CN109295079A - It is overexpressed AANAT gene and is improving the application in Chlamydomonas reinhardtii saline-alkaline tolerance - Google Patents
It is overexpressed AANAT gene and is improving the application in Chlamydomonas reinhardtii saline-alkaline tolerance Download PDFInfo
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Abstract
The invention discloses be overexpressed AANAT gene in the purposes for promoting Chlamydomonas reinhardtii saline-alkaline tolerance.The Chlamydomonas reinhardtii engineering algae that AANAT is overexpressed can help the enzyme activity for improving the antioxidant system of frustule and reduction membrane damage to improve the self-protection ability of Chlamydomonas reinhardtii with this come the undesirable stress from outside coped under condition of salt stress.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to be overexpressed AANAT gene and improving Chlamydomonas reinhardtii salt resistance
Application in ability.
Background technique
Salt stress is one of the Main Agricultural problem for causing crop-producing power to decline in the world.Salt stress passes through excessive sodium
Ion pair plant causes dehydration and osmotic stress injury to influence normal growth and the development of plant, is reply salt stress to plant
Ion toxicity and osmotic stress caused by object, Plant Evolution go out a variety of defense mechanisms to protect oneself.Wherein, oxidative stress is to plant
One of the mechanism of object early stage quick response salt stress, the active oxygen (ROS) that this process is formed includes: superoxide anion, peroxidating
Hydrogen and hydroxyl radical free radical etc..Moreover, ROS, as strong oxidizer, the ROS of high concentration can destroy film by lipid peroxidation, break
Bad DNA chain simultaneously inactivates various important enzymes.Plant forms a set of by antioxidase and non-anti- to cope with the injury of ROS
The antioxidant system of oxidizing ferment composition.Antioxidase mainly includes superoxide dismutase (SOD), catalase (CAT), mistake
Oxide enzyme (POD) etc., plant are removed or are reduced the accumulation of ROS by the effect of these enzymes, mitigate and resist salt stress pair
The injury of plant cell.
Summary of the invention
Technical problem to be solved by the present invention lies in view of the above shortcomings of the prior art, provide a kind of overexpression
AANAT gene is improving the application in Chlamydomonas reinhardtii saline-alkaline tolerance.
In order to solve the above technical problems, the technical solution adopted by the present invention is that, AANAT gene is overexpressed for promoting Lay
The purposes of mattress chlamydomonas saline-alkaline tolerance.
Further, using bead conversion method by the Matrix attachment region of AANAT channel genes Chlamydomonas reinhardtii.
The invention also discloses be overexpressed AANAT gene for promoting the effect of epiphysin overexpression in plant.
Further, which is Chlamydomonas reinhardtii.
The present invention is overexpressed AANAT gene and is improving the application in Chlamydomonas reinhardtii saline-alkaline tolerance, passes through technique for gene engineering
The Chlamydomonas reinhardtii engineering algae that obtained AANAT is overexpressed can help the anti-oxidant system for improving frustule under condition of salt stress
The enzyme activity and reduction membrane damage of system improve the self-protection ability of Chlamydomonas reinhardtii with this come the undesirable stress from outside coped with.
Detailed description of the invention
Fig. 1 Chlamydomonas reinhardtii nuclear expression vector construction flow chart；
Fig. 2 is PUCK-AANAT plasmid identification figure；
Wherein: APCR:M 15kb Marker, 1-4pUCK-AANAT, 5 blank control；
B Nde I, the verifying of EcoR I double digestion: M 10kb Marker, 1 blank control, 2-3 pUCK-AANAT；
Fig. 3 is pDBle-AANAT recombinant plasmid PCR verifying and double digestion proof diagram；
Wherein: APCR:M 15kb Marker, 1-8pDBle-AANAT, 9 positive control, 10 empty vector controls, 11 blank
Control；
B Nde I, EcoR I double digestion proof diagram: M 15kb Marker, 1 blank control, 2-3 pDBle-AANAT；
Fig. 4 converts culture figure of the algae strain on Ble resistance TAP plate；
Wherein: A adds 5.0 μ g/mL Ble TAP plate transformation chlamydomonas squamous subculture figures；
B adds 10.0 μ g/mL Ble TAP plate transformation chlamydomonas culture figure；
Fig. 5 is the molecular biological analysis by PCR analysis to Ble gene in different algal species bacterial strain: M 2kb
Marker, 1-8 are to convert algae strain, and 9 be positive control, and 10 convert Chlamydomonas reinhardtii for empty carrier, and 11 be unconverted algae plant, and 12 be empty
White control.
Fig. 6 detects the relative expression quantity of target gene AANAT in different algae strains by quantitative real-time RT-PCR.NT is not turn
The chlamydomonas of change；CK is the chlamydomonas of empty carrier conversion；T1, T2, T4, T5, T6 and T8 are the Chlamydomonas bacterial strains of conversion；Error bar is three
Average value (n=3) ± standard deviation of secondary measurement.
The variation of melatonin content in the algae strain of Fig. 7 difference, NT is unconverted scarce wall-shaped chlamydomonas；T1,T2,T4,T5,T6,T8
It is conversion algae strain.With Duncan's multiple comparative test, the error bar (mean+SD, n=3) of different letters is indicated
It is shown in the horizontal of P < 0.01 (capital latin) and significant difference is presented.
Influence of Fig. 8 salt stress to catalase activity in different algae strains, NT is unconverted scarce wall-shaped chlamydomonas；T1,T2,
T4, T5, T6, T8 are conversion algae strains.With Duncan's multiple comparative test, indicate different letters error bar (average value ±
Standard deviation, n=3) it is shown in the horizontal presentation significant difference of P < 0.01 (capital latin).
Influence of Fig. 9 salt stress to superoxide dismutase activity in different algae strains, NT is unconverted scarce wall-shaped chlamydomonas；
T1, T2, T4, T5, T6, T8 are conversion algae strains；With Duncan's multiple comparative test, the error bar for indicating different letters is (flat
Means standard deviation, n=3) it is shown in the horizontal presentation significant difference of P < 0.01 (capital latin).
Influence of Figure 10 salt stress to peroxidase activity in different algae strains, NT is unconverted scarce wall-shaped chlamydomonas；T1,
T2, T4, T5, T6, T8 are conversion algae strains；With Duncan's multiple comparative test, the error bar (average value of different letters is indicated
± standard deviation, n=3) it is shown in the horizontal presentation significant difference of P < 0.01 (capital latin).
Influence of Figure 11 salt stress to mda content in different algae strains, NT is unconverted scarce wall-shaped chlamydomonas；T1,T2,T4,
T5, T6, T8 are conversion algae strains；With Duncan's multiple comparative test, the error bar (average value ± standard of different letters is indicated
Difference, n=3) it is shown in the horizontal presentation significant difference of P < 0.01 (capital latin).
Specific embodiment
The present invention is overexpressed AANAT gene in the purposes for promoting Chlamydomonas reinhardtii saline-alkaline tolerance.It is converted using bead
Method will be in the Matrix attachment region of AANAT channel genes Chlamydomonas reinhardtii.
The invention also discloses the effects that AANAT gene is used to promote epiphysin overexpression in plant.The plant is
Chlamydomonas reinhardtii.
This experiment Chlamydomonas reinhardtii used is bought from the aquatic institute of Wuhan, China fresh water algae.
1. the building of Chlamydomonas reinhardtii nuclear expression carrier pDBle-AANAT；
The building process of Chlamydomonas reinhardtii nuclear expression carrier pDBle-AANAT is as shown in Figure 1.According to AANAT gene order
(NCBI GenBank, sequence number: AB474787.1) increases Nde I and EcoR I restriction enzyme site in upstream and downstream, is closed by company
At i.e. PUCK-AANAT, host strain is DH5 α.With 6.0 software Design primers of Primer Premier, sequence is as shown in table 1,
It is synthesized by company.PCR system is 20 μ L, contains upstream and downstream primer (AANAT-F, AANAT-R；20 μM) each 0.5 μ L, Premix
TaqTM10 μ L, 1 μ L of template.Amplification program is 95 DEG C of 8min of initial denaturation, is denaturalized 95 DEG C of 1min, and anneal 59 DEG C of 30s, extends 72 DEG C
50s, 30 circulations, 72 DEG C of 10min.
Plasmid PUCK-AANAT and pDBle restriction endonuclease Nde I and EcoR I, 37 DEG C of 3h are distinguished into enzyme
It cuts, recovery product is attached by T4-DNAligase, heat-shock transformed DH5 α competent cell, in Amp (100mg/mL) resistance
LB plate on carry out screening and culturing.Recombinant plasmid pDBle-AANAT is named as after obtaining positive monoclonal bacterium colony.
PCR and Nde I and EcoR I, 37 DEG C of double digestion 3h are carried out using PUCK-AANAT plasmid as template, then through 1% fine jade
Sepharose electrophoresis detection can observe the expection band of 576bp, as shown in Figure 2.
After culture obtains pDBle-AANAT positive monoclonal bacterium colony, picking single bacterium falls within 10mL containing Amp (100mg/mL)
It is incubated overnight in LB culture medium, extracts pDBle-AANAT plasmid with agent box.Through PCR and Nde I, EcoR I double digestion is verified all
The expection band of available 576bp, while there is not target stripe in empty vector control, as shown in Figure 3.10 μ L plasmids are taken to send
Sangon Biotech (Shanghai) Co., Ltd., which carries out DNA sequencing result, proves that the AANAT sequence on carrier is correct.
Each sequence is as follows:
1 gene order of table
2. bead conversion method converts Chlamydomonas reinhardtii cc-503；
Chlamydonomas reinhardtii cells wall missing 200 μ L of strain cc-503 original chlamydomonas algae solution is taken to be coated on TAP plate, wait grow uniformly
The backward picking list algae of algae falls to be inoculated in 100mL TAP fluid nutrient medium to be cultivated 5 days under 25 DEG C of 16h illumination conditions, activation training
Support logarithmic growth phase.Recombinant plasmid pDBle-AANAT is transferred in chlamydomonas genome using bead conversion method.It finally will be right
It is respectively coated on the TAP solid medium containing 5 μ g/mLBle according to the cell of group, experimental group, under 25 DEG C of 16h illumination conditions
Cultivate 15-20d.
After the conversion of bead conversion method chlamydomonas cc-503, about 15d, it is coated on containing the algae solution on 5 μ g/mL Ble TAP plates
Green algae can be seen to fall, continue picking list algae and fall within the progress secondary lineation culture of the TAP plate containing 10 μ g/mL Ble, green
Algae falls can be with continued growth, as shown in figure 4, illustrating that recombinant plasmid can help generation Ble resistance in the reinhardtii cell converted, life
It is long not inhibited by the Ble in TAP culture medium.The control of synchronous experimental group converts algae (not having Ble resistant gene), empty carrier
Conversion chlamydomonas and unconverted chlamydomonas cannot grow in resistant panel.
3. converting the screening and positive identification of Chlamydomonas reinhardtii algae strain；
Fall behind to grow algae in resistant panel, row number 1-8 (T is dropped into algae1-T8), continue at Ble resistance (5 μ g/mL)
TAP plate on squamous subculture to 3-4 generation, the algae of picking difference number, which falls in the TAP fluid nutrient medium containing 1 μ g/mLBle, to be trained
Support mid-log phase.Meanwhile NT is represented as conversion chlamydomonas strain.Take culture to the conversion algae strain of mid log phase, using changing
Good CTAB method extracts frustule genomic DNA, detects the integration of Ble by PCR come the whole of indirect proof target gene AANAT
It closes.TrizoL method extracts frustule RNA, with the II 1 st Strand cDNA Synthesis of PrimeScriptTM of TaKaRa
Kit kit reverse transcription cDNA, qRT-PCR detect the expression of AANAT.The reaction system for detecting Ble is 20 μ L, contains upstream and downstream
Primer ((Ble-F, Ble-R；20 μM) each 0.5 μ L, Premix TaqTM10 μ L, 1 μ L of template, 8 μ L of water；Amplification program is pre- becomes
Property 95 DEG C of 5min, be denaturalized 95 DEG C of 1min, anneal 59 DEG C of 30s, extend 72 DEG C of 1min, 32 circulations, 72 DEG C of 10min.Meanwhile it using
CDNA analyzes the expression of AANAT by real-time qRT-PCR as template.Gene specific for qRT-PCR amplification draws
Object is used for AANAT segment (aanat-F, aanat-R) and actin fragment (actin-F, actin-R).Reaction volume is 10-
μ L each contains 0.5 μ L upstream and downstream primer (10 μM), 5 μ L MasterSYRB Green Mix and 1 μ L template and 3 μ L
Water.Amplified reaction includes 95 DEG C of initial denaturations 10min, 95 DEG C of denaturation 10s, 62 DEG C of annealing 30s, 62 DEG C of extension 5s.Amplification includes total
Totally 39 circulations.
Specific step is as follows for above-mentioned CTAB method:
0.2g Chlamydomonas reinhardtii fresh weight is taken, liquid nitrogen is fully ground, and adding 650 2 × CTAB of μ L, (2 × CTAB is with preceding addition
0.2% mercaptoethanol) (2 × CTAB can be preheated in 65 DEG C of water-baths in advance) in 65 DEG C of water-bath 1h, during which mitigated every 10min
Overturning；Be cooled to room temperature after water-bath and isometric chloroform: isoamyl alcohol (24:1) be added, be placed at room temperature for 30min, 15 DEG C or more from
The heart, 12000rpm 10min take supernatant (with the pipette tips cut, avoiding damage to genomic DNA)；It is repeated once；Supernatant is added 2 times
The dehydrated alcohol of -20 DEG C of volume pre-coolings precipitates 2h or more, and 12000rpm is centrifuged 10min, abandons supernatant；With 70% dehydrated alcohol
Twice, 12000rpm is centrifuged 10min to washing precipitating, thoroughly blots residual ethanol, is placed in after superclean bench air-dries and 200 μ L are added
DdH2O, 4 DEG C of dissolutions overnight, sets -20 DEG C and saves backup.
Specific step is as follows for above-mentioned TrizoL method:
0.2g Chlamydomonas reinhardtii fresh weight is taken, liquid nitrogen is fully ground, and is transferred to rapidly in the EP pipe of the 1.5mL of pre-cooling, and 1mL is added
TRIzoL reagent mixes；15~30 DEG C of placement 5min, are kept completely separate nucleic acid-protein compound；4 DEG C, 10000rpm centrifugation
10min takes supernatant；It is every to add 0.2mL chloroform using 1mL TRIzoL, pipe lid is covered, 15s is acutely shaken, is placed at room temperature for 3min；4
DEG C, 10000rpm is centrifuged 15min, and sample is divided into three layers (bottom organic phases/interphase/upper layer colourless aqueous phase), and RNA is present in most
Upper strata aqueous phase, transfer water phase to new EP are managed；0.5mL isopropanol is added, mixes, is placed at room temperature for 10min, 4 DEG C, 10000rpm is centrifuged
10min abandons supernatant；75% ethyl alcohol of 1mL (being prepared with the processed water of DEPC) is added to wash precipitating, 4 DEG C, 7500rpm is centrifuged
5min abandons supernatant；It being placed at room temperature for and dries, be precipitated as RNA, add 25~200 μ L DEPC water, 55~60 DEG C of placement 10min dissolve,
- 80 DEG C are set to save backup.
The DNA and RNA (reverse transcription is at CDNA) of conversion chlamydomonas (Tn) are extracted respectively as template, carries out the PCR of Ble gene
And the detection of the methods of qRT-PCR of AANAT gene, wherein positive control is pDBle-AANAT plasmid, negative control is unloaded
Body turns chlamydomonas and unconverted scarce wall-shaped chlamydomonas.In the result that PCR is shown as shown in figure 5, conversion algae strain T1, T2, T4, T5, T6,
Available target stripe identical with pDBle-AANAT plasmid, size 523bp in T8.The knot analyzed according to real-time qRT-PCR
Fruit, as shown in fig. 6, observing the variation of mRNA expression in transgenic strain；AANAT in T1, T2, T4, T5, T6, T8
Expression be higher than NT and CK.After testing result illustrates target gene AANAT by genetic transformation operation, resistance screening,
It is smoothly inserted into and is incorporated into chlamydomonas nuclear DNA, and overexpression.
4. converting the measurement of algae strain and the intracellular epiphysin concentration of unconverted algae strain；
Expand and cultivate different algae strains, logarithmic growth phase 40mL frustule is collected by centrifugation.Sampling process are as follows: harvest the Lay of 40ml
Mattress reinhardtii cell is centrifuged 10 minutes at 4 DEG C, precipitating is resuspended in 1ml phosphate buffer (PBS), then in 8000rpm
2min is centrifuged at 4 DEG C；Finally sediment is transferred in fresh 1.5ml EP pipe, fresh weight about 0.1g~0.2g, liquid nitrogen is cold
Freeze, -80 DEG C of preservations.Extract the preprocessing process of epiphysin in reinhardtii cell are as follows: take out -80 DEG C of sample and 1ml is added
0.01M PBS.Mixture is ground to 10% homogenate on ice, and is transferred in the 2ml centrifuge tube for being enclosed with foil.The pipe is set
2 hours in oscillator, then with 10000rpm centrifugation 10 minutes.Supernatant is collected as test sample, is stored in 4 DEG C.It adopts
The measurement of intracellular epiphysin concentration is carried out with RNA isolation kit.
The different intracellular melatonin contents of algae strain are different, as shown in fig. 7, the mean concentration of the intracellular epiphysin of conversion algae strain
71.3pg/mL is the extremely significant concentration 40.2pg/mL higher than epiphysin in control group reinhardtii cell in the level of P < 0.01.Wherein
The concentration highest for converting the algae strain intracellular epiphysin of T6, is 86.7pg/mL, is 2.16 times of NT；T5, T8 are improved compared with NT respectively
82.1% and 83.8%；T1, T2, T4 are slightly lower, still improve 61.1%, 60.6%, 61.1% compared with NT respectively.It analyzes accordingly,
Overexpression of the epiphysin AANAT gene in conversion algae strain, can increase the melatonin content in chlamydonomas reinhardtii cells.
5. the measurement that salt stress handles chlamydomonas frustule and antioxidant system index of correlation, film Peroxidation Product malonaldehyde；
Expand different algae strain cultures, adds final concentration of 100mM NaCl in the medium in logarithmic growth phase, carry out
Salt stress processing is for 24 hours.40mL frustule is collected by centrifugation respectively.Extract the preprocessing process of antioxidase in reinhardtii cell are as follows: take
It is stored in -80 DEG C of sample out to be placed on ice, the phosphate buffer of 800 μ LpH=7 is added in sample, the even of sample is made
Starch excessive 4 times (example weight (g): volume buffer (ml)=1:4) of medium.It transfers the sample into mortar and carries out complete ice
Homogenate, is then transferred in new EP pipe by bath grinding.By 20% prepared homogenised sample in cooling low speed centrifuge
It is centrifuged 10~15min with 4000rpm, supernatant is collected as test sample, 4 DEG C is stored in, for measuring superoxide dismutase
Enzyme (SOD), catalase (CAT) and peroxidase (POD) activity.Intracellular antioxidant system is carried out using RNA isolation kit
The measurement of index of correlation, film Peroxidation Product mda content.
It is handled by the salt stress of 100mM NaCl, compared to the strain of NT algae, conversion algae strain T1、T2、T4、T5、T6、T8Intracellular three
The activity of kind antioxidase is all extremely significant raising in the level of P < 0.01, and as seen in figs. 8-10: CAT activity averagely mentions in Fig. 8
High 154%, T8The active highest of intracellular CAT, has reached 57.2U/mgprot, has been 3.227 times of NT.It is active in SOD
In measurement, as shown in figure 9, T2、T6The activity of middle SOD be it is highest, respectively reach 49.1,45.8U/mgprot, be NT
2.111 with 1.969 times；T1、T4、T5、T8The activity of middle SOD improves 59% compared with the strain of NT algae respectively, 86.3%, 89.3%,
67.5%.In the active measurement of POD (Figure 10), T1、T2、T6The active highest of middle POD, respectively reach 75.4,73.6,
76.6U/mgprot improves 67.8%, 63.9%, 70.5% compared with NT respectively；T4、T5The activity of middle POD is improved compared with NT algae strain
48.6%, 48.9%.It is handled by the salt stress of 100mM NaCl, the content of malonaldehyde is in unconverted scarce wall-shaped reinhardtii cell
5.45nmol/mgprot, extremely significant (P < 0.01) are higher than conversion algae strain.The content of the intracellular malonaldehyde of transgenic algae is more unconverted
The content for lacking malonaldehyde in wall-shaped frustule averagely reduces by 44.9% (Figure 11).So that malonaldehyde in the frustule of salt stress processing
Content significantly reduce, be more conducive to protect cell membrane system to damage from the external world.
In the present invention, the Chlamydomonas reinhardtii engineering algae being overexpressed by the AANAT that technique for gene engineering obtains, in salt stress item
Under part, the enzyme activity for improving the antioxidant system of frustule can be helped and reduce membrane damage, with this come the undesirable external world that copes with
Stress, improves the self-protection ability of Chlamydomonas reinhardtii.
Claims (4)
1. being overexpressed AANAT gene in the purposes for promoting Chlamydomonas reinhardtii saline-alkaline tolerance.
2. purposes according to claim 1, which is characterized in that use bead conversion method by AANAT channel genes Rhein
In the Matrix attachment region of chlamydomonas.
3.AANAT gene is used to promote the effect of epiphysin overexpression in plant.
4. purposes according to claim 3, which is characterized in that the plant is Chlamydomonas reinhardtii.
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| CN107326039A (en) * | 2017-02-21 | 2017-11-07 | 西北大学 | A kind of AANAT gene plants binary expression vector and its construction method for being used to convert Chlamydomonas reinhardtii |
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| CN107326039A (en) * | 2017-02-21 | 2017-11-07 | 西北大学 | A kind of AANAT gene plants binary expression vector and its construction method for being used to convert Chlamydomonas reinhardtii |
Non-Patent Citations (2)
| Title |
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| ZHANG，Y.J.等: "Exogenous melatonin confers salt stress tolerance to Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) by improving redox homeostasis", 《PHYCOLOGIA VOLUME》 * |
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| CN110117611A (en) * | 2019-05-16 | 2019-08-13 | 西北大学 | A kind of method and carrier for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function |
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