CN104195169A - Construction method for cold-resistant transgenic tobaccos based on fatty acid desaturase gene - Google Patents

Abstract

The invention provides a construction method for cold-resistant transgenic tobaccos based on a fatty acid desaturase gene. The construction method comprises the following steps: firstly, amplifying out stearoyl carrier protein desaturase gene (SAD) of spinach by virtue of an RT-PCR (reverse transcription-polymerase chain reaction) process, and connecting the stearoyl carrier protein desaturase gene to a plant genetic transformation carrier pBI121; cloning and recombining a correct carrier by virtue of nucleotide sequence analysis and transforming the correct carrier into agrobacterium tumefaciens LBA4404; transforming tobacco aseptic seedling explants by the agrobacterium tumefaciens containing recombinant plasmids by virtue of a leaf disc method, screening out a kanamycin resistant plant and transplanting the kanamycin resistant plant into a flower pot, and collecting seeds after the kanamycin resistant plant is mature; sowing the seeds on an MS culture medium containing kanamycin to germinate and grow; extracting nuclear DNA (deoxyribonucleic acid) from leaves of the kanamycin resistant plant to carry out PCR (polymerase chain reaction) detection, dot blotting and Southern cross identification; and finally, by taking molecularly-identified positive transgenic tobaccos as experimental materials, carrying out conductivity measurement and chlorophyll content measurement respectively under low temperature stress, wherein the cold resistance of SAD transgenic tobaccos is obviously improved.

Description

A kind of construction process of the cold-resistant transgene tobacco based on fatty acid desaturase gene

Technical field

The invention belongs to gene engineering technology field, relate in particular to a kind of construction process of the cold-resistant transgene tobacco based on fatty acid desaturase gene.

Background technology

The plant especially winter resistance problem of farm crop is the focus of studying both at home and abroad always.If farm crop also can normal growth at lower temperature, this is to farm crop opposing low temperature cold damage, especially to preventing that the agricultural disaster such as cold spell in later spring, early frost in China's proportion of crop planting has great economic implications.

Existing result of study shows, plant is resisted the ability of low temperature stress and the being proportionate property of film fat degree of unsaturation of vegetable cell, that is to say, the degree of unsaturation of film fat is higher, and plant is stronger to the resistibility of low temperature environment.Stearyl-carrier proteins desaturase (SAD) is the key enzyme of vegetable fatty acid desaturation metabolic process; the topmost lipid acid of its catalysis plant---stearic first desaturation reaction; and improve the degree of unsaturation of the fatty acyl group on vegetable cell, especially plant cell membrane by a series of desaturation reactions subsequently, thereby improve the cold tolerance of plant.

Physiology to plant cold damage and biochemical research had been illustrated plant and had suffered the process and mechanism of cold damage in the past, such as, chilling temperature is that cytolemma, film fat degree of unsaturation and cold damage degree are closely related to the main original site of plant injury.But, have not yet to see the report of improveing plant cold resistance both at home and abroad by importing high expression level stearyl-carrier proteins delta 8 desaturase genes.

Summary of the invention

The object of the present invention is to provide a kind of construction process of the cold-resistant transgene tobacco based on fatty acid desaturase gene, be intended to solve the problem that plant sustains an injury under chilling temperature condition.

The present invention is achieved in that a kind of construction process of the cold-resistant transgene tobacco based on fatty acid desaturase gene, comprises the following steps:

(1) utilize the spinach stearyl-carrier proteins delta 8 desaturase genes that RT-PCR method amplifies to be connected on Genetic Transformation in Higher Plants carrier pBI121;

(2) recombinant vectors correct nucleotide sequencing is converted in Agrobacterium Agrobacterium tumefaciens LBA4404 and the positive bacterium colony that transforms of qualification;

(3) positive transformant is contaminated to tobacco leaf disc, after formation callus differentiation are sprouted, (contain 100mg/L Km at resistance MS substratum, 500mg/L CB) on carry out root induction, in the time that root reaches several centimetres, transplant in filling the flowerpot of Nutrition Soil, under natural light, grow; Extract the leaf DNA of kalamycin resistance plant and carry out dot blot and Southern hybridization, determine the tobacco plant that SAD gene imports;

(4) transgene tobacco importing using SAD gene is as experiment material, and-20 DEG C are placed under room temperature for freezing 40 minutes, measures chlorophyll content over time; The lower transgene tobacco of cultivating of room temperature (27 DEG C) moves to 6 DEG C of growths, measures specific conductivity over time; Judge with the chlorophyll content and the conductivity variations that turn SAD genetic tobacco whether the winter resistance of transgene tobacco has clear improvement.

In step (1), described RT-PCR method amplification the primer is:

Upstream primer: 5 '-AGATTCGAACAATGGCTCTGAATCTCAACC-3 ',

Downstream primer: 5 '-AGATCTAGAACTCAAAGCTTTACTTG-3 '.

The present invention utilizes genetic engineering technique; a kind of preparation method of winter resistance transgene tobacco is provided, and is that spinach stearyl-carrier proteins delta 8 desaturase genes (SAD) is converted into tobacco plant and has carried out Cold Hardiness by the gene that can improve plant cold resistance by.Result shows, the winter resistance tool that turns SAD genetic tobacco of structure is significantly improved.

Brief description of the drawings

Fig. 1 is the agarose gel electrophoresis figure of spinach SAD gene RT-PCR amplified production.Wherein, 1:DNA molecular weight mark (BamH I+Hind III digested λ DNA); 2:RT-PCR amplified production.

Fig. 2 is recombinant plasmid pBI121-13 (containing forward SAD gene), the agarose gel electrophoresis figure of pBI121-6 (containing antisense SAD gene) after enzyme is cut.Wherein, 1: the agarose gel electrophoresis figure of recombinant plasmid pBI121-13 after Xba I enzyme is cut; The agarose gel electrophoresis figure of 2:pBI121-6 after Xba I enzyme is cut; 3:DNA molecular weight mark (BamH I+Hind III digested λ DNA).

Fig. 3 is the DNA dot blot figure of kalamycin resistance tobacco plant, taking NOS terminator region as probe.Wherein, A: No. 1 to 6, the tobacco plant that recombinant vectors pBI121-13 transforms; B: No. 8 to 13, the tobacco plant that recombinant vectors pBI121-13 transforms; C:a → b is No. 14 to 15, the tobacco plant that recombinant vectors pBI121-13 transforms; C → f is No. 1 to 4, the tobacco plant that recombinant vectors pBI121-6 transforms; D:a → b is No. 1 to 2, the sample that recombinant vectors pBI121-13 transforms; C → d is unconverted tobacco plant (negative control), and f is the tobacco plant (positive control) that the unloaded plasmid of pBI121 transforms.

Fig. 4 is the Southern hybridization analysis figure of transgene tobacco.Wherein, swimming lane 1: contain GUS+NOS _terthe positive control of fragment; Swimming lane 2:pBI121 plasmid; Swimming lane 3,4,7,8: No. 2, No. 13, No. 9, No. 14 plant that transform with recombinant plasmid pBI121-13; Swimming lane 5,6,9,10: No. 3, No. 4, No. 1, No. 2 plant that transform with recombinant plasmid pBI121-6.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.

The structure of embodiment 1 Genetic Transformation in Higher Plants carrier

Stearyl-carrier proteins desaturase (SAD) is the key enzyme of vegetable fatty acid desaturation metabolic process, and the degree of unsaturation of cell membrane lipid and the winter resistance of plant are proportionate.SAD gene is expressed lower in plant, and SAD enzyme is the desat the first step of stearyl, is also the rate-limiting step that a series of desaturations react subsequently.So the present invention is inserted into the spinach SAD gene being cloned in the 35S promoter downstream of CaMV, be built into can high efficient expression SAD gene Genetic Transformation in Higher Plants carrier pBI121-13.The SAD gene of antisense is also built up to pBI121 simultaneously, obtain recombinant plant genetic transformation carrier pBI121-6, as negative control.

First, with the total RNA of phenol/chloroform extraction method extraction spinach, according to the cDNA sequences Design of the spinach SAD gene of GenBank synthetic a pair of Auele Specific Primer, the clone for SAD gene:

Upstream primer: 5 '-AGATTCGAACAATGGCTCTGAATCTCAACC-3 ',

Downstream primer: 5 '-AGATCTAGAACTCAAAGCTTTACTTG-3 ';

Carry out RT-PCR amplification with this synthetic pair of primers and the total RNA of spinach of extraction.Detect through 0.8% agarose gel electrophoresis, obtain the amplified production (Fig. 1) of a treaty 1.2Kb.

By the RT-PCR product after plant binary expression vector pBI121 and purifying respectively through CSP45I and XbaI double digestion, with T4DNA ligase enzyme, linearizing carrier and PCR product being carried out to 16 spends night and is connected, again connection product is converted into intestinal bacteria (Escherichia coli) DH5 α bacterial strain, 37 DEG C of overnight incubation.Selecting positive bacteria next day drops into row cultivation, prepares recombinant plasmid.Recombinant plasmid carries out 0.8% agarose gel electrophoresis after enzyme is cut, and detects and has or not Insert Fragment and clip size (Fig. 2).By the DNA fragmentation inserting on recombinant plasmid being carried out to nucleotide sequencing and comparing with SAD gene order, result shows that amplification DNA fragmentation is out the cDNA of SAD gene.The recombinant plasmid called after pBI121-13 obtaining.Meanwhile, utilize aforesaid method, the SAD gene of antisense is also cloned on pBI121 carrier, gained recombinant plasmid called after pBI121-6.

The Genetic Transformation in Higher Plants carrier transformation of tobacco such as embodiment 2pBI121, pBI121-13, pBI121-6

Taking-up is stored in the Agrobacterium Agrobacterium tumefaciens LBA4404 of-70 DEG C of refrigerators, lines on YEB flat board, and 28 DEG C of cultivations, until grow single bacterium colony.Picking list bacterium colony in 5ml YEB substratum, 28 DEG C, 250rpm overnight incubation.Get 2ml and be inoculated in continuation cultivation in 40ml YEB.After approximately 3 hours, pour in the centrifuge tube of precooling, ice bath 30 minutes, centrifugal 10 minutes of 6000rpm, precipitation is suspended in 1ml0.1M CaCl ₂in solution, draw 200 μ l and be sub-packed in the centrifuge tube of precooling, add respectively about 800ng pBI121, pBI121-13, pBI121-6.Ice bath 5 minutes, be placed in liquid nitrogen 8 minutes, after taking-up, put into immediately 37 DEG C of water-baths 5 minutes, add YEB to 1ml, 28 DEG C, 250rpm are cultivated 4～5 hours, centrifugal, precipitation is suspended in 200 μ l YEB, coat on the LB flat board that contains kantlex (50 μ g/ml), 28 DEG C of cultivations, until grow single bacterium colony.Picking list colony inoculation, in the liquid LB substratum that contains kantlex (50 μ g/ml), until grow to logarithmic phase, extracts plasmid electrophoresis detection.

Import Genetic Transformation in Higher Plants carrier pBI121 by above-mentioned with freeze-thaw method, pBI121-13, the agrobacterium strains of pBI121-6 screens and identifies positive transformant.The single bacterium colony of the positive Agrobacterium of picking is inoculated in respectively 5ml containing in the YEB substratum of kantlex (50 μ g/ml), and 28 DEG C, 250rpm are cultured to OD ₆₀₀=0.8～1.0, centrifugal collection thalline, with after the washing of MS substratum, regulates cell concentration to OD ₆₀₀=0.5～0.6.The aseptic blade of tobacco (reforming No. 29) is cut into the square fritter of 1cm, be dipped in different Agrobacterium suspensions, 5～7 minutes, be then placed on MS flat board, 28 DEG C of illumination cultivation are after three days, the leaf dish of conversion processing is placed in to MS nutrient solution, after 5～7 minutes, then forward MS flat board to, 25 DEG C of cultivations, light/be secretly 12h/12h, sprout until form callus and break up.The bud differentiating is smoothly cut and is forwarded in MS solid medium, and root induction, in the time that root reaches several centimetres, transplants to filling in the flowerpot of Nutrition Soil, makes its growth under the natural lighting of laboratory.In the time growing to 6～7 leaves, get plant leaf extraction core DNA and carry out Molecular Identification.

Embodiment 3 dot blots and Southern screening by hybridization transgene tobacco

Prepare hybridization nylon membrane, nylon membrane is soaked with distilled water, in 2X SSC solution, soak 5 minutes, suction filtration point sample, adds 150 μ l2X SSC and rinses, and treats DNA complete drying, be placed on sex change on the saturated filter paper of 0.4MNaOH 5 minutes, and then with in the saturated filter paper of 2XSSC and 5 minutes, roasting film 1 hour in 80 DEG C of vacuum drying ovens, dry place deposits for subsequent use.

By BamHI and EcoRI double digestion for pBI121 plasmid, after electrophoretic examinations enzyme cuts entirely, with low melting-point agarose gel electrophoresis recovery GUS+NOS _terfragment is as DNA probe.The radio-labeling of DNA probe, adopts Random Primer method mark.

Hybridization: 100 DEG C of radiolabeled DNA probes are boiled 5 minutes, and ice bath is cooling, adds EDTA to 20mM.Pour out prehybridization solution, add hybridization solution, add probe, hybridize 16～24 hours for 42 DEG C.Wash film and radioautograph: discard hybridization solution, Hybond membrane first rinses once with 2X SSC+0.5%SDS solution, at room temperature wash again 5 minutes, with 2X SSC+0.1%SDS washing 5 minutes, finally in 0.1X SSC+0.5%SDS, wash 30 minutes at 68 DEG C again, and wash away SDS with 0.1X SSC, then suck nylon membrane debris with filter paper, wrap preservative film, press X-mating plate and intensifying screen in-70 DEG C of radioautograph, 2～3 days development X-mating plates afterwards.

Choose the pBI121-13 screening through kalamycin resistance, pBI121-6, pBI121 transformed plant and 2 strains not genetic transformation plant contrast, and extract leaf DNA and carry out dot blot experiment.According to dot blot result (Fig. 3), select pBI121-13 transformed plant that hybridization signal is the strongest 2, No. 13, what pBI121-6 transformed extract core DNA for 3, No. 4 does double digestion; 9, No. 4 of pBI121-13 conversion and the core DNA that extracts for 1, No. 2 of pBI121-6 conversion do single endonuclease digestion.With Gus+NOS _terfor probe carries out Southern hybridization check.Southern results of hybridization shows, double digestion sample has a hybrid belt in the position of 2.0Kb, and single endonuclease digestion sample has many hybrid belts (Fig. 4) at different positions.Illustrate that these plants are transgenic plant, and be multiple copies.

Embodiment 4 contrasts tobacco and turns the Cold Hardiness of SAD genetic tobacco

Conductivity variations measure: by Southern hybridize be positive 6 ², 6 ³, 6 ⁴, 13 ⁹, 13 ¹⁵, 13 ¹⁴plant and without the plant (contrast) of genetic transformation, from room temperature (approximately 27 DEG C), light are displaced downwardly to 6 DEG C, dark incubator.Respectively at the specific conductivity of surveying plant under room temperature and under low temperature after 40 hours, 88 hours.Then plant is transferred to room temperature (approximately 27 DEG C), under natural light, after 48 hours, surveys specific conductivity.

Get the blade that size is close, beat and get 10～15 sequins with punch tool, put into 10mlddH ₂in O, be tiltedly placed on shaking table and shake 1h, survey electricity and lead, then boiling water boiling 15 minutes, cooling rear survey electricity is led, and accounts for the percentage that boils rear electric conductivity value as the relative conductivity of every kind of plant to boil front electric conductivity value.

Measuring chlorophyll content: from Southern hybridize be positive 6 ², 6 ³, 6 ⁴, 13 ⁹, 13 ¹⁵, 13 ¹⁴plant and upper without the plant (contrast) of genetic transformation, each clip size, two leaves that leaf age is consistent, put into disposable Thin film glove, sealing.Wherein a slice be placed in-20 DEG C freezing 40 minutes, after taking-up, be put under room temperature (approximately 27 DEG C), another sheet leaf is held under room temperature and compares.After 4 days, survey chlorophyll content.

Get 1g left and right leaf tissue, add a small amount of CaCO ₃and 80% acetone, be ground to homogenate, filter.Wash mortar for several times with 80% acetone again and washing lotion filtered and merged, with 80% acetone constant volume filtrate to 10ml.Under 652nm wavelength, survey OD value, calculate chlorophyllous total amount.Respectively organize the variation of chlorophyll content.

Before subzero treatment, the relative conductivity of each group tobacco leaf is basically identical.6 degree are after lower 40 hours, and the specific conductivity of each group occurs difference but be relatively little.After subzero treatment 88 hours, the relative conductivity of control group and pBI121-6 transformed plant group all obviously raises.And the relative electric conductivity of pBI121-13 transformed plant group remains at lower level (table 1).

The specific conductivity of control group and treatment group under table 1 differing temps

Note: CK ¹, CK ²: No. 1, No. 2 plant that do not carry out genetic transformation; 13 ⁹, 13 ¹⁴, 13 ¹⁵: No. 9, No. 14, No. 15 plant that transform with recombinant plasmid pBI121-13; 6 ², 6 ³, 6 ⁴: No. 2, No. 3, No. 4 plant that transform with recombinant plasmid pBI121-6

Tobacco leaf, after of short duration freezing (20 DEG C, 40 minutes) are processed, is placed in to room temperature lower 4 days, measures the difference of chlorophyll content in processing and non-processing tobacco leaf.Although some individual difference, the Chlorophyll loss amount of pBI121-13 transformed plant is starkly lower than contrast and pBI121-6 transformed plant (table 2).

The difference of the each group of table 2 tobacco leaf chlorophyll content before and after freezing treatment

Each treatment group	Before processing	After processing	After processing/before processing (%)
				CK 1	1.423	0.4767	33.50
CK 2	2.271	0.7653	33.70
				CK 3	2.471	0.6342	25.67
CK 4	1.927	0.5604	29.08
				13 13	1.886	0.9639	51.11
13 2	1.665	0.825	49.73
				13 14	2.161	0.7995	36.99
13 15	2.602	0.6867	26.39
				6 1	2.337	0.6315	27.02
6 2	2.463	0.4485	18.21
				6 3	2.618	0.7905	30.19
6 4	2.338	0.4182	17.89

Note: 13:pBI121-13; 6:pBI121-6

Can find out from the experimental data of table 1 and table 2, the winter resistance of transfer-gen plant (the high expression level justice SAD gene) group of pBI121-13 genetic transformation is obviously better than without the control group of genetic transformation and the transfer-gen plant of pBI121-6 genetic transformation (high expression level antisense SAD gene) group.Therefore, the present invention, from positive and negative two aspects, has proved to strengthen the expression of SAD gene in plant materials, can significantly improve the winter resistance of plant.

Compared with the shortcoming and defect of prior art, the present invention has following beneficial effect: for obtaining cold-resistant high-yield crop kind, and ordinary method (cross-breeding) length consuming time, blindness is large.It is an important and popular domain of international genetically engineered research with genetic engineering technique directional transformation plant cold resistance.For a long time, the winter resistance of known plants and membrane lipids of plant cell degree of unsaturation are proportionate, but the winter resistance that how to improve the film fat degree of unsaturation of plant and then improve plant is still the problem that current genetically engineered and molecular biology are explored.In all higher plant fatty acid desaturases; stearyl-carrier proteins desaturase (SAD) is the lipid desaturase of the solubility of current unique qualification; and the SAD synthetic the first step that is unsaturated fatty acids, its active height is a key factor that determines saturated and unsaturated fatty acids ratio in vegetable cell.Yet there are no the report about turning SAD gene plant and winter resistance relation.What experiment showed, that the present invention builds turns SAD genetic tobacco and can improve significantly its winter resistance.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims (2)

1. a construction process for the cold-resistant transgene tobacco based on fatty acid desaturase gene, is characterized in that, comprises the following steps:

(1) utilize the spinach stearyl-carrier proteins delta 8 desaturase genes that RT-PCR method amplifies to be connected on Genetic Transformation in Higher Plants carrier pBI121;

(2) recombinant vectors correct nucleotide sequencing is converted in Agrobacterium Agrobacterium tumefaciens LBA4404 and the positive bacterium colony that transforms of qualification;

(3) positive transformant is contaminated to tobacco leaf disc, formation callus differentiation (contain 100mg/L Km at resistance MS substratum after sprouting, 500mg/LCB), carry out root induction, in the time that root reaches several centimetres, transplant in filling the flowerpot of Nutrition Soil, under natural light, grow; Extract the leaf DNA of kalamycin resistance plant and carry out dot blot and Southern hybridization, determine the tobacco plant that SAD gene imports;

(4) transgene tobacco importing using SAD gene is as experiment material, and-20 DEG C are placed under room temperature for freezing 40 minutes, measures chlorophyll content over time; The lower transgene tobacco of cultivating of room temperature (27 DEG C) moves to 6 DEG C of growths, measures specific conductivity over time; Judge with the chlorophyll content and the conductivity variations that turn SAD genetic tobacco whether the winter resistance of transgene tobacco has clear improvement.

2. the construction process of the cold-resistant transgene tobacco based on fatty acid desaturase gene as claimed in claim 1, is characterized in that, in step (1), described RT-PCR method amplification the primer is:

Upstream primer: 5 '-AGATTCGAACAATGGCTCTGAATCTCAACC-3 ',

Downstream primer: 5 '-AGATCTAGAACTCAAAGCTTTACTTG-3 '.