous phase organic synthesis
(Candida antarctica Lipase B, CALB, aminoacid sequence such as NCBI UniProtKB/Swiss-Prot:
Shown in P41365.1), for food processing Fusarium oxysporum lipase (Fusarium oxysporum Lipase, FOL and
FOL1 aminoacid sequence is respectively as shown in SEQ ID NO.3, SEQ ID NO.4) as target enzyme, by rhizopus chinensis genome
Lipase and esterase gene carry out Blast comparison with target enzyme gene order.Using standard Blastp comparison process result not
Time preferable, use the sensitiveest Position-Specific Iterated (PSI)-BLAST, be conducive to finding distant relative's species
Similar protein or the newcomer of certain protein family.With Query cover, Score, Identities and Positives it is
Index is analyzed, and selects most possible purpose enzyme candidate gene.For each target enzyme main candidate and alignment parameters
As shown in table 1, wherein, for Rhizopus oryzae lipase ROL, most possible candidate's lipase gene be followed successively by A07506,
A06672 and A03113 (http://genome.jgi.doe.gov/pages/search-for-genes.jsf？organism =Rhich1)。
Table 1 each target enzyme candidate gene and alignment parameters
The construction and expression of embodiment 3 candidate's lipase gene colibacillus engineering
Separately designing specific primer for the candidate gene in table 1 and mature peptide fragment thereof, pcr amplification product connects
PMD19-T, is transformed in escherichia coli Jm109,37 DEG C of overnight incubation, picking positive transformant, extracts plasmid, through double digestion and
Check order after PCR checking.Through double digestion after order-checking is correct, respectively genes of interest is connected pET-22b, convert escherichia coli
Jm109,37 DEG C of incubated overnight, picking positive transformant, extracts plasmid, checks order after double digestion and PCR are verified.Through order-checking
Recombinant expression plasmid after Zheng Que Transformed E .coli BL21 (DE3) respectively, picking monoclonal spreads cultivation, and in LB culture medium
Middle cultivation, bacterial concentration OD600Add IPTG when 0.6～0.8 and carry out purpose to 0.4mmol/L30 DEG C of inducing culture 4h of final concentration
The heterogenous expression of gene.Supernatant and precipitation are collected, as thick enzyme after ultrasonication by thalline after abduction delivering respectively
It is further analyzed.
The lipase active analysis of embodiment 4 candidate's lipase gene expression product and the screening of lipase gene
For each candidate gene recombination bacillus coli difference expression product (including soluble protein and inclusion body), according to fat
Enzyme purposes is respectively adopted different lipase activity determination methods and is analyzed, and measures fat including olive oil indicator titration method
Enzyme hydrolysis activity, organic facies Lipase absobed method measure synthesizing activity, methanol-water solution measures alcoholysis activity.Simultaneously not carry candidate
The Host Strains of gene is as comparison bacterium.Comparison bacterium only shows faint lipase hydrolysis activity (< 5U/mL), and other activity are not
Can detect.And recombinant bacterium each lipase expression product all shows hydrolysing activity in various degree, wherein A07506 expresses
Soluble fraction hydrolysing activity is of a relatively high.But unexpectedly show more significantly lipase synthesis activity and first
Alcoholysis activity, be but the inclusion body part of A06672 gene expression, after renaturing inclusion bodies, above two loss of activity.This
The activity analysis result of two gene expression products is as shown in table 2.
The lipase active analysis of the different expression product of table 2
In conjunction with the result of Tables 1 and 2, for the Rhizopus oryzae lipase ROL synthesized for biodiesel, candidate gene
In A07506 with A06672 sequence more closely, methanol solution activity analysis to also indicate that the expression product of the two gene has similar
Function, but only inclusion body has such activity, and wherein the expression product activity of A06672 is higher, shows that this gene is for having
The lipase function of alcoholysis activity, has potential using value in fields such as biodiesel synthesis.
For the candida antarctica lipase B (CALB) for nonaqueous phase organic synthesis, gene comparison does not finds very
Similar rhizopus chinensis lipase gene, thus the rhizopus chinensis lipase gene expression product higher with CALB homology fails to show
Go out higher synthesizing activity.But have been reported that ROL also has higher lipase synthesis activity, thus the highest with ROL homology
A06672 express inclusion body also show higher synthesizing activity, have association area application potentiality.
For the Fusarium oxysporum lipase FOL for food processing, although gene comparison does not finds that homology is higher
Rhizopus chinensis lipase gene, but the most general lipase of lipase hydrolysis activity that FOL has can show, and activity height is just
Seem the most important.Thus activity analysis shows, rhizopus chinensis lipase A 07506 has the application potential of association area.
Embodiment 5 specific lipase gene activity expression in recombination bacillus coli and the preparation of lipase
Will be with the single bacterium colony of the E.coli BL21 (DE3) of expression plasmid pET22-RCL1 (rhizopus chinensis lipase A 06672)
Add 37 DEG C of 200rpm in the 5ml LB fluid medium test tube containing Amp resistance and cultivate 3-4 hour as seed liquor, 1L LB
Adding 3 test tube seed liquor in fluid medium shaking flask, 37 DEG C of 200rpm cultivate 4-5 hour, add IPTG to final concentration
0.4mmol/L induces, and 28 DEG C of 180rpm induce 3-5 hour, and centrifugal collection thalline, at 20mM phosphate buffer (pH6.2)
Middle ultrasonication 5min (400w, interval time 3s), centrifuged deposit is inclusion body protein, and its nonaqueous phase Lipase absobed activity is
0.42U/mg, methanol solution activity is 201.7U/mg (comparison bacterium activity is 0U/mg), can close directly as catalysis nonaqueous phase ester
Become or the lipase of alcoholysis reaction uses.
Will be with the single bacterium colony of the E.coli BL21 (DE3) of expression plasmid pET22-RCL2 (rhizopus chinensis lipase A 07506)
Add 37 DEG C of 200rpm in the 5ml LB fluid medium test tube containing Amp resistance and cultivate 3-4 hour as seed liquor, 1L LB
Adding 3 test tube seed liquor in fluid medium shaking flask, 30 DEG C of 200rpm cultivate 1 hour, add IPTG to final concentration 0.4mmol/L
Inducing, 16 DEG C of 180rpm induce 2 hours, centrifugal thalline of collecting, ultrasonic in 20mM Tris-HCl (pH8.0) buffer
Broken 5min (400w, interval time 3s), centrifugal after the supernatant that obtains be lipase solution, measure through hydrolysing activity, live
Power is 109U/mg.Can use as common lipase.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (1)
1. the method for a specificity screening lipase gene, it is characterised in that be to combine based on rhizopus chinensis whole genome sequence
The lipase gene candidate gene that bioinformatics screening is potential, by candidate gene respectively at expression in escherichia coli, by dividing
Jian Ce not screen the different catalytically active of soluble protein and inclusion body part, it is thus achieved that there is the rhizopus chinensis of specific catalytic performance
Lipase gene；Described method mainly comprises the steps that
(1) potential lipase and esterase gene are obtained according to rhizopus chinensis full-length genome gene annotation, and to lipase and esterase
Classify, there is known to selection the lipase gene of specific catalysis characteristics, the fat potential with gained rhizopus chinensis genome
Fat enzyme and esterase gene carry out sequence analysis, select lipase or esterase gene that homology is higher, it was predicted that its catalysis characteristics,
As candidate gene；
(2) candidate gene is set up lipase gene library, at expression in escherichia coli, detection soluble protein and inclusion body respectively
Different catalysis characteristicses, screening obtains has the rhizopus chinensis lipase gene of specific catalytic performance；
Step (1) described rhizopus chinensis is rhizopus chinensis (Rhizopus chinensis) CCTCC No:M201021；Described known
The lipase with specific catalysis characteristics includes the Rhizopus oryzae lipase for biodiesel synthesis, for nonaqueous phase organic synthesis
Candida antarctica lipase B, for the Fusarium oxysporum lipase of food processing；
Step (2) described detection soluble protein and the different catalysis characteristicses of inclusion body respectively, be the catalysis different according to lipase
Characteristics and uses, use different lipase activity determination methods；Lipase hydrolysis is measured including olive oil indicator titration method
Activity, organic facies Lipase absobed method measure lipase synthesis activity, methanol-water solution measures lipase methanol solution activity；Step (2)
With e. coli bl21 (DE3) as host, with pET-22b as expression vector.
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| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN102140426A (en) * | 2010-09-20 | 2011-08-03 | 江南大学 | High-yield lipase gene engineering bacterium and construction method therefor |
| CN102851263A (en) * | 2011-07-01 | 2013-01-02 | 丰益(上海)生物技术研发中心有限公司 | High-throughout screening method of lipase gene mutation database and lipase mutation gene |
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| CN1681942A (en) * | 2002-09-13 | 2005-10-12 | 韩国生命工学研究院 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
| CN102140426A (en) * | 2010-09-20 | 2011-08-03 | 江南大学 | High-yield lipase gene engineering bacterium and construction method therefor |
| CN102851263A (en) * | 2011-07-01 | 2013-01-02 | 丰益(上海)生物技术研发中心有限公司 | High-throughout screening method of lipase gene mutation database and lipase mutation gene |
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| GenBank Accession NO.:EF405962.2;NCBI;《NCBI GenBank》;20070827;具体序列信息 * |
| 华根霉全基因组序列分析及其初步应用;吴荣;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20140215;B024-346，论文全文，特别是摘要以及正文第3.2.1节 * |
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