CN102827849B - Method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene BAN and DFR - Google Patents
Method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene BAN and DFR Download PDFInfo
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- CN102827849B CN102827849B CN 201210295306 CN201210295306A CN102827849B CN 102827849 B CN102827849 B CN 102827849B CN 201210295306 CN201210295306 CN 201210295306 CN 201210295306 A CN201210295306 A CN 201210295306A CN 102827849 B CN102827849 B CN 102827849B
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Abstract
The invention discloses a method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene BAN and DFR. According to the invention, an anthocyanidin reductase BAN gene and a 4-flavanonol reductase DFR gene are cloned from cDNA of brassica juncea purple-leaf mustard seed coat; a prokaryotic expression vector pET-BAN-DFR containing the BAN and DFR genesis constructed; and the BAN and the DFR genes are introduced into DH5alpha escherichia coli simultaneously. The transformed strain is cultured in an LB liquid medium at 25-37 DEG C at 150-250 rpm for6-12 hours, then cultured under shaking in an LB liquid medium containing black soybean seed coat extracts (0.5-1.0 g/L) at 25-37 DEG C at 150-250 rpm for 36-54 hours; the strain is collected by centrifugation; and the proanthocyanidin content in escherichia coli is determined by a DMACA-HCl method. The transgenic escherichia coli obtained in the invention has significantly increased proanthocyanidin content, and the maximum content can be 19.8 times higher than that of a non-transformed control strain.
Description
Technical field
What the present invention relates to is a kind of method in genetically engineered and microbial fermentation technology field, particularly a kind of method that adopts two key gene cotransformations and add the synthetic proanthocyanidin of crude extract raising intestinal bacteria of key gene catalyzed reaction precursor in substratum.
Background technology
Proanthocyanidin (Proathocyanidins), also be pycnogenols, is a kind of of flavonoid.Proanthocyanidin is to remove one of antioxidant that free radical is the strongest, can protect brain and nervous tissue, improves blood circulation, the effect of flexible joint and young skin.The long-term edible high food containing proanthocyanidin, can alleviate the diseases such as cardiovascular.Scientist also finds that proanthocyanidin and derivative thereof also have anti-inflammatory, degeneration-resistant border, antitumor and immunoregulatory function, and visible proanthocyanidin element is a kind of natural drug that has potentiality, and consumers demand is increasing.
At present the main source of proanthocyanidin is from the plant tissue extracting section, but the time that plant-growth need to be longer, the not high and extraction process more complicated of the content of proanthocyanidin, and productive rate is low, makes the large-scale commercial production of proanthocyanidin be restricted.Because proanthocyanidin synthetic difficulty is larger, yield poorly, cost is high, and feasibility is not strong.
We find mustard type rape gene DFR(dihydroflavonol 4-reductase in the research in early stage, 4-flavanonol reductase enzyme) and BAN(anthocyanidin reductase, expression amount cyanidin(e) reductase enzyme) and the content of rape kind skin proanthocyanidin are proportionate.Through the prior art literature search being found to YanY J etc. (2008) is at " biotechnology and biotechnology " (Biotechnol. Bioeng. 2008; 100:126 – 140) reported on and utilized in expression in escherichia coli Arabidopis thaliana, African chrysanthemum (Gerbera), gene in cyanidin(e) (Anthocyanin) route of synthesis in petunia (Petunia), Common Snapdragon (Antirrhinum) can improve the resultant quantity of intestinal bacteria cyanidin(e), cyanidin(e) and proanthocyanidin all belong to the flavonoid class material, but there are no adopting rape proanthocyanidin synthetic gene to improve the report of the resultant quantity of intestinal bacteria proanthocyanidin.
Cyanidin(e) reductase enzyme BAN is a key enzyme in the proanthocyanidin route of synthesis; the activity of cyanidin(e) reductase enzyme is higher; more be conducive to synthetic proanthocyanidin; adopt genetic engineering means; with gene BAN and the synthetic key enzyme DFR cotransformation intestinal bacteria of precursor substance thereof; to break the bottleneck of proanthocyanidin biosynthesizing speed limit, thereby obtain the bacterial strain of proanthocyanidin high yield, for large-scale production proanthocyanidin element provides a new way.
summary of the invention
The object of the invention is to overcome deficiency of the prior art, a kind of method that adopts gene BAN and DFR cotransformation to improve intestinal bacteria Procyanidins content is provided.The key gene clone who the present invention relates to, vector construction, genetic transformation, Molecular Detection, proanthocyanidin extract and assay.The present invention has set up and has improved the method for producing proanthocyanidin in intestinal bacteria, for utilizing transgenosis intestinal bacteria scale operation proanthocyanidin, establishes solid basis.
The present invention is achieved by the following technical solutions: a kind of method that adopts mustard type rape gene BAN and DFR cotransformation to improve intestinal bacteria Procyanidins content, it is characterized in that, and comprise the following steps:
(1) clone's cyanidin(e) reductase enzyme BAN gene and 4-flavanonol reductase enzyme DFR gene from the cDNA of mustard type rape kind skin;
(2) BAN gene and DFR gene are connected in to expression regulation sequence operably, form the prokaryotic expression carrier pET-BAN-DFR containing BAN gene and DFR gene;
(3) BAN and DFR gene are imported to DH5 α intestinal bacteria simultaneously, transformed bacteria is in the LB liquid nutrient medium, 25-37 ℃, 150-250rpm (rev/min) cultivate after 6-14 hour, again containing in the LB liquid nutrient medium of black soybean peel extract, 25-37 ℃, 150-250rpm concussion training is after 36-54 hour, at room temperature, centrifugal force is the centrifugal collection thalline of 10000-12000g;
(4) adopt the DMACA-HCl method to measure the amount of the contained proanthocyanidin of intestinal bacteria, screening obtains proanthocyanidin content and improves the gene coli strain.
In step (3), contain 0.5-1.0g/L black soybean peel extract in described LB liquid nutrient medium.
The extracting method of described black soybean peel extract: the black soybean peel is pulverized with pulverizer, cross the sieve of 1.0 mm, will be with the ethanol (v/v) of 30%-70% as extraction solvent, the soybean peel amount is 1 with the ratio of solvent load: 4-8 (w/w), extraction temperature is 60 ℃-70 ℃, and extraction time is 4-8 hour; Concentrated solution centrifugal 10-15min in the whizzer of 8000-10000g after recovery ethanol, filter impurity elimination, then add acetone in concentrated solution, concentrated solution is 1 with the ratio of acetone: 1-3 (v/v), stirring and evenly mixing 2-5min, remove by filter impurity, concentrated solution, after reclaiming acetone, obtains the soybean peel extract 40 ℃ of-60 ℃ of lower vacuum-dryings.
BAN of the present invention and DFR cotransformation improve the method for intestinal bacteria Procyanidins content, to adopt gene engineering method, key gene BAN and DFR are imported in intestinal bacteria, obtained the transgenosis e. coli strains that proanthocyanidin content significantly improves, the content of corotation BAN and DFR gene intestinal bacteria Procyanidins can reach 12.8mg/g FW (fresh weight), be the 8.8-19.8 of (the 0.4-0.9mg/g FW) of non-transformed Bacillus coli communis under substratum of the same race doubly, this invention is for for the proanthocyanidin large-scale production, providing high yield, stablize source new drugs significant.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to the present embodiment.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, " molecular cloning: laboratory manual " (New York: Cold Spring Harbor Laboratory Press such as Sambrook etc., 1989), " modern molecular biology experiment " (Beijing: Higher Education Publishing House of Zheng Weijuan etc., 2010) condition described in, or the condition of advising according to manufacturer.
(1) clone of rape BAN and DFR gene
1. mustard type rape (Brassica juncea) is planted the extraction of the total RNA of Pi
The kind skin of 15-25 days after the purple leaf mustard pollination of the mustard type rape that takes a morsel, strict and impartial reason etc. is pressed in concrete operations
(strict and impartial reason, Liu Zhongsong, official's spring cloud, Chen member, Liu Xianjun. a kind of method of extracting the high quality Semen Brassicae campestris and planting skin RNA. the biotechnology circular. 2007, method 6:97-100) is carried outthe extraction of total RNA
.
2. the clone of mustard type rape BAN and DFR gene
The obtained total RNA of rape kind Pi is obtained to the first chain cDNA by ThermoScript II (AMV) reverse transcription, according to the encoding sequence (sequence 1 in sequence table) of described mustard type rape DFR gene and the encoding sequence (sequence 2 in sequence table) of BAN gene, design amplifies the upstream and downstream primer of complete encoding sequence respectively, and introduce respectively restriction endonuclease sites (determining according to the carrier of selecting) on the upstream and downstream primer, so that construction of expression vector.Described the first chain cDNA of take is template, and after the PCR amplification, product is checked order after being connected to the pMD18-T cloning vector.The DNA sequencing adopts 3730 sequenators to complete by biotech firm.Sequencing result shows, the sequence of cloning is consistent with the encoding sequence of the DFR of prediction (sequence 1 in sequence table) and BAN gene (sequence 2 in sequence table).
Adopt gene clone method to obtain proanthocyanidin biosynthesizing key gene BAN and the DFR that sequence is correct from mustard type rape, for improve intestinal bacteria Procyanidins content by two key gene cotransformation strategies, provide two important key genes.
(2) contain the structure of the plant binary expression vector of BAN and DFR gene
Selecting pMD18-BAN, pMD18-DFR and pET is primary element, carrier construction pET-BAN-DFR, concrete operation reference (Yan YJ, Li Z, Koffas MA. High-Yield Anthocyanin Biosynthesis in Engineered Escherichia coli. Biotechnol Bioeng, 2008,100:126 – 140) in, the method for report is carried out.
Proanthocyanidin biosynthetic pathway key gene BAN and DFR are connected in to expression regulation sequence operably, form the prokaryotic expression carrier containing BAN and DFR gene, this expression vector can be used for improving by the metabolic engineering strategy content of intestinal bacteria Procyanidins.
(3) extraction of black soybean peel extract
The black soybean peel of having selected is weighed, pulverize with pulverizer, cross the sieve of 1.0 mm, will be with 50% ethanol (v/v) as extraction solvent, the soybean peel amount is 1: 5 (w/w) with the ratio of solvent load, and when extraction temperature is 65 ℃, extraction time is 8 hours.Concentrated solution after recovery ethanol is 10000g centrifugal settling 15min at centrifugal force, filter impurity elimination, then add acetone in concentrated solution, concentrated solution is 1: 2 (v/v) with the ratio of acetone, stirring and evenly mixing 5min, remove by filter impurity, and concentrated solution is after reclaiming acetone, under 60 ℃, vacuum-drying obtain the soybean peel extract.
(4) carrier pET-BAN-DFR genetic transformation obtains the transgenosis e. coli strains of high yield proanthocyanidin
1. the acquisition of transgenosis e. coli strains
Containing BAN and DFR prokaryotic expression vector, by the heat shock conversion method, being transformed into DH5 α intestinal bacteria, by the resistance performing PCR of selecting to go forward side by side, verify.Result shows, containing BAN and DFR expression vector, oneself successfully is transformed into DH5 α intestinal bacteria.
2. colibacillary cultivation:
Choose the mono-bacterium colony of DH5 α containing carrier pET-BAN-DFR, the unconverted DH5 α of take is contrast, in the liquid nutrient medium of 1.0ML, 37 ℃, 200rpm (rev/min) cultivate 6 as a child after, be transferred in the LB liquid nutrient medium containing black soybean peel extract (1.0g/L), 37 ℃, 200rpm cultivates 48 hours again.
3. adopt the DMACA-HCl method to measure intestinal bacteria Procyanidins content
By cultured bacterium liquid under room temperature, 10000g centrifugal 10 minutes, collect coli somatic.The extraction of thalline Procyanidins and assay are pressed document (Li YG, Tanner GJ, Larkin PJ, The DMACA-HCl Protocol and the Threshold Proanthocyanidin Content for Bloat Safety in Forage Legumes, Journal of Science of Food and Agriculture, 1996, method 70:89-101) is carried out, and test arranges 3 repetitions.
The content of corotation BAN and DFR gene intestinal bacteria Procyanidins reaches 7.9mg/g FW, is (0.4mg/g FW) 19.8 times of non-transformed Bacillus coli communis.Corotation BAN of the present invention and DFR gene have significantly improved intestinal bacteria Procyanidins content.
The present embodiment is the intestinal bacteria that the metabolic engineering strategy of employing BAN and DFR gene has obtained the proanthocyanidin high yield, for the large-scale production proanthocyanidin provides a kind of comparatively ideal method.
<110 > University Of Science and Technology Of Hunan
<120 > adopt mustard type rape gene BAN and DFR cotransformation to improve the method for intestinal bacteria Procyanidins content
<160>?2
<210>?1
<211>?1158
<212>?DNA
<213 > mustard type rape (Brassica juncea)
<400>?1
ATGGTAGCTC?ACAAAGAGAC?CGTGTGCGTA?ACCGGCGCAT?CAGGATTCAT?TGGTTCATGG?60
CTCGTGATGC?GGCTACTGGA?ACGTGGTTAT?TTTGTCCGTG?CCACTGTTCG?CGATCCTGGA?120
AATTTGAAGA?AAGTGCAACA?TCTTCTTGAT?TTGCCAAACG?CGAAGACGCA?ACTCACTTTA?180
TGGAAAGCCG?ATTTATCTGA?CGAAGGAAGC?TACGATGACG?CCATAAACGG?ATGCGACGGC?240
GTTTTCCACA?TAGCAACTCC?CATGGATTTT?GAATCTAAGG?ACCCCGAGAA?CGAAGTGATA?300
AAACCGACAG?TGAATGGAGT?GTTGGGGATA?ATGAAAGCAT?GTGATAAGGC?AAAGACCGTA?360
CGAAGAATTG?TGTTTACTTC?GTCTGCTGGA?ACGGTTAATG?TTGAGGAACA?CCAGAAAAAT?420
GTCTATGATG?AAAACGATTG?GAGTGATCTT?GACTTTATCA?TGTCCAAGAA?GATGACAGGA?480
TGGATGTATT?TCATGTCGAA?AACGTTAGCC?GAGAAAGCAG?CTTGGGATTA?CGCGAAGGAA?540
AAAGGAATAG?ATTTCATTAG?TATTATCCCG?ACATTGGTGA?TCGGTCCATT?TATAACAACA?600
TCTATGCCGC?CTAGCCTTAT?TACCGCGCTC?TCTCCTATCA?CTCGTAACGA?GGCACATTAC?660
TCCATCATAA?GACAAGGACA?GTATGTCCAC?TTGGACGATT?TATGCAATGC?CCATATATTC?720
TTGTACGAAC?AAGCTGCTGC?CAAGGGACGT?TATGTTTGTT?CCTCTCACGA?TGCAACGATT?780
CTTACTATCT?CCGAGTTTCT?CAGGCAAAAA?TATCCAGAAT?ATAACGTGCC?TTCAACGTTT?840
GAAGGAGTGG?ATGAGAATCT?AAAGAGCATT?ATGTTCAGTT?CCAAGAAGCT?GATTGATATG?900
GGATTTAACT?TCAAGTATAG?TCTCGAGGAT?ATGTTGGTGG?AATCGATTGA?GACATGTCGT?960
CAAAAGGGTT?TTCTCCCTGT?CACTTTACCG?GAACATTTGA?AATCTGAGGA?CAAAGTTCCG?1020
GGCAGTGATG?ATAATAAGGA?GATTAAAAAC?GGATCTGCAG?GTTTAACTGA?TGGTATGGTA?1080
GCTTGTAAGA?AGACCGAACC?AGGGATGGCC?GGCGAGAAAG?CCGATAGTCA?CATGTCGGCA?1140
CAGCAGATCT?GTGCTTAG?1158
<210>?2
<211>?1038
<212>?DNA
<213 > mustard type rape (Brassica juncea)
<400>?2
ATGGCAACCG?TTGATCAGAC?CGCTGTAACC?ACCGGAACTA?AGAAGGCTTG?TGTCATCGGT?60
GGCACAGGAA?ACTTAGCCTC?TATTCTGATC?AAGCATTTGC?TTCAAAGCGG?CTACAAAGTT?120
AACACCACAG?TTAGAGATCC?AGAGAATGAG?AAGAAAATGG?CTCACCTTAA?GGTACTTCAA?180
GAGCTCGGGG?ATCTCAAGAT?CTTCAAGGCG?GATTTAACGG?ATGAAGGGAG?TTTCACTTCA?240
CCAATCTCGG?GCTGTGAATA?TGTGTTCCAT?GTCGCAACAC?CAATCAGCTT?TACATCTCAA?300
GATCCCGAGA?AAGACATGAT?CAAACCAGCG?GTACGAGGAG?TGATCAACGT?GTTGAAACCT?360
TGCTTAAAAT?CGAACTCGAT?CAAGCGCGTG?ATCTACACTT?CTTCAGCTGC?TGCGGTTTCT?420
ATCAACAACC?TTTCGGAACC?TGGACTTGTG?ATGACCGAAG?AAAACTGGTC?TGACGTTGAT?480
TTTCTCACAA?AGGAGAAGCC?GTTTAACTGG?GTTTACCCAG?TCTCAAAGAC?CTTAGCAGAA?540
AAGGAAGCTT?ATAAATATGC?GGAAGAGAAT?AAGATTGATC?TCGTTACTGT?GGTTCCAGCA?600
CTCATAGCCG?GAAACTCTCT?CCTCTCTGAT?CCTCCGAGCA?GTTTATCTCT?CTCGATGTCT?660
TTAATCACTG?GGAAAGAAAT?GCATCTGAGC?GGTCTCAAGG?AAATGCAGAA?GCTATCTGGA?720
TCCATCTCGT?TCATCCACGT?GGACGACCTA?GCTCGTGCAC?ATATGTTTCT?TGCGGAGAAA?780
GAAACAGCTT?CTGGCCGTTA?CATTTGCTGT?TATTACAACA?CAAATGTTCC?AGAGATTGCG?840
GATTTTCTCA?GGCGAAGATA?TACTAAGTAC?AATGTTTTGT?CAGAATTTGA?AGAGTGCTTA?900
TCAAGTGCGA?AACTGACGCT?ATCCTCGGAA?AAACTCATCA?AGGAAGGCTT?TCGATTTGAA?960
TATGGGATCA?GTGAGATGTA?TGATGAGATG?ACGGAGTACT?CCGAGTCAAA?AGGATTGATC?1020
AAACCTAAAG?TATCTTGA?1038
Claims (1)
1. a method that adopts mustard type rape gene BAN and DFR cotransformation to improve intestinal bacteria Procyanidins content, is characterized in that, comprises the following steps:
(1) clone's cyanidin(e) reductase enzyme BAN gene and 4-flavanonol reductase enzyme DFR gene from the cDNA of mustard type rape kind skin;
(2) BAN gene and DFR gene are connected in to expression regulation sequence operably, form the prokaryotic expression carrier pET-BAN-DFR containing BAN gene and DFR gene;
(3) BAN and DFR gene are imported to DH5 α intestinal bacteria simultaneously, transformed bacteria is in the LB liquid nutrient medium, 25-37 ℃, 150-250rpm (rev/min) cultivate after 6-14 hour, again containing in the LB liquid nutrient medium of black soybean peel extract, 25-37 ℃, the 150-250rpm concussion was cultivated after 36-54 hour, at room temperature, centrifugal force is the centrifugal collection thalline of 10000-12000g;
(4) adopt the DMACA-HCl method to measure the amount of the contained proanthocyanidin of intestinal bacteria, screening obtains proanthocyanidin content and improves the gene coli strain;
In step (3), contain 0.5-1.0g/L black soybean peel extract in described LB liquid nutrient medium;
The extracting method of described black soybean peel extract: the black soybean peel is pulverized with pulverizer, cross the sieve of 1.0 mm, will be with the ethanol of 30%-70% (v/v) as extraction solvent, the soybean peel amount is 1 with the ratio of solvent load: 4-8 (w/w), extraction temperature is 60 ℃-70 ℃, and extraction time is 4-8 hour; Concentrated solution centrifugal 10-15min in the whizzer of 8000-10000g after recovery ethanol, filter impurity elimination, then add acetone in concentrated solution, concentrated solution is 1 with the ratio of acetone: 1-3 (v/v), stirring and evenly mixing 2-5min, remove by filter impurity, concentrated solution, after reclaiming acetone, obtains the soybean peel extract 40 ℃ of-60 ℃ of lower vacuum-dryings.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273985A (en) * | 1999-05-17 | 2000-11-22 | 中国科学院华南植物研究所 | Process for preparing proanthocyandin |
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| CN1273985A (en) * | 1999-05-17 | 2000-11-22 | 中国科学院华南植物研究所 | Process for preparing proanthocyandin |
Non-Patent Citations (5)
| Title |
|---|
| High-Yield Anthocyanin Biosynthesis in Engineered Escherichia coli;Yajun Yan et.al;《Biotechnology and Bioengineering》;20080501;第100卷(第1期);第2页左栏最后一段，第4页左栏最后一段，第5页左栏最后一段 * |
| The DMACA-HC1 Protocol and the Threshold Proanthocyanidin Content for Bloat Safety in Forage Legumes;Yu-Guang Li et.al;《Journal of the Science of Food and Agriculture》;19961231;第70卷(第1期);全文 * |
| Yajun Yan et.al.High-Yield Anthocyanin Biosynthesis in Engineered Escherichia coli.《Biotechnology and Bioengineering》.2008,第100卷(第1期),第126-140页. |
| Yu-Guang Li et.al.The DMACA-HC1 Protocol and the Threshold Proanthocyanidin Content for Bloat Safety in Forage Legumes.《Journal of the Science of Food and Agriculture》.1996,第70卷(第1期), |
| 严明理.芥菜型油菜黄籽形成的分子机理研究.《中国博士学位论文全文数据库（农业科技辑）》.2008,(第7期),D047-9. * |
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