CN104911180B - Potato class T DNA elements and Cisgenesis system and its application - Google Patents
Potato class T DNA elements and Cisgenesis system and its application Download PDFInfo
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Abstract
The invention discloses 25 potato classes " T DNA " elements and Cisgenesis system and its application.Inventor is studied and explored by lot of experiments, and being finally obtained 25 potato classes, " T DNA " elements, their nucleotides sequence are classified as shown in the sequence 1 to sequence 25 in sequence table.Genetic transformation shows that " the potato Cisgenesis carrier of T DNA " elements and its structure can be applied potato class in the safe transgene genetic breeding of potato, and a kind of unmarked exoskeletal Cisgenesis system is provided for potato class plant.
Description
Technical field
This patent belongs to field of plant genetic.More particularly to class " T-DNA " element in 25 potato sources
With 1 Cisgenesis system that can be applied to potato and the safe transgenosis of other plant and application.Wherein 25 potatos
Class " T-DNA " element in source and the Cisgenesis system thus built are improving all kinds of plant transgene safety such as potato
Property etc. has notable function, can be applied in the safe transgenic field of plant.
Background technology
As population in the world is skyrocketed through, " the first time green revolution " of the 60's of 20th century starting is faced now more than 60
The food problem of hundred million people can't bear the heavy load.Promote meeting in the sections of the U.S. of 2 months 2010（AAAS）In annual meeting, there is scholarly forecast, the whole world
Population will be expanded to 9,000,000,000 in or so the year two thousand fifty.However, while population rapid growth, the cultivable farmland in the whole world,
For the fresh water used and other agricultural resources but without great change.To ensure food security, reducing environmental nuisance, break through and plough
The resource constraints such as ground, water, world food safety and the permanently effective supply of agricultural product are ensured, to lean on scientific and technical innovation after all and answer
With.Transgenic technology research and application are promoted, is to ensure that the important channel of world food safety.
Nevertheless, since the end of last century, the security of genetically modified crops causes striving for fierceness in the world
By.In general, bone of contention is mainly that other species genes that can not determine to be shifted completely are carried on the back in receptor biological heredity
Whole performances in scape, particularly whole show of the gene with function element sequence in plant from microorganism can not be complete
Full prediction, genetically modified organism once enter natural environment, will take root procreation, plus transgenes escape phenomenon etc., once confirmation is deposited
In risk, to be withdrawn from environment will be extremely difficult（The risk and pipe of the genetically modified organisms such as Li Weimin, Yue Ning and products thereof
Manage biotechnologys circular 2000,4: 41-44）.
To improve the security of genetically modified crops, scientists propose a kind of new transgenosis strategy in recent years --- and it is interior
Source transgenosis (Cisgenesis/Intragenisis).It is different according to the source of target gene, transgenosis can be divided into endogenous
Transgenosis (Cisgenesis/Intragenisis) and heterologous transgene (Transgenesis), endogenous transgenosis be utilize by
Body species in itself or its Wild related germplasm gene carry out genetic modification transgenic technology, heterologous transgene then be using difference
The functional gene of species, is conducted into cultivar by transgenosis, is bred as new varieties.Gene used in endogenous transgenosis
I.e. endogenous gene (Cisgene) is already present in acceptor with species or its Wild related germplasm, therefore does not change receptive material
The gene pool of kind.Compared with conventional breeding, the endogenous more conventional breeding of transgenosis can simplify operation, cost-effective, and have no volume
Outer risk, this viewpoint have all reached common recognition all over the world, have included the opinion poll of the country such as New Zealand, North America and Europe
As a result show that endogenous transgenic technology generally believes to be received.Therefore, in being used using plant functional gene inherently
The strategy of source transgenosis carries out genetic improvement to crop, can not only effectively avoid various risks caused by heterologous gene, beat
Disappear doubt of the public to safety of transgenic crops, and simplifies the numerous and diverse field hybridization of conventional breeding, option program, shortens
Breeding cycle, the final efficiency for improving crop breeding.
Potato is the crops of grain, dish, feed and the dual-purpose that processes raw material, and its wide adaptability, yielding ability are good, nutrition is rich
Rich, high financial profit, it has also become the fourth-largest cereal crops after rice, wheat and corn in the world.China is in the world
Potato produces the first big country, and development Potato Industry is for ensureing China's grain security, promoting increasing peasant income and promoting agricultural
It is significant with sustainable development of countryside economy.Cultigen potato（Solanum tuberosumL.）Hereditary basis
It is narrow, it is often necessary to the genetic improvement that disease-resistant, Fineness gene is used for cultivar is introduced from wild species and nearly edge cultigen.But
Potato cultivar is autotetraploid crop, and heredity separation is complicated, and reproduction be present with many wild species and original cultigen
Isolation, therefore, traditional Potato Breeding technology --- crossbreeding technology limits the utilization to these precious resources.With
The progress of modern science and technology interpenetrates with Bu Tong interdisciplinary, and genetic engineering breeding technology has been dissolved into Potato Breeding
In, generate it is large quantities of be difficult to caused breeding material by conventional breeding mode, enrich breeding resources, accelerate breeding and enter
Journey.Particularly nearly coming year more than ten, Chinese scholar have carried out disease-resistant, degeneration-resistant and quality-improving of potato etc. using technique for gene engineering
Many research, certain achievement is had been achieved for, however, the security as described above, the public improves the breed genetic engineering
Worry, significantly limit the commercialization process of transgenic potato.
To solve the potential safety hazard of potato transgenic product, some scholars have started to set about the endogenous transgenosis skill of potato
The research of art, existing report highlights the endogenous of purpose character gene mostly, but the DNA sequence dna of construction of expression vector is still
So microorganism can not be still derived from using the DNA sequence dna of homologous or near edge source of species, many DNA sequence dnas completely, such as：
The T-DNA border sequences in Agrobacterium source etc..Heterologous sequence on transgene carrier can be inserted into plant base with target gene
Because in group, its potential influence is still unable to estimate, and triggers safe worry of the public to transgenic product.In general, at present
Query of the public to Transgene-safty mainly includes two aspects, and one is derived from the purpose character gene phase between different plant species
Issuable threat is mutually converted, second, the allogeneic dna sequence element on transgene carrier（Such as Agrobacterium T-DNA sequence）With
Purpose character gene is inserted into the potential threat in Plant Genome.Clone's research for homologous purpose character gene at present is
Have many but also extremely limited for the safety research of carrier sequence.Therefore, source DNA in safer transgenosis is developed
Sequential element is used for genetic improvement, is the task of top priority of the plant transgenic technology research and development including potato.
In view of importance of the DNA sequence endogeneous element for transgenosis safe, some scholars are in summary class text in the world
Some perspective discussion [Conner A J, Barrell P J, et al.. Erasmuson A have been carried out in chapter to it
K, Nap J P, Jacobs J M. E. Intragenic vectors for gene transfer without foreign DNA. Euphytica. 2007, 154: 341-353；Rommens, C M, Haring M A, et al..
"The intragenic approach as a new extension to traditional plant breeding."
Trends Plant Sci.2007, 12(9):397-403], and Liang Ge research groups internal source DNA sequential element
Some correlative studys are done.[Baldwin, S., the A. such as the Baldwin of research institute of imperial family of New Zealand crop and Food Research Inst.
Lokerse, et al..Intragenic vectors: mining plant EST databases for T-DNA
border-like sequences. Proceedings of the 13th Australasian Plant Breeding
The pp. 946-951 of Conference, Christchurch, New Zealand. 2006] by analyzing agrobatcerium T-DNA member
Part, it is found that the element sequences have two important conserved domains, search plant est database with the two domains respectively
It was found that conserved domain is all widely present in the transcript of most plants, search for, analyze and area of computer aided splicing after obtain
Much " T-DNA " sequences of bar plant origin.Verify that " T-DNA " sequence of which part plant origin has through endogenous conversion
Biological function.The plant " T-DNA " of the researchs such as Baldwin is although sequence is entirely from plant transcription sheet and has biology work(
Can, but because they are to be searched for through computer to splice again respectively by two conserved domains to form, therefore, can they
The endogenous DNA element for being defined as plant origin is still disputable.
[Rommens, C. M., O. Bougri, et the al.. Plant- such as Rommens of Xin Pulao companies of the U.S.
derived transfer DNAs. Plant Physiol 2005, 139 (3):1338-49] then according to agrobatcerium T-DNA
Two conserved domains design degenerate primer, in multiple Plant Genomes such as potato, rice respectively clone obtained number
Bar class " T-DNA " element, is studied through transgene tobacco, it was confirmed that class " T-DNA " element in 5 potato gene group sources has
Originate forwarding function.Transfer efficiency is originated compared to agrobatcerium T-DNA, wherein 4 metastasis sequence starting transfer efficiencies arrive 50%
Between 105%.Thereafter Rommens laboratories continue with these class " T-DNA " elements and construct potato Cisgenesis body again
System, and quality [Chawla, R., the R. such as high acrylamide after low temperature saccharification for transgene improvement potato and frying
Shakya, et al.. Tuber-specific silencing of asparagine synthetase-1 reduces
the acrylamide-forming potential of potatoes grown in the field without
affecting tuber shape and yield. Plant Biotechnol J 2012, 10 (8):913-24；
Rommens, C. M., H. Yan, K. Swords, et al.. Low-acrylamide French fries and
potato chips. Plant Biotechnol J 2008, 6 (8):843-53； Rommens, C. M., C. M.
Richael, et al.. Engineered native pathways for high kaempferol and
caffeoylquinate production in potato. Plant Biotechnol J 2008, 6 (9):870-86；
Rommens, C. M., J. M. Humara, et al.. Crop improvement through modification
of the plant's own genome. Plant Physiol 2004, 135 (1):421-31.]。
In summary, although it is recognized that the identification and utilization of DNA sequence endogeneous element have for transgenosis safe
Make a difference, but the report being related in the world at present is mostly review papers, correlative study class report is less and relatively preliminary,
Only Rommens laboratories are made that certain achievement in research in the field.However, used in the research in Rommens laboratories
Degenerate primer amplification method can not find potato class " T-DNA " element comprehensively, and therefore, the research needs further comprehensively
Expansion.
The content of the invention
With the completion of potato gene group sequencing frame diagram, the present inventor's more adding system, potato is comprehensively analyzed
Class " T-DNA " function element, obtain 25 by Protocols in Molecular Biology separation and transgenosis functional verification and have no report
Potato class " T-DNA " element, and the starting transfer efficiency of wherein 7 elements is have detected, and the termination transfer effect of 25 elements
Rate.These class " T-DNA " elements are also constructed endogenous Transgenics by this final research together with plant anti-salt Gene A tNHX1
System, and endogenous transgenic potato plant is obtained by the system, the system can be applied to safe transgenic research with educating
Kind, certainly will be as the important application platform of the safe transgenosis of plant such as potato.
In consideration of it, first purpose of the present invention is to provide and a series of can carry out the important of potato Cisgenesis
Function element.
To achieve these goals, inventor is studied and explored by lot of experiments, is finally obtained 25 potato classes
" T-DNA " element, their nucleotides sequence are classified as shown in the sequence 1 to sequence 25 in sequence table.
25 above-mentioned potato class " T-DNA " elements of the present invention, wherein the potato class " T- with starting forwarding function
DNA " elements 7, preferably tool starting forwarding function class " T-DNA " element 3, their nucleotides sequence is classified as shown in table 1.
25 above-mentioned potato class " T-DNA " elements of the present invention, wherein with the potato class " T- for terminating forwarding function
DNA " elements 23, preferably tool terminate forwarding function class " T-DNA " element 9, and their nucleotides sequence is classified as shown in table 2.
Potato class " T-DNA " sequence of the preferred tool starting forwarding function of table 1
| Numbering | Sequence information |
| StTL01 | CACTACAACTCAATATATCCTGTAC |
| StTL02 | TAATACTCTGATATATATCCTGCAT |
| StTL03 | AAGTACAGCATCATATATCCTGAGT |
The preferred tool of table 2 terminates potato class " T-DNA " sequence of forwarding function
Second object of the present invention is the provision of the starting transfer table comprising above-mentioned 7 classes " T-DNA " element up to load
Body, wherein preferable expression vector 3；And provide the termination transfer expression vector comprising above-mentioned 23 classes " T-DNA " element；
Wherein preferable expression vector has 9.
The present invention is had found by experimental study, and agrobatcerium T-DNA right border sequence is changed and makees three preferred potato class " T-
One of DNA " elements, it is remarkably improved the transformation efficiency of plant transgene；It is excellent that agrobatcerium T-DNA left margin sequence is changed into nine, work
One of potato class " T-DNA " element is selected, can reduce carrier framework in transfer-gen plant brings frequency into.After element is improved
Transgene expression vector has higher transformation efficiency and more excellent security.Especially suitable for structure potato and corresponding plants
Cisgenesis system and safe transformation system, provided safeguard to improve plant transformation efficiency and security, so as to favourable
In the commercialization process for accelerating transgenic product, traditional breeding method cost is reduced, shortens the traditional breeding method time, is advantageous to improve breeding
Efficiency, the manpower and materials for saving breeding, reduce environmental pollution.
Therefore, third object of the present invention is to provide and replaced by above-mentioned preferably potato class " T-DNA " element StTL01
T-DNA right margins, 4 StTL04 replacement T-DNA left margins and salt resistant gene AtNHX1 are changed as Security selection marker gene, it is common
With the endogenous transformation system of potato of structure.The present invention is studied by scientific experiment and confirmed, the system can be applied in Ma Ling
Endogenous marker-free transformation is carried out in potato, the Cisgenesis plant of marker-free can be obtained in potato, safety applications in
Potato transgenic breeding, help speed up Potato Breeding process and the commercialization process of related transgenic product.
Compared with prior art, the starting transfer efficiency of potato class " T-DNA " element that the present invention clones is compared with Rommens
Gained St02 improves 1.17 ~ 2.93 times, and verifies the termination forwarding function of potato class " T-DNA " element first, finds
To the termination transfer element more excellent compared with Agrobacterium " T-DNA " element, and the termination transfer efficiency of 9 class " T-DNA " elements is compared with agriculture
Bacillus T-DNA is high.Also, carried by the way that these elements to be constructed to potato Cisgenesis together with Security selection marker gene
Body, the carrier obtain transfer-gen plant by converting potato, can effective expression target gene, to obtain potato safety
Transfer-gen plant provides technical guarantee.
Brief description of the drawings
Fig. 1 is that potato class " T-DNA " sequence originates transfer efficiency analysis result figure.
Fig. 2 is potato class " T-DNA " sequence ends transfer efficiency analysis result figure.
Fig. 3 is 1 ~ 4 repetition StTL04 sequence ends transfer efficiency analysis result figure.
Fig. 4 is that plasmid vector pXR0072 builds schematic diagram.
Fig. 5 is that plasmid vector pXR0073 builds schematic diagram.
Fig. 6 is that digestion detects pXR0074 plasmid electrophoretograms；Wherein swimming lane M：DL15000 marker；Swimming lane 1-3：HindIII single endonuclease digestion plasmids pXR0074.
Fig. 7 is that plasmid vector pXR0074 builds schematic diagram.
Fig. 8 is the state diagram of explant under different NaCl concentrations；A：75 mM NaCl B：100 mM NaCl C：150 mM
NaCl。
Fig. 9 is the state diagram of explant under hormon concentration；A：S2 differential mediums, B：S2-1 differential mediums, C：
S2-2 differential mediums；Black arrow points to position caused by Bud Differentiation.
Figure 10 is that GUS active masses chemical staining analyzes transformation efficiency figure；A schemes：pBI121；B schemes：pCAMBIA1302；C
Figure：pCAMBIA 1305；D schemes：Negative control.
Figure 11 is the vector construction schematic diagram for changing carrier framework.
Figure 12 is that double digestion detects pXR0094 plasmid electrophoretograms；Swimming lane M：DL15000 marker；Swimming lane 1-2：AndI/
SmaI double digestion plasmids pXR0094.
Figure 13 is that the PCR of regeneration plant is detected（At-L/R primers）Electrophoretogram；Swimming lane M：DL2000 marker；Swimming lane 1：
Positive control；Swimming lane 2：Negative control；Swimming lane 3：E3；Swimming lane 4-15：Regeneration plant.
Figure 14 is that the PCR of regeneration plant is detected（GUS-L/R primers）Electrophoretogram；Swimming lane M：DL2000 marker；Swimming lane 1：
Positive control；Swimming lane 2：Negative control；Swimming lane 3：E3；Swimming lane 4-15：Regeneration plant.
Figure 15 is the GUS detection figures of transgenic line；1-2：Transgenic line；E3：Negative control E3.
Figure 16 is 94-LBS-F/R（A）And 94-LBS-2F/R（B）The carrier framework electrophoretogram of primer detection positive plant；
Swimming lane M：DL2000 marker；Swimming lane 1：Positive control；Swimming lane 2：Negative control；Swimming lane 3：E3；Swimming lane 4-7：GUS dyeing sun
Property plant.
Embodiment
More detailed description is made to the present invention below in conjunction with specific embodiment.According to following description and these implementations
Example, those skilled in the art can determine the essential characteristic of the present invention, and in the situation without departing from spirit and scope of the invention
Under, various changes and modifications can be made to the present invention, so that it is applicable various uses and condition.
Present invention clone amounts to clone's potato class " T-DNA " element 25, wherein 3 class " T-DNA " elements are compared with forefathers
The St02 of research has more excellent starting transfer efficiency, and 9 class " T-DNA " elements have more excellent termination compared with agrobatcerium T-DNA
Transfer efficiency.In addition, StTL01 and StTL05 elements and plant anti-salt Gene A tNHX1 are used for structure, potato is homologous turns base
Because of carrier, the expression vector can turn target gene by agrobcterium-mediated transformation or other transgenic methods
Enter in all kinds of plants such as potato, unmarked, the exoskeletal Cisgenesis plant or strain obtained by Screening and Identification is equal
Belong to protection scope of the present invention.The present invention can be obtained unmarked, exoskeletal by carrying out Cisgenesis in potato body
Cisgenesis potato plant, in every respect lifted potato transgenosis security, the present invention research find it is each excellent
First class " T-DNA " element and the potato Cisgenesis carrier built by it and its application in each field belong to the present invention
The scope of protection.
The bioinformatic analysis of potato class " T-DNA " sequence of the present invention
Reported according to documents and materials, and the T-DNA sequences of the various Agrobacteriums of software analysis are compared using homologous sequence, found
To 2 functions conserved sequence TAC and ATATATCCTG of T-DNA elements, simple perl LISP program LISPs and blastn mono- are designed
Rise and compare degenerate sequence in each potato Relational databases of NCBI and potato gene group database of download
NNNTACNNNNNNATATATCCTGNNN（N=A/T/G/C）, respectively obtained in ncbi database and potato gene group database
It is newfound class " T-DNA " sequence that 5 class " T-DNA " sequences, wherein ncbi database comparison result, which have 3, potato-based
Because group database comparison result has 4 class " T-DNA " sequences for being new hair style, will a little researchs such as sequence and Rommens originate
The higher St02 of transfer efficiency and agrobatcerium T-DNA enter the analysis of start of line transfer efficiency together.
In addition, being reported according to documents and materials, contain 2 degeneracies more powerful conserved sequence DAC and AKAHRTCCTG couples
The function effect of class " T-DNA " element is smaller, and can obtain more classes " T-DNA " element in database comparison.Therefore,
The present invention in each potato Relational databases of NCBI and potato gene group database by comparing degenerate sequence
NNNDACNNNNNNAKAHRTCCTGNNN（R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, H=A/C/, B
=C/G/T, V=A/C/G, D=A/G/T, N=A/C/G/T）, remove wherein with identical class " T-DNA " sequence such as Rommens
Afterwards, 61 and 129 class " T-DNA " sequences of new discovery are distinguished in two databases.
The clone of potato class " T-DNA " sequence and the structure of Function detection carrier
According to the influence of each base-pair function of conserved sequence, 7 class " T-DNA " sequences and St02 are chosen（Rommens etc.
Study transformation efficiency highest class " T-DNA " element）Structure starting forwarding function detection carrier together.Specific behaviour's step is as follows.
Addition HindIII restriction enzyme sites on the outside of 2.1 pCAMBIA1302 plasmid vector T-DNA right border sequences
According to pCAMBIA1302 plasmid vector sequences, following 4 primers are designed by its T-DNA right border sequence：
overF1：AACTGCAGAGCAGCTTGAGCTTGGATCAGAT
Pst Ⅰ
overR1：AACGCTCTTTTCTCTTAGAAGCTTGTTTACCCGCCAATATATCCTGTCAA
Hind Ⅲ
overF2：TATATTGGCGGGTAAACAAGCTTCTAAGAGAAAAGAGCGTTTATTAGAATAACG
Hind Ⅲ
overR2：CGCGTAACTTAGGACTTGTGCGA
By overlapping pcr, go out specific PCR products using pCAMBIA1302 DNAs template amplification.Over-lap PCR
Amplification method is specially：First use overF1/overR1 and overF2/overR2 two to primer respectively with pCAMBIA1302 plasmids
Carrier is DNA profiling, and pfu high-fidelity enzymes are used in PCR instrument（Purchased from MBI Fermentas companies, republic of Lithuania）Amplification
Go out single band, with glue reclaim kit（Purchased from Shanghai Sheng Gong companies, China, detailed process is referring to specification）Reclaim target bar
Band.Then above-mentioned 2 kinds of PCR primers are mixed as template, is primer with overF1 and overR2, protected in PCR instrument with pfu is high
True enzyme（Purchased from MBI Fermentas companies, republic of Lithuania）Amplify single band.With glue reclaim kit（Purchased from upper
Marine growth Engineering Co., Ltd, China, detailed process is referring to specification）Target stripe is reclaimed, is usedPstI HeSphI double digestion, with
Same warpPstI HeSphPCAMBIA1302 plasmid vectors large fragment connection after I double digestion.5 μ L connection products are taken to carry out thermal shock
Convert bacillus coli DH 5 alpha competence（Write with reference to J. Pehanorm Brookers etc., Huang Peitang etc. is translated, Molecular Cloning:A Laboratory guide（3rd
Version）, Science Press, 2002 editions）, be coated on newly configure contain 50 mg/L kanamycins（Kam）LB solid mediums
On, 37 DEG C of overnight incubations select that bacterial plaque is some, in the LB liquid medium of the mg/L containing Kam 50, in 37 DEG C of 200 r/
Min shaken cultivations are overnight to muddy.A small amount of method extraction plasmids（Write with reference to J. Pehanorm Brookers etc., Huang Peitang etc. is translated, molecular cloning
Experiment guide（The third edition）, Science Press, 2002 editions）, send 2 positive colonies supreme marine growth engineering finite after digestion detection
It is sequenced, the correct plasmid vector of sequencing result is named as pCAMBIA1302R, saves backup.
Agrobatcerium T-DNA right border sequence obtains in 2.2 potato class " T-DNA " sequence replacing pCAMBIA1302R carriers
Forwarding function detection carrier must be originated
According to 7 preferred potato class " T-DNA " element sequences and St02 sequences, primer needed for design vector structure is such as
Under：
RB-F：CATGGCATGGATGAACTATACAAAG
RB-StTL01：CCCAAGCTTCACTACAACTCAATATATCCTGTACAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL01
RB- StTL02：CCCAAGCTTTAATACTCTGATATATATCCTGCATAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL02
RB- StTL03：CCCAAGCTTAAGTACAGCATCATATATCCTGAGTAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL03
RB- StTL04：
CCCAAGCTTGCATACCTCTGAATATATCCTGCGGAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL04
RB- StTL05：CCCAAGCTTCTCTACCCCCTAATATATCCTGTGTAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL05
RB- StTL06：CCCAAGCTTCCATACTATCAAATATATCCTGGTGAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL06
RB- StTL07：CCCAAGCTTCCATACTTCACCATATATCCTGTCAAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ StTL07
RB-St02
CCCAAGCTTCATTACCAACAAATATATCCTGGCCAACACTGATAGTTTAATTCCCGATC
Hind Ⅲ St02
By round pcr, specific PCR is amplified respectively by template and above-mentioned primer of pCAMBIA1302R DNAs
Product.With HindIII andBstEIIDouble digestion PCR primer and plasmid vector pCAMBIA1302R respectively, reclaim PCR digestion products
With pCAMBIA1302R plasmid large fragments.The large fragment of PCR digestion products and pCAMBIA1302R plasmids is through being connected, converting, carrying
Plasmid, digestion detection and sequencing（Specific method is same as above）Afterwards, obtain and correct plasmid vector 8 is sequenced, be respectively designated as pRB-
StTL01 ~ 07 and pRB-St02.Wherein pRB-StTL01 ~ 07 grade, 7 carriers be originate Function detection carrier, pRB-St02 and
PCAMBIA1302R is respectively the starting Function detection control vector comprising potato class " T-DNA " element and agrobatcerium T-DNA.
Addition MluI restriction enzyme sites on the inside of 2.3 pCAMBIA1302R plasmid vector T-DNA left margins sequences
According to pCAMBIA1302R plasmid vector sequences, following 4 primers are designed by its T-DNA right border sequence.
LoverF1：CCGGTGATATTCTCATTTTAGCCATT
LoverR1：
TTAAGTTGTCTAAGCGTCAATTACGCGTTGTTTACACCACAATATATCCTGCCA
Mlu I
LoverF2：
GGATATATTGTGGTGTAAACAACGCGTATTGACGCTTAGACAACTTAATAACACATT
Mlu I
LoverR2：GTCCGAGGGCAAAGAAATAGAGTAGAT
Ibid, by overlapping pcr, 2 short-movie section PCR are first amplified using pCAMBIA1302R DNAs template
Product, finally go out the specific DNA of a long segment using short-movie section PCR primer as template amplification again（Specific method is the same）.
With glue reclaim kit（Purchased from Shanghai bioengineering Co., Ltd, China, detailed process is referring to specification）Reclaim target stripe.
WithPsp Ⅺ HeSacII double digestion, with same warpPsp Ⅺ HeSacPCAMBIA1302R plasmid vectors after II double digestion are large stretch of
Section is connected, converted, paving plate, upgrading grain, digestion（Ditto）.The supreme marine growth engineering of 2 positive colonies is sent to have after digestion detection
Limit is sequenced, and the correct plasmid vector of sequencing result is named as pCAMBIA1302RL, saves backup.
Agrobacterium T-DNA sequence is terminated in 2.4 potato class " T-DNA " sequence replacing pCAMBIA1302RL carriers
Forwarding function detects carrier
According to 25 preferred potato class " T-DNA " element sequences and St02 sequences, design terminates forwarding function detection and carried
Primer needed for body structure is as follows.
LB-F TTCCGGTGATATTCTCATTTTAGCC LB-St02：
ATCGACGCGTTcattaccaacaaatatatcctggccCCAGCCAGCCAACAGCTCC
MluⅠ St02
LB-StTL01：
ATCGACGCGTTcactacaactcaatatatcctgtacCCAGCCAGCCAACAGCTCC
MluⅠ StTL01
LB-StTL02：
ATCGACGCGTTtaatactctgatatatatcctgcatCCAGCCAGCCAACAGCTCC
MluⅠ StTL02
LB-StTL03：
ATCGACGCGTTaagtacagcatcatatatcctgagtCCAGCCAGCCAACAGCTCC
MluⅠ StTL03
LB-StTL04：
ATCGACGCGTTgcatacctctgaatatatcctgcggCCAGCCAGCCAACAGCTCC
MluⅠ StTL04
LB-StTL05：
ATCGACGCGTTctctaccccctaatatatcctgtgtCCAGCCAGCCAACAGCTCC
MluⅠ StTL05
LB-StTL06：
ATCGACGCGTTccatactatcaaatatatcctggtgCCAGCCAGCCAACAGCTCC
MluⅠ StTL06
LB-StTL07：
ATCGACGCGTTccatacttcaccatatatcctgtcaCCAGCCAGCCAACAGCTCC
MluⅠ StTL07
LB-StTL08：
ATCGACGCGTTcattacgttctaatacatcctgcatCCAGCCAGCCAACAGCTCC
MluⅠ StTL08
LB-StTL09：
ATCGACGCGTTatgtactgataaataaatcctgaaaCCAGCCAGCCAACAGCTCC
MluⅠ StTL09
LB-StTL10：
ATCGACGCGTTtaagacctggtaatatatcctggtcCCAGCCAGCCAACAGCTCC
MluⅠ StTL10
LB-StTL11：
ATCGACGCGTTtattacgttctgatacatcctgcatCCAGCCAGCCAACAGCTCC
MluⅠ StTL11
LB-StTL12：
ATCGACGCGTTgggaactcgaatatatatcctgactCCAGCCAGCCAACAGCTCC
MluⅠ StTL12
LB-StTL13：
ATCGACGCGTTggtaacaactcaatatatcctgaaaCCAGCCAGCCAACAGCTCC
MluⅠ StTL13
LB-StTL14：
ATCGACGCGTTatgtactgagccatatgtcctgcatCCAGCCAGCCAACAGCTCC
MluⅠ StTL14
LB-StTL15：
ATCGACGCGTTttctactacttgatatgtcctgtacCCAGCCAGCCAACAGCTCC
MluⅠ StTL15
LB-StTL16：
ATCGACGCGTTtcgtacaaataaataaatcctgcaaCCAGCCAGCCAACAGCTCC
MluⅠ StTL16
LB-StTL17：
ATCGACGCGTTggggaccaccccatatatcctgttgCCAGCCAGCCAACAGCTCC
MluⅠ StTL17
LB-StTL18：
ATCGACGCGTTttctacaaagcaatacatcctgcaaCCAGCCAGCCAACAGCTCC
MluⅠ StTL18
LB-StTL19：
ATCGACGCGTTgcatacacaaagataaatcctgtaa CCAGCCAGCCAACAGCTCC
MluⅠ StTL19
LB-StTL20：
ATCGACGCGTTaaggacaaatatatatatcctgaacCCAGCCAGCCAACAGCTCC
MluⅠ StTL20
LB-StTL21：
ATCGACGCGTTgcatacagaaacatacatcctgatcCCAGCCAGCCAACAGCTCC
MluⅠ StTL21
LB-StTL22：
ATCGACGCGTTagaaaccacttaatatatcctgtcaCCAGCCAGCCAACAGCTCC
MluⅠ StTL22
LB-StTL23：
ATCGACGCGTTgacaactacataatatatcctgttcCCAGCCAGCCAACAGCTCC
MluⅠ StTL23
LB-StTL24：
ATCGACGCGTTtggtactattatatatgtcctgtctCCAGCCAGCCAACAGCTCC
MluⅠ StTL24
LB-StTL25：
ATCGACGCGTTatttacaattaaatatgtcctgcatCCAGCCAGCCAACAGCTCC
MluⅠ StTL25
By round pcr, specific PCR is amplified respectively by template and above-mentioned primer of pCAMBIA1302RL DNAs
Product.With HindIII andBstEIIDouble digestion PCR primer and plasmid vector pCAMBIA1302RL respectively, recovery PCR digestion productions
Thing and pCAMBIA1302RL plasmid large fragments.PCR digestion products with the large fragment of pCAMBIA1302RL plasmids through being connected, turn
Change, upgrading grain, digestion detection and sequencing（Specific method is same as above）Afterwards, obtain and correct plasmid vector 25 is sequenced, name respectively
For pLB-StTL01 ~ 25 and pLB-St02.Wherein pLB-StTL01 ~ 25 grade, 25 carriers are expiry feature detection carrier,
PLB-St02 and pCAMBIA1302RL is respectively the starting function inspection comprising potato class " T-DNA " element and agrobatcerium T-DNA
Survey control vector.
Transgenosis identifies potato class " T-DNA " functional nucleotide sequence and analysis transfer efficiency
3.1 starting forwarding function detections
By build 7 starting forwarding functions detection carriers and 2 control plasmid carrier pRB-St02 and
PCAMBIA1302R is transferred to agrobacterium strains GV3101 respectively, each Function detection that then will be built by Agrobacterium flower-dipping method
Carrier stable conversion Columbia Arabidopsis thaliana ecotypes.The seed of transformed plant is collected, positive turn is screened on resistance culture base
Gene plant, whether there is right margin starting forwarding function with this analysis classes " T-DNA " sequence, and analyze its starting transfer effect
Rate.
Transgenic research result shows that 7 class " T-DNA " sequences can convert plant, obtains transgenic positive plant.
Shown after repeating Study on Transformation through statistical analysis, wherein 5 classes such as StTL01, StTL02, StTL03, StTL04, StTL07
The starting transfer efficiency of " T-DNA " reaches more than the 50% of agrobatcerium T-DNA transfer efficiency（Fig. 1）.Compared to control Agrobacterium T-
DNA right margins, StTL01 and StTL02 transfer efficiency is significantly higher than control in class " T-DNA " element（p<0.01）, St02,
StTL03, StTL04 and StTL07 are with compareing no significant difference（p>0.05）, and StTL05 and StTL06 is substantially less than and compareed（p<
0.01）.Wherein StTL01 originates transfer efficiency highest, originate transfer efficiency for agrobatcerium T-DNA 2.67 times, is St02 startings
2.93 times of transfer efficiency.
Through analysis, there is StTL01 ~ potato class " T-DNA " sequence of 07 grade 7 right margin to originate forwarding function, wherein
3 class " T-DNA " element starting transfer efficiencies such as StTL01, StTL02 and StTL03 are compared with 2 control St02 and agrobatcerium T-DNA
Significantly rise.Wherein StTL01 originates transfer efficiency highest, originate transfer efficiency for agrobatcerium T-DNA right margin 2.67
Times.
3.2 terminate forwarding function detection
By build 25 terminate forwarding functions detection carriers and 2 control plasmid carrier pLB-St02 and
PCAMBIA1302RL is transferred to agrobacterium strains GV3101 respectively, each termination work(that then will be built by Agrobacterium flower-dipping method
Carrier stable conversion Columbia Arabidopsis thaliana ecotypes can be detected.The arabidopsis seed received is spread into resistant panel, screening is positive
Transfer-gen plant.The DNA of about 50 parts of transfer-gen plants of each plasmid extraction, the side of plasmid vector frame sequence is expanded by PCR
Method identification T-DNA terminates transfer efficiency.Pcr amplification primer thing is：
LBS-2F CCGCCTTACAACGGCTCTC LBS-R CCGTGGTTGGCTTGTATGGAG
PCR amplifications show there is the class " T- for terminating forwarding function in 25 detected potato classes " T-DNA "
DNA " has 24, and only StTL03 does not possess termination transferance.Wherein, StTL02, StTL04, StTL05, StTL07,
The termination transfer efficiency of 9 potato class " T-DNA " elements such as StTL11, StTL12, StTL16, StTL17, StTL18 is compared with agriculture
Bacillus T-DNA left margin sequence efficiency highs（Fig. 2）, wherein StTL04 stopping transfer efficiency highest（47.1%）, secondly
3 class " T-DNA " elements such as StTL05, StTL07, StTL17 stop transfer efficiency also up to more than 20%（Fig. 2）.
3.3, which repeat series connection LB sequences, further improves termination transfer efficiency
Transfer efficiency is terminated further to improve, reduce plasmid vector skeleton brings frequency into, and StTL04 is entered in this research
Series connection is repeated several times in row, analyzes the influence that multiple right margins are brought into potato carrier framework.
This research is as follows according to primer of the pLB-StTL04 carrier sequences design comprising StTL04 first.
2*StTL04-F：CGCGCCGCAGGATATATTCAGAGGTATGCACGCGT
StTL04
2* StTL04-R：CGCGACGCGTGCATACCTCTGAATATATCCTGCGG
StTL04
Above-mentioned PCR primer is dissolved in water to final concentration 100uM, 1:1 mixing two primers of Direct/Reverse.Then, in PCR
The steps such as 95 DEG C of denaturation 30sec, 72 DEG C of -37 DEG C -25 DEG C each 2min are sequentially completed on instrument, the progressively annealing of primer is completed, makes to draw
Slowly annealing forms dimer double-strand to thing.By MluI single endonuclease digestion pLB-StTL04 plasmid vectors, recovery digestion products with it is above-mentioned
Primer dimer is together through connecting, converting, spreading Kam resistant panels（Specific method is same as above）Afterwards, direct 10 positive colonies of picking
The sequencing of Shanghai bioengineering Co., Ltd is delivered to after shaking bacterium, the correct plasmid vector of sequencing is obtained and is named as pLB-2*StTL04.
Thereafter, pLB-2*StTL04 is subjected to MluI single endonuclease digestions, recovery digestion products are connected with primer dimer, converted, spreading Kam resistances
Flat board（Specific method is same as above）, 10 positive colonies of picking, which shake, delivers to the sequencing of Shanghai bioengineering Co., Ltd after bacterium, is surveyed
The correct plasmid vector of sequence is named as pLB-3*StTL04.The acquisition that repeats the above steps is sequenced correct plasmid vector and is named as
pLB-4*StTL04。
Ditto transgenosis plan is carried out to 3 plasmid vectors such as pLB-2*StTL04, pLB-3*StTL04, pLB-4*StTL04
Southern mustard analysis carrier framework brings frequency into.As a result show, 2 times, 3 times, 4 repetitions respectively by terminate transfer efficiency bring up to
51.85%th, 83.02%, 88.71%, and compare agrobatcerium T-DNA left margin sequence termination transfer efficiency be 12.8%, 1
StTL04 termination transfer efficiency is 47.1%（Fig. 3）.As a result illustrate, series connection potato class " T-DNA " element is repeated several times
What StTL04 reduced carrier framework brings frequency into, improves termination transfer efficiency..
The structure of the endogenous expression vector of potato
Successively clone 1617 bp's respectively from arabidopsis cDNA, gDNAAtNHX1Gene, 292 bpAtNHX1Terminate
Subsequence and 794 bp'sAtACT11Promoter, insert in plasmid vector pXR0057 and successfully construct pXR0072 carriers, specifically
Construction step is shown in Fig. 4.
To remove marker gene and integrating class " T-DNA " border sequence, and target gene GUS is added, constructedpXR0073Carrier, specific construction method are useAseI singly cutspXR0062Large fragment is connected after plasmid vector to obtainpXR0069Carrier,pXR0069Plasmid vector is usedBglII HeBstEII pair cut back to close large fragment withpCAMBIA1305Plasmid vectorBglII HeBstE
II pair of GUS small fragment cut connectspXR0073（Fig. 5）.
WithEcoRI andPmeI double digestion pXR0072 carriers, reclaim small fragment（2787 bp）,EcoRI andSmaI double digestions
PXR0073 carriers, reclaim large fragment（9570 bp）, in the presence of T4-ligase, 16 DEG C of connections, two fragments, connection product
Heat-shock transformed bacillus coli DH 5 alpha, used after choosing monoclonal extraction plasmidHindIII single endonuclease digestions detect, and digestion detects obtained fragment
Size is with being expected unanimously（Fig. 6）, that is, obtain plasmid vector pXR0074 carriers（pCAMBIA1302-StTL01-AtNHX1-GUS-
4*StTL04）（Fig. 7）.
Potato salt screens system optimization
After building endogenous transgene carrier, carrier is converted into agrobacterium strains GV3101, because using resistant gene of saltAtNHX1Instead of routine antibiotic marker genes, so experiment in take NaCl alternatively agent carry out transformed cells sieve
Choosing, is groped endogenous transformation system.
Various concentrations NaCl genetic transformation result
The endogenous transgene carrier pXR0074 built is entered by the fast genetic transformation system of this laboratory foundation early stage
Row conversion, concrete operation step are as follows：
A. the Agrobacterium GV3101 containing plant expression carrier plasmid is inoculated in added with 50 mg/L Km and 50 mg/L
In Rif LB fluid nutrient mediums, in culture on 28 DEG C, 200 r/min shaking tables to OD600For 0.5 or so.
B. 7 min are centrifuged in 4000 r/min, its precipitation is suspended again with MS fluid nutrient mediums.2-3 will be grown individual month, be straight
Footpath is about that 0.5 cm Potato microtuber is cut into 1-2 mm thin slice, and 10 min are soaked in above-mentioned Agrobacterium bacterium solution, nothing is used after taking-up
Bacterium filter paper blots surface bacterium solution, is transferred to and co-cultures culture medium S1（MS+0.2 mg/L IAA+0.2 mg/L GA3+0.5 mg/L
6-BA+2 mg/L ZT）In, in 24 DEG C of light culture 2d.
C. the potato chips after light culture 2d are cleaned 2-3 times with the single bacterium water of sterilizing, potato chips surface liquid is blotted with aseptic filter paper
Body, then clean potato chips are transferred to additional NaCl/Km and Cef differential medium S2（MS+0.2 mg/L IAA+0.2
mg/L GA3+0.5 mg/L 6-BA+2 mg/L ZT+ NaCl/ Km +400 mg/L Cef）In, it is placed in intensity of illumination 2000
Lx, the h/d of photoperiod 16,23 ± 1 DEG C of temperature illumination box in cultivate.
D. lateral bud growth is had after cultivating 1 week around potato chips, lateral bud need to be cut off, after 3-4 weeks, from Potato microtuber thin slice
Centre can differentiate resistant budses, cut and be transferred to root induction on additional 400 mg/L Cef screening and culturing medium of taking root, so as to
Obtain intact plant.
The NaCl that various concentrations are added in differential medium S2 carries out the screening of transformed cells, wherein NaCl concentration point
Wei not 100 mM, 150 mM, 175 mM, 200 mM, 225 mM.The selection result shows higher in salinity（225 mM、200
mM、175 mM）Differential medium in explant potato chips there is different degrees of browning, in the differential medium of low concentration（100
mM、150 mM）Middle explant does not produce browning, but has more serious callus to produce around explant.
The result of genetic transformation after increase 6-BA concentration
The problems such as in order to solve Brown and more surrounding callus, take increase differential medium S2 culture mediums
The scheme of middle 6-BA concentration carries out screening differentiation culture.6-BA concentration in S2 culture mediums is improved to original 2 times（1mg/L）,
And take the NaCl of low concentration（75 mM、100 mM、150 mM）Carry out the screening of transformed cells.Fig. 8 is after 40d is cultivated in differentiation
Growth conditions of the explant under 3 kinds of NaCl concentrations.Under 3 kinds of different NaCl concentration condition of culture, Brown substantially mitigates,
The callus of surrounding is also slightly reduced, but has no that obvious Bud Differentiation produces.
Change the result of hormone concentration ratio genetic transformation
Further to solve the problems, such as that callus is largely produced and non-resistant bud is formed, using 50 mM NaCl screenings simultaneously
The scheme for reducing hormone concentration in S2 differential mediums carries out genetic transformation（Table 3）.As a result show, NaCl screening concentration is dropped
As little as 50 mM, screening differentiation can obtain resistant budses when cultivating 40 days（Fig. 9）.S2 with adding normal hormonal concentration, which breaks up, to be trained
Foster base is compared, and adds the S2-1 of 1/2 hormone concentration and not add Callus formation in the S2-2 differential mediums of hormone less,
Caused Bud Differentiation is more（Table 5）.
The hormone concentration of the differential medium of table 3
| Hormone | S2 | S2-1 | S2-2 |
| IAA | 0.2 mg/L | 0.1 mg/L | 0 mg/L |
| GA3 | 0.2 mg/L | 0.1 mg/L | 0 mg/L |
| 6-BA | 0.5 mg/L | 0.25 mg/L | 0 mg/L |
| ZT | 2.0 mg/L | 1.0 mg/L | 0 mg/L |
6. influence of the carrier framework to transformation efficiency is analyzed in instantaneous conversion
Studied due to transformation efficiency of the pCAMBIA series plasmids carriers in Transformation of potato and have no report, and this research
It was found that the endogenous transgene carrier pXR0074 of potato based on pCAMBIA1302 carrier frameworks converted in potato compared with
Hardly possible obtain transgenic positive plant, therefore, this research take instantaneous conversion method analysis pBI121, pCAMBIA1302,
The transformation efficiency of 3 plasmid vectors such as pCAMBIA1305.Specific method is, first by pBI121, pCAMBIA1302,
This 3 plasmid conversion agrobacterium strains GV3101 of pCAMBIA1305, the potato chips rapid conversion system established by this laboratory are carried out
The Agrobacterium of potato is infected and co-cultured（The same step of 5.4 A, B two）, clean Agrobacterium 3-4 times with aqua sterilisa after co-culturing 3 days,
GUS dyeing is observed and counts coloration result.As a result show, 3 carrier transformation efficiencies have notable difference, and wherein pBI121 carriers turn
Potato chips almost all after change can be dyed blueness by GUS dyeing liquors and coloring is deeper（Figure 10 A）, and pCAMBIA1302 and
PCAMBIA1305 carriers conversion after potato chips pigmentable potato chips it is less, dyeing it is shallower（Figure 10 B, C）, it is completely not colored to compare E3
（Figure 10 D）.Statistics shows that the transformation efficiency of pBI121 plasmid vectors is 89.37 ± 1.18%, and pole is significantly higher than
PCAMBIA1302 and the plasmid vectors of pCAMBIA 1305 transformation efficiency（Respectively 1.57 ± 1.36% and 27.40 ± 9.66%,p
<0.01）, it is shown in Table 4.
The transformation efficiency of different carriers is analyzed in the instantaneous conversion of table 4
| Carrier | Number of repetition | Average conversion efficiency | Standard error | The significance of difference |
| pBI121 | 3 | 89.37% | 1.18% | A |
| pCAMBIA1302 | 3 | 1.57% | 1.36% | B |
| pCAMBIA1305 | 3 | 27.40% | 9.66% | C |
Note：LSD is test, and different letters represent that difference reaches the pole level of signifiance（p<0.01）.
The optimization of endogenous expression vector and the preliminary foundation of expression system
The optimization of 7.1 endogenous expression vectors
The result analyzed according to above-mentioned instantaneous conversion, to improve transformation efficiency, take and replace above-mentioned endogenous turn of pXR0074
The scheme of the frame sequence of genophore.Added first near pBI121 carrier left marginsAvrII restriction enzyme sites, structure
pXR0090；PBI121 RB sequences are replaced with to the StTL01 sequences in potato source again, build pXR0091；Exist simultaneously
The left margin of pXR0074 carriers nearby addsAvrII restriction enzyme sites, build pXR0092；Then useAvrII andPmeI is double respectively
Digestion pXR0091 and pXR0092 carrier, reclaim pXR0091 large fragment（8599 bp）With pXR0092 small fragment（6151
bp）, through connecting, converting,AhdI/SmaI double digestions detect（Figure 12）, show that it is the endogenous of 14750 bp successfully to build size
Transgene carrier pXR0094（pBI121-StTL01-AtNHX1-GUS-4*StTL04）.To incite somebody to actionAtNHX1Mark marks with Km
Two carriers carry out genetic transformation efficiency comparison, added on the basis of pXR0094 carriersnptIIEncoder block, construct
PXR0096 carriers.Specific construction strategy is shown in Figure 11.
The foundation of endogenous expression system
PXR0094 the and pXR0096 vector plasmids built are transferred to agrobacterium strains GV3101, horse by electric shocking method
The two carriers are converted Hubei Province potato No. 3 and turn black two Potatoes of the heart by bell potato fast genetic transformation system（Specific behaviour
It is the same to make method）.The explant that pXR0094 carriers convert is placed in 50 mM NaCl of addition differential medium and screened, will
The explant of pXR0096 carriers conversion, which is placed in 50 mg/L Km of addition differential medium, to be screened, statistical correlation data
（Table 5）.As a result show, in Km screening systems, 26.28% explant can produce Bud Differentiation, under NaCl screening systems not
There is 4.67%, 7.56%, 22.61% explant to produce Bud Differentiation respectively with hormone combination S2, S2-1, S2-2.Screened in NaCl
208 plants of complete plant are obtained under system altogether, 250 plants of resistant budses are obtained in Km screening systems.In Km screening systems,
10.8% Bud Differentiation can the root induction in screening and culturing medium of taking root because the resistant budses obtained under NaCl screening systems
Growth potential is weaker, and the screening that take root will cause plant dead, therefore be not provided with selection of salt tolerance in link of taking root, but directly
Cultivated in common MS culture mediums, finally take PCR modes to detect positive plant.
The safety label of table 5 conversion statistics
Note： -：Represent shortage of data；*：Come from a part for total resistant budses number, other strains are lost.
In order to be detected to obtained intact plant PCR, respectively in resistant gene of saltAtNHX1WithGUSRespectively set inside reporter gene
Count pair of primers At-L/R and GUS-L/R.
At-L：TGCGTTCTGGTGCGGTAATAGG
At-R：GACTTGGGTGATTATCTTGCTATTGG
GUS-L： CTTGTAGCCGAAATCTGGAATG
GUS-R：TACCTGGGAGAAGATTCGGAC
Using the gDNA of intact plant as template, enter performing PCR detection, wherein At-L/R amplifications to it respectively with these two pair primer
Obtain 921 bp specific fragment（Figure 13）, GUS-L/R expands to obtain 667 bp specific fragment (Figure 14).As a result count such as
PCR detection positive strain coefficients in table 5, obtain 22 plants of positive plants in the complete regenerated plant of 93 plants of resistant gene of salt mark, and 11
The equal test positive of regeneration plant after screening of taking root of strain Km marks.GUS work has been carried out to the strain of PCR test positive
Property histochemical stain detection, GUS detection positive strain coefficient row, in Hubei Province potato No. 3 and turn to obtain respectively in heart crow material
1 plant of positive strain（Figure 15）, 1 plant of transfer-gen plant of wherein E3 materials obtains in S2-2 culture mediums, and turns heart crow material
The transgenic line of material obtains in S2-3 culture mediums（Table 5）.And 11 plants of PCR positive strains that Km screenings obtain can
Success is dyed（Table 5）.
PCR testing results are shown, Km is taken root the obtained all PCR positives strains of plant of screening, and in S2, S2-1 and
Screen obtained plant by NaCl on tri- kinds of different differential mediums of S2-2 has 0.00%, 29.41% and 22.97% plant respectively
Strain is detected as PCR positive strains.GUS coloration results find that Km screening gained PCR positive plants can be all colored, and
PCR positive plants obtained by NaCl have 2 plants can substantially be coloured.
The skeleton of transgenic line brings analysis into
In order to which the transgenic line marked to the resistant gene of salt of PCR test positive analyzes the feelings that its carrier framework brings into
Condition, reverse primer 94-LBS-R is designed at distance LB left ends 418bp, is separately designed just at the bp of LB right-hand members 45 and 141 bp
To primer 94-LBS-F and 94-LBS-2F.
94-LBS-F：CCTGTATCGAGTGGTGATTTTGTG
94-LBS-2F：TAGTTGCCGTTCTTCCGAATAGC
94-LBS-R：GCTTCTTGACTCTTTTGTATTCTATGG
Using the gDNA of 4 plants of NaCl screening gained GUS stained positive transgenic lines as template, with this 94-LBS-F/94-
LBS-R and 94-LBS-2F/94-LBS-R carries out specific amplification, amplifies 561 bp and 656 bp specific fragment respectively（Figure
16）, as a result showing in 4 plants of transgenic lines has 1 plant not bring carrier framework into.
The salt tolerance detection of transgenic line
Take the transgenic line of 2 GUS test positive（PXR0074-9 and pXR0074-36）And control（E3 cultigens）
Carry out Salt-Tolerance Identification.Concrete operation method is the various concentrations NaCl that 0 mM, 250 mM are added in MS culture mediums, cultivates 15
Observed after it and count take root state and Reducing sugar of the test tube seedling in different culture media.As a result show, do not add
NaCl, plant can all take root and growing way is preferable, without dead plant, shows to test material therefor growth normally（Table 6）.
Under 250 mM concentration, E3 rooting rate is 3.5%, the death rate 26.2%；PXR0074-9 and pXR0074-36 rooting rate difference
For 13.6% and 14.8%, higher than control, the death rate is respectively 13.6%, 21.5%, less than control（Table 6）.
The Salt-Tolerance Identification of the transgenic line of table 6
。
Sequence table
<110>Hua Zhong Agriculture University
<120>Potato class T-DNA elements and Cisgenesis system and its application
<160> 25
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
cactacaact caatatatcc tgtac 25
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<400> 2
taatactctg atatatatcc tgcat 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
aagtacagca tcatatatcc tgagt 25
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
gcatacctct gaatatatcc tgcgg 25
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
ctctaccccc taatatatcc tgtgt 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<400> 6
ccatactatc aaatatatcc tggtg 25
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence
<400> 7
ccatacttca ccatatatcc tgtca 25
<210> 8
<211> 25
<212> DNA
<213>Artificial sequence
<400> 8
cattacgttc taatacatcc tgcat 25
<210> 9
<211> 25
<212> DNA
<213>Artificial sequence
<400> 9
atgtactgat aaataaatcc tgaaa 25
<210> 10
<211> 25
<212> DNA
<213>Artificial sequence
<400> 10
taagacctgg taatatatcc tggtc 25
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<400> 11
tattacgttc tgatacatcc tgcat 25
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence
<400> 12
gggaactcga atatatatcc tgact 25
<210> 13
<211> 25
<212> DNA
<213>Artificial sequence
<400> 13
ggtaacaact caatatatcc tgaaa 25
<210> 14
<211> 25
<212> DNA
<213>Artificial sequence
<400> 14
atgtactgag ccatatgtcc tgcat 25
<210> 15
<211> 25
<212> DNA
<213>Artificial sequence
<400> 15
ttctactact tgatatgtcc tgtac 25
<210> 16
<211>
<212> DNA
<213>Artificial sequence
<400> 16
tcgtacaaat aaataaatcc tgcaa 25
<210> 17
<211> 25
<212> DNA
<213>Artificial sequence
<400> 17
ggggaccacc ccatatatcc tgttg 25
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence
<400> 18
ttctacaaag caatacatcc tgcaa 25
<210> 19
<211> 25
<212> DNA
<213>Artificial sequence
<400> 19
gcatacacaa agataaatcc tgtaa 25
<210> 20
<211> 25
<212> DNA
<213>Artificial sequence
<400> 20
aaggacaaat atatatatcc tgaac 25
<210> 21
<211> 25
<212> DNA
<213>Artificial sequence
<400> 21
gcatacagaa acatacatcc tgatc 25
<210> 22
<211> 25
<212> DNA
<213>Artificial sequence
<400> 22
agaaaccact taatatatcc tgtca 25
<210> 23
<211> 25
<212> DNA
<213>Artificial sequence
<400> 23
gacaactaca taatatatcc tgttc 25
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence
<400> 24
tggtactatt atatatgtcc tgtct 25
<210> 25
<211> 25
<212> DNA
<213>Artificial sequence
<400> 25
atttacaatt aaatatgtcc tgcat 25
Claims (4)
- Potato class 1. " T-DNA " element, its nucleotides sequence are classified as shown in the sequence 2 in sequence table.
- 2. including, the potato of potato class " T-DNA " element and Security selection marker gene described in claim 1 is homologous to turn base Because of system.
- 3. potato Cisgenesis system according to claim 2, it is characterised in that：Described Security selection marker base Because plant anti-salt Gene A tNHX1.
- 4. the carrier framework in transfer-gen plant is reduced of potato class " T-DNA " element described in claim 1 is brought into frequency Application.
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