CN1618976A - Method for cultivating low-temperature saccharification resistant potato strains by introducing AcInv antisense gene - Google Patents
Method for cultivating low-temperature saccharification resistant potato strains by introducing AcInv antisense gene Download PDFInfo
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- CN1618976A CN1618976A CN 200410068868 CN200410068868A CN1618976A CN 1618976 A CN1618976 A CN 1618976A CN 200410068868 CN200410068868 CN 200410068868 CN 200410068868 A CN200410068868 A CN 200410068868A CN 1618976 A CN1618976 A CN 1618976A
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Abstract
A method for configuring the plant expression carrier of AcInV antisense gene and culturing the low temp tolerant saccharified potato variety includes cloning the low-temp induction type Rd29A promotor from mouseearcress leaf and the acidic transferase gene cDNA from potato tuber, using Rd29A to replace CaMV 35S promotor on plant expression carrier, reversely inserting the AcInV gene cDNA fragment between Rd29A promotor and NOS terminator to configure low-temp induction type AcInv antisense gene plant expression carrier pBIAc, directly transferring it to agrobacterium LBA 4404 to obtain the engineering bacterium, and using it to transfer the variety of potato to obtain two target varieties.
Description
Technical field the present invention relates to a kind of recombinant DNA and structure thereof, and is mediated transformation potato kind with the Agrobacterium, cultivates the method for the anti-low temperature saccharification strain of potato tuber.Specifically, the present invention relates to a kind of Arabidopis thaliana (Arabidopsis thaliana) rd29A promotor (rd29A promoter) and potato common cultivation kind (Solanumer tuberosum of including, 2n=4X) acid invertase (acidinvertase, AcInv) cDNA reverse sequence, can in vegetable cell, be subjected to low temperature induction to transcribe or express the plant expression vector construction of acid invertase inverted defined gene, and be mediated transformation potato common cultivation kind (2n=4X) with the Agrobacterium, cultivate the method for the anti-low temperature saccharification strain of potato tuber.
The background technology potato is important grain dish dual-purpose and insutrial crop, is one of the world four big crops.Because it is barren-resistant, stable high yield, wide adaptability, nutritive ingredient is complete and the industry chain length, and is subjected to global great attention.According to the guestimate of International Potato Center (CIP), the whole world is used for directly edible potato not as good as 1/2 of ultimate production, and all the other all are used for deep processing.In some developed countries, potato processing proportion is generally all 70%, even the country that has is up to more than 80%, and a series of supporting processed-type kinds are arranged.Though China's potato planting area accounts for nearly 1/4 of whole world potato sown area, gross output accounts for 1/5, its cultivated area and gross output all occupy first place in the world (Zhang Changsheng. Chinese Potato Industry one Potato Industry and development of the West Regions geared to the 21st century. Harbin, press of Harbin Engineering University, 2000,10-13), but processing industry is relatively backward always, processing proportion is less than 20% of ultimate production, and the rich in natural resources advantage is fully developed, and potato production benefit is still lower.The advantage that keeps China potato to produce must be support with the processing industry, and rising in value by the deep processing of product drives the development of Potato Industry.
The raw potatoes kind that is used to process requires than general grain dish dual-purpose type kind height, not only want high yield, disease-resistant, and to stem tuber outward appearance and the strict in addition requirement of inclusion, wherein stem tuber dry matter content and reducing sugar content are two important indicators of processed-type kind.General requirement is greater than 1.080 as its stem tuber proportion of kind that processes raw material, and dry matter content is more than 20%.The kind that is used for potato chips, potato piece, its stem tuber reducing sugar (mainly being glucose, fructose) content should be less than 0.1% (fresh weight), the upper limit be no more than 0.3% (Fan civilian worker, high blunt Kui. Chinese Potato Industry-Potato Industry geared to the 21st century and development of the West Regions .2001,84-87).
Because the traditional potato consumption habit of China is to be used to eat raw, breed breeding also with high yield, disease-resistant be major objective, improved variety mostly is grain dish dual-purpose type greatly, starch content is many between 12%～16%, the high starch kind that is suitable for processing is in great shortage.In recent years, by introducing and self-fertile has filtered out the kind of some high-content of starch, satisfied the requirement of processing enterprise basically.But these kind reducing sugar content are generally higher, and especially the reducing sugar content of stem tuber increases under the low temperature storage condition, badly influence the converted products quality.Therefore, the kind of low reducing sugar content of seed selection and anti-low temperature saccharification has become a urgent task of current potato breeding work.
Potato tuber reducing sugar content height both by inherited genetic factors control, was subjected to the influence of holding conditions again.Low temperature is the main affecting factors that causes reducing sugar content to increase in many environmental factorss.After the potato tuber that is used to the process results, generally all will be through one period storage period, due to illness evil invasion and attack during this period, sprout and dehydration easily causes the loss of rotting.The measure that loss is stored sth. in a cellar in control sprays short dormancy chemical agent or adopts the low temperature storage method.Because the chemical agent processing can cause residual hazard, generally bans use of; Utilize peasant's cellar storage, with have conveniently, safety and the low outstanding advantage of cost, but since the cellar storage utilization be natural low temperature, often be lower than 10 ℃ at severe winter, tend to cause the increase of stem tuber reducing sugar content, promptly so-called cold mashing (cold-sweetening).The stem tuber that saccharification takes place is used for starch and processes that then flour extraction rate is lower; During as the fried food product raw material, the Mai Laerde reaction (Maillar-typereaction) of non-enzymatic then takes place in high temperature fry process, it is the material that reducing sugar and free amino acid react and generate brown and have bitter taste in the cell, badly influence flavours in food products and impression (Shailenberger et al..J.Agric.FoodChem., 1959,7:274-277; Pressy et al..Arvh.Biochem.Biophys., 1966,133:667-674; Samotus et al..Potato Res., 1974,17:64-81; Eileen.PlantPhysiol., 1991:335-341; Vayda et al..Potato Genetics, CBA InternationPress, Oxon UK, 1994:250-251).Therefore, the reducing sugar content in the control stem tuber is very important to the quality that improves its converted products.
The general measure that reduces the stem tuber reducing sugar content has two kinds: the one, get warm again after a cold spell (reconditioning), and the 2nd, breed breeding (Samotus et al., Potato Res., 1994,17:64-81).After improvements are meant that the stem tuber of low temperature under storing sth. in a cellar goes out the cellar for storing things, placing for some time in the environment more than 10 ℃ again, making in the body reducing sugar fall (Bruton et al..Eur.Potato J., 1969 by self respiration consumption, 12:81-95), thus reduce reducing sugar content in the stem tuber.Implementing the improvements measure not only needs certain site facility and increases production process, and can cause the meaningless consumption of raw material, will inevitably increase production cost, generally is difficult to processed enterprise and adopts.Comparatively speaking, cultivate the kind that reducing sugar does not raise under low reducing sugar content and the low temperature, utilize natural low temperature ground kiln to preserve,, processing enterprise's production cost is descended significantly according to the demand of processing enterprise base feed according to plan by the breeding approach.Should say that this is to solve the most cost-effective measure of stem tuber low temperature saccharification.
Though there is bigger difference in potato tuber reducing sugar content height between kind, might cultivate the low strain of reducing sugar content by the breeding approach.But unfortunately, utilize low reducing sugar clone and common variety hybridization, its offspring distributes and tends to the high-reducing sugar parent, the low shared ratio of reducing sugar offspring is extremely low, come the kind of seed selection reducing sugar content and the anti-low temperature saccharification of stem tuber by the conventional breeding method, to be extremely difficult and very consuming time (Eileen et al..Plant Physiol, 1991,335～341).So people's attempt is controlled the low temperature saccharification metabolic process by genetic engineering technique, cultivate the strain of low reducing sugar content of stem tuber and anti-low temperature saccharification.
Molecular biology research shows that sense-rna (antisense RNA) technology is the effective way that control specific gene (target gene) is expressed.First cultivation that example is the storage tolerance tomato variety of Antisense RNA Technique successful Application.At present this technology has been applied to that melon cauliflower grass is fresh-keeping, the aspects such as control of some human diseases, its successful example is also more and more, this just edifies us and adopts Antisense RNA Technique to suppress key gene in the stem tuber low temperature saccharification process, to cultivate the kind of low reducing sugar content of stem tuber and anti-low temperature saccharification.
Shift to new management mechanisms according to carbohydrate in the potato tuber, in the starch degradation metabolic process, UDPglucose pyrophosphorylase (uridine diphosphate glucose pyrophosphorylase, UGPase) and saccharase (invertase) be two important enzymes (Bruton et al..Eur.Potato J., 1969,12:81-95; Samotus et al..Potato Res., 1974,17:64-81).1993, the Zrenner clone also made up UGPase inverted defined gene plant expression vector, was subjected to obvious inhibition by UGPase activity in the transfer-gen plant of genetic transformation acquisition, and its active contrast has descended 95%～96%.But regrettably, remaining UGPase activity still makes starch---the reducing sugar conversion process is carried out as usual, illustrate control UGPase activity can not reduce effectively the low temperature saccharification metabolic process (Zrenner et al..Planta, 1993,190:247-252).Saccharase comprises that (acid invertase, AcInv), its effect all is that the degraded of catalysis sucrose produces hexose for neutral saccharase (nautral invertase) and acid invertase.Neutral saccharase is higher at the plant in-vivo content of growth, and its effect is closely related with anabolism, and the content fluctuation in the refrigeration stem tuber is very little, activity is also very low, almost have nothing to do with the effect of refrigeration stem tuber low temperature saccharification (Zrenner et al..Planta, 1996,198:246-252).AcInv is then closely related with the effect of stem tuber cold mashing, the research of Pressey point out in the low temperature storage stem tuber AcInv have maximum activity (Pressey.Arvh.Biochem.Biophys., 1966,133:667-674).In the low temperature saccharification metabolic process, AcInv catalysis sucrose hydrolysis is that the reaction of Nulomoline is irreversible.Biochemical metabolism regulation and control theory thinks that the enzyme of general catalysis irreversible reaction influences the rate-limiting enzyme of whole pathways metabolism often, also is the key position of metabolic regulation, so AcInv is considered to the key enzyme in the low temperature saccharification metabolic process.We think in view of the above, suppress the breach that the AcInv activity might become control stem tuber low temperature saccharification.
Up to the present, the molecular biology research of relevant AcInv report is less.Zhou etc. from the low temperature storage stem tuber, cloned the earliest AcInv cDNA full length sequence (Plant Physiol., 1994,106:397-398).After this, Zrenner etc. have made up by cauliflower mosaic virus 35S promoter (cauliflower mosaic virus 35S promoter, CaMV 35S) the AcInv inverted defined gene plant expression vector of Qu Donging, transform the potato kind and obtain transfer-gen plant, though its stem tuber soluble sugar (sucrose, glucose and fructose) total amount under the low temperature storage condition does not change, (the Planta but the ratio of reducing sugar/sucrose descends, 1996,198:246-252), the degraded that can control sucrose to the Antisense Suppression of AcInv gene effectively is described.But the CaMV 35S promoter is a kind of composition type expression promoter, all can start the genetic expression that it is controlled under any tissue of plant, any condition.This expression pattern not only is a kind of meaningless waste in energy metabolism, also might have a negative impact to growth and development of plant.On producing, potato many with stem tuber as reproductive material, in the potato seed germination process in the body starch of storage must be converted into soluble sugar and could be utilized by cambium and organ, wherein the effect of saccharase is very important.Although there is research to point out, sprout and the plant strain growth phase at potato tuber, with the master that act as of neutral saccharase, do not get rid of AcInv effect (Isherword.Phytochemistry, 1976,15:33-41).If in the normal growth phase, the intravital AcInv effect of plant still is suppressed, and perhaps has a negative impact to growing.Because the environmental induction factor of stem tuber generation saccharification is a low temperature, if the AcInv inverted defined gene is placed under the low temperature induction promotor, transcribing of inverted defined gene stopped or transcriptional level extremely low, then very little or do not exert an influence to endogenous AcInv activity influence; In case stem tuber is under the cold condition, low temperature induction type promotor then drives transcribing of AcInv inverted defined gene, suppress endogenous AcInv gene transcription or expression, the saccharification that AcInv is caused is reduced to minimum, so just might prevent stem tuber generation low temperature saccharification.
In view of above consideration, we are at the general thought of research: the AcInv inverted defined gene expression pattern that designs a kind of low temperature induction, the AcInv inverted defined gene is in a kind of silence state in the render transgenic plant under normal temperature conditions, and under the low temperature storage condition, efficiently transcribe, to suppress stem tuber endogenous AcInv expression of gene, reach the purpose of anti-low temperature saccharification.
Up to the present, the low temperature induction promotor that isolation identification goes out from plant mainly contains the bn115 of rape, rd29A, cor15A and adh, the wes120 of wheat and the gene promoters such as mlip15 of corn of Arabidopis thaliana, wherein the rd29A promotor is paid attention to (Yamaguchi-Shinozaki et al..Plant Cell by people in the effect aspect the gene expression in plants regulation and control, 1994,6:251-264).Many results of study show, the rd29A promotor can drive genetic transcription and the translation that it is controlled at low temperatures, it also is subjected to arid in addition, high salt penetration is coerced or inducing of Exogenous Abscisic Acid (ABA) and raise expression (the Xiong et al..Plant Physiol. of institute's controlling gene, 1999,119:205-212).Therefore, we have selected the AcInv inverted defined gene expression pattern of rd29A promotor control for use.
Utilize the genetic transformation technology exogenous gene transfered plant cell, and to be incorporated in the genome be the key link of plant genetic engineering.At present, the method that plant genetic transforms mainly contains agrobacterium-mediated transformation, pollen tube passage method, particle bombardment, supersonic method, electrization and laser microbeam introductory technique etc., wherein the genetic transformation system of Agrobacterium (Agrobacterium tumefaciens) mediation is that research is maximum at present, mechanism is the clearest, technological method is the most ripe, most widely used conversion system, the transgenic plant of first energy expression alien gene obtain by agrobacterium tumefaciens-mediated transformation, in the nearly 200 kinds of transgenic plant that obtained so far, have 80% be utilize that the agrobacterium tumefaciens conversion system produces (Li Wei etc. Science Bulletin, 2000,45 (8): 798-807).Utilize genetic engineering technique to cultivate in the research of potato kind, example with agriculture bacillus mediated genetic transformation success is also maximum, but also there is the not high problem of genotype dependency and transformation efficiency, how further improves transformation efficiency and remain the current subject matter that needs solution.
Summary of the invention an object of the present invention is to provide a kind of construction process of AcInv inverted defined gene plant expression vector of low temperature induction type promoters driven, promptly make up and contain rd29A promotor (rd29A promoter), it can be subjected to low temperature induction to drive potato common cultivation kind (Solanum tuberosum, 2n=4X) the carrier of acid invertase (AcInv) cDNA reverse sequence transcript and expression, transform potato common cultivation kind and obtain transfer-gen plant, its stem tuber AcInv inverted defined gene under normal temperature conditions is not transcribed, and should efficiently transcribe when being subjected to low temperature induction, to suppress endogenous AcInv expression of gene, thereby improve the ability of the anti-low temperature saccharification of stem tuber, reduce the stem tuber reducing sugar content.
Another object of the present invention provides a kind of agrobacterium-mediated transformation that utilizes goal gene is imported the optimization method of potato common cultivation kind, to improve transformation efficiency, obtains transgenic strain.
For achieving the above object, the present invention at first from Arabidopis thaliana (Arabidopsis thaliana) genomic dna cloning rd29A promotor (SEQ ID NO 1), cloned AcInv gene cDNA (SEQ ID NO 2) potato common cultivation kind under low temperature induction " No. 3, the agricultural potato " stem tuber, with CaMV 35S promoter and the gus gene on rd29A promotor and the AcInv gene cDNA fragment replacement plant expression vector pBI121, be built into the AcInv inverted defined gene plant expression vector pBIAc of low temperature induction type rd29A promoters driven.Transform Agrobacterium LBA4404 with direct guiding method then, acquisition has the AcInv inverted defined gene Agrobacterium engineering bacteria of rd29A promoters driven, utilize the AcInv inverted defined gene Agrobacterium engineering bacteria of low temperature induction type rd29A promoters driven to transform the potato kind again, cultivate the anti-low temperature saccharification strain of stem tuber.
The present invention studies agriculture bacillus mediated potato genetic conversion system, by to nutrient media components, acceptor material, acceptor to the susceptibility of Kan, pre-incubation time, Agrobacterium concentration, immerged time, the brown chemoprevention of incubation time and transformation receptor material such as ends at the research of aspect altogether, set up the better conversion system, make the potato AcInv inverted defined gene transformation efficiency of agriculture bacillus mediated rd29A promoters driven reach 0.501%, be higher than the Transformation of potato rate (0.1%) of present report.Utilize this method, obtained 2 transgenic strains, wherein " Ac changes the Atlantic Ocean " stem tuber dry-matter, starch and reducing sugar content are respectively 24.53%, 19.96% and 0.05%, and its reducing sugar content comparison is according to kind " Atlantic Ocean " low 89.5%; " Ac changes sweet 2 " stem tuber dry-matter, starch and reducing sugar content are respectively 27.93%, 21.30% and 0.44%, and its reducing sugar content comparison is according to kind low 59.8%.After 4 weeks of storage, the stem tuber reducing sugar content of " Ac changes the Atlantic Ocean " strain becomes 0.08% at low temperature (4 ℃ ± 2 ℃), and comparison is according to low 91.3%; " Ac changes sweet 2 " strain stem tuber reducing sugar content is 0.48%, and comparison is according to having reduced by 79.6%.Having proved the AcInv inverted defined gene plant expression vector that adopts the low temperature induction promoters driven, by agriculture bacillus mediated genetic transforming method, is a kind of effective scheme of cultivating the anti-low temperature saccharification strain of potato tuber.At home and abroad there is no identical report at present.
Description of drawings
Fig. 1 is from the PCR product agarose gel electrophoresis result of Arabidopis thaliana DNA cloning Rd29A promotor
Lane?1：DGL2000?DNA?Markers
Fig. 2 cloning vector pUCRD enzyme is cut (Hind III+Bam HI) product electrophoresis result
Lane?1：DGL2000?DNA?Markers
Lane 2:Hind III+Bam HI enzyme is cut the product of pUCRD
The pcr amplification product agarose gel electrophoresis result of Fig. 3 cloning vector pUCRD
Lane?1：DGL2000?DNA?Markers
The cloning vector collection of illustrative plates of Fig. 4 low temperature induction type promotor rd29A
The GFP gene plant expression vector collection of illustrative plates of Fig. 5 CaMV35S promoters driven
The GFP gene plant expression vector collection of illustrative plates of Fig. 6 rd29A low temperature induction promoters driven
The expression microscopic examination (20X) of GFP in the onion epidermis cell that Fig. 7 transforms
The expression of GFP in the onion epidermis cell under A～B:28 ℃, the low light level (1000lux) condition
The expression of GFP in the onion epidermis cell under C～D:4 ℃, the low light level (1000lux) condition
A and C:pBIG transformant; B and D:pBIRG transformant.
Fig. 8 potato tuber RNA RT-PCR product agarose gel electrophoresis result
Lane?1：DGL2000?DNA?Markers
Lane 2: be the RT-PCR product of template with potato tuber RNA
The cloning vector collection of illustrative plates of Fig. 9 AcInV gene
Figure 10 AcInv gene antisense plant expression vector (pBIRA) enzyme is cut detected result
Lane?1：DGL2000?DNA?Markers
Lane 2:(HindIII+BamHI) the electrophoresis detection result of double digestion pBIRA
The plant expression vector figure of the AcInv inverted defined gene of Figure 11 rd29A low temperature induction type promoters driven
Spectrum
The PCR of Figure 12 AcInv inverted defined gene Agrobacterium engineering bacteria detects the agarose gel electrophoresis result
Lane?1：λDNA/EcoR?I+Hind?III?Markers
Figure 13 is to the changing effect of the different acceptor materials of potato
A. test-tube plantlet stem section B. test tube potato potato chips C. test-tube plantlet blade D. test-tube plantlet stem section
E. micro potato potato chips F. test-tube plantlet stem section transformation tissue culture seedling
Figure 14 transforms the Southern bloting detected result of seedling
1.Dig DNA Markers 2. Atlantic Ocean transformed plant DNA of mark
3. the gram transformed plant DNA of No. 2 transformed plant DNA of sweet agricultural potato 4. boolean classes
5. Xia Baidi transformed plant DNA 6.pBIRA/Bam HI+SacI
7. the unconverted plant DNA in the Atlantic Ocean
Figure 15 " Ac changes the Atlantic Ocean " and " Ac changes sweet 2 " strain micro potato
A.Ac changes Atlantic Ocean B.Ac and changes sweet 2
Figure 16 " Ac changes the Atlantic Ocean " and the performance of " Ac changes sweet 2 " strain land for growing field crops
A.Ac changes Atlantic Ocean B.Ac and changes sweet 2
The potato piece of Figure 17 transgenic strain and check variety relatively
Figure 18 plant expression vector construction schema of the present invention
Embodiment
The clone of embodiment 1. Arabidopis thaliana rd29A promotors
According to (1994) such as Yamaguchi-Shinozaki the report rd29A gene promoter nucleotide sequence (GenBank:D13044 gi:285614) designs and synthesizes following paired primer:
Hind?III
Primer R1:5 '-
AAG CTTAAC GCA TGA TTT GAT GGA GGA-3 '
Bam?HI
Primer R2:5 '-
GGA TCCCTT TCC AAT AGA AGT AAT CAA ACC-3 '
With the total DNA of Arabidopis thaliana is template, by pcr amplification (94 ℃, the pre-sex change of 3min; 94 ℃ of 40sec, 55 ℃ of 40sec, 72 ℃ of 1.5min, 30 amplifications circulate; 72 ℃ of 10min mend flat) obtain the specific fragment (Fig. 1) of about 0.95kb.
Reclaim the specific fragment of 0.95kb with PCR Fragment Recovery Kit, under the effect of T4 dna ligase, the specific fragment that reclaims is connected with pUCm-T Vector, Transformed E .coli DH5 α competent cell, the back 37 ℃ of incubated overnight of coated plate (the LB solid medium that contains 50mg/L Amp).Select the good white single bacterial plaque of isolation and shake bacterium.Collect thalline, adopt the alkaline lysis trace to extract plasmid.Cut (Fig. 2) and PCR evaluation (Fig. 3) by the screening of hysteresis plasmid, enzyme, the recon of screening submits to Shanghai United Gene Science Co., Ltd to check order, and sequencing result is SEQ ID NO 1.With the rd29A gene promoter sequence (D13044 among sequencing result SEQ ID NO1 and the GenBank, gi:285614) carry out homology relatively, identical rate reaches 98.12%, it mainly regulates and control the zone as TATA box and low temperature response element (lowtemperature response element, LTRE) any change does not take place, conclusive evidence obtains the rd29A gene promoter.With this recombinant plasmid called after pUCRD (Fig. 4).
The GFP gene plant expression vector establishment of embodiment 2. Arabidopis thaliana rd29A promoters driven and rd29A promoter activity detect
Green fluorescent protein (green-fluorescent protein, GFP) be the peculiar bioluminescence fibroins of some coelenteratess, the expression study of its gene in the allos cell shows: under blue-light excited, GFP in the transformant can both send green glow, the formation of i.e. emission group does not have species specificity, does not need special cofactor yet.Various biologies have been widely used in based on these characteristics GFP gene as novel markings or reporter gene.In our research, adopt the transient expression level detection of GFP gene, to determine the activity of rd29A promotor.
1. the GFP gene plant expression vector establishment of Arabidopis thaliana rd29A promoters driven
Plant expression vector pBIG (Fig. 5) (CaMV 35S+GFP gene) with we have made up with Hind III and Bam HI double digestion, reclaims big fragment; Cut pUCRD with two same restriction enzyme, reclaim small segment; Two fragments that reclaim are mixed, connect with the T4 dna ligase; Connect product Transformed E .coli DH5 α competent cell, the back 37 ℃ of incubated overnight of coated plate (the LB solid medium that contains Amp); Select white single bacterial plaque and shake bacterium, adopt the alkaline lysis trace to extract plasmid; By hysteresis plasmid screening, enzyme is cut and the PCR evaluation and screening goes out recon, with this recombinant plasmid called after pBIRG (Fig. 6).Because this plasmid is with the CaMV35S on the rd29A gene promoter replacement pBIG, is the connection of double digestion product, can reach directed purpose of connecting, makes the rd29A promotor be positioned at the upstream of GFP gene in addition.So this carrier is the GFP gene plant expression vector of rd29A promoters driven.So far, obtained to have the GFP gene two kind of plant expression vectors (pBIG and pBIRG) that the CaMV 35S promoter drives and the rd29A gene promoter drives.The structural representation of these two kinds of carriers is as follows:
2.Rd29A promoter activity detects
Promotor is added in the upstream of reporter gene, and examining report expression of gene level can be used to weigh the activity of promotor then.The transient expression of gene is to detect promotor and the active common method of enhanser.Owing to all have the GFP gene in pBIG that we make up and the pBIRG carrier, detect the fluorescence intensity of GFP, just can determine the activity of promotor.This research is bombarded the onion epidermis tissue with bag by the tungsten powder of pBIG and pBIRG plasmid DNA respectively with particle gun, under fluorescent microscope, observe fluorescence intensity in the processed tissue, in order to the GFP gene expression dose of comparison rd29A promotor and the driving of CaMV 35S promoter, and then the activity of definite rd29A promotor and characteristics.
Before particle gun bombardment onion epidermis tissue, earlier the onion epidermis tissue is carried out sterilization, be inoculated into then and contain permeate agent (0.25mol/L N.F,USP MANNITOL+0.25mol/L sorbyl alcohol, be used for keeping the normal osmotic pressure of cell) MS solid medium (Φ 9cm culture dish), in growth cabinet, the pre-4～6h that cultivates under aseptic, 28 ℃ ± 2 ℃, the low light level (1000lux) condition.To wrap respectively by the pre-incubated onion epidermis tissue of the tungsten powder of pBIG, pBIRG plasmid DNA (Φ 1.2nm, Bio-Rad product) bombardment with particle gun then.Then the onion epidermis tissue after the particle gun bombardment is carried out transition cultivation 3h, the recovery that is beneficial to be bombarded cell with under 28 ℃ ± 2 ℃, the low light level (1000lux) condition.After this, the converting material that transition is cultivated respectively is divided into two batches, and a collection of continuation is cultivated under 28 ℃ ± 2 ℃, the low light level (1000lux) condition, and another batch then places under 4 ℃ ± 2 ℃, the low light level (1000lux) condition and cultivate 10h, observes with fluorescent microscope.
Result according to the observation, rd29A promotor and CaMV 35S promoter drive GFP genetic expression under condition of different temperatures notable difference.Under 28 ± 2 ℃, the low light level (1000lux) condition, (Fig. 7-A) is higher than epidermic cell (Fig. 7-B), illustrate that the activity of CaMV 35S promoter under the normal temperature conditions will be higher than the rd29A promotor that the pBIRG plasmid DNA transforms to the green fluorescence intensity of the onion epidermis cell that the pBIG plasmid DNA transforms; Opposite, under 4 ℃ ± 2 ℃, the low light level (1000lux) condition, in the epidermic cell that the pBIRG plasmid DNA transforms, (Fig. 7-C) obviously is better than the epidermic cell that the pBIG plasmid DNA transforms, and (Fig. 7-D) illustrates that the rd29A promotor is one to be subjected to the promotor of low temperature induction to green fluorescence intensity.
The clone of embodiment 3 potato tuber AcInv genes and the AcInv inverted defined gene plant expression vector construction of rd29A promoters driven
1. the clone of potato tuber AcInv gene
In order to induce the AcInv gene transcription,, extract total RNA then with potato kind " No. 3, agricultural potato " stem tuber storage 1-2 week under 2 ℃ of conditions.With the total RNA of stem tuber is template, Oligo (dT)
15Make primer, synthetic cDNA first chain under AMV Reverase (AMV-RT) effect.
According to known AcInv gene nucleotide series (GenBank:L29099.1, GI:529515) design a pair of special primer:
PAC-1：
Sac?I
5’-
GAG?CTC?ATG?GCC?ACG?CAG?TAC?CAT?TCC?AG-3’
PAC-2：
BamH?I
5’-
GGA?TCC?TTA?CAA?GTC?TTG?CAA?GGG?GAA?GGA?TC-3’
The reverse transcription product dilution is made template for 50 times, add synthetic primer (PAC-1 and PAC-2), dNTP and Taq DNA polymerase, the laggard performing PCR amplification of mixing.The pcr amplification reaction parameter is:
30cycles
Amplification obtains the fragment (Fig. 8) of the about 1.9kb of molecular weight.
The specific band that is about 1.9kb with molecular weight in the PCR Fragment Recovery Kit recovery PCR product.Under the effect of T4 dna ligase, (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product Cat.No.D0211) connects transformed competence colibacillus E.coli DH5 α will to reclaim fragment and pUCm-T Vector.Select white single bacterial plaque, shake bacterium, adopt the alkaline lysis trace to extract plasmid.By hysteresis plasmid screening, enzyme is cut and PCR identifies, tentatively is defined as recon, submits " Bioisystech Co., Ltd is contained in match north " order-checking to.Sequencing result is shown in SEQ ID NO 2, (GenBank:L29099.1 GI:529515) carries out homology relatively, and homology reaches 98.96% with AcInv gene order among the GenBank, conclusive evidence obtains the AcInv gene, with this recon called after pGEMA (Fig. 9).
2.rd29A the structure of the AcInv inverted defined gene plant expression vector that gene promoter drives
Cultivate pBIRG/E.coli DH5 α and pUCAc/E.coli DH5 α respectively, extract plasmid.In order to replace the GFP gene fragment on the pBIRG with the AcInv cDNA fragment on the pUCAc, guarantee simultaneously AcInv cDNA fragment oppositely is connected after the rd29A promotor, according to the restriction enzyme site comparative analysis on pBIRG and the pUCAc, select for use Sac I and BamH I respectively two kinds of plasmids to be carried out enzyme and cut earlier; From pBIRG plasmid and pUCAc plasmid enzyme restriction product, reclaim 13.05kb fragment and 1.92kb fragment respectively; Two fragments that will reclaim with T4 DNA Ligase connect; Connect product transformed competence colibacillus E.coli DH5 α, be applied to the LB solid culture primary surface that contains Kan (50mg/L), 37 ℃ of incubated overnight; Next day, picking was isolated 8 of good single bacterium colonies, shook bacterium on a small quantity, and the upgrading grain carries out enzyme and cuts evaluation (Figure 10), and one of two fragments that produce after the recombinant plasmid enzyme is cut as a result conform to the molecular size of AcInv gene; Be template with the recombinant plasmid simultaneously, carry out pcr amplification with primer PAC-1 and PAC-2, the fragment that amplification obtains also conforms to the molecular size of AcInv gene, proves that AcInv cDNA oppositely is inserted into rd29A promotor downstream.AcInv inverted defined gene upstream is plant-derived rd29A gene promoter on this plasmid, and therefore, it is the plant expression vector of AcInv inverted defined gene.With this plant expression vector called after pBIRA (Figure 11).
The potato genetic transformation that embodiment 4 is agriculture bacillus mediated and the acquisition of transgenic strain
1.pBIRA importing to agrobacterium tumefaciens
Adopt direct guiding method (work such as Wang Guanlin, the plant genetic engineering philosophy and technique, Beijing, Science Press, 1998:472-473) pBIRA is imported Agrobacterium LBA4404, make the AcInv inverted defined gene of rd29A promoters driven be incorporated into Ti-plasmids, obtain the genetic transformation that the Agrobacterium engineering bacteria is used for potato.Owing to have battery limit (BL), the T-DNA left side and battery limit (BL), the right (the AcInv inverted defined gene is expressed unit and unitary outer survey of npt-II genetic expression) on the pBIRA, after in case pBIRA imports Agrobacterium, just can with the T-DNA homology segment generation double exchange on the Ti-plasmids, section displacement between battery limit (BL), the T-DNA left side and battery limit (BL), the right is incorporated on the Agrobacterium Ti-plasmids, and makes Ti-plasmids become cointegrates.Its concrete operation method is: the pBIRA/LBA4404 Agrobacterium that transforms is applied to contains kantlex (Kan, 100 μ g/ml)+Streptomycin sulphate (Str, 25 μ g/ml)+Li Fuping (Rif, 25 μ g/ml) on the YEP solid medium, cultivate 14～18h for 28 ℃, picking is separated good several single bacterium colony at random, extracts Ti-plasmids.Do template with the Ti-plasmids that extracts, special primer during with clone AcInv gene carries out pcr amplification, the result has all amplified the dna molecular fragment (Figure 12 swimming lane 2-5) of 1.92kb from four single bacterium colony Ti-plasmids templates selecting, illustrate to transform and succeed, the Agrobacterium engineering bacteria called after LBRA after this conversion is identified.
2. the foundation of agriculture bacillus mediated Transformation of potato system
By to nutrient media components, acceptor Kan susceptibility, different acceptor material, pre-incubation time, Agrobacterium concentration, immerged time, incubation time and the research that prevents the aspects such as brownization of transformation receptor material altogether, tentatively set up the better conversion system.The main contents of this transformation system have:
1. the substratum of substratum test-tube plantlet stem section and blade conversion adopts 1/2MS, and hormone concentration and proportioning see Table 1.Potato chips cultivate that institute adds hormone kind and working concentration has certain difference with test-tube plantlet stem section and the cultivation of leaf dish, also need increase the organism supplementary component, comprise kinetin (KT), folic acid, vitamin H, 2,4-D and caseinhydrolysate etc.
2. acceptor material be suitable for as converting material be test-tube plantlet stem section, the ability of its callus induction and differentiation strong (Figure 13-A, D and F); Next is potato chips (Figure 13-B and E), and wherein (Figure 13-B) is better than land for growing field crops potato piece potato chips to the micro potato potato chips, the poorest (Figure 13-C) of leaf dish.
3. the working concentration stem section 75mg/L of Kan, potato chips 75mg/L, leaf dish are 25mg/L.
4. pre-incubation time is cultivated 2d in advance with the stem section, and it is the highest that potato chips are cultivated the 3d transformation efficiency in advance.
5. Agrobacterium concentration is that 0.5 concentration ratio is more suitable with OD600, immerged time with the stem section infect 10min, leaf dish 5min, potato chips 3min effect is better.
6. incubation time stem section is cultivated 1d altogether with 2d, leaf dish 3d, potato chips altogether.
7. inductor is coated with proline(Pro) with acetyl butyryl ketone (AS) and 1 μ mol/L at the solid culture primary surface, and conversion is had obvious facilitation.
8. the prevention of brownization of converting material prevents brownization of converting material, and is very important to improving transformation efficiency.Cultivation stage (comprise pre-cultivation, be total to and cultivate and select to cultivate initial stage) places the dark place can effectively prevent brownization in the early stage.If in substratum, add 2%PVP or caseinhydrolysate, prevent that then the effect of brownization is better.
Make the AcInv inverted defined gene of agriculture bacillus mediated rd29A promoters driven transform the stem section with above transformation system, its transformation efficiency has on average reached 0.501%, the Transformation of potato rate (0.1%) that ratio is reported at present [work such as Wang Guanlin. plant genetic engineering philosophy and technique (second edition). Beijing: Science Press, 2002:389-402] improved 5 times.
Adopt agrobacterium-mediated transformation, potato kind " No. 1, sweet agricultural potato ", " No. 2, sweet agricultural potato ", " No. 3, agricultural potato ", " Favorita ", " platform is red ", " Atlantic Ocean ", " Novaly ", " the sweet SK13 of academy of agricultural sciences " and 9 strains such as " the sweet 92-24-114 of academy of agricultural sciences " of cultivating in the present production have been carried out genetic transformation, from " Favorita ", " platform is red ", " Atlantic Ocean " and " the sweet SK13 of academy of agricultural sciences strain " and five kinds such as " No. 2, sweet agricultural potatos " obtain transgenic seedling.Through Southern hybridization [work such as Wang Guanlin. plant genetic engineering philosophy and technique (second edition). Beijing: Science Press, 2002:844-854] detect, conversion seedling from " Atlantic Ocean " and " No. 2, sweet agricultural potato " is positive (Figure 14), with this transformation plant called after " Ac changes the Atlantic Ocean " and " Ac changes sweet 2 ".
Transgenic strain " Ac changes the Atlantic Ocean " and " Ac changes sweet 2 " are carried out tissue rapid propagation, be planted in the greenhouse vermiculite, the periodically sprinkle nutritive medium is gathered in the crops micro potato (Figure 15), as the land for growing field crops potato seed behind growth 60d.Micro potato is planted in the Tianzhuzangzu Autonomous County, Gansu Province in 2003 and has carried out strain evaluation (Figure 16).From two transgenic strains in the field growing situation, its plant type, blade profile do not change, but compare with original kind, its growth potential is more prosperous, and the leaf look more dark green, and it is big that blade becomes, potato type, pattern produce variation, change Atlantic Ocean strain system as Ac, its pattern is that lavender (Atlantic Ocean kind is for spending in vain), potato type become oblong, bud eyebrow obviously elongated and slightly outstanding (Figure 17).
After the ripe stem tuber results, from each transgenic strain and corresponding check variety, get 8 stem tubers at random, clean peeling, respectively take by weighing 1g potato meat, immediately with liquid nitrogen flash freezer, grind to form powdery, forward a 2ml centrifuge tube to, (contain 10mmol/L Hepes-KOH, pH7.4) 80 ℃ are extracted 1～2h to add 1ml 80% ethanol, then according to described methods such as Stitt (Methods Enzymol, 1989,174:518-552), get supernatant liquor and measure reducing sugar (glucose, fructose) and sucrose content.Last precipitation is carried out drying with after twice of the cold wash, provides method according to Chinese analyzing food nutrition components of mirowave inspection center then---enzyme hydrolysis method (
Http:// www.fh21.com.cn) the mensuration starch content.Dry matter content is measured and is adopted the oven dry weighting method.
Transgenic strain and check clone stem tuber dry matter content, starch content and reducing sugar content measurement result are shown: the dry matter content of check variety " Atlantic Ocean " stem tuber is 20.19%, and starch content is 17.7%, and reducing sugar content is 0.49%; The dry matter content of transgenic strain " Ac changes the Atlantic Ocean " stem tuber is 24.53%, and starch content is 17.9%, and reducing sugar content is 0.08%; Its reducing sugar content comparison is according to having reduced by 83.7%.The dry matter content of check variety " No. 2, sweet agricultural potato " stem tuber is 26.5%, and starch content is 19.6%, and reducing sugar content is 1.09%; Transgenic strain " Ac changes sweet 2 " strain stem tuber dry matter content is 27.93%, and starch content is 21.30%, and reducing sugar content is 0.44%; Its reducing sugar content comparison is according to having reduced by 59.6%.
Select " Ac changes the Atlantic Ocean ", " Ac changes sweet 2 " that is of moderate size, the potato piece in " Atlantic Ocean " and " No. 2, sweet agricultural potato ", be housed in 6 weeks under low temperature (4 ± 2 ℃) condition, then the reducing sugar content of stem tuber is measured." Atlantic Ocean " stem tuber reducing sugar content after the refrigeration is 0.92%, and " Ac changes the Atlantic Ocean " is 0.12%, and " No. 2, sweet agricultural potato " is 2.35%, and " Ac changes sweet 2 " is 0.52%.Compare with the stem tuber reducing sugar content before the low temperature storage, the reducing sugar content of transgenic strain has increased by 18%～50%, and the reducing sugar content of check variety has increased by 1 times; The reducing sugar content of " Ac changes the Atlantic Ocean " stem tuber is lower by 86.9% than " Atlantic Ocean ", and " Ac changes sweet 2 " is than " No. 2, sweet agricultural potato " low 77.8%.Illustrate that it is an effective way of cultivating the anti-low temperature saccharification strain of stem tuber that the AcInv inverted defined gene plant expression vector that adopts the low temperature induction promoters driven transforms the potato kind.
Three kinds of hormone concentrations that table 1 test-tube plantlet stem section, leaf dish are cultivated and proportioning optimizing result
The differentiation of explant tethelin (mg/L) callus of induce root differentiation bud
NAA 0.2 0.4 0
6-BA 1.125 0 3.4
The stem section
GA
3 0 0 4.5
NAA∶6-BA∶GA
3 1∶5∶0 1∶0∶0 0∶1∶1.2
NAA 0.3 0.4 -
6-BA 2.25 0 -
The leaf dish
GA
3 0 0 -
NAA∶6-BA∶GA
3 1∶6∶0 1∶0∶0 -
The information of SEQ ID NO 1
Sequence signature:
(A) (length): 958 bases
(B) type: nucleic acid
(C) chain: two strands
(D) (topological framework): linearity
Sequence description: SEQ ID NO 1
Hind?III
1
AAGCTTAACG?CATGATTTGA?TGGAGGAGCC?ATAGATGCAA?TTCAATCAAA
51 CTGAAATTTC TGCAAGAATC?TCAAACACGG?AGATCTCAAA?GTTTGAAAGA
101 AAATTTATTT CTTCGATTCA?AAACAAACTT?ACGAAATTTA?GGTAGAACTT
151 ATATACATTA TATGTGTAAT?TTTTTGTAAC?AAAATGTTTT?TATTATTATT
201 ATAGAATTTT ACTGGTTAAA?TTAAAAATGA?ATAGAAAAGG?TGAATTAAGA
251 GGAGAGAGGA GGTAAACATT?TTCTTCTATT?TTTTCATATT?TTCAGGATAA
301 ATTATTGTAG AAGTTTAAAA?GATTTCCATT?TGACTAGTGT?AAATGAGGAA
351 TATTCTCTAG TAAGATCATT?ATTTCATCTA?CTTCTTTTAT?CTTCTACCAG
401 TAGAGGAATA AACAATATTT?AGCTCCTTTG?TAAATACAAA?TTAATTTTCG
451 TTCTTGACAT CATTCAATTT?TAATTTTACG?TATAAAATAA?AAGATCATAC
501 CTATTAGAAC GATTAAGGAG?AAATACAATT?CGAATGAGAA?GGATGTGCCG
551 TTTGTTATAA TAAACAGCCA?CACGACGTAA?ACGTAAAATG?ACCACATGAT
601 GGGCCAATAG ACATGGACCG?ACTACTAATA?ATAGTAAGTT?ACATTTTAGG
High salt/dehydration/low temperature/ABA response element 1
651 ATGGAATAAA?TATCA
CAGTTT?GAAAGAAAAG?GGAAAAAAAG
High salt/dehydration/low temperature/ABA response element 2
701 AAAAAATAAA?TAAAAGATAT?AC
GAGTTCCAA?AAAGCAAAAA
751 AAAAGATCAA?GCCGATACAG?ACACGCGTAG?AGAGCAAAAT?GACTTTGACG
801 TCACACCACG?AAAACAGACG?CTTCATACGT?GTCCCTTTAT?CTCTCTCAGT
TATA?box
851 CTCTC
CTTAGTGAG?ACCCTCCTCT?GTTTTACTCA?CAAATATGCA
901 AACTAGAAAA?CAATCATCAG?GAATAAAGGG?TTTGATTACT?TCTATTGGAA
BamH?I
951 AG
GGATCC
The information of SEQ ID NO 2
Sequence signature:
(A) (length): 1920 bases
(B) type: nucleic acid
(C) chain: two strands
(D) (topological framework): linearity
Sequence description: SEQ ID NO 2
1 ATGGCCACGC?AGTACCATTC?CAGTTATGAC?CTGGAAAACT?CCGCCTCCCA
51 TTACACATTC?CTCCCGGATC?AACCTGATTC?CGGCCACCGG?AAGTCCCTTA
101 AAATCATCTC?CGGCATTTTC?CTCTCCTCTT?TCCTTTTGCT?TTCTGTAGCC
151 TTCTTTCCGA?TCCTCAACAA?CCAATCACCG?GACTTGCAGA?GTAACTCCCG
201 TTCGCCGGCG?CCGCCGTCAA?GAGGTGTTTC?TCAGGGAGTC?TCCGATAAGA
251 CTTTTCGAGA?TGTCGTCAAT?GCTAGTCACG?TTTCTTATGC?GTGGTCCAAT
301 GCTATGCTTA?GCTGGCAAAG?AACTGCTTAC?CATTTTCAAC?CTCAAAAAAA
351 TTGGATGAAC?GATCCTAATG?GTCCATTGTA?CCACAAGGGA?TGGTATCATC
401 TTTTTTATCA?ATACAATCCA?GATTCAGCTA?TTTGGGGAAA?TATCACATGG
451 GGCCATGCCG?TATCCAAGGA?CTTGATCCAC?TGGCTCTACT?TGCCTTTTGC
501 CATGGTTCCT?GATCAATGGT?ACGATATAAA?CGGTGTCTGG?ACTGGGTCCG
551 CTACCATCCT?ACCCGATGGT?CAGATCATGA?TGCTTTATAC?CGGTGACACT
601 GATGATTATG?TACAAGTGCG?AAATCTTGCG?TACCCCACCA?ACTTATCTGA
651 TCCTCTCCTT?CTAGACTGGG?TCAAGTACAA?AGGCAACCCG?GTTCTGGTTC
701 CTCCACCCGG?CATTGGTGTC?AAGGACTTTA?GAGACCCGAC?CACTGCTTGG
751 ACCGGACCCC AAAATGGGCA?ATGGCTTTTA?ACAATCGGGT?CTAAGACTGG
801 TAAAACGGGT ATTGCACTTG?TTTATGAAAC?TTCCAACTTC?ACAAGCTTTA
851 AGCTATTGGA TGAAGTGCTG?CATGCGGTTC?CGGGTACGGG?TATGTGGGAG
901 TGTGTGGACT TTTACCCGGT?ATCGACTGAA?AAAACAAACG?GGTTGGACAC
951 ATCATATAAC GGCCCGGGTG?TAAAGCATGT?GTTAAAAGCA?AGTTTAGATG
1001 ACAATAAGCA AGATCACTAT?GCTATTGGGA?CGTATGACTT?GACAAAGAAC
1051 AAATGGACAC CCGATAACCC?GGAATTGGAT?TGTGGAATTG?GGTTGAAGCT
1101 GGATTATGGG AAATATTATG?CATCAAAGAC?ATTTTATGAC?CCGAAGAAAC
1151 AACGAAGAGT ACTGTGGGGA?TGGATTGGGG?AAACTGATAG?TGAATCTGCT
1201 GACCTGCAGA AGGGATGGGC?ATCTGTACAG?AGTATTCCAA?GGACAGTGCT
1251 TTACGACAAG AAGACAGGGA?CACATCTACT?TCAGTGGCCA?GTTGAAGAAA
1301 TTGAAAGCTT AAGAGCGGGT?GATCCTATTG?TTAAGCAAGT?CAATCTTCAA
1351 CCAGGTTCAA TTGAGCTACT?CCATGTTGAC?TCAGCTGCAG?AGTTGGATAT
1401 AGAAGCCTCA TTTGAAGTGG?ACAAAGTCGC?GCTCCAGGGA?ATAATTGAAG
1451 CAGATCATGT AGGTTTCAGC?TGCTCTACTA?GTGGAGGTGC?TGCTAGCAGA
1501 GGCATTTTGG GACCATTTGG?TGTCGTTGTA?ATTGCTGATC?AAACGCTATC
1551 TGAGCTAACG CCAGTTTACT?TCTACATTTC?TAAAGGAGCT?GATGGCCGAG
1601 CTGAGACTCA CTTCTGTGCT?GATCAAACCA?GATCCTCAGA?GGCTCCGGGA
1651 GTTGCTAAAC AAGTTTATGG?TAGTTCAGTA?CCCGTGTTGG?ACGGTGAAAA
1701 ACATTCGATG AGATTATTGG?TGGACCACTC?AATTATGGAG?AGCTTTGCTC
1751 AAGGAGGAAG AACAGTCATA?ACATCGCGAA?TTTACCCAAC?AAAGGCAGTG
1801 AATGGAGCAG CACGACTCTT?CGTTTTCAAC?AATGCCACAG?GGTCTAGCGT
1851 GACTGCCTCC GTCAAGATTT?GGTCACTTGA?GTCGGCTAAT?ATTCAATCCT
1901 TCCCCTTGCA AGACTTGTAA
Claims (7)
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| CN 200410068868 CN1618976A (en) | 2004-07-13 | 2004-07-13 | Method for cultivating low-temperature saccharification resistant potato strains by introducing AcInv antisense gene |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100199386A1 (en) * | 2009-02-03 | 2010-08-05 | Wisconsin Alumni Research Foundation | Control of cold-induced sweetening and reduction of acrylamide levels in potato or sweet potato |
| CN104919047A (en) * | 2012-11-09 | 2015-09-16 | 杰.尔.辛普洛公司 | Use of invertase silencing in potato to minimize losses from zebra chip and sugar ends |
| US10513698B2 (en) | 2012-12-21 | 2019-12-24 | Cellectis | Potatoes with reduced cold-induced sweetening |
| CN112725367A (en) * | 2021-03-01 | 2021-04-30 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato sucrose invertase gene IbINV and application thereof |
| CN114829587A (en) * | 2019-10-01 | 2022-07-29 | 菲利普莫里斯生产公司 | Regulating reducing sugar content (INV) in plants |
-
2004
- 2004-07-13 CN CN 200410068868 patent/CN1618976A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100199386A1 (en) * | 2009-02-03 | 2010-08-05 | Wisconsin Alumni Research Foundation | Control of cold-induced sweetening and reduction of acrylamide levels in potato or sweet potato |
| US20160194648A1 (en) * | 2009-02-03 | 2016-07-07 | Wisconsin Alumni Research Foundation | Control of cold-induced sweetening and reduction of acrylamide levels in potato or sweet potato |
| CN104919047A (en) * | 2012-11-09 | 2015-09-16 | 杰.尔.辛普洛公司 | Use of invertase silencing in potato to minimize losses from zebra chip and sugar ends |
| US10513698B2 (en) | 2012-12-21 | 2019-12-24 | Cellectis | Potatoes with reduced cold-induced sweetening |
| CN114829587A (en) * | 2019-10-01 | 2022-07-29 | 菲利普莫里斯生产公司 | Regulating reducing sugar content (INV) in plants |
| CN112725367A (en) * | 2021-03-01 | 2021-04-30 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato sucrose invertase gene IbINV and application thereof |
| CN112725367B (en) * | 2021-03-01 | 2022-07-15 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato sucrose invertase gene IbINV and its application |
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