CN109777822A - A kind of method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene - Google Patents
A kind of method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene Download PDFInfo
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Abstract
The invention discloses a kind of methods of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene, and this method includes: the test of plasmid single endonuclease digestion are carried out using restriction endonuclease, to prepare linear plasmid；Wherein, restriction endonuclease includes: any one in BamH I, Xba I, EcoR I and Sac I；Linear plasmid is converted using glass bead method: the Chlamydomonas reinhardtii of mid-log phase being resuspended using culture medium, the bead of 3~5 μ g plasmids and sterilizing is added, the chlamydomonas after shaking 24~26s is transferred in new culture medium, dark recovery；After recovering, using culture medium successively illumination and dark culturing containing 14~16 μ g/mL bleomycins, Chlamydomonas reinhardtii transformant is obtained.Method of the invention can be improved conversion ratio, realize that linear plasmid is integrated into Matrix attachment region.
Description
Technical field
The present invention relates to a kind of methods of nucleus Efficient Conversion foreign gene, and in particular to a kind of chlamydonomas reinhardtii cells core
The method of Efficient Conversion foreign gene.
Background technique
Chlamydomonas reinhardtii (Chlamydomonas reintmrdtii) is eucaryote unicellular, with flagellum, is under the jurisdiction of green
Algae door volvocales Chlamydomonas, 3 sets of genomes can carry out genetic transformation, realize the conversion of single foreign gene, such as
Ble, aadA, CRY1-1, nptII, GAT and ALS, and a variety of high added value albumen of successful expression, such as have reported HbcAg,
The pharmaceutical proteins such as HIVP24, EGF, the functional proteins such as FAO1, people's selenoprotein and functional enzyme Xylanase1.Therefore, by Rhein
Chlamydomonas exploitation is that " bioreactor " has great application value.
Based on the Integration Mode of the non-homogeneous recombination (Non-homologous End Joining, NHEJ) in end, Rhein clothing
Algae in conversion exogenous DNA by randomly, non-homogeneously be integrated into different loci, this mode may cause gene delection,
Recombination and dystopy, exogenous genetic fragment quantity, length and the integrality of integration have uncertainty.Moreover, chlamydonomas reinhardtii cells
Circular plasmids are used in the research of Nuclear transformation foreign gene mostly, resistance screening gene, endogenesis promoter and terminator are constituted
Fixed expression cassette, this structure make foreign gene be not easy to be integrated into chlamydomonas Matrix attachment region, further increase external source base
Because of the difficulty of conversion.Therefore, start to probe into linear plasmid conversion, in nineteen ninety, Kindle Karen L studies Chlamydomonas reinhardtii glass
It is proposed when glass pearl method transformation system: linear plasmid conversion can get preferable changing effect, and conversion ratio is up to 103A transformant/μ
G plasmid, the significantly larger than transformation efficiency of super spirial plasmid.
But the key conditions such as plasmid single endonuclease digestion mode and linear plasmid conversion are not yet illustrated, therefore, research is linear
The conversion of plasmid is of great significance to the Nuclear transformation efficiency of chlamydomonas is improved.In addition, the problems such as transgene efficiency is low, gene silencing
It is not solved effectively also, exogenous gene expression amount, recombinant protein expression quantity and activity level are high not enough.Therefore, need be
System comprehensively studies the Nuclear transformation system of reinhardtii cell, probes into efficient, mass expressing external gene method.
Summary of the invention
The object of the present invention is to provide a kind of method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene, this method is solved
Existing method transformation efficiency low problem can be improved conversion ratio, realize that linear plasmid is integrated into Matrix attachment region.
In order to achieve the above object, the present invention provides a kind of sides of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene
Method, this method include: the test of plasmid single endonuclease digestion are carried out using restriction endonuclease, to prepare linear plasmid；Wherein, described
Restriction endonuclease includes: any one in BamH I, Xba I, EcoR I and Sac I；Using glass bead method transition lines
Property grain: the Chlamydomonas reinhardtii of mid-log phase is resuspended using culture medium, the bead of 3~5 μ g plasmids and sterilizing, concussion 24 is added
Chlamydomonas after~26s is transferred in new culture medium, dark recovery；After recovering, using 14~16 μ g/mL bleomycins of addition
Culture medium successively illumination and dark culturing obtain Chlamydomonas reinhardtii transformant.
Preferably, the culture medium is TAP culture medium.
Preferably, when using glass bead method conversion linear plasmid, the Chlamydomonas reinhardtii of mid-log phase is centrifuged, supernatant is abandoned
Afterwards, it is resuspended with TAP culture medium.
Preferably, the centrifugal rotational speed is 12,000r/min.
Preferably, when using glass bead method conversion linear plasmid, the dark recovery and illumination and dark culturing exist
It is carried out at 20 DEG C.
Preferably, the illumination cultivation 16h, dark culturing 8h.
Preferably, when using glass bead method conversion linear plasmid, the mass ratio of the plasmid and bead is 1:1*
105, the plasmid and Chlamydomonas reinhardtii amount ratio are 4 g:3 × 10 μ6It is a.
Preferably, when preparing linear plasmid, using pSP108 plasmid.
Preferably, when preparing linear plasmid, 10 × restriction enzyme Buffer in plasmid single endonuclease digestion reaction system: limit
Property restriction endonuclease processed: pSP108 plasmid: H2The volume ratio of O is 4:1:15:20.
Preferably, when preparing linear plasmid, plasmid single endonuclease digestion reaction condition is 37 DEG C of water-bath 3h.
The method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene of the invention, it is low to solve existing method transformation efficiency
The problem of, it has the advantage that
(1) method of the invention passes through BamH I, I digestion of Xba I, EcoR I or Sac, the equal energy of the linear plasmid of acquisition
Successful conversion, I restriction enzyme site of BamH are located at terminator, and the conversion ratio highest of linear plasmid, shows digestion after terminator after digestion
The linear plasmid of preparation is integrated into Matrix attachment region most useful for conversion, and ble gene is expressed on molecule and transcriptional level, verifying
The transformation efficiency of linear plasmid is higher；
(2) method of the invention, when conversion best plasmid dosage be 4 μ g, the best concussion time be 25s, most it is suitable it is rich come it is mould
Plain screening concentration is 15 μ g/mL, provides certain experiment basis and theoretical direction for building " chlamydomonas bioreactor ".
Detailed description of the invention
Fig. 1 is the flow chart of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene of the invention.
Fig. 2 is pSP108 plasmid enzyme restriction site schematic diagram.
Fig. 3 is the PCR testing result figure of transformant after I digestion of BamH.
Fig. 4 is the PCR testing result figure of transformant after I digestion of Sac.
Fig. 5 is the PCR testing result figure of transformant after I digestion of Xba.
Fig. 6 is the PCR testing result figure of transformant after I digestion of EcoR.
Fig. 7 is the RT-PCR testing result figure of positive algae strain.
Fig. 8 is the chlamydomonas conversion results figure under different plasmid dosages.
Fig. 9 is the chlamydomonas conversion results figure under the different concussion times.
Figure 10 is chlamydomonas transformant blasticidin resistance the selection result figure in TAP solid medium.
Note: in Fig. 3-7, M is 2000 DNA marker of DL, and 1 is positive control, and 2 be negative control, and 3 be blank control,
Remaining is transformant；In Fig. 8,1,2,3,4 respectively represent the plasmid dosage of 2,3,4,5 μ g；In Fig. 9,1,2,3,4 are respectively represented
15s, 20s, 25s, 30s concussion；In Figure 10,1,2,3,4 respectively represent 0,15,20,30 μ g/mL bleomycins.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
A kind of method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene, as shown in Figure 1, being Chlamydomonas reinhardtii of the invention
The flow chart of nucleus Efficient Conversion foreign gene, this method includes: carrying out plasmid single endonuclease digestion using restriction endonuclease
Test, to prepare linear plasmid；Wherein, the restriction endonuclease includes: BamH I, Xba I, EcoR I and Sac I
In any one；Linear plasmid is converted using glass bead method: the Chlamydomonas reinhardtii of mid-log phase being resuspended using culture medium, is added 3
The bead of~5 μ g plasmids and sterilizing, the chlamydomonas after shaking 24~26s are transferred in new culture medium, dark recovery；Wait recover
Afterwards, using culture medium successively illumination and the dark culturing that 14~16 μ g/mL bleomycins are added, Chlamydomonas reinhardtii transformant is obtained.
It is carried out below in the method for 1~4 pair of experimental example chlamydonomas reinhardtii cells core Efficient Conversion foreign gene of the invention detailed
It describes in detail bright.
Experimental material:
Cell wall deficiency Chlamydomonas reinhardtii CC-400, purchased from Duke Univ USA (TAP culture medium, 20 DEG C, brightness duration
16h/8h, shaking speed 120r/min)；The Nuclear transformation expression vector pSP108 (E.coli) of chlamydomonas is (sweet purchased from Duke Univ USA
Oil is preserved in -20 DEG C, LB culture medium, 37 DEG C, shaking speed 150r/min).
Experiment reagent:
Restriction endonuclease (Takara)；Bleomycin Zeocin (100mg/mL, Invitrogen)；Bead
(0.5mm,Sigma)；10×PCR buffer,0.25mmol/L MgCl2, dNTPs, Taq enzyme, DL2000 DNA marker,
DNA of bacteria extracts kit and the small extraction reagent kit of plasmid are purchased from Tiangeng biochemical technology Co., Ltd.
Laboratory apparatus:
MGC-100P type illumination box (is purchased from Shanghai Yiheng Scientific Instruments Co., Ltd)；PowerPac Basic type electricity
Swimming instrument, Gel Doc type gel imaging system, S1000 type PCR instrument (Bio-Rad)；The desk-top room temperature supercentrifuge of PiCo21 type
(Thermo)；3 type vortex oscillator (IKA) of Genius；ZWYR-D2403 type constant-temperature shaking incubator (Shanghai intelligence city analysis instrument
Manufacturing Co., Ltd).
Assay medium:
TAP (Tris-acetate-phosphate) culture medium: 0.4g NH4Cl、0.026g CaCl2·2H2O、
0.0935g K2HPO4、0.062g KH2PO4、0.3465g MgSO4·7H2O, 2.423g Tris (trishydroxymethylaminomethane),
1mL Hunter ' s trace element is added appropriate glacial acetic acid and adjusts pH value of solution to 7.0,15g/ is added in solid medium
L agar powder, 121 DEG C of high pressure sterilization 20min.
1 single endonuclease digestion of experimental example prepares linear plasmid
As shown in Fig. 2, being pSP108 plasmid enzyme restriction site schematic diagram, according to pSP108 plasmid construction map, limited using 8 kinds
Property endonuclease processed carries out the test of plasmid single endonuclease digestion, to prepare linear plasmid.
Plasmid single endonuclease digestion system (80 μ L of total volume) includes: in 8 μ 10 × restriction enzyme of L Buffer, 2 μ L are restricted
Enzyme cutting, 30 μ L pSP108 plasmids, 40 μ L H2O, single endonuclease digestion condition are 37 DEG C of water-bath 3h.
The dehydrated alcohol of two volumes is added in plasmid after digestion, in freezing 2h in -80 DEG C of refrigerators after mixing well,
12,000r/min centrifugation 4min abandon supernatant, then are rinsed with 70% ethyl alcohol, dry up remaining ethyl alcohol and 40 μ L ddH are added2O is (pre-
Heat is to 60 DEG C) dissolution plasmid.
Experimental result: the linear plasmid energy successful conversion after BamH I, Xba I, EcoR I, I digestion of Sac, wherein
The digestion point of BamH I is located at after terminator, and transformant number is most, and 1 digestion point of Xba is located at after termination codon, EcoR
I restriction enzyme site is located at before the end of promoter 5 ', and the restriction enzyme site of Sac I is located at the end of promoter 5 '.It is tested and is made by single endonuclease digestion
Standby linear plasmid, obtains the linear plasmid through BamH I, I single endonuclease digestion of Xba I, EcoR I and Sac after converting using glass bead method
40 transformants are obtained in equal successful conversion.
The conversion of 2 linear plasmid of experimental example and resistance screening transformant
(1) linear plasmid is converted using glass bead method
3mL is taken to cultivate to the chlamydomonas of mid-log phase, 12,000r/min centrifugation 2min abandon supernatant, cultivated with 300 μ L TAP
Base weight hangs reinhardtii cell, and the bead of 3 μ g plasmids and 300mg sterilizing, drying is added, and the chlamydomonas after 30s will fully shake and be transferred to
In 20mL fresh TAP culture medium, 20 DEG C of dark recoveries are overnight.
Chlamydomonas centrifugal concentrating is to 150 μ L after recovery, is spread evenly across containing on 15 μ g/mL bleomycin TAP plates, Yu Guangzhao
It is cultivated in incubator, temperature is 20 DEG C, illumination 16h, dark 8h, screens positive chlamydomonas, and single algae, which falls about 2~4 weeks, to be occurred.
Choose the single algae grown on blasticidin resistance plate to fall, be incubated in TAP fluid nutrient medium, while cultivating one group
Non-transformed chlamydomonas is cultivated to mid-log phase as control and extracts the DNA of chlamydomonas according to DNA of bacteria extracts kit specification.
(2) the PCR identification of positive chlamydomonas
Based on the gene order of ble, design primer is as follows:
ble-sense:5′-GGATCCATGGCCAAGCTGACCAGCGCCGTTCC-3′；
ble-anti-sense:5′-AGCTTACCTCCGCCGTCCTGCTCCTCGGCCACGAAGTG-3′。
Genomic DNA is fallen as template using single algae of extraction, and pSP108 plasmid is positive control, and unconverted chlamydomonas is negative right
According to ddH2O is blank control, and using ble-sense/ble-anti-sense primer, the integration of ble gene is detected by PCR.
PCR amplification system are as follows: 10 × Taq buffer, 2.5 μ L, 0.25mmol/LMgCl22.0 μ L, dNTPs2.0 μ L, on
Each 1 μ L of downstream primer, 0.25 μ L of Taq enzyme, 1 μ L of template DNA add ddH2O water supplies 25 μ L.
Pcr amplification reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C extend
2min, totally 35 recycle；72 DEG C of extension 10min.
Experimental result: as illustrated in figures 3-6, occur single, bright band, the piece with positive control at 361bp
Segment length is consistent, and negative and blank control illustrates that ble gene is successfully integrated into clothing in transformant without target stripe
In algae Matrix attachment region；Wherein 15 positive sons, conversion ratio highest is obtained in linear plasmid conversion after I digestion of BamH.
The RT-PCR identification of the positive chlamydomonas of experimental example 3
7 plants of positive sons through PCR identification are selected at random, and the chlamydomonas of culture to mid log phase is collected by centrifugation, uses
Trizon reagent method extracts chlamydomonas total serum IgE, and reverse transcription obtains cDNA, and using cDNA as template, pSP108 plasmid is positive control, not
Conversion chlamydomonas is negative control, ddH2O is blank control, using ble-sense/ble-anti-sense primer, passes through RT-
The expression of PCR detection ble gene.With experimental example 2, amplified production carries out 1.5% agar for RT-PCR amplification system and reaction condition
Sugared gel electrophoresis.
Experimental result: as shown in fig. 7, for the RT-PCR testing result figure of positive algae strain, it can be seen that expanded through RT-PCR
Occur single, bright band at 361bp afterwards, it is consistent with the height of target gene band, demonstrate again that ble gene
It is successfully integrated among chlamydomonas genome, and in transcriptional level successful expression.
4 glass bead method of experimental example converts the optimization of linear plasmid conversion condition
(1) determination of best plasmid dosage
PSP108 plasmid is extracted by the small extraction reagent kit of plasmid, initial concentration is 0.5 μ g/mL, passes through cryoprecipitation
It is concentrated, and single endonuclease digestion is carried out by BamH I and prepares linear plasmid, take culture to the chlamydomonas of mid log phase, concussion
20s, bleomycin concentration are 20 μ g/mL, are converted using the linear plasmid of 2,3,4,5 μ g, 3 levels of every group of setting, are remembered
Record conversion situation.
Experimental result: when existing glass bead method converts, using the chlamydomonas (1 × 10 of logarithmic growth phase6/ mL), plasmid and algae
Body quantity ratio is 2 g:3 × 10 μ7It is a, positive rate highest after conversion.As shown in Figure 8, it can be seen that with the increase of plasmid dosage,
The number of transformant successively increases, but plasmid dosage be 4 μ g and 5 μ g when, transformant number difference is little, therefore the best matter of the present invention
Grain 4 μ g of dosage, frond number are 3 × 106It is a.
(2) the most preferably determination of concussion time
Take culture to the chlamydomonas of mid log phase, bleomycin concentration is 20 μ g/mL, using 15s, 20s, 25s, 30s
Carry out concussion conversion, 3 levels of every group of setting, record conversion situation.
Experimental result: the concussion time is an important factor for influencing the conversion of chlamydomonas glass bead method, if concussion time deficiency, cell
Film opening is too small, and plasmid is difficult to import；Overlong time is shaken, eucaryotic cell structure is even dead by destroying.It shakes as can be seen from Figure 9
When 15s, occurring almost without transformant, when shaking 30s, only obtains 3 transformants, transformant quantity is most when shaking 25s, therefore most
The good concussion time is 25s.
(3) determination of most suitable bleomycin concentration
Bleomycin will lead to the non-specific damage of chlamydomonas DNA, and the resistance screening gene ble codified used is to rich next
Mycin albumen has the Ble albumen of very big compatibility, when the two combines, then chlamydomonas energy normal growth；And chlamydomonas itself is to rich next
Mycin is very sensitive, it is virtually impossible to generate spontaneous resistant mutation.In order to determine the best screening concentration of positive chlamydomonas, takes and cultivate to right
The chlamydomonas of number growth mediums, plasmid concentration are 5 μ g/mL, and bleomycin concentration is 15 μ g/mL, shake 25s, using 0,15,20,
30 μ g/mL bleomycins are tested, 3 levels of every group of setting, record conversion situation.
Experimental result: containing screening chlamydomonas transformant in various concentration bleomycin solid medium, it can be seen that clothing
Algae is very sensitive for bleomycin, and as can be seen from Figure 10, when not adding bleomycin, chlamydomonas can not form single algae and fall, with
Bleomycin concentration increases, and chlamydomonas transformant number gradually decreases, when bleomycin concentration is 30 μ g/mL, almost without turning
Beggar's growth it is most to screen resulting transformant quantity by 15 μ g/mL bleomycins, accordingly, it is determined that screening chlamydomonas transformant
Most suitable bleomycin screening concentration be 15 μ g/mL.
It is discussed in detail although the contents of the present invention have passed through above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read above content, for of the invention
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. a kind of method of chlamydonomas reinhardtii cells core Efficient Conversion foreign gene, which is characterized in that this method includes:
The test of plasmid single endonuclease digestion is carried out using restriction endonuclease, to prepare linear plasmid；Wherein, the restriction nuclease
Restriction endonuclease includes: any one in BamH I, Xba I, EcoR I and Sac I；
Linear plasmid is converted using glass bead method: the Chlamydomonas reinhardtii of mid-log phase being resuspended using culture medium, 3~5 μ g matter are added
The bead of grain and sterilizing, the chlamydomonas after shaking 24~26s are transferred in new culture medium, dark recovery；After recovering, using containing
The culture medium of 14~16 μ g/mL bleomycins successively illumination and dark culturing obtain Chlamydomonas reinhardtii transformant.
2. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 1, which is characterized in that described
Culture medium is TAP culture medium.
3. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 1, which is characterized in that adopting
When converting linear plasmid with glass bead method, the Chlamydomonas reinhardtii of mid-log phase is centrifuged, after abandoning supernatant, is resuspended with TAP culture medium.
4. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 3, which is characterized in that described
Centrifugal rotational speed is 12,000r/min.
5. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 1, which is characterized in that adopting
When converting linear plasmid with glass bead method, the dark recovery and illumination and dark culturing are carried out at 20 DEG C.
6. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 5, which is characterized in that described
Illumination cultivation 16h, dark culturing 8h.
7. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 1, which is characterized in that adopting
When converting linear plasmid with glass bead method, the mass ratio of the plasmid and bead is 1:1*105, the plasmid and Chlamydomonas reinhardtii
Amount ratio is 4 g:3 × 10 μ6It is a.
8. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to any one of claims 1-7,
It is characterized in that, when preparing linear plasmid, using pSP108 plasmid.
9. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 8, which is characterized in that making
When standby linear plasmid, 10 × restriction enzyme Buffer in plasmid single endonuclease digestion reaction system: restriction enzyme: pSP108 matter
Grain: H2The volume ratio of O is 4:1:15:20.
10. the method for chlamydonomas reinhardtii cells core Efficient Conversion foreign gene according to claim 9, which is characterized in that
When preparing linear plasmid, plasmid single endonuclease digestion reaction condition is 37 DEG C of water-bath 3h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0342824A1 (en) * | 1988-05-05 | 1989-11-23 | The Scripps Research Institute | Methods and systems for transforming chlamydomonas reinhardtii |
| CN103757044A (en) * | 2013-11-04 | 2014-04-30 | 刘军 | Experimental method for transforming chlamydomonas by glass bead method |
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2019
- 2019-03-21 CN CN201910218911.4A patent/CN109777822A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0342824A1 (en) * | 1988-05-05 | 1989-11-23 | The Scripps Research Institute | Methods and systems for transforming chlamydomonas reinhardtii |
| CN103757044A (en) * | 2013-11-04 | 2014-04-30 | 刘军 | Experimental method for transforming chlamydomonas by glass bead method |
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| 刘芮均等: "莱茵衣藻外源基因整合特征分析及蛋白表达", 《四川大学学报(自然科学版)》 * |
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