CN110117611A - A kind of method and carrier for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function - Google Patents
A kind of method and carrier for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function Download PDFInfo
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- CN110117611A CN110117611A CN201910406105.XA CN201910406105A CN110117611A CN 110117611 A CN110117611 A CN 110117611A CN 201910406105 A CN201910406105 A CN 201910406105A CN 110117611 A CN110117611 A CN 110117611A
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Abstract
The invention discloses a kind of methods for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function, Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported in chlamydonomas reinhardtii cells Matrix attachment region, screening obtains the transgenic algae strain of AANAT gene silencing；Step 3: choosing transgenic algae strain described in wild Chlamydomonas reinhardtii and step 2；Wild Chlamydomonas reinhardtii and transgenic algae strain are cultivated in the TAP culture solution containing same concentrations NaCl respectively, obtain algae solution；Step 4: taking each algae solution in part, after processing, detection determines the saline-alkaline tolerance of transgenic algae strain, verifies Chlamydomonas reinhardtii AANAT gene salt resistance function.Using this method for the later period, epiphysin research provides frustule material in chlamydomonas, synthesize convenient for accurately studying AANAT gene with epiphysin between relationship.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of verifying Chlamydomonas reinhardtii AANAT gene salt resistance function
Method and carrier.
Background technique
The soil salinization is stepped up at present, may cause the following arable land loss about 30%, under the salt stress environment,
The seed of plant is sprouted, and root elongation, seedling growth etc. is suppressed all significantly, the salt marsh ingredient higher level in stressful environmental
When, plant cell cannot maintain ionic homeostasis and growth, ion toxicity and osmotic stress will lead to oxidative stress and it is a series of after
Hair property stress, endangers the normal growth of plant.
Under condition of salt stress, resistance protection mechanism has fraction of research in one-celled plants Chlamydomonas reinhardtii.Wherein take off
The resistance effect that melanocyte has the influence of protecting Chlamydomonas reinhardtii from salt stress, and the key enzyme in epiphysin biosynthesis it
First is that aromatic yl alkyl amine N-acetyl-transferase AANAT, whether it directly determines epiphysin synthesis.
Epiphysin MT is handled in the function of condition of salt stress frequently with external source in verifying Chlamydomonas reinhardtii at present, but frustule
Illumination needed for growth, easily causes external source epiphysin to decompose, while external source epiphysin is consumed in frustule growth course, also results in
Epiphysin function and effect are of short duration inhomogenous, stablize lasting effect without endogenous epiphysin.
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 verifying Rhein clothing
The method and carrier of algae AANAT gene salt resistance function obtain AANAT gene silencing by the silent technology of endogenous AANAT gene
Transgenic algae strain, in algae epiphysin research provide AANAT gene silencing frustule material, convenient for accurately research
AANAT gene synthesized with epiphysin between relationship.
In order to solve the above technical problems, the technical solution adopted by the present invention is that, a kind of verifying Chlamydomonas reinhardtii AANAT gene is anti-
The method of salt functional, this method comprises the following steps:
Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported chlamydonomas reinhardtii cells Matrix attachment region
In, screening obtains the transgenic algae strain of AANAT gene silencing；
Step 3: choosing transgenic algae strain described in wild Chlamydomonas reinhardtii and step 2；By the wild Chlamydomonas reinhardtii
It is cultivated in the TAP culture solution containing same concentrations NaCl respectively with transgenic algae strain, obtains algae solution；
Step 4: taking each algae solution in part, after processing, it is placed in the form of microscopically observation chlamydonomas reinhardtii cells,
Compare the algae solution of the wild Chlamydomonas reinhardtii and transgenic algae strain, cell quantity is reduced and cataplastic for transgenic algae strain
Algae solution；Meanwhile MT content drop it is much lower for transgenic algae strain.
Further, the concentration of NaCl is 50mM~100mM in the TAP culture solution.
The invention also discloses a kind of carriers for Chlamydomonas reinhardtii AANAT gene silencing, are connected by aanat genetic fragment
PDBle wins Lay carrier and obtains.
The invention also discloses a kind of preparation method of the transgenic algae strain of AANAT gene silencing, this method includes as follows
Step: Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported chlamydonomas reinhardtii cells Matrix attachment region
In, screening obtains the transgenic algae strain of AANAT gene silencing.
A kind of method for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function of the present invention and carrier have the advantages that realization
The silencing of endogenous AANAT gene in chlamydomonas in Chlamydomonas reinhardtii obtains the transgenic algae strain of AANAT gene silencing, makes chlamydomonas
Interior epiphysin has more stable dauer effect than external source processing, and epiphysin research provides algae in chlamydomonas for the later period
Cell material, and lay a good foundation for correlative studys such as content, function of the epiphysin in Chlamydomonas reinhardtii or even algae.
Detailed description of the invention
Fig. 1 is Chlamydomonas reinhardtii nuclear expression vector construction flow chart；
Fig. 2 is influence of the NaCl of various concentration to Chlamydomonas reinhardtii algae strain growth；
Fig. 3 is the observation on Growth figure of Chlamydomonas reinhardtii difference phytem under condition of salt stress in the present invention；
Fig. 4 is that NaCl is coerced to melatonin content in Chlamydomonas reinhardtii difference phytem and AANAT gene expression feelings in the present invention
The influence of condition；
Fig. 5 is influence of the NaCl stress to the activities of antioxidant enzymes of Chlamydomonas reinhardtii difference phytem in the present invention；
Fig. 6 is influence of the NaCl stress to the reactive oxygen species variation of Chlamydomonas reinhardtii difference phytem in the present invention.
Specific embodiment
The invention discloses the algaes that a kind of method for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function, this experiment use
Kind CC-503 purchase is in Chinese Academy of Sciences's algae library.
This method comprises the following steps:
Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；Structure figures such as Fig. 1
It is shown.
Specifically: aanat gene is cloned from Chlamydomonas reinhardtii CC-503, passes through Chlamydomonas reinhardtii on Chlamydomonas reinhardtii on NCBI
AANAT gene order, sequence number: AB474787.1, design primer, and increase EcoRI and NdeI restriction enzyme site in upstream and downstream, it uses
Chlamydomonas reinhardtii cDNA is template through PCR amplification purpose band, recycles target fragment aanat with DNA plastic recovery kit, and by its
It is connect with pMD18-T carrier, converts escherichia coli DH5a competent cell.Through PCR, double digestion identification purpose bacterial strain and surveyed
Sequence constructs correct cloning vector pMD18-aanat；With NdeI and EcoRI double digestion plasmid pMD18-aanat and pDBle, turn
Change escherichia coli DH5a competent cell, through PCR, double digestion identification purpose bacterial strain and be sequenced, constructs correctly expression and carry
Body pDBle-aanat.
A kind of above-mentioned carrier for Chlamydomonas reinhardtii AANAT gene silencing is carried by aanat genetic fragment connection pDBle
Body obtains.
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported in chlamydonomas reinhardtii cells Matrix attachment region, sieve
Choosing obtains the transgenic algae strain of AANAT gene silencing；It is imported using bead introductory technique.
Step 3: choosing transgenic algae strain described in wild Chlamydomonas reinhardtii and step 2；By the wild Chlamydomonas reinhardtii
It is cultivated in the TAP culture solution containing same concentrations NaCl respectively with transgenic algae strain, obtains algae solution.In above-mentioned TAP culture solution
The concentration of NaCl is 50mM~100mM.
Step 4: taking each algae solution in part, after processing, it is placed in the form of microscopically observation chlamydonomas reinhardtii cells,
Compare the algae solution of the wild Chlamydomonas reinhardtii and transgenic algae strain, cell quantity is reduced and cataplastic for transgenic algae strain
Algae solution；Meanwhile MT content drop it is much lower for transgenic algae strain.
Meanwhile each algae solution in part is taken, measure antioxidase in the wild Chlamydomonas reinhardtii and transgenic algae strain cell
Activity and active o content, wherein activities of antioxidant enzymes reduces, the increased algae solution for transgenic algae strain of active o content.
For determination wild Chlamydomonas reinhardtii can be carried out first to the size for the NaCl concentration that Chlamydomonas reinhardtii has an impact, the present invention
The screening of the NaCl resistance concentration of CC-503, specific as follows:
The Chlamydomonas reinhardtii CC-503 for lacking wall-shaped strain is selected, with 250mL TAP culture solution culture, with containing different dense after 7d
The TAP culture solution culture of the NaCl of degree, the concentration of NaCl are respectively 0,20mM, 40mM, 60mM, 80mM and 100mM.Cultivate 3d
Afterwards, it takes pictures and determines suitable NaCl concentration for later period salt stress.
As a result as shown in Fig. 2, in figure 0,20,40,60,80 and 100 be NaCl concentration, unit mM.Be attached with 0,
In the TAP culture solution of 20 and 40mM NaCl, without apparent variation bottle green is still presented, in 60,80 and 100mM in algae solution
In the stressful environmental of NaCl, algae solution color is gradually changed into light green or yellow green by dark color, and growth is successively restricted；And 40 Hes
Algae solution color gradient in 60mMNaCl environment, but change slightly, frustule growth conditions are good.So determining 50mM
The salt stress of NaCl influences the strain growth of Chlamydomonas reinhardtii algae, but does not have a adverse impact.
The invention also discloses a kind of preparation method of the transgenic algae strain of AANAT gene silencing, this method includes as follows
Step: Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported chlamydonomas reinhardtii cells Matrix attachment region
In, screening obtains the transgenic algae strain of AANAT gene silencing.
The method of verifying Chlamydomonas reinhardtii AANAT gene salt resistance function in the present invention, specific as follows:
The algae strain that obtained transgenic algae strain is AANAT gene silencing is determined first, and screening and qualification process are as follows:
The molecular biology verifying of aanat genetic transformation algae strain:
The DNA and RNA for extracting conversion chlamydomonas, then respectively as template, carry out Ble gene by RNA reverse transcription at cDNA
The methods of PCR and aanat gene qRT-PCR detection.In the result that PCR is shown, the available target in conversion algae strain
Band, size 849bp.According to real-time qRT-PCR analysis as a result, observing the change of mRNA expression in transgenic strain
Change；The expression of aanat is lower than wild algae strain in transgenosis phytem.Testing result illustrates that target gene aanat passes through heredity
After conversion operation, resistance screening, smoothly insertion is incorporated into chlamydomonas nuclear DNA, and low expression.PCR and qRT-PCR
After testing result illustrates target gene epiphysin key synthase gene AANAT by genetic transformation operation, resistance screening, Ke Yishun
Benefit insertion is incorporated into chlamydomonas nuclear DNA, and low expression.
The screening of glass bead mediated Chlamydomonas reinhardtii CC-503 conversion and transgenic algae strain:
Bead conversion method converts reinhardtii cell CC-503, flat in the solid TAP containing 6 μ g/mLBle after applying plate 13d
Recombinant plasmid group has grown single algae and has fallen on plate, this is because recombinant plasmid can help the reinhardtii cell converted to generate Ble resistance,
Growth is not inhibited by the Ble in TAP culture medium.And the control conversion algae of wild type chlamydomonas CC-503 and synchronous experimental group is not
It can be grown in resistant panel, there is no Ble resistant gene in control conversion algae.Picking list algae falls within the liquid containing 2.0 μ g/mL Ble
After cultivating 7d in body TAP, collects algae solution and be coated on the solid TAP plate of different Ble concentration, Ble concentration is respectively
4,6,8 and 10 μ g/mL, green algae, which falls, to be successfully transferred to continued growth, illustration purpose gene aanat through bead conversion
Chlamydonomas reinhardtii cells.
Then, when verifying the experiment of Chlamydomonas reinhardtii AANAT gene salt resistance function, wild Chlamydomonas reinhardtii group and screening are selected
Obtained above-mentioned transgenic algae strain group uses 250mL TAP culture solution culture respectively, cultivates 7 days.It is chosen in TAP culture solution and trains
Support wild Chlamydomonas reinhardtii and transgenic algae strain in 7 days.
Analyze influence of the NaCl stress to different phytem cellular morphologies:
Wild Chlamydomonas reinhardtii and transgenic algae strain group are coerced into 3 days, as shown in Figure 3a, algae at various concentration NaCl respectively
Without apparent difference in liquid color；Wherein, the concentration of the NaCl of selection is respectively 0,50mM and 100mM.
Then they are distinguished under inverted microscope 100X oil mirrors, in the case where no NaCl is coerced, in Fig. 3 b, shown in B and E,
Cellular morphology variation display shows: under non-salt stress environment, the size of the cell of wild Chlamydomonas reinhardtii and transgenic algae strain is appointed
For homogenization, reinhardtii cell shows vigorous vitality, and movement velocity is very fast, high-visible wild Chlamydomonas reinhardtii and turns
Gene algae strain flagellum.
In 50mM NaCl processing group, in Fig. 3 b, C and F, S51And S52Cytochromes quantity is reduced in group, Geng Duozao
There is atrophy in cell, but still keeps intact cell body, S51The cell growth state ratio S5 of group2It organizes, S52The frustule of group
Atrophy phenomenon becomes apparent；Wherein: S51Wild Chlamydomonas reinhardtii is referred to cultivate in the TAP liquid of 50mM NaCl；S52Reference turns base
Because algae strain is cultivated in the TAP liquid of 50mMNaCl.
In 100mM NaCl processing group, in Fig. 3 b, shown in D and G, S101And S102In group wild Chlamydomonas reinhardtii and turn
The cell density of gene algae strain is substantially reduced, and cell volume becomes larger, and most cells lose flagellum, and movement velocity slows down, cell
Vitality be substantially reduced.S101Group frustule growth conditions are better than S102S10 can be observed in group2Group has cell dissolution, with
The excessive phenomenon of intracellular molten substance.Observe transgenic phytem it is more sensitive to the wilder Chlamydomonas reinhardtii of salt stress, this with turn
The low expression of epiphysin synthase gene AANAT in gene phytem is related.Wherein S101Wild Chlamydomonas reinhardtii is referred in 100mM
It is cultivated in the TAP liquid of NaCl, S102Transgenic algae strain is referred to cultivate in the TAP liquid of 100mM NaCl.
Influence of the NaCl stress conditions to MT Content in different phytems:
Wild Chlamydomonas reinhardtii and transgenic algae strain are chosen, then salt stress 3 days in various concentration NaCl respectively by them
Afterwards, the melatonin content of different Chlamydomonas reinhardtii systems and the case where epiphysin synthase gene AANAT it is measured.Wherein, selection
The concentration of NaCl is respectively 0,50mM and 100mM.
Under conditions of no NaCl is handled, transgenic algae strain is compared with wild Chlamydomonas reinhardtii, melatonin content and AANAT
Though expression have reduction and downward, there is no conspicuousness differences with wild Chlamydomonas reinhardtii；However, at the NaCl of various concentration
After reason, compared with wild Chlamydomonas reinhardtii, melatonin content is significantly reduced in transgenic algae strain, p < 0.05, as shown in figure 4, in figure
WT indicates that wild Chlamydomonas reinhardtii, AR3 represent transgenic algae strain.Wherein, after 50mM NaCl processing, compared with wild Chlamydomonas reinhardtii,
The expression quantity that melatonin content reduces 7.48%, AANAT gene significantly in transgenic algae strain has lowered 0.38 times；And 100m
M NaCl processing after compared with wild Chlamydomonas reinhardtii, in transgenic algae strain MT content reduce extremely significantly 16.87%, p <
The expression quantity of 0.01, AANAT gene has lowered 0.61 times.These results show that in transgenic algae strain AANAT low expression shadow
The content of intracellular epiphysin is rung.
NaCl stress conditions influence the activities of antioxidant enzymes of different phytems:
Wild Chlamydomonas reinhardtii and transgenic algae strain are after various concentration NaCl processing, compared with the algae strain of unused NaCl processing
Compared with SOD, POD, CAT and APX activity increase, and POD activity change becomes apparent, compared with SOD, CAT and APX sensitivity, such as Fig. 5
Shown in middle 5a, 5b, 5c and 5d, WT represents wild Chlamydomonas reinhardtii in figure, and AR3 represents transgenic algae strain.Kit measurement is used respectively
The activity of SOD, CAT, POD and APX.It is measured under 550nm wavelength with erythrocuprein SOD assay kit-WST-1 method
SOD activity, cat catalase assay kit-visible light method measure CAT activity, peroxidase under 420nm wavelength
POD assay kit measures POD activity under 405nm wavelength, and ascorbate peroxidase APX kit is under 290nm wavelength
Measure the activity of APX.Wherein, the concentration of the NaCl of selection is respectively 0,50mM and 100mM, as shown in abscissa in figure.
Wild Chlamydomonas reinhardtii and transgenic algae strain are anti-oxidant in transgenic algae strain after the processing of 100mM NaCl concentration
Enzyme SOD, POD, CAT activity is substantially reduced, p < 0.05, such as Fig. 5；SOD and POD activity has been even up to extremely significant reduction, p <
0.01.Compared with wild Chlamydomonas reinhardtii, SOD and POD activity reduces 19.93% and 13.86% respectively for transgenic algae strain.?
Under condition of salt stress, various antioxidant enzymes remove the ability decline of ROS, the reduction of this and melatonin content in transgenic algae strain
There is relationship.Side illustration epiphysin has the ability of active oxygen radical in scavenger-cell.
The influence that NaCl stress conditions change different phytem reactive oxygen species:
After wild Chlamydomonas reinhardtii and transgenic algae strain are handled in 50 or 100mM NaCl, transgenic algae strain and wild Lay
Mattress chlamydomonas is compared, H in transgenosis phytem2O2And O2Content increase, as shown in figure 6 a and 6b.With phase under no condition of salt stress
Than H in wild Chlamydomonas reinhardtii2O2And O2The stringer of content fifty-fifty increases by 93.37% and 92.21% respectively；Transgenic algae strain
Middle H2O2And O2The stringer of content fifty-fifty increases 94.97% and 98.83% respectively.
And under 50 or 100mM NaCl stress, transgenic algae strain increases with MDA content in wild Chlamydomonas reinhardtii；Turn base
Because algae strain does not change significantly with wild Chlamydomonas reinhardtii, such as Fig. 6 c.The result of Fig. 6 implies, and salt stress can induce generation
Intracellular a large amount of ROS generate injury to cell；These results show that salt stress can induce a large amount of generations of intracellular ROS,
Injury is generated to cell；And epiphysin has the ability for removing ROS, reduces damage of the ROS to cell.And it is taken off in transgenic algae strain
The reduction of melanocyte cannot protect ROS to damage caused by reinhardtii cell as shown in figure 4, reducing the ability that body removes ROS
Wound.
Using the verification method in the present invention, the drawbacks of consuming external source epiphysin in frustule growth course is avoided, is
Later period, epiphysin research provided frustule material in chlamydomonas, closed convenient for accurately research AANAT gene and epiphysin
Relationship between.
Claims (4)
1. a kind of method for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function, which is characterized in that this method comprises the following steps:
Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported in chlamydonomas reinhardtii cells Matrix attachment region, sieve
Choosing obtains the transgenic algae strain of AANAT gene silencing；
Step 3: choosing transgenic algae strain described in wild Chlamydomonas reinhardtii and step 2；By the wild Chlamydomonas reinhardtii and turn
Gene algae strain is cultivated in the TAP culture solution containing same concentrations NaCl respectively, obtains algae solution；
Step 4: taking each algae solution in part, after processing, it is placed in the form of microscopically observation chlamydonomas reinhardtii cells, is compared
The algae solution of the wild Chlamydomonas reinhardtii and transgenic algae strain, cell quantity reduces and the cataplastic algae for transgenic algae strain
Liquid；Meanwhile MT content drop it is much lower for transgenic algae strain.
2. a kind of method for verifying Chlamydomonas reinhardtii AANAT gene salt resistance function according to claim 1, which is characterized in that
The concentration of NaCl is 50mM~100mM in the TAP culture solution.
3. a kind of carrier for Chlamydomonas reinhardtii AANAT gene silencing, which is characterized in that connect pDBle by aanat genetic fragment
Rich Lay carrier obtains.
4. a kind of preparation method of the transgenic algae strain of AANAT gene silencing, which is characterized in that this method comprises the following steps:
Step 1: the Chlamydomonas reinhardtii nuclear expression carrier pDBle-aanat of building AANAT containing target gene；
Step 2: the nuclear expression carrier pDBle-aanat in step 1 is imported in chlamydonomas reinhardtii cells Matrix attachment region, sieve
Choosing obtains the transgenic algae strain of AANAT gene silencing.
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