CN100374567C - Process for culturing brownness resistant sweet potatoes utilizing gene engineering technology - Google Patents
Process for culturing brownness resistant sweet potatoes utilizing gene engineering technology Download PDFInfo
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- CN100374567C CN100374567C CNB2005100570701A CN200510057070A CN100374567C CN 100374567 C CN100374567 C CN 100374567C CN B2005100570701 A CNB2005100570701 A CN B2005100570701A CN 200510057070 A CN200510057070 A CN 200510057070A CN 100374567 C CN100374567 C CN 100374567C
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- sweet potato
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- polyphenol oxidase
- polyphenoloxidase
- brownness
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Abstract
The present invention provides a method for culturing brownness resisting sweet potatoes by utilizing a gene engineering technology; the present invention relates to a method of obtaining the key segment of a polyphenol oxidase gene of sweet potatoes and culturing brownness resisting transgenic sweet potatoes by utilizing an antisense RNA technology. The present invention clones the key segment of the polyphenol oxidase gene from the sweet potatoes and establishes a high-efficiency antisense expressing carrier of plants; the expressing carrier is led to agrobacteria and genetically transforms the sweet potatoes. The method can reduce the expression level of the polyphenol oxidase gene in the tuberous roots of the sweet potatoes and improve the quality of the sweet potatoes, and the processed products of the sweet potatoes.
Description
Technical field
The invention belongs to fields such as molecular biology, zymetology, physiology, thremmatology and genetically engineered, relate to a kind of method of utilizing genetic engineering technique to cultivate brownness resistant sweet potatoes, be specifically related to sweet potato polyphenol oxidase genes cDNA and antisense strand sequence thereof, and the efficient expression vector that utilize to make up and engineering strain are expressed the specific procedure of this gene and inverted defined gene, screening transformant in plant.The present invention also provides commentaries on classics polyphenoloxidase and the cell of antisense polyphenoloxidase and filial generation, regeneration plant, plant tissue or the seed of hairly root and cultivation thereof that utilizes genetically engineered to obtain.
Background technology
Polyphenoloxidase is that a class is distributed widely in the plant materials, can catalytic unit phenol, dihydric phenol, polyphenol dehydrogenation reaction to the hydroxylation of xenol and hydroxyl phenol to quinone, its special catalysis is the basic reason that causes plant brown stain, reduction quality.It and sweet potato process for processing and transportation are closely related.Sweet potato has the characteristics of food crop and cash crop concurrently, starch content height in the sweet potato root tuber, and its starch can be used as the industrial raw material of products such as bean vermicelli, modified starch, Dian Fentang and citric acid.But the utilization of China sweet potato at present is still based on human consumption or cook cattle food, and the deep processing of sweet potato is restricted, and wherein a Guan Jian problem is exactly that sweet potato starch is compared with W-Gum, yam starch, the poor quality of sweet potato starch, and whiteness is low.Major cause is: a spot of pectin, tannin, polyphenols are after the sweet potato fragmentation in the sweet potato, be subjected to polyphenoloxidase (polyphenol oxidase, PPO) effect forms melanochrome, is adsorbed on the starch granules, and this is the major cause that causes the sweet potato starch whiteness low; The oxidation products of polyphenoloxidase also can produce bad local flavor simultaneously, reduces the sweet potato starch product quality.
In order to prevent brownization of sweet potato, the multiple ordinary method of utilization reduces polyphenol oxidase activity and content in the sweet potato in the production, for example, with xitix, sodium sulfate, citric acid etc. is the composite color fixative of host, scald with boiling water, bagging etc., but these all can not tackle the problem at its root.
By contrast, utilize genetic engineering technique improvement sweet potato, the sweet potato of cultivating anti-brownization just has very fiery advantage: (1) is in the biotechnology research field, the researchist is to the long-term extensive and deep research of sweet potato isolated culture and the success repeatedly of sweet potato genetic transformation, for the transgenosis sweet potato that utilizes biotechnology to cultivate anti-brownization has been established good theory and practice basis; (2) owing to illustrated the brown stain mechanism of sweet potato at present substantially, a plurality of species polyphenol oxidase genes have been cloned, utilize Antisense RNA Technique to obtain the transgenic Rhizoma Solani tuber osi of anti-brownization, so utilize genetic engineering technique on gene level, to improve sweet potato fast and efficiently, obtaining genetic modification transgenosis sweet potato (comprising seedling, clone etc.) and other life entities becomes possibility, thereby to utilize Antisense RNA Technique be to cultivate the optimal path that resists brownization transgenosis sweet potato.
At present, there is not using gene engineering technique mentioned in any and the present patent application to cultivate the relevant report of the transgenosis sweet potato of anti-brownization as yet, comprises patent and document.
Summary of the invention
First purpose of the present invention just provides a kind of method of utilizing genetic engineering technique to cultivate brownness resistant sweet potatoes, this method with multiple transfer antisense polyphenol oxidase genes core segment in sweet potato.
In another aspect of this invention, also provide a kind of antisense expression vector, it comprises above-mentioned polyphenol oxidase genes core segment.
In another aspect of this invention, also provide a kind of usefulness above-mentioned antisense expression vector transformed host cells.This host cell is a sweet potato in example.
Technical scheme of the present invention is as follows:
The isolated dna molecular of the present invention has the core fragment of the nucleotide sequence of coding sweet potato polyphenoloxidase.This core fragment derives from the nucleotide sequence of the sweet potato polyphenol oxidase genes of being logined by the applicant (AY822711) in GenBank, use primer P1:5 '-cgagctctatgttcagaccgattacccc-3 ' and P2:5 '-cggatccatggtccacattcggtcgac-3 ' can amplify this core fragment from sweet potato, sequence is seen SEQ ID NO:1.
A kind of method of utilizing Antisense RNA Technique to reduce sweet potato polyphenoloxidase content, be characterised in that the antisense expression vector that utilizes nucleotide sequence core fragment, adopt any transgenic method antisense expression in sweet potato cell, tissue, organ, plant with coding sweet potato polyphenoloxidase:
Its step is as follows:
(1) adopts any possible core segment that obtains the sweet potato polyphenol oxidase genes in section (as methods such as gene clone, population's synthetic genes);
(2) polyphenol oxidase genes core segment operationally is connected in expression regulation sequence, forms antisense expression vector;
(3) adopt any transgenic method to shift polyphenol oxidase genes core segment in sweet potato cell, tissue, organ, plant;
(4) screen and identify transformant under given conditions;
(5) transformant of cultivating under the condition that is fit to obtains transgenic progeny.
The carrier that the present invention relates to comprises the DNA of the core fragment of the nucleotide sequence with coding sweet potato polyphenoloxidase.
With the life entity that aforesaid method obtains, it is to produce cell, tissue, organ, the plant that polyphenoloxidase content reduces sweet potato.
In the present invention, can select various carrier known in the art for use, the carrier as commercially available comprises plasmid etc.In the present invention, term " life entity " refers to cell, tissue, organ, the plant of sweet potato.
In the present invention, term " any possible means " specifically comprises homology clone (as RT-PCR, RACE etc.), library screening then synthetic sweet potato polyphenol oxidase genes and varient thereof for obtaining the method for sweet potato polyphenol oxidase genes.
In the present invention, term " any transgenic method " comprises that the conversion of agrobacterium tumefaciens Ti-plasmids mediated gene, the plasmid-mediated gene transformation of Agrobacterium rhizogenes Ri, plant viral vector mediated gene transform, transform as the conversion of PEG mediated gene, liposome-mediated gene transformation, the conversion of electric shocking method mediated gene, ultrasonic-mediated gene transformation, the conversion of microinjection mediated gene, the conversion of laser microbeam mediated gene, the conversion of particle bombardment mediated gene, the conversion of pollen tube channel mediated gene, sexual cell infusion method mediated gene.
In the present invention, term " screen under given conditions and identify transformant " is meant the transformant of selecting antibiotics resistance under the condition that is used in isolated culture with microbiotic (kantlex, Totomycin, G418 etc.); Can use methods such as PCR, Southern hybridization, Northern hybridization and Western trace to identify transformant.
In the present invention, term " is cultivated the transformant that the sweet potato polyphenol oxidase activity reduces; obtain transgenic progeny " and is meant the transformant isolated culture through identifying under the condition that is fit to, and detect polyphenoloxidase content with active, the low good transformant of screening polyphenol oxidase activity is cultivated, and obtains transgenic progeny.
In the present invention, we clone polyphenol oxidase genes core segment from sweet potato, and have made up the plant efficient antisense expression vector, import Agrobacterium and genetic transformation sweet potato, to reduce the expression level of polyphenol oxidase genes in the sweet potato root tuber, improved the quality of sweet potato and converted products thereof.
Embodiment
In conjunction with specific embodiments, further set forth the present invention down.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, condition described in for example " molecular cloning " (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Embodiment 1
The pulsating synthetic structure that reaches binary expression vector and rules bacterium of sweet potato polyphenol oxidase genes core
1. the acquisition of antisense polyphenol oxidase genes core fragment
Full length sequence according to polyphenol oxidase genes; between 488 bases of the 1st base to the in the inside of polyphenol oxidase genes, design primer; amplification 502bp sequence is as the inverted defined gene fragment; and according to the polyclone restriction enzyme site of plant binary carrier pCAMB1A1304; design two pcr amplification primers that contain SacI and BamHI restriction endonuclease sites and protection base respectively, its primer sequence is:
P1：5’-cgagctctatgttcagaccgattacccc-3’
P2：5’-cggatccatggtccacattcggtcgac-3’
Carry total RNA (RNA of Shanghai China Shun biotechnology company limited extracts test kit) from the stem tuber of sweet potato, reverse transcription becomes cDNA (TaKaRa RNA PCR Kit), carries out pcr amplification then; the PCR reaction system is 50ul, comprises deionized water, 10 * PCR buffer 5ul; dNTP 1ul, MgCl
23ul, each 1ul of primer, cDNA touches plate 1ul, Taq enzyme 0.5ul.The PCR condition be 94 ℃ 3 minutes, carried out 29 circulations in 1 minute with 94 ℃ 45 seconds, 58 ℃ 45 seconds and 72 ℃ thereupon, extended 8 minutes with 72 ℃ at last.1% agarose electrophoresis detects pcr amplification product, and the acquisition expanding fragment length is 502bp.
After the electrophoretic separation, reclaim (match hundred victory PCR products and reclaim test kit) PCR product, get and change DH5 α over to an amount of recovery product is connected (TaKaRa PMD 18-T Vector test kit) with PMD18-T easy Vector carrier after, resistance screening positive colony on LB+ penbritin (100mg/l) solid medium, PCR send order-checking (the hundred victory companies that match finish) after identifying.
2.CaMV35S constitutive promoter expression vector pCAMBIA1304
+The structure of-ppoF.
Selecting pBI121 and pCAMBIA1304 for use is primary element, makes up double base trivalent plant expression vector pCAMBIA1304
+(p1304
+).The structure flow process is as follows: 1) HindIII and EcoRI double digestion pBI121 and pCAMBIA1304; Reclaim the pBI121 expression cassette, reclaim the big fragment of pCAMBIA1304; Connect these two and reclaim product, transform DH5 α, screening obtains mono-clonal on kantlex LB flat board, shakes the bacterium enlarged culturing, extracting plasmid, HindIII and the checking of EcoRI double digestion; Promptly build pCAMBIA1304
+
To shake bacterium through the DH5 α (having changed the polyphenol oxidase genes core fragment over to) after the order-checking correctly, enlarged culturing is extracted plasmid, behind BamHI and sacI double digestion, reclaims small segment after 1% agarose electrophoresis.
To build pCAMBIA1304
+After the polyphenol oxidase genes core fragment is connected with the T4 ligase enzyme, transform DH5 α competent cell, screen resistance clone on LB+Kan (100mg/l) solid medium, PCR and double digestion are identified.Obtain to change the recombinant plasmid and the called after pCAMBIA1304 of polyphenoloxidase core fragment
+-ppoF.
3.pCAMBIA1304
+-ppoF transforms Agrobacterium LBA4404
With pCAMBIA1304
+-ppoF is transformed into agrobacterium strains LBA4404, draws the positive single bacterium colony of plate picking and shakes bacterium, and the bacterium liquid extracting plasmid that takes a morsel carries out PCR and enzyme and cuts evaluation.Positive colony is defined as LBA4404-ppoF.
Embodiment 2
Obtain the active anti-brownization transgenosis sweet potato that reduces of PPO
1, Agrobacterium LBA4404-ppoF.Take out from refrigerator before using, be inoculated in 50ml YEB liquid (Rif
+, Str
+, Kan
+), 28 degree, twice of 200rpm shaking culture;
2, activate OD for the second time
600Reach at 0.3 o'clock and add 100 μ mol/mL Syringylethanones, continue 28 degree, 200rpm shaking culture, OD
600Reach the centrifugal 10min of 4000r under 0.6 o'clock room temperature;
3, abandon supernatant, thalline suspends with MS (100 μ mol/mL Syringylethanone) liquid nutrient medium, is diluted to 5-20 times of original volume, makes the OD of bacterium liquid
600About=0.3; Claim conversion fluid;
4, get plant different sites such as aseptic sweet potato terminal bud, lateral bud, leaf, stem, root, stem is cut into the 1cm segment, or blade is cut into about 1cm2, draw with "+" font wound with aseptic scalper, put into above-mentioned conversion fluid, take out after infecting 2-10min, blot with aseptic toilet paper, insert the MS+1mg/LNAA of 100 μ mol/mL Syringylethanones, cultivated altogether in the substratum 2 days and under illumination condition, carry out common cultivation, both prevent the hypertrophy of agrobacterium tumefaciens effectively, helped surviving of stem explants and sprouting and growth of resistant buds again.Be transferred in the MS+1mg/LNAA substratum that contains 250mg/L cephamycin (Cef), 50mg/L Totomycin (Hygromycin) after cultivation finishes altogether and cultivate, shift 4～5 times till no bacterium, obtain resistant buds;
5, the resistant buds of length more than 1cm is inserted into into seedling substratum (MS+50mg/L Hygormycin+250mg/LCef) and goes up cultivation.Obtain complete hygromycin resistance plant;
6, detect the hygromycin resistance plant with PCR and Southern blotting, detect the expression level of polyphenol oxidase genes with Northern blotting;
7, the transgenosis sweet potato is carried out field test, detect the activity of polyphenoloxidase with the potato piece.Further screen the low transgenosis sweet potato of polyphenol oxidase activity;
8, the mensuration of polyphenol oxidase activity.That polyphenoloxidase in the sweet potato (comprising seedling, hairly root, clone etc.) adopts colorimetric to send out to measure is active (Jiang Shaotong, Luo Zhigang, etc., 2001).
Embodiment 4: transgenosis sweet potato and non-transgenic sweet potato polyphenol oxidase activity compare and anti-brown voltinism can compare
1, the activity that compares transgenosis and non-transgenic sweet potato potato piece polyphenoloxidase, the method (2001) that adopts the logical people of grade of Jiang Shao to set up;
2, the activity 0.09 of polyphenoloxidase in the transgenosis sweet potato root tuber, polyphenol oxidase activity is 0.89 in the non-transgenic sweet potato root tuber;
3, measure the intensity of brownization of sweet potato according to the method (1984) of V.Sciancale and V.Zongne invention, brownization of transgenosis sweet potato root tuber intensity is 0.11, and polyphenol oxidase activity is 0.58 in the non-transgenic sweet potato root tuber.
SEQ?ID?NO：1
＜110〉Southwestern Normal University
＜120〉a kind of method of utilizing genetic engineering technique to cultivate brownness resistant sweet potatoes
<160>1
<210>1
<211>569
<212>DNA
＜213〉sweet potato (Ipomoea batatas)
<220>
<221>misc_feature
<222>(1)...(569)
＜223〉sweet potato polyphenol oxidase genes core segment
<400>1
tatgttcaga?ccgattaccc?cgacaaggaa?atccaagtcc?acaattcatg?gctgtttttc?60
cctttccaca?gatggtactt?atatttctac?gagagaatct?tggggaagct?aatcaacgac?120
cccaccttcg?ggcttccgtt?ctggaactgg?gatacccccg?cgggaatgct?gattcctcag?180
tatttccgaa?accaaaactc?gccgttgtac?gatgagaatc?gtttgcaatc?ccatctccca?240
ctcgttatgg?acctcggcta?cgccgggacc?gacactgacg?tcacggatga?tgagagaatc?300
tctaacaacc?tggccttgat?gtacaagagt?atggtgacta?acgccggaac?cgccgagctt?360
ttcctcggga?aaccgtacaa?agccggcgac?gacccggtca?acaaaggcgg?cgggtcgatc?420
gagaatatcc?cgcatacccc?ggtccaccgc?tgggtcggcg?acgttcaac?cgagaacacaa?480
aatggggagg?atatggggaa?cttttactcg?gccgggcggg?atattttgtt?ttactgtcac?540
cattccaacg?tcgaccgaat?gtggaccat 569
Claims (3)
1. an isolated dna molecular is characterized in that, it is the core fragment of the nucleotide sequence of coding sweet potato polyphenoloxidase, and sequence is SEQ IDNO:1.
2. method of utilizing Antisense RNA Technique to reduce sweet potato polyphenoloxidase content, be characterised in that the antisense expression vector that utilizes nucleotide sequence core fragment, adopt transgenic method antisense expression in sweet potato cell, tissue, organ, plant again with coding sweet potato polyphenoloxidase; The sequence of the nucleotide sequence core fragment of coding sweet potato polyphenoloxidase is SEQ IDNO:1;
Its step is as follows:
(1) adopt gene clone or synthetic gene method to obtain the core segment of sweet potato polyphenol oxidase genes;
(2) the disconnection of polyphenol oxidase genes core sheet in expression regulation sequence, form antisense expression vector;
(3) adopt transgenic method to shift polyphenol oxidase genes core segment in sweet potato cell, tissue, organ, plant;
(4) screening and evaluation transformant;
(5) cultivate transformant, obtain transgenic progeny.
3. a carrier is characterized in that, it comprises the described DNA of claim 1.
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| CNB2005100570701A CN100374567C (en) | 2005-05-18 | 2005-05-18 | Process for culturing brownness resistant sweet potatoes utilizing gene engineering technology |
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| CN100374567C true CN100374567C (en) | 2008-03-12 |
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| CN107653256A (en) * | 2017-11-21 | 2018-02-02 | 云南省烟草农业科学研究院 | A kind of Polyphenol Oxidase in Tobacco gene NtPPO1 and its directed mutagenesis method and application |
| CN112544440A (en) * | 2020-12-23 | 2021-03-26 | 江苏师范大学 | Method for cultivating new transgenic or gene-edited seedlings of root tuber plants |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996037617A1 (en) * | 1995-05-23 | 1996-11-28 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from lettuce and banana |
| WO1998053080A1 (en) * | 1997-05-19 | 1998-11-26 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from banana, tobacco and pineapple |
-
2005
- 2005-05-18 CN CNB2005100570701A patent/CN100374567C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996037617A1 (en) * | 1995-05-23 | 1996-11-28 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from lettuce and banana |
| WO1998053080A1 (en) * | 1997-05-19 | 1998-11-26 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from banana, tobacco and pineapple |
Non-Patent Citations (3)
| Title |
|---|
| Control of Enzymatic Browning in Potato (Solanum tuberosumL.) by Sense and Antisense RNA from Tomato PolyphenolOxidase. Chris Coetzer et al.J. Agric. Food Chem.,Vol.49 No.2. 2001 * |
| Expression of antisense polyphenol oxidase inhibition ofbrowning in transgenic potato. Bachem C W B et al.Bio/Technology,Vol.12 . 1994 * |
| 甘薯多酚氧化酶cDNA 的克隆及序列分析. 彭世清等.农业生物技术学报，第10卷第3期. 2002 * |
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