CN105907733B - A kind of Sophora alopecuroide inositol transmethylase and its encoding gene and application - Google Patents
A kind of Sophora alopecuroide inositol transmethylase and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to a kind of Sophora alopecuroide inositol transmethylase and its encoding gene and applications, belong to gene engineering technology field.The entitled SaIMT of Sophora alopecuroide inositol methyl transferase gene, size is 1095bp, 364 amino acid are encoded, using the cDNA Yeast expression library of the Sophora alopecuroide root constructed under salt (NaCl) stress conditions, are screened out from it gene order SaIMT relevant in salt stress；The bioinformatic analysis of SaIMT shows, the IMTs albumen of SaIMT and other species has higher homology and closer affiliation, SaIMT and soybean GmIMT simultaneously, it is very conservative on protein sequence structural domain, and the tertiary protein structure of the two is all closely similar, shows that SaIMT and its homologous gene have similar function；Real-time fluorescence quantitative PCR is the result shows that SaIMT gene has apparent raising in salt stress initial stage expression quantity；The salt tolerance of the soybean Hairy root and soybean Hairy root plant complex that turn SaIMT significantly improves, and illustrates that SaIMT can improve the salt tolerance of genetically modified plants, is of great significance to abundant resistant gene resource and cultivation salt tolerant soybean varieties.
Description
Technical field
The invention belongs to gene engineering technology field, be related to Sophora alopecuroide inositol transmethylase and its gene C DS sequence and
Using.
Background technique
The soil salinization is a current global problem for restricting agricultural production, and there are about 20% arable lands by salt in the whole world
Evil threatens, and 43% arable land is arid, semiarid zone.Salt damage seriously affects the growth and development of plant, causes crop failure, and
Keep ecological environment worsening.Under field conditions (factors), crop growth has been seriously affected due to environment-stress, heredity is latent
Power is difficult to play, and salt marsh not only affects the yield of crop, but also limits the widely distributed of plant, therefore, improves crop
Salt resistance ability has become one of breeding for stress tolerance critical issue urgently to be solved.With the development of molecular biology and transgenic technology
Maturation, using transgenic technology improve plant Saline alkali tolerance, be widely used in terms of salt affected soil improvement.Together
When, it is to obtain resistance that using non-irrigated life, saline alkali tolerant plant as research material, therefrom separation clone, which obtains the significant gene of Saline alkali tolerance,
The effective ways of genetic resources.
Sophora alopecuroide (Sophora alopecuroides.L) is cassia leguminous plant, and alias, herba sophorae alopecuroide, Europe kuh-seng etc. are
The raw salt-tolerant plant of perennial herb, rhizome underground bud drought.Its is drought-enduring, salt tolerance is significant, is a resistant gene gene abundant
Resources bank.Therefore, the screening and cloning salt stress related genes from Sophora alopecuroide analyze its salt tolerance, illustrate its correlation function, help
In the further utilization to salt resistant gene.
Pine camphor (O- methyl-inositol) is the methyl-derivatives of inositol, is a kind of polyalcohol, it is by inositol transmethylase
(IMT) it catalyzes and synthesizes.Accumulation and plant of the pine camphor in plant cell are closely related to arid, the tolerance of salt stress.?
In many species, also has including bacterium, yeast, algae and animal and observed similar stress-tolerance phenomenon.Plant is coerced in salt
These compounds can be largely generated and accumulate under the conditions of compeling, these metabolites play a role as osmotic adjustment, pass through
Osmotic potential is reduced to increase the water holding capacity of plant.Some metabolism, which generate polyalcohol, can also be used as active oxygen scavenger, Neng Gouqing
Except to the extremely strong hydroxyl radical free radical of toxicity.And their another critical function is the water that protein is kept under water stress
It closes, guarantee is normally carried out for what cell was thanked.
Alcohol is via four step synthesis once.In L- inositol 1- phosphate synthase (MIPS) catalysis G-6-P synthesis
Inositol, the enzyme is by INO1 negative gene responsible editor's code [98].The L- inositol 1- phosphorus that the inositol catalyzed and synthesized through MIPS is relied on by Mg2+ again
Acidification enzymatic forms free inositol.Under condition of salt stress, free inositol is methylated by inositol transmethylase (IMT) again,
And then inositol methyl esters is synthesized, inositol methyl esters is final to synthesize osmotic adjustment organic matter again by inositol methyl esters isomerase epimerization
Pine camphor.S-adenosylmethionine (SAM) is depended in the catalytic process of IMT, the circulation of SAM and methyl is closely related.It closes at present
It clones and obtains from various plants at the IMT1 gene of inositol transmethylase, including arabidopsis, wild rice
(Porteresia coarctata), soybean, sesame (Sesamum indicum) and halophytes ice Ye Ju
(Mesembryanthemum crystallinum)
In the entire approach of pine camphor synthesis, IMT is key enzyme therein, and inositol is most important substrate.Research hair
Existing, it is special that the MIPs albumen encoded in wild rice by PcINO1 gene has the function of, though the enzyme under high-salt stress still
Very strong activity is so kept, ensure that the sufficient supply of the substrate inositol in salt damage.Simultaneously under condition of salt stress, IMT
Synthesis also increase accordingly, inositol is largely converted into pine camphor.
Summary of the invention
The present invention provides a kind of Sophora alopecuroide inositol transmethylase and its encoding gene and application, can improve plant salt tolerance
Property.
Sophora alopecuroide inositol transmethylase of the present invention, amino acid sequence are SEQ ID No.2.
The present invention encodes the gene of Sophora alopecuroide inositol transmethylase, and nucleotides sequence is classified as SEQ ID No.1.
Sophora alopecuroide inositol transmethylase of the present invention is improving the application in plant salt endurance.
We screen Sophora alopecuroide salt resistance related gene using Sophora alopecuroide cDNA Yeast expression library, have obtained a salt
Stress-related genes, inositol methyl transferase gene, are named as SaIMT.
In Sophora alopecuroide, up to the present, the effect about SaIMT is had not been reported.
The carrier that foreign gene can be guided to express in plant using any one, by SaIMT provided by the present invention
Encoding gene imports plant cell, can get transgenic cell line and transgenic plant that salt tolerance improves.Using of the invention
When gene constructed plant expression vector, it can be opened plus any enhancing promoter or induction type before its transcription initiation nucleotide
Mover.For the ease of transgenic plant cells or plant are identified and screened, used carrier can be processed, such as
Plant alternative label (gus gene, luciferase genes etc.) is added or resistant antibiotic marker (is celebrated big mould
Element, kanamycins etc.).The expression vector for carrying SaIMT of the present invention can be carried by using Ti-plasmids, Ri plasmid, plant virus
The conventional biology methods such as body, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus convert plant cell or tissue, and will
The plant tissue of conversion is cultivated into plant.The host being converted is either monocotyledon, is also possible to dicotyledon.This
The gene pairs of invention improves plant salt endurance, and mentioning is not to cultivate salt tolerant soybean varieties to be of great significance.
Detailed description of the invention
Fig. 1 is SD/-Ura (0.68mol/l containing NaCl) screening and culturing medium bacterium colony figure；
Fig. 2 is yeast-positive clone PCR detection figure, M:2000bp marker, 1-10: yeast amplification；
Fig. 3 is expression result chart of the SaIMT gene in Sophora alopecuroide different tissues；
Fig. 4 is expression result chart of the SaIMT gene under NaCl stress, reference gene Sophora
Alopecuroides lectin (Gen Bank searching number: DQ011517.1)；Data are mean ± SD of three repeated experiments；
Fig. 5 is the Agrobacterium bacterium solution PCR of SaIMT gene as a result, M:2000bpmarker, 1-6:SaIMT gene；
Fig. 6 is induction and the GUS detection figure of soybean Hairy root, wherein A: negative control, B: genetically engineered soybean Hairy root
GUS dyeing；
Fig. 7 is the qRP-PCR detection figure of SaIMT gene in genetically engineered soybean Hairy root；1,2,3 is 3 repetitions, PCHF-
1301 be negative control；
Fig. 8 is band cotyledon genetically engineered soybean Hairy root salt-tolerant phenotype figure；
Fig. 9 is that the dry weight of Hairy root compares figure, * table under NaCl stress with cotyledon genetically engineered soybean Hairy root
Show and reaches the 0.05 probability level of signifiance；
Figure 10 is in vitro genetically engineered soybean Hairy root salt-tolerant phenotype figure；The left side: control (zero load), the right: transgenosis hair-like
Root；
Figure 11 is that weight in wet base of the in vitro genetically engineered soybean Hairy root under NaCl stress compares figure, and * expression reaches
0.05 probability level of signifiance * * expression reaches the extremely significant level of 0.01 probability；
Figure 12 is that survival number of the in vitro genetically engineered soybean Hairy root under NaCl stress compares figure, and * expression reaches
To the 0.05 probability level of signifiance；
Figure 13 is the salt-tolerant phenotype figure for turning SaIIMT transgenic soybean hair-like root complex plant；
Figure 14 is the weight in wet base for turning SaIMT transgenic soybean hair-like root complex plant Hairy root under NaCl stress
Compare, * expression reaches the 0.05 probability level of signifiance.
Specific embodiment
Embodiment 1, the screening of Sophora alopecuroide SaIMT gene and clone
The Sophora alopecuroide seed 10g of full seed is chosen, the concentrated sulfuric acid immersion treatment 20min of 5ml concentration 98% is added.It cleans
Potting in flower soil is seeded in after seed.Condition of culture are as follows: 16h illumination, 26 DEG C of temperature, humidity 65%, 30000 lux of light intensity.
After sprouting four weeks, it is 200mmol, Na that seedling, which is transferred to NaCl concentration, respectively2CO3Concentration is 140mmol and PEG6000 concentration
To handle 3h, 12h, for 24 hours, 72h respectively in 8% hoagland nutrient solution.The Sophora alopecuroide root under each processing is taken, is extracted respectively
The totalRNA of Sophora alopecuroide root under the conditions of different disposal.Take 4 Sophora alopecuroide root RNA handled of said extracted, etc. quality it is mixed
Each group sample is closed to construct for cDNA library.The cDNA library plasmid that building is completed is extracted, Library plasmid is converted to wine on a large scale
Brewer yeast INVSC1 competent cell constructs Sophora alopecuroide seedling stage Yeast expression cDNA library.Utilize yeast plant stress-resistance genescreen
System For Screening Sophora alopecuroide salt resistance related gene, screening technique are as follows:
It takes appropriate library bacterium solution (making 5-10 times that clones total Da Wenku titre), coating SD/-Ura (contains
NaCl0.68mol/l) screening flat board, 30 DEG C of inversions cultivate 2-4 days to bacterium colony appearance, as shown in Figure 1；Save the yeast screened
Bacterial strain, according to Yeast expression carrier primers, primer are as follows:
PCR detection is carried out according to the reaction of table 1 and 2 program of table:
1 PCR reaction system of table
2 PCR program of table
As shown in Figure 2；Extract the matter of above-mentioned yeast liquid respectively referring to the method for Sangon yeast plasmid extracts kit
Grain, chooses the snippet extraction plasmid greater than 700, and Transformed E coli DH5 α saves bacterium solution and is simultaneously sequenced, by the sequence after sequencing
Carrier sequence is removed, the sequence less than 500bp is eliminated, utilizes ncbi database Blast
(http://blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn&PAGE_TYPE=
BlastSearch&LINK_LOC=blasthome sequence alignment analysis) is carried out, Sophora alopecuroide inositol transmethylase is obtained
(SaIMT) gene.It is made of 1095 base-pairs, reading frame encodes one by 364 from the 1st, the end 5' to the 1095th bit base
The protein of a amino acid residue composition, SaIMT albumen include an O- methyl transferase domains, activity and the S- gland of the enzyme
Glycosides methionine is related；Also there is a Dimerized structural domain, which often appears in O- methyl transferase gene family
N-terminal, it can mediate the Dimerized of O- methyl transferase gene.Illustrate SaIMT gene and O- transmethylase family gene
With similar function.
The tissue specific expression of embodiment 2, Sophora alopecuroide SaIMT gene
The processing of NaCl salt stress is carried out to Sophora alopecuroide, processing method is the same as embodiment 1.The processing time is respectively 1h, 2h, 4h,
8h, 12h, for 24 hours, 48h.Take Sophora alopecuroide root under each processing, at the same take the Sophora alopecuroide cultivated in hoagland nutrient solution root,
Stem, leaf.Referring to the total serum IgE of the pillar plant total serum IgE extracting and purifying kit extraction process material of sangon company, through 1% fine jade
The integrality of lipolysaccharide electrophoresis detection RNA.The synthesis of cDNA is according to ReverseTranscriptase M-MLV (RNase H-)
Specification carries out.Using real-time fluorescence quantitative PCR to SaIMT gene in Sophora alopecuroide different tissues and salt treatments time root
Expression detected.Experimental implementation is according to sangong company SGExcel FastSYBR Mixture (With ROX)
Specification is carried out in real-time fluorescence quantitative PCR instrument ABI 7500.Using Sophora alopecuroide Lectin as reference gene, primer is as follows:
PCR reaction system and program such as table 3:
3 PCR reaction system of table and response procedures
PCR response procedures
Using 2-ΔΔCTMethod analyzes data, determines the relative expression quantity of gene.Test sets 3 technologies altogether and repeats, 3 secondary pollutants
It learns and repeats.
As a result (Fig. 3) shows that SaIMT gene has expression in the root, stem apex, blade of Sophora alopecuroide, wherein the table in root
Up to amount highest, other tissue expression amounts are relatively low, and expression quantity of the SaIMT gene in blade is minimum.While SaIMT gene
Expression does not have too big variation in salt treatment early stage, but when treated between to 4h when, SaIMT gene expression dose significantly mentions
Height, expression quantity is gradually reduced later, is finally restored to untreated preceding expression (Fig. 4).This is with pine camphor as osmotic adjustment
Substance is consistent the response time of salt stress.
The expression of embodiment 3, SaIMT in soybean and the analysis to salt tolerance
Construct plant expression vector pCHF-1301-SaIMT.The soybean embryo point genetic transformation mediated using agrobacterium rhizogenes
GUS is carried out by plant expression vector pCHF-1301-SaIMT (Fig. 5) soybean transformation Jilin 35, and to genetically engineered soybean Hairy root
Detection of expression, while destination gene expression amount in positive plant is detected, and divide the salt tolerance of genetically engineered soybean Hairy root
Analysis.Specific method and result are as follows:
The genetic transformation of 3.1 soybean root of hairs and screening
1) processing of vegetable material
Full, disease-free 35 seed of soybean Jilin is chosen, lies against in culture dish, culture dish is placed in closed drying
In device, and be put into drier one added with the dense HCl of 96mlNaClO and 4ml beaker, Cl2Sterilize 14-h16h.
2) soya seeds germinate
35 seed of soybean Jilin for taking above-mentioned sterilizing is inoculated on germination culture medium, in 16h illumination, 26 DEG C of temperature, and humidity
65%, the tissue culture room of 30000 lux of light intensity is germinateed 4-6 days.
3) preparation of bacterium solution is infected
I. taken out from -80 DEG C of refrigerators preservation Agrobacterium rhyzogenesK599 (carrier be respectively pCHF-1301-SaIMT and
PCHF-1301), cross on YEP (containing 100 μ g/mlKan) solid plate, 28 DEG C of cultures are for 24 hours；
Ii. the single colonie on picking plate is inoculated in 50ml YEP (containing 100 μ g/mlKan) fluid nutrient medium,
250rpm, 28 DEG C of shaken cultivation 8h-10h, makes OD600=0.8-1.0；
Iii. cultured bacterium solution 5ml is taken, is added in 500ml YEP (containing 100 μ g/mlKan) fluid nutrient medium,
250rpm, 28 DEG C of shaken cultivation 4h-6h, makes OD600=0.6-0.8, the bacterium solution are to infect bacterium solution.
4) soybean infect and the induction of Hairy root
The conversion of a soybean cotyledon node
I. good 35 seed of soybean Jilin of above-mentioned germination is chosen, kind of a skin is gently peelled off, cuts away part radicle, leave and take 3-
5mm hypocotyl；
Ii. it is cut vertically from centre by two panels cotyledon along hypocotyl, removes cotyledon, drawn in parallel with scalpel along cotyledonary node
Wound 10 times, then by explant be placed in it is above-mentioned infect in bacterium solution impregnate infect 30min；
Iii. explant is taken out from infecting in bacterium solution, is inside lain against in the co-culture medium for being lined with filter paper downwards,
In 16h illumination, 26 DEG C of temperature, the tissue culture room of humidity 65%, 30000 lux of light intensity is co-cultured 4-5 days.
Iv. the explant after co-cultivation is cleaned 3-4 times with root induction fluid nutrient medium, then impregnates 30min, then with filter
Paper draws explant surface liquid, removes bud with scalpel, makes in explant upwardly, to tilt 45 ° of oblique cuttings and enter root induction solid
In culture medium, in 16h illumination, 26 DEG C of temperature, the tissue culture room of humidity 65%, 30000 lux of light intensity is cultivated 15 days.
B soybean complex plant transformation
I. good 35 seed of soybean Jilin of above-mentioned germination is chosen, kind of a skin is gently peelled off, cuts away part radicle, leave and take 3-
5mm hypocotyl；
Ii. cross is cut 2-3 times among cotyledon hypocotyl with scalpel, explant is then placed in above-mentioned infect in bacterium solution
30min is infected in immersion；
Iii. explant is taken out from infecting in bacterium solution, is inside lain against in the co-culture medium for being lined with filter paper downwards,
In 16h illumination, 26 DEG C of temperature, the tissue culture room of humidity 65%, 30000 lux of light intensity is co-cultured 4-5 days.
Iv. the explant after co-cultivation is cleaned 3-4 times with root induction fluid nutrient medium, then impregnates 30min, then with filter
Paper draws explant surface liquid, and explant radicle is downward, is inserted vertically into root induction solid medium, in 16h illumination,
26 DEG C of temperature, the tissue culture room of humidity 65%, 30000 lux of light intensity is cultivated 15 days.
3.2 genetically engineered soybean Hairy root GUS detection of expression
Part Hairy root is randomly selected, is dyed with X-Gluc, 37 DEG C of dark dyeing 12-14h, with 70% ethanol decolorization number
It is secondary, detect the expression of gus gene.Fig. 6 is the GUS dyeing qualification result for filling the soybean Hairy root of SaIMT gene.The result shows that
SaIMT gene is expressed in soybean Hairy root.
The detection of expression of the target gene of 3.3 genetically engineered soybean Hairy roots
Choose the hair-like for turning SaIMT gene explant Hairy root and converting pCHF-1301 of above-mentioned GUS test positive
Root extracts RNA, reverse transcription cDNA, and by the expression quantity of qRT-PCR testing goal gene, internal reference is soybean β-actin, side
Method is referring to embodiment 2.As a result as shown in fig. 7, compared with the control of conversion pCHF-1301, SaIMT table in transgenosis Hairy root
It is significantly increased up to amount, illustrates that SaIMT is successfully transferred in soybean.
The Salt Tolerance Analysis of 3.4 turns of SaIMT transgenic soybean Hairy roots
1) band cotyledon turns SaIMT transgenic soybean Hairy root Salt Tolerance Analysis
Agrobacterium rhizogenes infect after soybean explant in being cultivated 5-7 days on root induction culture medium, will have root of hair sign
Subband leaf explant and control are transferred in solid MS (hygromycin containing 16mg/L) culture medium containing various concentration NaCl, are cultivated
It is counted after 15 days.The results show that with the increase of NaCl concentration, the growing state of genetically engineered soybean Hairy root is better than pair
According to group, and the dry weight of Hairy root is also significantly greater than and compares；And when NaCl concentration reaches 200mmol/L, turn SaIMT base
The growth of the Hairy root of cause is suppressed, and difficulty or ease induction generates normal Hairy root, and explant chlorosis turns yellow.See Fig. 8 and figure
9。
2) turn SaIMT transgenic soybean Hairy root Salt Tolerance Analysis in vitro
It will test that (tide containing 16mg/L is mould in the solid MS of various concentration NaCl for positive genetically engineered soybean Hairy root culture
Element) in culture medium, processing carried out observation statistics after 15 days.As a result, it has been found that in low concentration, (concentration is 50mmol/L's or less)
When NaCl processing, the upgrowth situation for turning the Hairy root of SaIMT gene is compared in control has no significant difference, with NaCl concentration
It increases, the growth of transgenosis Hairy root and weight in wet base are shown in Figure 10 obviously higher than control；When NaCl concentration reaches 150mmol/L,
Compared with the control group, the weight in wet base increment rate for turning the Hairy root of SaIMT gene reaches maximum, about the 3.5 of control group times, sees figure
11.And when NaCl concentration reaches 200mmol/L, transgenosis Hairy root is suppressed with the growth compareed, but transgenosis is sent out
The survival rate of shape root is all remarkably higher than control, and the survival rate for turning the Hairy root of SaIMT gene is 37.8%, the survival rate of control group
It is 15.6%, sees Figure 12.
3) turn the Salt Tolerance Analysis of SaIMT transgenic soybean hair-like root complex plant
The solid MS that the dicotyledonous explant of entire soybean that agrobacterium rhizogenes is infected is incubated at various concentration NaCl (is contained
16mg/L hygromycin) in culture medium, processing carried out observation statistics after 30 days.When NaCl concentration is 50,100,150mmol/L,
The growing way for turning the Hairy root plant complex of SaIMT gene is better than control, sees Figure 13；It is 100,150mmol/ in NaCl concentration
Under L stress conditions, the weight in wet base of the Hairy root plant complex of SaIMT gene is all remarkably higher than control.See Figure 14.
We carry out salt stress processing to the soybean Hairy root for turning SaIMT gene, analyze its shadow to soybean root system salt tolerant
It rings, the results showed that, it is certain resistance to that overexpression of the SaIMT gene in Hairy root shows genetically engineered soybean Hairy root
Salt.To transgenosis Hairy root, the growth conditions of Hairy root, survival rate, weight gain etc. are carried out under various concentration condition of salt stress
Statistics, the results show that growth conditions are good, salt tolerant significant effect when NaCl concentration is 100,150mmol/L；In NaCl
When concentration is 200mmol/L, although its growth is suppressed, survival rate is significantly higher than control.Illustrate the mistake of SaIMT and gene
The salt tolerance of the different degrees of raising genetically modified plants of expression energy.
Claims (3)
1. a kind of Sophora alopecuroide inositol transmethylase, it is characterised in that: its amino acid sequence is SEQ ID No.2.
2. a kind of gene for encoding Sophora alopecuroide inositol transmethylase, it is characterised in that: its nucleotides sequence is classified as SEQ ID
No.1。
3. Sophora alopecuroide inositol transmethylase as described in claim 1 is improving the application in plant salt endurance.
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| CN111118043B (en) * | 2020-01-13 | 2022-07-05 | 吉林大学 | Sophora alopecuroides SaMET6 gene clone and application thereof |
| CN111154789B (en) * | 2020-01-13 | 2023-04-07 | 吉林大学 | Cloning and application of Sophora alopecuroides SaENO2 gene |
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| Isolation and Characterization of a myo-inositol-1-phosphate Synthase Gene from Yellow Passion Fruit (Passiflora edulis f. flavicarpa) Expressed During Seed Development and Environmental Stress;EMANUEL F.M.ABREU等;《Annals of Botany》;20061130;第99卷;全文 |
| 转肌醇甲基转移酶基因烟草的耐盐性及其遗传分析;董云洲等;《农业生物技术学报》;20001231;第8卷(第1期);全文 |
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