CN105906695A - Sophora alopecuroides aquaporin and encoding gene and application thereof - Google Patents
Sophora alopecuroides aquaporin and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a sophora alopecuroides aquaporin and an encoding gene and application thereof and belongs to the technical field of genetic engineering. The amino acid sequence of the sophora alopecuroides aquaporin is as shown in SEQ ID No.2, the nucleotide sequence of the gene encoding the sophora alopecuroides aquaporin is as shown in SEQ ID No.1, and the sophora alopecuroides aquaporin is applied to improvement of plant salt resistance. Salt resistance of transgenic plants can be improved by the sophora alopecuroides aquaporin, and the sophora alopecuroides aquaporin plays an important role in enriching resistance gene resources and cultivating salt-resistant soybean varieties.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the application of Herba Sophorae alopecuroidis water channel protein gene CDS sequence.
Background technology
The soil salinization is the global problem restricting agricultural production at present, and the whole world there are about the arable land of 20% and threatened by salt damage, and the arable land of 43% is arid, semiarid zone.Salt damage has a strong impact on the growth promoter of plant, causes crop failure, and makes ecological environment go from bad to worse.Under field conditions (factors), having had a strong impact on crop growth due to environment-stress, its genetic potential is difficult to play, salting not only have impact on the yield of crop, and limit the widely distributed of plant, therefore, the salt resistance ability improving crop has become one of breeding for stress tolerance key issue being badly in need of solution.Along with development and the maturation of transgenic technology of molecular biology, utilize transgenic technology to improve the Saline alkali tolerance of plant, be widely used in terms of salt affected soil improvement.Meanwhile, with drought life, saline alkali tolerant plant as research material, therefrom separating clone obtains the significant gene of Saline alkali tolerance, is the effective ways obtaining resistant gene resource.
Herba Sophorae alopecuroidis (Sophora alopecuroides.L) is cassia leguminous plant, another name, Sophora Alopecuroides L., Europe Radix Sophorae Flavescentis etc., for perennial herb, the raw salt-tolerant plant of rhizome underground bud drought.It is drought-enduring, salt tolerance is notable, is the abundant genetic resources storehouse of a resistant gene.Therefore, screening and cloning salt stress related genes from Herba Sophorae alopecuroidis, analyze its salt tolerance, illustrate its correlation function, help against salt gene further with.
Aquaporin (aquaporins, AQPs) be a class can the embrane-associated protein of efficient special transhipment hydrone, under multiple abiotic stress, such as arid, saline and alkaline, low temperature etc., all can there is cell moisture imbalance in plant.Intracellular hydropenia be coerce in common physiological reaction, AQPs transmembrane transport efficient to moisture, it is possible to play maintenance cellular osmotic balance, keep the effect of cell moisture.
Within 1988, it is isolated to first AQP albumen by Agre etc., in xenopus leavis oocytes expression system, then demonstrates the function of its mediation Water Transportation.The AQPs all ratios of the structure in different species are more conservative.
In aquaporin, the opening and closing in water hole is the phosphorylation by specific site upper amino acid and protonates.And the opening and closing in AQPs water hole is regulated and controled by multiple extracellular signal, including Ca2+Signal, PH, Premeabilisation of cells pressure etc..Meanwhile, AQPs is also by reorientating its hydraulic conductivity on Endomembrane system.Research to arabidopsis finds, AtPIP2；1 can transfer to endoplasmic reticulum from cell membrane internalization under extraneous salt stress so that the hydraulic conductivity of root declines, and reduces water loss.
The salt stress initial stage, the infringement that first plant occurs is osmotic stress, now plant water absorbing capacity declines, internal water lacks, now AQP gene reduces the permeability of plasma membrane by reducing expression, AQP albumen by the AQPs albumen internalization on phosphorylation closedown water hole and plasma membrane, reduces the loss of moisture under osmotic imbalances.
The later stage is coerced, due to intracellular Na at salt damage+And Cl-Accumulation, cause Ion toxicity.In the research discovery to halophytes frost flower, under salt stress, can produce a kind of little vacuole in frost flower mesophyll cell, its effect is separating Na+, tonoplast exists substantial amounts of AQPs, shows that AQPs participates in Na+Separating process, and then maintain the ionic equilibrium of cell.
Under condition of salt stress, the expression of AQPs gene is regulated and controled by ABA.Fructus Lycopersici esculenti spraying ABA carry out processing 24h, the hydraulic conductivity of its root significantly raises, and multiple AQPs gene expression amounts of Fructus Lycopersici esculenti root significantly rise simultaneously.
Summary of the invention
The present invention provides a kind of Herba Sophorae alopecuroidis aquaporin and encoding gene thereof and application, can improve plant salt endurance.
Herba Sophorae alopecuroidis aquaporin of the present invention, its aminoacid sequence is SEQ ID No.2.
The present invention encodes the gene of Herba Sophorae alopecuroidis aquaporin, and its nucleotides sequence is classified as SEQ ID No.1.
The Herba Sophorae alopecuroidis aquaporin of the present invention application in improving plant salt endurance.
We utilize Herba Sophorae alopecuroidis cDNA yeast expression library salt-related gene anti-to Herba Sophorae alopecuroidis to screen, and have obtained a salt stress related genes, water channel protein gene, named SaAQP.
In Herba Sophorae alopecuroidis, up to the present, the effect about SaAQP have not been reported.
Utilize the carrier that any one can guide exogenous gene to express in plant, SaAQP encoding gene provided by the present invention is imported plant cell, transgenic cell line and transfer-gen plant that salt tolerance improves can be obtained.When using the gene constructed plant expression vector of the present invention, promoter or inducible promoter can be strengthened plus any one before its transcription initiation nucleotide.For the ease of transgenic plant cells or plant being identified and screening, the carrier used can be processed, as added plant alternative labelling (gus gene, luciferase genes etc.) or there is the antibiotic marker thing (gentamycin, kanamycin etc.) of resistance.The expression vector carrying SaAQP of the present invention can convert plant cell or tissue by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, the conventional biology methods such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.The host being converted both can be monocotyledon, it is also possible to be dicotyledon.The gene pairs of the present invention improves plant salt endurance, and carrying is not that cultivation salt tolerant soybean varieties is significant.
Accompanying drawing explanation
Fig. 1 is SD/-Ura (containing NaCl 0.68mol/l) screening culture medium bacterium colony figure；
Fig. 2 is yeast-positive clone PCR detection figure, M:2000bp marker, 1-6: yeast amplification；
Fig. 3 is SaAQP gene expression of results figure in Herba Sophorae alopecuroidis different tissues；
Fig. 4 is SaAQP gene expression of results figure under NaCl coerces；
Fig. 5 is the Agrobacterium bacterium solution PCR result figure of SaAQP gene, M:2000bpmarker, 1-5:SaAQP gene；
Fig. 6 A is induction and the GUS detection negative control figure of Semen sojae atricolor Hairy root；
Fig. 6 B is induction and the GUS detection figure of genetically engineered soybean Hairy root；
Fig. 7 is the qRP-PCR detection figure of SaAQP gene in genetically engineered soybean Hairy root, and 1,2,3 is to repeat for 3 times, and PCHF-1301 is negative control；
Fig. 8 is band cotyledon genetically engineered soybean Hairy root salt-tolerant phenotype figure；
Fig. 9 is band cotyledon genetically engineered soybean Hairy root dry weight comparison diagram of Hairy root under NaCl stress, and * represents and reaches 0.05 probability significant level；
Figure 10 is in vitro genetically engineered soybean Hairy root salt-tolerant phenotype figure, the left side: comparison (unloaded), the right: transgenic Hairy root；
Figure 11 is in vitro genetically engineered soybean Hairy root weight in wet base comparison diagram under NaCl stress, and * represents that reaching 0.05 probability significant level * * represents and reach 0.01 probability pole significant level；
Figure 12 is in vitro genetically engineered soybean Hairy root survival number comparison diagram under NaCl stress, and * represents and reaches 0.05 probability significant level；
Figure 13 is the salt-tolerant phenotype figure turning SaIAQP transgenic soybean Hairy root complex plant；
Figure 14 is to turn SaAQP transgenic soybean Hairy root complex plant weight in wet base of Hairy root under NaCl stress to compare, and * represents and reaches 0.05 probability significant level.
Detailed description of the invention
The screening of embodiment 1 Herba Sophorae alopecuroidis SaAQP gene and clone
Choose the Herba Sophorae alopecuroidis seed 10g of full seed, add concentrated sulphuric acid immersion treatment 20min of 5ml concentration 98%.Clean be seeded in after seed spend in soil potted plant.Condition of culture is: 16h illumination, temperature 26 DEG C, humidity 65%, light intensity 30000 lux.After sprouting surrounding, it is 200mmol, Na that seedling is transferred to NaCl concentration respectively2CO3Concentration be 140mmol and PEG6000 concentration be 8% hoagland nutritional solution in process 3h, 12h, 24h, 72h respectively.Take the Herba Sophorae alopecuroidis root under each process, the respectively totalRNA of Herba Sophorae alopecuroidis root under the conditions of extraction different disposal.Take the Herba Sophorae alopecuroidis root RNA of 4 process of said extracted, wait mass mixing respectively to organize sample and build for cDNA library.Extract the cDNA library plasmid built, extensive for Library plasmid transformed saccharomyces cerevisiae INVSC1 competent cell is built Herba Sophorae alopecuroidis yeast expression in seedling stage cDNA library.Utilizing the yeast plant stress-resistance genescreen anti-salt-related gene of System For Screening Herba Sophorae alopecuroidis, screening technique is as follows:
Take appropriate library bacterium solution (making 5-10 times of clone's sum Da Wenku titre), coating SD/-Ura (containing NaCl 0.68mol/l) screening flat board, be inverted cultivation for 30 DEG C and occur to bacterium colony, as shown in Figure 1 for 2-4 days；Preserving the yeast strain screened, according to Yeast expression carrier primers, primer is:
PCR detection is carried out according to table 1 reaction and table 2 program:
Table 1PCR reaction system
Table 2PCR program
As shown in Figure 2；Method with reference to Sangon yeast plasmid extraction test kit extracts the plasmid of above-mentioned yeast liquid respectively, choose the snippet extraction plasmid more than 700, Transformed E coli DH5 α, preserve bacterium solution and check order, sequence after order-checking is removed carrier sequence, eliminate the sequence less than 500bp, utilize ncbi database Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM=blastn&PAGE_TYPE=BlastSearch &LINK_LOC=blasthome) carry out sequence alignment analysis, obtain Herba Sophorae alopecuroidis aquaporin (SaAQP) gene.It is made up of 753 base pairs, reading frame from the 1st, 5' end to the 753rd bit base, encode the protein S aAQP albumen being made up of 250 amino acid residues and there is integral protein (MIP) family structure territory, MIPs belongs to an albuminoid of aquaporin, with six transbilayer helixs after its N end, plasma membrane has the effect of selectivity water delivery.Illustrate that SaAQP falls within MIP family, have similar function to MIP.
The tissue specific expression of embodiment 2 Herba Sophorae alopecuroidis SaAQP gene
Herba Sophorae alopecuroidis carries out NaCl salt stress process, and processing method is with embodiment 1.The process time is respectively 1h, 2h, 4h, 8h, 12h, 24h, 48h.Take Herba Sophorae alopecuroidis root under each process, be taken in hoagland nutritional solution the root of Herba Sophorae alopecuroidis of cultivation, stem, leaf simultaneously.With reference to the total serum IgE of the pillar plant total serum IgE extracting and purifying test kit extraction process material of sangon company, detect the integrity of RNA through 1% sepharose electrophoresis.The synthesis of cDNA is according to Reverse Transcriptase M-MLV (RNase H-) description carry out.Utilize real-time fluorescence quantitative PCR that SaAQP gene expression in Herba Sophorae alopecuroidis different tissues and salt treatments time root is detected.Experimental implementation is being carried out in real-time fluorescence quantitative PCR instrument ABI 7500 according to sangong company SGExcel FastSYBR Mixture (With ROX) description.With Herba Sophorae alopecuroidis Lectin as reference gene, primer is as follows:
PCR reaction system and program such as table 3:
Table 3PCR reaction system and response procedures
Use 2- ΔΔ CTMethod analytical data, determines the relative expression quantity of gene.Test sets 3 technology altogether and repeats, and 3 secondary pollutants repeat.
Result (Fig. 3) shows that SaAQP gene all has expression in the root of Herba Sophorae alopecuroidis, stem apex, blade, wherein, expression in root is the highest, other tissue expression amounts are relatively low, SaAQP gene expression in stem apex is minimum, most AQPs albumen are all positioned on the plasma membrane of root system of plant, therefore the expression that SaAQP gene is in root is significantly higher than its hetero-organization.The expression of SaAQP gene rises the most rapidly in salt treatment 1h simultaneously, and 4h reaches maximum, declines the most rapidly.(Fig. 4).This is owing to the expression of SaAQP gene is regulated and controled by ABA, and under condition of salt stress, root system of plant can produce substantial amounts of ABA in a short period of time, and the expression of the gene regulated and controled by ABA changes the most therewith.
The expression in Semen sojae atricolor of embodiment 3SaAQP and the analysis to salt tolerance
Build plant expression vector pCHF-1301-SaAQP.Use the Semen sojae atricolor embryo point genetic transformation of Agrobacterium rhizogenes mediation by plant expression vector pCHF-1301-SaAQP (Fig. 5) soybean transformation Jilin 35, and genetically engineered soybean Hairy root is carried out GUS detection of expression, destination gene expression amount in detection positive plant simultaneously, and the salt tolerance of genetically engineered soybean Hairy root is analyzed.Concrete grammar and result are as follows:
The genetic transformation of 3.1 Semen sojae atricolor root of hairs and screening
1) process of vegetable material
Choose full, disease-free Semen sojae atricolor Jilin 35 seed, lie against in culture dish, culture dish is placed in airtight exsiccator, and in exsiccator, put into one added with the beaker of the dense HCl of 96mlNaClO and 4ml, Cl2Sterilizing 14-h16h.
2) soybean seed germinates
Take Semen sojae atricolor Jilin 35 seed of above-mentioned sterilizing, be inoculated in germination culture medium, in 16h illumination, temperature 26 DEG C, humidity 65%, the group training room germination of light intensity 30000 lux 4-6 days.
3) preparation of bacterium solution is infected
I. from-80 DEG C of refrigerators, take out the Agrobacterium rhyzogenesK599 (carrier is respectively pCHF-1301-SaAQP and pCHF-1301) of preservation, YEP (containing 100 μ g/mlKan) solid plate is rule, cultivate 24h for 28 DEG C；
Ii. the single bacterium colony on picking flat board, is inoculated in 50ml YEP (containing 100 μ g/mlKan) fluid medium, 250rpm, 28 DEG C of shaken cultivation 8h-10h, makes OD600=0.8-1.0；
Iii. take cultured bacterium solution 5ml, join in 500ml YEP (containing 100 μ g/mlKan) fluid medium, 250rpm, 28 DEG C of shaken cultivation 4h-6h, make OD600=0.6-0.8, this bacterium solution is for infecting bacterium solution.
4) the infecting and the induction of Hairy root of Semen sojae atricolor
A soybean cotyledon node converts
I. choose Semen sojae atricolor Jilin 35 seed that above-mentioned germination is good, peel off seed coat gently, cut away part radicle, leave and take 3-5mm hypocotyl；
Ii. vertically cut from centre by two panels cotyledon along hypocotyl, remove cotyledon, with scalpel along parallel scuffing at cotyledonary node 10 times, then outer implant is placed in above-mentioned infect bacterium solution is soaked infect 30min；
Iii. outer implant is taken out from infecting bacterium solution, inside face down lie against be lined with filter paper co-culture in culture medium, in 16h illumination, temperature 26 DEG C, humidity 65%, the group training room of light intensity 30000 lux co-cultures 4-5 days.
Iv. the outer implant root induction fluid medium after co-culturing cleans 3-4 time, soak 30min again, then outer planting surface liquid is drawn with filter paper, remove bud with scalpel, make outer planting body interior towards upper, tilt 45 ° of oblique cuttings and enter in root induction solid medium, in 16h illumination, temperature 26 DEG C, humidity 65%, the group training room of light intensity 30000 lux is cultivated 15 days.
B Semen sojae atricolor complex plant transformation
I. choose Semen sojae atricolor Jilin 35 seed that above-mentioned germination is good, peel off seed coat gently, cut away part radicle, leave and take 3-5mm hypocotyl；
Ii. in the middle of cotyledon hypocotyl, cut cross 2-3 time with scalpel, then outer implant is placed in above-mentioned infect in bacterium solution soak infect 30min；
Iii. outer implant is taken out from infecting bacterium solution, inside face down lie against be lined with filter paper co-culture in culture medium, in 16h illumination, temperature 26 DEG C, humidity 65%, the group training room of light intensity 30000 lux co-cultures 4-5 days.
Iv. the outer implant root induction fluid medium after co-culturing cleans 3-4 time, soak 30min again, then outer planting surface liquid is drawn with filter paper, by downward for outer implant radicle, it is inserted vertically in root induction solid medium, in 16h illumination, temperature 26 DEG C, humidity 65%, the group training room of light intensity 30000 lux is cultivated 15 days.
3.2 genetically engineered soybean Hairy root GUS detection of expression
Randomly selecting part Hairy root, dye with X-Gluc, 37 DEG C of dark dyeing 12-14h, the ethanol decolorization with 70% for several times, detects the expression of gus gene.Fig. 6 A, Fig. 6 B is the GUS dyeing qualification result of the Semen sojae atricolor Hairy root of dress SaAQP gene.Result shows, SaAQP gene is expressed in Semen sojae atricolor Hairy root.
The detection of expression of the genes of interest of 3.3 genetically engineered soybean Hairy root
Choosing turning the outer implant Hairy root of SaAQP gene and converting the Hairy root of pCHF-1301 of above-mentioned GUS test positive, extract RNA, reverse transcription is cDNA, and by the expression of qRT-PCR testing goal gene, internal reference is Semen sojae atricolor β-actin, and method is with reference to embodiment 2.Result is as it is shown in fig. 7, compared with the comparison converting pCHF-1301, SaAQP expression in transgenic Hairy root is all significantly increased, and illustrates that SaAQP the most successfully proceeds in Semen sojae atricolor.
The Salt Tolerance Analysis of 3.4 turns of SaAQP transgenic soybean Hairy root
1) band cotyledon turns SaAQP transgenic soybean Hairy root Salt Tolerance Analysis
The outer implant of Semen sojae atricolor after Agrobacterium rhizogenes is infected is cultivated 5-7 days in root induction culture medium, outer for the subband leaf having root of hair sign implant and comparison are transferred in solid MS (containing the 16mg/L hygromycin) culture medium containing variable concentrations NaCl, add up after cultivating 15 days.Result shows, along with the increase of NaCl concentration, the growing state of genetically engineered soybean Hairy root is better than matched group, and the dry weight of Hairy root is also significantly greater than comparison；And when NaCl concentration reaches 200mmol/L, the growth of the Hairy root turning SaAQP gene is suppressed, difficulty or ease induction produces normal Hairy root, and outer implant chlorosis turns yellow.See Fig. 8 and Fig. 9.
2) SaAQP transgenic soybean Hairy root Salt Tolerance Analysis is turned in vitro
By the genetically engineered soybean Hairy root culture of test positive in solid MS (containing the 16mg/L hygromycin) culture medium of variable concentrations NaCl, carry out after processing 15 days observing statistics.Found that, when the NaCl of low concentration (concentration is at below 50mmol/L) processes, the upgrowth situation of the Hairy root turning SaAQP gene is compared in comparison and be there is no significant difference, along with the rising of NaCl concentration, the growth of transgenic Hairy root and weight in wet base, obviously higher than comparison, are shown in Figure 10；The weight in wet base of the Hairy root turning SaAQP gene increases the most notable, and weight in wet base increment is 3 times of comparison, sees Figure 11.And when NaCl concentration reaches 200mmol/L, transgenic Hairy root is all suppressed with the growth compareed, but the survival rate of transgenic Hairy root is all remarkably higher than comparison, and the Hairy root survival rate turning SaAQP gene is 38.4%, the survival rate of matched group is 15.6%, sees Figure 12.
3) Salt Tolerance Analysis of SaAQP transgenic soybean Hairy root complex plant is turned
The dicotyledonous outer implant of whole Semen sojae atricolor infected by Agrobacterium rhizogenes is incubated in solid MS (containing the 16mg/L hygromycin) culture medium of variable concentrations NaCl, carries out observing statistics after processing 30 days.When NaCl concentration be 50,100,150mmol/L time, the growing way of the Hairy root plant complex turning SaAQP gene is all better than comparison, sees Figure 13；NaCl concentration be 100, under 150mmol/L stress conditions, the weight in wet base of the Hairy root plant complex of SaAQP gene is all remarkably higher than comparison.See Figure 14.
We carry out salt stress process to the Semen sojae atricolor Hairy root turning SaAQP gene, analyze its impact on soybean root system salt tolerant, and result shows, SaAQP gene process LAN in Hairy root all makes genetically engineered soybean Hairy root show certain salt tolerance.Being added up transgenic the Hairy root growth conditions of Hairy root, survival rate, weightening finish etc. under variable concentrations condition of salt stress, result shows, NaCl concentration be 100,150mmol/L time, its growth conditions is good, and salt tolerant effect is notable；When NaCl concentration is 200mmol/L, although its growth is suppressed, but survival rate is significantly higher than comparison.Illustrate that the process LAN of SaAQP and gene all can the salt tolerance improving transgenic plant in various degree.
Claims (3)
1. a Herba Sophorae alopecuroidis aquaporin, it is characterised in that: its aminoacid sequence is SEQ ID No.2.
2. the gene encoding Herba Sophorae alopecuroidis aquaporin, it is characterised in that: its nucleotides sequence is classified as SEQ
ID No.1。
3. the Herba Sophorae alopecuroidis aquaporin as claimed in claim 1 application in improving plant salt endurance.
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