CN108739348A

CN108739348A - A kind of method of quick discriminating transgenic positive strain

Info

Publication number: CN108739348A
Application number: CN201810351835.XA
Authority: CN
Inventors: 谢鑫; 孙涛; 王勇; 蒋君梅; 丁海霞
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2018-04-19
Filing date: 2018-04-19
Publication date: 2018-11-06

Abstract

The invention discloses a kind of methods of quick discriminating transgenic positive strain, include the following steps：(1) mixed solution containing screening antibiotic and basic element of cell division 6-BA is prepared, the screening antibiotic is the corresponding antibiotic of resistant gene that transfer-gen plant to be analyzed contains；(2) mixed solution is moved into liquid container；(3) clip rotaring gene plant blade to be analyzed, is dipped into liquid container；(4) liquid container is placed in illumination box, 28 DEG C are cultivated 10 days；(5) the downright bad situation of record blade flavescence is examined；Compared with prior art, the present invention may be implemented the quick screening to transgenic seedling, while the present invention has the characteristics that high-throughput, easy to operate, of low cost, widely applicable, can be applied to the screening and identification of the genetically modified plants such as corn, rice, soybean.

Description

A kind of method of quick discriminating transgenic positive strain

Technical field

The present invention relates to a kind of methods of quick discriminating transgenic positive strain, belong to the discriminating field of transgenic line.

Background technology

With the development of biotechnology, the gene of biology is transformed and has been become increasingly prevalent, this generally use The means of genetic engineering (DNA recombinant techniques) are completed, by being transformed in vitro to hereditary material DNA, according to people Set wish is integrated, and is realized the variation of its final character and function, is finally passed through agriculture bar with the help of external source " carrier " Bacterium is infected or the modes such as particle gun (plant) or microinjection (animal) are recombinated in organism and expressed.This use The plant that the means of genetic manipulation obtain is commonly referred to as " genetically engineered plants or genetically modified plants ".From nineteen eighty-three in the world first Since strain genetically modified plants (tobacco for turning antibiotic resistance gene) come out, the development of great-leap-forward has occurred in plant genetic engineering, arrives mesh Before until people transgenosis is successfully carried out on more than 60 kinds plants, the wherein crops such as rice, corn and soybean, tomato have been criticized Quasi- listing, this has absolutely proved that transgenosis shows great potential in the following crop improvement.

Plant transgene generally carries out genetic transformation by agriculture bacillus mediated method, and the T-DNA entrained by Agrobacterium can Radom insertion finally generates transformed cells and non-transformed cell to host cell genome, that is, genetic transformation offspring Some is " false positive ".This just needs to exclude these false positive transformed plants, often at present using some identification methods Identification method has, the methods of Southern hybridization, Northern hybridization, Western hybridization, Real-time PCR, still These methods are complicated for operation, cumbersome, and need specialized instrument and equipment, are unsuitable for Large-scale Screening transgenic progeny, therefore Need to find a kind of easy, quick, efficient screening technique, the present invention directly uses transgenosis riddled basins, using from Body blade is screened, and has the characteristics that be simple and efficient.

Invention content

The technical problem to be solved by the present invention is to：A kind of method of quick discriminating transgenic positive strain is provided, there is sieve Choosing is quick, of low cost, easy to operate, it is widely applicable the features such as, with overcome the deficiencies in the prior art.

The technical scheme is that：A kind of method of quick discriminating transgenic positive strain, includes the following steps：

(1) mixed solution containing screening antibiotic and basic element of cell division 6-BA is prepared, the screening antibiotic is waits for point The corresponding antibiotic of resistant gene that the transfer-gen plant of analysis contains；

(2) mixed solution is moved into liquid container；

(3) clip rotaring gene plant blade to be analyzed, is dipped into liquid container；

(4) liquid container is placed in illumination box, 26 DEG C~28 DEG C are cultivated 10~12 days；

(5) the downright bad situation of record blade flavescence is examined, is positive strain if blade keeps green.

The liquid container is 96 orifice plates.

The rotaring gene plant blade to be analyzed of the clip is 2.5-3.5cm.

The mixed solution is matched in screening 40~50ug/ml of antibiotic and basic element of cell division 6-BA 1~2ug/ml ratios System.

The screening antibiotic is hygromycin HPH, G418, bleomycin or gentamicin.

The beneficial effects of the invention are as follows：Compared with prior art, the quick screening to transgenic seedling may be implemented in the present invention, Simultaneously the present invention have the characteristics that it is high-throughput, easy to operate, of low cost, widely applicable, can be applied to corn, rice, greatly The screening and identification of the genetically modified plants such as beans.In addition, screening antibiotic in addition to other than hygromycin HPH, can also use G418 etc. as Antibiotic is screened, it is with high application prospect.

Description of the drawings

Fig. 1 is rice leaf of the hygromycin selection after 1 week, wherein+it is expressed as hygromycin selection positive plant ,-indicate For negative plant；

Fig. 2 is that PCR identifies transgenic paddy rice strain；

Fig. 3 is table 1, i.e. hygromycin and PCR qualification results, wherein in table-indicate uncertain.

Specific implementation mode

Below in conjunction with the accompanying drawings and invention is described further in specific embodiment：

By taking the screening of hygromycin resistance transgenic paddy rice as an example：

Overexpression green fluorescent protein (GFP, nucleotide series such as SEQ ID in rice Nipponbare (Nipponbare) NO：Shown in 5), and the T1 of acquisition is screened for the method for the transgenic line present invention, the transgenic paddy rice in present case Resistance screening, the wherein nucleotide series of hygromycin HPH such as SEQ ID are carried out with hygromycin resistance label, therefore with hygromycin NO：Shown in 6.It in practice, also can be according to species of discriminating, such as selection G418, bleomycin or gentamicin etc..

First cultivate the seed of transgenic paddy rice：In T1 generations to be detected, are turned GFP gene seeds to be placed in superclean bench It centrifuges on tube sheet；With liquid-transfering gun 1 minute is stood to 1 milliliter of 75% alcohol, upper and lower mixing are added in each centrifuge tube；Draw wine Essence；It is rinsed 3 times with sterile purified water；40% sodium hypochlorite is added, turn upside down mixing, stands 30 minutes；Use sterile purified water It rinses 3 times, draws waste liquid；Seed to be detected is moved into 1/2MS culture mediums with tweezers, is placed in illumination box culture 7 days, so It is transferred to the flowerpot culture in greenhouse afterwards, in hot-house culture one month.

Differentiate positive strain therein using the present invention, steps are as follows：

1, the mixed solution of hygromycin molten (50ug/ml) and basic element of cell division 6-BA (1ug/ml) are prepared, wherein mixing is molten Liquid is prepared in screening 40~50ug/ml of antibiotic and basic element of cell division 6-BA 1~2ug/ml ratios.

2, mixed solution is dispensed into 96 hole screen plates.

3, clip rice leaf to be measured (3 strains) about 3cm is put into step 2 in ready screening liquid.

4, screen plate is sealed with preservative film, is placed in 26 DEG C~28 DEG C illumination box cultures 10~12 days.

5, blade, the situation (as shown in Figure 1) of statistics blade flavescence necrosis are gripped from screen plate with tweezers.

The verification of reliability of the present invention：The DNA of plant is extracted with CTAB methods, then uses hygromycin HPH primer pairs respectively HPH-F/HPH-R, GFP special primer are to GFP-F/GFP-R detection samples (as shown in Figure 2).Testing result (such as Fig. 3, i.e. table 1 It is shown) prove that the method for the present invention is consistent with PCR results, reliability is high.Wherein, HPH-F nucleotide series such as SEQ ID NO：1 institute Show, i.e.,：ATTGACTGGAGCGAGGCG.HPH-R nucleotide series such as SEQ ID NO：Shown in 2, i.e.,： GCTTCTGCGGGCGATTT.GFP-F nucleotide series such as SEQ ID NO：Shown in 3, i.e.,：TCCAGGAGCGCACCATCT.GFP- R nucleotide series such as SEQ ID NO：Shown in 4, i.e.,：TGCCGTTCTTCTGCTTGTCG.Aforementioned primer from 5 ' to 3 '.

CTAB methods are as follows to the extraction step for being inoculated with rice leaf total DNA：

1, rice leaf sample is put into 2.0ml centrifuge tubes, steel ball is added, 30s is placed in liquid nitrogen；

2, sample is put into sample grinding machine Tissue lyser (Qiagen), is crushed with 28 times/second, concussion 20s frequencies；

3, plus in 600 μ l CTAB extracting solutions to centrifuge tube, 65 DEG C of 15 points of water-baths incubations are put into after acutely shaking sample Clock, incubation period overturn mixing sample 3 times；

4, the 600 violent mixing samples of μ l chloroforms are added into the sample being cooled to room temperature, -20 degree place 30min, then 12000rpm is centrifuged 10 minutes；

5, DNA precipitations are washed with 75% ethyl alcohol of 1ml, 12000rpm centrifuges 10min, the drying 10min in draught cupboard；

6, in the aqua sterilisa of 0.5%RNase, -20 DEG C save backup dissolving DNA.

Wherein CTAB buffer solutions are formulated as follows：

25g Sorbitol

10g Sarkosyl(N-lauryl sarcosine)

8g CTAB

47g NaCl

8g EDTA

10g PVPP(Polyvinylpyrrolidone)

It will be spare in the mentioned reagent constant volume to the aqua sterilisa of 1L weighed up.

Nucleotide series table：

SEQUENCE LISTING

Sequence table

<110>Guizhou University

<120>A kind of method of quick discriminating transgenic positive strain

<160> 6

<210> 1

<211> 18

<212> DNA

<213>Artificial series

<400> 1

ATTGACTGGA GCGAGGCG 18

<210> 2

<211> 17

<212> DNA

<213>Artificial series

<400> 2

GCTTCTGCGG GCGATTT 17

<210> 3

<211> 18

<212> DNA

<213>Artificial series

<400> 3

TCCAGGAGCG CACCATCT 18

<210> 4

<211> 20

<212> DNA

<213>Artificial series

<400> 4

TGCCGTTCTT CTGCTTGTCG 20

<210> 5

<211> 720

<212> DNA

<213>Green fluorescent protein DNA

<400> 5

ATGGTGAGCA AGGGCGAGGA GCTGTTCACC GGGGTGGTGC CCATCCTGGT CGAGCTGGAC 60

GGCGACGTAA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAGGGCGA TGCCACCTAC 120

GGCAAGCTGA CCCTGAAGTT CATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC 180

CTCGTGACCA CCCTGACCTA CGGCGTGCAG TGCTTCAGCC GCTACCCCGA CCACATGAAG 240

CAGCACGACT TCTTCAAGTC CGCCATGCCC GAAGGCTACG TCCAGGAGCG CACCATCTTC 300

TTCAAGGACG ACGGCAACTA CAAGACCCGC GCCGAGGTGA AGTTCGAGGG CGACACCCTG 360

GTGAACCGCA TCGAGCTGAA GGGCATCGAC TTCAAGGAGG ACGGCAACAT CCTGGGGCAC 420

AAGCTGGAGT ACAACTACAA CAGCCACAAC GTCTATATCA TGGCCGACAA GCAGAAGAAC 480

GGCATCAAGG TGAACTTCAA GATCCGCCAC AACATCGAGG ACGGCAGCGT GCAGCTCGCC 540

GACCACTACC AGCAGAACAC CCCCATCGGC GACGGCCCCG TGCTGCTGCC CGACAACCAC 600

TACCTGAGCA CCCAGTCCGC CCTGAGCAAA GACCCCAACG AGAAGCGCGA TCACATGGTC 660

CTGCTGGAGT TCGTGACCGC CGCCGGGATC ACTCTCGGCA TGGACGAGCT GTACAAGTGA 720

<210> 6

<211> 1026

<212> DNA

<213>Hygromycin HPH

<400> 6

ATGAAAAAGC CTGAACTCAC CGCGACGTCT GTCGAGAAGT TTCTGATCGA AAAGTTCGAC 60

AGCGTCTCCG ACCTGATGCA GCTCTCGGAG GGCGAAGAAT CTCGTGCTTT CAGCTTCGAT 120

GTAGGAGGGC GTGGATATGT CCTGCGGGTA AATAGCTGCG CCGATGGTTT CTACAAAGAT 180

CGTTATGTTT ATCGGCACTT TGCATCGGCC GCGCTCCCGA TTCCGGAAGT GCTTGACATT 240

GGGGAATTCA GCGAGAGCCT GACCTATTGC ATCTCCCGCC GTGCACAGGG TGTCACGTTG 300

CAAGACCTGC CTGAAACCGA ACTGCCCGCT GTTCTGCAGC CGGTCGCGGA GGCCATGGAT 360

GCGATCGCTG CGGCCGATCT TAGCCAGACG AGCGGGTTCG GCCCATTCGG ACCGCAAGGA 420

ATCGGTCAAT ACACTACATG GCGTGATTTC ATATGCGCGA TTGCTGATCC CCATGTGTAT 480

CACTGGCAAA CTGTGATGGA CGACACCGTC AGTGCGTCCG TCGCGCAGGC TCTCGATGAG 540

CTGATGCTTT GGGCCGAGGA CTGCCCCGAA GTCCGGCACC TCGTGCACGC GGATTTCGGC 600

TCCAACAATG TCCTGACGGA CAATGGCCGC ATAACAGCGG TCATTGACTG GAGCGAGGCG 660

ATGTTCGGGG ATTCCCAATA CGAGGTCGCC AACATCTTCT TCTGGAGGCC GTGGTTGGCT 720

TGTATGGAGC AGCAGACGCG CTACTTCGAG CGGAGGCATC CGGAGCTTGC AGGATCGCCG 780

CGGCTCCGGG CGTATATGCT CCGCATTGGT CTTGACCAAC TCTATCAGAG CTTGGTTGAC 840

GGCAATTTCG ATGATGCAGC TTGGGCGCAG GGTCGATGCG ACGCAATCGT CCGATCCGGA 900

GCCGGGACTG TCGGGCGTAC ACAAATCGCC CGCAGAAGCG CGGCCGTCTG GACCGATGGC 960

TGTGTAGAAG TACTCGCCGA TAGTGGAAAC CGACGCCCCA GCACTCGTCC GAGGGCAAAG 1020

GAATAG 1026

Claims

1. a kind of method of quick discriminating transgenic positive strain, includes the following steps：

(1) mixed solution containing screening antibiotic and basic element of cell division 6-BA is prepared, the screening antibiotic is to be analyzed The corresponding antibiotic of resistant gene that transfer-gen plant contains；

(2) mixed solution is moved into liquid container；

2. the method for quick discriminating transgenic positive strain according to claim 1, it is characterised in that：The liquid container For 96 orifice plates.

3. the method for quick discriminating transgenic positive strain according to claim 1, it is characterised in that：The clip waits for Analysis rotaring gene plant blade is 2.5-3.5cm.

4. the method for quick discriminating transgenic positive strain according to claim 1, it is characterised in that：The mixed solution It is prepared in screening 40~50ug/ml of antibiotic and basic element of cell division 6-BA 1~2ug/ml ratios.

5. the method for quick discriminating transgenic positive strain according to claim 1, it is characterised in that：The screening antibiosis Element is hygromycin HPH, G418, bleomycin or gentamicin.