CN105255936A - Method for establishing salinispora arenlocola CNS-205 gene genetic manipulation system - Google Patents
Method for establishing salinispora arenlocola CNS-205 gene genetic manipulation system Download PDFInfo
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Abstract
The invention discloses a method for establishing a salinispora arenlocola CNS-205 gene genetic manipulation system. The method includes the steps that a sterilized SFMS culture medium is melted, the temperature is lowered to be 40-50 DEG C, an appropriate amount of antibiotic is added in the SFMS culture medium, a flat plate is inverted, salinispora arenlocola CNS-205 spore suspension coats the flat plate, and culture is conducted for 4-5 days at the temperature of 25-30 DEG C; the unified plasmid is introduced into a conjugational transfer donor through a CaCl2 method, and the unified plasmid is introduced into a salinispora arenlocola CNS-205 spore obtained in the step A from the donor in a conjugational transfer mode to be integrated by the aid of tra genes on the donor. According to the technical scheme, the probability of introducing exogenous DNA fragments into the salinispora arenlocola CNS-205 is realized.
Description
Technical field
The present invention relates to technical field of biological genetic engineering, particularly relate to a kind of construction process of marine actinomycete gene genetic operating system.
Background technology
Streptomycete (Streptomyces) belongs to Prokaryota, actinomycetales, Streptomycetaceae on taxonomy, is the aerobic gram positive bacterium that a class has branch filament, is one of microbe groups main in soil.Streptomyces gene group mean size is 8000kb, is about the twice of genome of E.coli.With other biophase ratio, one of maximum feature of its genome is exactly that its DNA has high G+Cmol%, can up to 69 ~ 78%, is one of biological group that known up to now G+Cmol% content is the highest.Streptomycete is one of monoid with commercial value most in industrial microorganism.People have recognized that occurring in nature has the microbiotic of nearly 70% to be produced by streptomycete and nearly edge actinomycetes thereof.First microbiotic is found from nineteen twenty-eight AlexanderFleming--since penicillin, ten hundreds of microbiotic be it is found that just continuously and creates out and bring benefit to the mankind.
As bioactive natural product important sources Lu Sheng microorganism along with research continuous expansion with deeply, its species diversity and corresponding structure, active diversity gradually be familiar with by people, but as biogenesis with cover the microorganism contained in the ocean of earth surface about 70% and know little about it.Ocean residing for marine microorganism is a very unique environment, has the feature of high salt, high-alkali, low temperature, result in the diversity of microbe groups and the diversity of microbial gene resource.The microorganism cultivated at present only accounts for 0.1% to 1% of all microorganisms, and therefore abundant Living marine resources are undoubtedly the important sources of natural drug screening.It is reported, US National institute of oncology screens in new anticancer compound every year has 5% from marine organisms, and the compound extracted from marine organisms has 10% to have antitumour activity.Therefore, based on the exploration of the bioactive natural product biosynthetic pathway of marine microorganism and discovery be important supplement and the extension of new drug development.
An important colony in marine microorganism is exactly specificity marine actinomycete.Fenical in 1991 etc. found first rely on seawater growth special actinomycetes family and by its called after " MAR1 ".2005, after analyzing in conjunction with comprehensive morphological feature analysis and gene order-checking, " MAR1 " was accredited as first specificity marine actinomycete, and formally renames as " Salinispora ".Subsequently, actinomyces are in succession separated is accredited as specificity marine actinomycete for Demequina, Marinispora, Solwaraspora, Lamerjespora, Serinicoccus, Salinibacterium, Aeromicrobium, Willamsia, Marinactinospora and Sciscionella etc.Wherein, marine actinomycete SalinisporaarenlocolaCNS-205 is exactly the representative bacterial strain of specificity marine actinomycete Salinispora.Marine actinomycete SalinisporaarenlocolaCNS-205 is widely distributed, and due to the singularity of its living environment, this bacterium generation various active is various, novel structure secondary metabolite, becomes the new resources of new drug development research.But its biosynthesizing mechanism is but always not revealed.Not yet about the construction process of Yu Haiyang actinomycetes gene genetic operating system in prior art.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of construction process of marine actinomycete gene genetic operating system, be intended to solve in prior art not yet about the problem of the construction process of Yu Haiyang actinomycetes gene genetic operating system.
Technical scheme of the present invention is as follows:
A construction process for marine actinomycete gene genetic operating system, wherein, comprises step:
A, sterilized SFMS substratum melted, temperature is down to 40 ~ 50 DEG C, adds appropriate microbiotic, be down flat plate in SFMS substratum, by SalinisporaarenlocolaCNS-205 spore suspension spread plate, cultivates 4 ~ 5d for 25 ~ 30 DEG C;
B, integrative plasmid is passed through CaCl
2method imports in donor bacterium, and by means of tra gene on donor bacterium, described integrative plasmid to import to the SalinisporaarenlocolaCNS-205 spore that steps A obtains from donor bacterium in conjugal transfer mode and integrates.
The construction process of described marine actinomycete gene genetic operating system, wherein, in described steps A, described microbiotic is apramycin.
The construction process of described marine actinomycete gene genetic operating system, wherein, in described step B, described integrative plasmid is pSET152 and pIB139.
The construction process of described marine actinomycete gene genetic operating system, wherein, in described step B, described donor bacterium is pUZ8002.
The construction process of described marine actinomycete gene genetic operating system, wherein, in described step B, the ratio of donor bacterium and recipient bacterium SalinisporaarenlocolaCNS-205 spore is (15 ~ 1): 1.
The construction process of described marine actinomycete gene genetic operating system, wherein, in described step B, the ratio of donor bacterium and recipient bacterium SalinisporaarenlocolaCNS-205 spore is 8:1.
The construction process of described marine actinomycete gene genetic operating system, wherein, comprise after described step B: with apramycin resistance gene aac (3) IV on integrative plasmid pSET152 and pIB139 for template, design primer CP1 and CP2, verify conjugal transfer, extracts SalinisporaarenlocolaCNS-205 genomic dna performing PCR of going forward side by side and verifies.
The construction process of described marine actinomycete gene genetic operating system, wherein, described CP1 is 5 '-TTTATCACCACCGACTATTTGC-3 '; Described CP2 is 5 '-TCATCTCGTTCTCCGCTCA-3 '.
The construction process of described marine actinomycete gene genetic operating system, wherein, the conditioned response program of described PCR checking is 98 DEG C of 1min; 98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C extend 10min.
Beneficial effect: integrative plasmid to be imported to recipient bacterium SalinisporaarenlocolaCNS-205 spore from donor bacterium by conjugal transfer mode and integrates by the present invention, achieves the possibility importing exogenous dna fragment to SalinisporaarenlocolaCNS-205.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the PCR checking of pIB139 and pSET152 conjugal transfer in the embodiment of the present invention 1.
Embodiment
The invention provides a kind of construction process of marine actinomycete gene genetic operating system, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The invention provides a kind of construction process of marine actinomycete gene genetic operating system, it comprises step:
A, sterilized SFMS substratum melted, temperature is down to 40 ~ 50 DEG C, adds appropriate microbiotic, be down flat plate in SFMS substratum, by SalinisporaarenlocolaCNS-205 spore suspension spread plate, cultivates 4 ~ 5d for 25 ~ 30 DEG C;
B, integrative plasmid is passed through CaCl
2method imports in donor bacterium, and by means of tra gene on donor bacterium, described integrative plasmid imports to SalinisporaarenlocolaCNS-205 spore from donor bacterium in conjugal transfer mode, and integrates.
Pass through technique scheme, the present invention successfully by site-specific integration type plasmid integration in SalinisporaarenlocolaCNS-205 spore, on follow-up SalinisporaarenlocolaCNS-205 karyomit(e), the functional study of each gene and the transformation of biosynthesis gene are laid a good foundation.
In described steps A, described microbiotic is apramycin.
In described step B, described integrative plasmid is pSET152 and pIB139.Plasmid is a kind of closed hoop double-stranded DNA, and it does not belong to genomic dna, is free on outside chromosomal DNA.In streptomycete genetic manipulation, people often it can be used as carrier to import to mediate foreign DNA to host cell.Carrier can whether the feature that can be incorporated on streptomycete chromosome DNA be divided into integrating vector and non-integrating vectors according to it, and such as, integrating vector is integrative plasmid pIB139 and pSET152.Such as, and whether non-integrating vectors can be divided into suicide type carrier and episomal vector, episomal vector pYH7 containing streptomycete replicon according to it.
The mode of Exogenous DNA transfered host cell is mainly contained: calcium transfer, conjugal transfer, electricity conversion and microinjection etc.In step B of the present invention, select the mode of conjugal transfer to import in SalinisporaarenlocolaCNS-205 spore by external source integrative plasmid, this be due to conjugal transfer import that efficiency is higher because having, height is across species variation, the advantage such as cheap, easy to operate.
In described step B, described donor bacterium is pUZ8002.
In described step B, the donor bacterium (intestinal bacteria) of conjugal transfer and the ratio of recipient bacterium SalinisporaarenlocolaCNS-205 spore are (15 ~ 1): 1.Preferably, in described step B, the donor bacterium (intestinal bacteria) of conjugal transfer and the ratio of recipient bacterium SalinisporaarenlocolaCNS-205 spore are 8:1.This is due in conjugal transfer process, and the donor bacterium containing foreign vector and recipient bacterium (SalinisporaarenlocolaCNS-205 spore), when same grow on plates, exist between the two and inevitably vie each other.Because the speed of growth of recipient bacterium SalinisporaarenlocolaCNS-205 spore is slower than the speed of growth of donor bacterium, excessive donor bacterium can make the growth of chain SalinisporaarenlocolaCNS-205 spore be suppressed, and causes conjugal transfer rate to reduce.When the described donor bacterium of conjugal transfer and the ratio of described SalinisporaarenlocolaCNS-205 spore are 8:1, conjugal transfer rate reaches the highest.
Describe the present invention below by specific embodiment.
Embodiment
Experiment material
Gene clone acceptor E.coliDH10B, conjugal transfer donor bacterium E.coliET12567 (pUZ8002), integrative plasmid pSET152 and pIB139(all carry apramycin resistance gene aac (3) IV, conjugal transfer site oriT).
Reagent
Thiostrepton and apramycin purchased from American Sigma company; Paraxin, penbritin, kantlex, thiostrepton, Lambda/Hind, restriction enzyme, DNA reclaim test kit, plasmid extraction kit etc. purchased from the precious biotechnology company limited in Dalian; Other common agents are all purchased from Shanghai traditional Chinese medicines group.
Substratum and microbiotic
The plate culture medium of SalinisporaarenlocolaCNS-205 is SFMS, culturing bacterium for LB substratum, the pre-germination medium of spore be 2 × YT, conjugal transfer uses SFMS substratum.
In LB, antibiotic concentration is: apramycin 25 μ g/mL, paraxin 25 μ g/mL, kantlex 25 μ g/mL, penbritin 100 μ g/mL; In conjugal transfer substratum, antibiotic concentration is apramycin 25 μ g/mL, nalidixic acid 25 μ g/mL.
(1), antibiotics sensitivity experiment
Melted by sterilized SFMS substratum, temperature is down to 45 DEG C, adds microbiotic (kantlex 0 ~ 100 μ g/mL of suitable mass concentration; Paraxin 0 ~ 100 μ g/mL; Apramycin 0 ~ 100 μ g/mL; Thiostrepton 0 ~ 100 μ g/mL), be down flat plate, by the spore suspension spread plate of SalinisporaarenlocolaCNS-205 bacterial strain, cultivate 5d, according to the susceptibility of SalinisporaarenlocolaCNS-205 strain growth state evaluation SalinisporaarenlocolaCNS-205 strains for 28 DEG C.
(2) conjugal transfer, between intestinal bacteria and SalinisporaarenlocolaCNS-205
Integrative plasmid pSET152 and pIB139 is passed through CaCl
2method imports in donor bacterium pUZ8002, and by means of tra gene on pUZ8002, above-mentioned two kinds of plasmids import to SalinisporaarenlocolaCNS-205 spore from pUZ8002 in conjugal transfer mode, and integrate.
(3), the checking of conjugal transfer
With apramycin resistance gene aac (3) IV on integrative plasmid pSET152 and pIB139 for template, design primer CP1 and CP2 is to verify positive conjugal transfer.
CP1：5’-TTTATCACCACCGACTATTTGC-3；
CP2：5’-TCATCTCGTTCTCCGCTCA-3’。
Extract SalinisporaarenlocolaCNS-205 genomic dna to go forward side by side performing PCR checking.PCR conditioned response program used is 98 DEG C of 1min; 98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C extend 10min.
(4), the stability of plasmid pSET152 and pIB139 in SalinisporaarenlocolaCNS-205 conjugal transfer
Conjugal transfer is inoculated in not containing on antibiotic SFMS flat board, after its spore maturation (about 15d), again be inoculated in not containing on antibiotic SFMS flat board, separation and purification is carried out after continuous switching 3 times, random choose list bacterium colony, be transferred on the SFMS flat board containing 25 μ g/mL apramycins, can grow according to single bacterium colony after 5d and determine the genetic stability of plasmid pSET152 and pIB139 in zygote.
Result shows:
(1), the susceptibility of SalinisporaarenlocolaCNS-205 strains as shown in Table 1, SalinisporaarenlocolaCNS-205 bacterial strain can not grow containing on the substratum of 12.5 μ g/mL kantlex and apramycin, show SalinisporaarenlocolaCNS-205 bacterial strain to kantlex and apramycin very responsive; SalinisporaarenlocolaCNS-205 bacterial strain, not growing containing on the substratum of 25 μ g/mL paraxin, shows that SalinisporaarenlocolaCNS-205 bacterial strain is more responsive to paraxin; SalinisporaarenlocolaCNS-205 bacterial strain can grow on the substratum containing thiostrepton 100 μ g/mL, illustrates that SalinisporaarenlocolaCNS-205 is insensitive to thiostrepton.In view of the conjugal transfer integrative plasmid used in the embodiment of the present invention carries apramycin resistance gene aac (3) IV, therefore with 25 μ g/mL apramycin screening conjugal transfer.
Table 1, the SalinisporaarenlocolaCNS-205 growing state on four kinds of different concns microbiotic flat boards, wherein, raised growth (++); A small amount of growth (+); Do not grow (-).
(2) the fresh mycelia, respectively integrative plasmid pSET152 and pIB139 being imported to SalinisporaarenlocolaCNS-205 from donor bacterium pUZ8002 by amphiphilic conjugal transfer method and spore, result shows: the amphiphilic conjugal transfer carried out with the pUZ8002 bacterial strain containing integrative plasmid pSET152 and pIB139 as recipient bacterium at SalinisporaarenlocolaCNS-205 bacterial strain sprouting spore, can obtain the resistant clones of apramycin; And in the amphiphilic conjugal transfer that SalinisporaarenlocolaCNS-205 bacterial strain fresh mycelia carries out with the pUZ8002 bacterial strain containing integrative plasmid pSET152 and pIB139 as recipient bacterium, do not obtain the resistant clones of apramycin.
(3), in order to determine that recipient bacterium in conjugal transfer process (SalinisporaarenlocolaCNS-205 spore) and donor bacterium (pUZ8002) inoculate optimum proportion, pUZ8002 and the SalinisporaarenlocolaCNS-205 spore of different ratios carries out mixing spread plate.The results are shown in Table 2 to show: when the ratio of intestinal bacteria and SalinisporaarenlocolaCNS-205 spore reaches 8:1, the conjugal transfer subnumber of acquisition is maximum.
Table 2, based on streptomycete spore conjugal transfer flat board statistics
| The ratio of intestinal bacteria and spore | 15:1 | 8:1 | 5:1 | 1:1 |
| pSET152 | 8 | 11 | 2 | 2 |
| pIB139 | 84 | 85 | 56 | 12 |
(4), with CP1 and CP2 for primer, carry out pcr amplification checking, and using the amplification of starting strain SalinisporaarenlocolaCNS-205 genomic dna as negative control, the amplification of integrative plasmid pSET152 and pIB139 is as positive control, the result of amplification as shown in Figure 1,2 strain conjugal transfer obtained can amplify the 882bp band of prediction, reclaim the sub-amplified band of 2 strain conjugal transfer respectively, send order-checking after TA clone, sequencing result also demonstrate that 2 strains are all positive colony.Fig. 1 verifies from the PCR of pIB139 and pSET152 conjugal transfer, in Fig. 1, and 1:1kbladder; 2: integrative plasmid pIB139; 3: integrative plasmid pSET152; 4: wild-type SalinisporaarenlocolaCNS-205; 5: conjugal transfer containing pIB139; 6: conjugal transfer containing pSET152.1:1kbladder;2:pIB139plasmid;3:pSET152plasmid;4:wild-type;5:wild-type/pIB139;6:wild-type/pSET152。
(5), in order to detect the stability of integrative plasmid pIB139 and pSET152 in SalinisporaarenlocolaCNS-205 bacterial strain, random picking verifies 200 correct conjugal transfer, find that they can grow on the SFMS culture medium flat plate being added with apramycin and antibiotic-free, do not find the bacterium colony that apramycin resistance disappears.Result shows that integrative plasmid pIB139 and pSET152 Absorbable organic halogens in SalinisporaarenlocolaCNS-205 bacterial strain exists.
From above-described embodiment 1, by to SalinisporaarenlocolaCNS-205 antibiotics sensitivity and the mensuration based on SalinisporaarenlocolaCNS-205 spore and mycelium conjugal transfer system, successfully site-specific integration type plasmid pIB139 and pSET152 is incorporated in SalinisporaarenlocolaCNS-205 karyomit(e), the exactness of conjugal transfer obtained is proved at molecular level, this illustrates that the marine actinomycete gene genetic operating system set up is effective and feasible, for on follow-up SalinisporaarenlocolaCNS-205 karyomit(e), the functional study of each gene and the transformation of biosynthesis gene are laid a good foundation.
In sum, the construction process of a kind of marine actinomycete gene genetic operating system provided by the invention, by setting up the gene transfer system of SalinisporaarenlocolaCNS-205, import exogenous dna fragment by conjugal transfer to SalinisporaarenlocolaCNS-205.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (9)
1. a construction process for marine actinomycete gene genetic operating system, is characterized in that, comprises step:
A, sterilized SFMS substratum melted, temperature is down to 40 ~ 50 DEG C, adds appropriate microbiotic, be down flat plate in SFMS substratum, by SalinisporaarenlocolaCNS-205 spore suspension spread plate, cultivates 4 ~ 5d for 25 ~ 30 DEG C;
B, integrative plasmid is passed through CaCl
2method imports in donor bacterium, and by means of tra gene on donor bacterium, described integrative plasmid to import to the SalinisporaarenlocolaCNS-205 spore that steps A obtains from donor bacterium in conjugal transfer mode and integrates.
2. the construction process of marine actinomycete gene genetic operating system according to claim 1, is characterized in that, in described steps A, described microbiotic is apramycin.
3. the construction process of marine actinomycete gene genetic operating system according to claim 1, is characterized in that, in described step B, described integrative plasmid is pSET152 and pIB139.
4. the construction process of marine actinomycete gene genetic operating system according to claim 1, is characterized in that, in described step B, described donor bacterium is pUZ8002.
5. the construction process of marine actinomycete gene genetic operating system according to claim 1, is characterized in that, in described step B, the ratio of donor bacterium and recipient bacterium SalinisporaarenlocolaCNS-205 spore is (15 ~ 1): 1.
6. the construction process of marine actinomycete gene genetic operating system according to claim 1, is characterized in that, in described step B, the ratio of donor bacterium and recipient bacterium SalinisporaarenlocolaCNS-205 spore is 8:1.
7. the construction process of marine actinomycete gene genetic operating system according to claim 1, it is characterized in that, comprise after described step B: with apramycin resistance gene aac (3) IV on integrative plasmid pSET152 and pIB139 for template, design primer CP1 and CP2, verify conjugal transfer, extracts SalinisporaarenlocolaCNS-205 genomic dna performing PCR of going forward side by side and verifies.
8. the construction process of marine actinomycete gene genetic operating system according to claim 7, is characterized in that, described CP1 is 5 '-TTTATCACCACCGACTATTTGC-3 '; Described CP2 is 5 '-TCATCTCGTTCTCCGCTCA-3 '.
9. the construction process of marine actinomycete gene genetic operating system according to claim 7, is characterized in that, the conditioned response program of described PCR checking is 98 DEG C of 1min; 98 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C extend 10min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754882A (en) * | 2016-12-29 | 2017-05-31 | 佛山科学技术学院 | A kind of method of amidase PCR |
| CN110408641A (en) * | 2019-07-23 | 2019-11-05 | 湖北大学 | A kind of genetic transforming method of Selective medium strain and its application |
| CN115960791A (en) * | 2023-01-08 | 2023-04-14 | 天津大学 | Marine-source actinomycete gene genetic operation system and construction method |
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2015
- 2015-10-15 CN CN201510663567.1A patent/CN105255936A/en active Pending
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| HUILING WU等: "SlnM gene overexpression with different promoters on natamycin production in Streptomyces lydicus A02，", 《J IND MICROBIOL BIOTECHNOL》 * |
| 林钦恒 等: "浅蓝霉素产生菌海洋异壁放线菌 WH1-2216-6 遗传操作体系的建立", 《微生物学报》 * |
| 陆胜利 等: "海洋放线菌Salinispora arenlocola CNS-205腺苷化结构域基因的克隆、表达和纯化", 《华中师范大学学报（自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754882A (en) * | 2016-12-29 | 2017-05-31 | 佛山科学技术学院 | A kind of method of amidase PCR |
| CN110408641A (en) * | 2019-07-23 | 2019-11-05 | 湖北大学 | A kind of genetic transforming method of Selective medium strain and its application |
| CN115960791A (en) * | 2023-01-08 | 2023-04-14 | 天津大学 | Marine-source actinomycete gene genetic operation system and construction method |
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Application publication date: 20160120 |