CN109486858A - The construction method of required carrier in a kind of initiative of third generation sterile line - Google Patents

Abstract

This application discloses the construction methods of required carrier in a kind of initiative of third generation sterile line, the following steps are included: building double T-DNA carrier, three linked genes are contained in one of area T-DNA, and resistant gene is contained in another area T-DNA, for callus conversion and the screening in plant regeneration stage.Three linked gene is respectively as follows: DsRed gene, EAT1 gene, ZmAA gene.The resistant gene is the resistant gene of hygromycin and/or kanamycins.Hygromycin gene and red fluorescent gene (DsRed) gene are carried out chain rear rice transformation mature embryo-derived callus by this forwarding method, hygromycin is added in callus cell culture screening stage, it detects in combination with red fluorescence, quickly and effectively screens.

Description

The construction method of required carrier in a kind of initiative of third generation sterile line

Technical field

The present invention relates to genetic engineering genetic thremmatology and biological heredity improving technology field, in particular to a kind of third generation The construction method of carrier needed for sterile line is formulated.

Background technique

Rice is one of most important cereal crops at past half as the staple food for being in the world more than half population In more centuries, rice breeding achieves huge success.

The specific yield of rice realizes multiplication, and some areas are even improved to 3 times, this does safely for guarantee world food Huge contribution but in recent ten years the yield stagnation of rice are gone out, this aspect is due to not having on breeding technique New breakthrough and genetic diversity gradually narrowing in cultivar, on the other hand also in that the pest and disease damage frequently occurred And the natural calamities such as drought make Rice Production suffer heavy losses.

However, the sustainable growth of world population and the fast development of social economy cause the demand to grain constantly to increase Add.For these problems, Chinese scholar proposes the imagination for cultivating green super hybridization rice, high around Rice Resistance disease pest, drought resisting, nutrition The five big important characters such as utilization, high-quality, high yield are imitated, improvement comprehensively is carried out to realize the sustainable development of agricultural to rice varieties. And the breeding technique that transgenic technology is emerging as one kind, will play a significant role on realizing green super hybridization rice target

The transgenic research of rice starts from phase late 1980s, has a large amount of transgenic paddy rice research so far and is reported Road.

Transgenic Rice technology starts from Protoplast cuhnre, and 1985 successfully complete from rice protoplast regeneration for the first time Plant；First transgenic rice plant is obtained in japonica rice variety within 1988；Nineteen ninety is from rice variety Chinsurah First case transgenic indica type rice plant is obtained in Boro II；Nineteen ninety Li Baojian etc. infects rice tissue with Agrobacterium and is converted Callus；Transgenic plant is successfully obtained with particle bombardment using Rice Young Embryo as acceptor material, and is turned within 1991 Change efficiency to significantly improve.

From this, rataria is widely studied application as transgenic acceptor.1993 with agrobacterium co-cultivation in japonica rice product Transgenic plant is obtained on kind, hereafter, particle bombardment and agrobacterium-mediated transformation are wide as the path for transformation of most worthy It is general to be studied for Transgenic Rice.

According to the principle of third generation sterile line, the sterile line that we finally take does not contain any transgenic element, and obtains To holding system then only contain: the riddled basins-pollen lethal gene-gene of restoring gene three, wherein screening mark Remember that gene is red fluorescent protein (DsRed), comes from reef coral (Discosoma sp.), utilize the starting of seed specific expression Son makes DsRed gene and screens in seed kind specifically expressing the seed of sterile line；Pollen lethal gene is corn alpha amylase gene (ZmAA1), corn is come from, the pollen sterility for carrying the gene can be made；Restoring gene is usually common hidden in rice Property genic male sterile gene, come from rice itself, can make corresponding gene mutant strain restore fertility.

Summary of the invention

Technical problem to be solved by the present invention lies in provide the structure of required carrier in a kind of initiative of third generation sterile line Construction method.This forwarding method by hygromycin gene and red fluorescent gene (DsRed) gene carry out it is chain after rice transformation at The callus of cooked flake induction is added hygromycin in callus cell culture screening stage, detects in combination with red fluorescence, Quickly and effectively screen.

In order to solve the above technical problems, the present invention provides the building sides of required carrier in a kind of initiative of third generation sterile line Method, comprising the following steps: three linked genes are contained in building double T-DNA carrier, one of area T-DNA, another area T-DNA contains Resistant gene, for callus conversion and the screening in plant regeneration stage.

Three linked gene is respectively as follows: DsRed gene, EAT1 gene, ZmAA gene.

The resistant gene is the resistant gene of hygromycin and/or kanamycins.

Building the area T-DNA containing three linked genes method include:

A, the DNA of rice is extracted with CTAB method；

B, three kinds of linked gene espression boxes are obtained respectively；

C, the acquisition of the pCAMBIA1300 plasmid of Hpt gene is removed；

D, the connection of each expression cassette；

E, the connection of segment and pCAMBIA1300-hpt- plasmid is merged.

Building the area T-DNA containing EAT1 gene method include: using step A obtain DNA as template, with For primer amplification rice EAT1 expression casette.

Building the area T-DNA containing DsRed gene method include: using the pGDR plasmid containing DsRED segment as template, It utilizes Primer amplification DsRED expression casette is obtained,

Building the area T-DNA containing ZmAA gene method include: using maize dna as template, with Gene ZmAA1 expression casette is obtained for primer amplification.

The step A extracts the DNA of rice with CTAB method, further includes steps of

1.1. liquid nitrogen grinding 0.8-1.2g plant tissue is used, the powder after grinding is transferred in the centrifuge tube of pre-cooling；

1.2. 500 μ 2 × CTAB of L are added into centrifuge tube, place 30~60min after being mixed by inversion in 65 DEG C of water-baths, Period will turn upside down mixing one to repeatedly；

1.3. centrifuge tube is taken out, chloroform: isoamyl alcohol, mixing of turning upside down is added；

1.4. 12000rpm is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6. 12000rpm is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, turns upside down for several times, washes Wash precipitating；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7. 12000rpm is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and blows It is dry；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placements make for 4-12 hours It is sufficiently dissolved.

In order to solve the above technical problems, the present invention also provides in a kind of aforementioned any one third generation sterile line initiative The carrier of the construction method building of required carrier.

In order to solve the above technical problems, invention further provides a kind of methods for extracting paddy DNA with CTAB method, including with Lower step:

1.1. liquid nitrogen grinding 0.8-1.2g plant tissue is used, the powder after grinding is transferred in the centrifuge tube of pre-cooling；

1.2. 500 μ 2 × CTAB of L are added into centrifuge tube, place 30~60min after being mixed by inversion in 65 DEG C of water-baths, Period will turn upside down mixing one to repeatedly；

1.3. centrifuge tube is taken out, chloroform: isoamyl alcohol, mixing of turning upside down is added；

1.4. 12000rpm is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6. 12000rpm is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, turns upside down for several times, washes Wash precipitating；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7. 12000rpm is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and blows It is dry；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placements make for 4-12 hours It is sufficiently dissolved.

Beneficial effect of the present invention includes: the construction method of required carrier in third generation sterile line initiative of the present invention, is constructed Three above-mentioned linked genes are contained in double T-DNA carrier, one of area T-DNA, and hygromycin or card are contained in another area T-DNA The resistant genes such as that mycin, for callus conversion and the screening in plant regeneration stage.

Detailed description of the invention

Fig. 1 is the p CAMBIA1300 plasmid map after removal Hyg resistant gene described in the embodiment of the present invention；

Fig. 2 is DsRED expression cassette electrophoresis detection figure described in the embodiment of the present invention；

Fig. 3 is EAT1 expression cassette electrophoresis detection figure described in the embodiment of the present invention；

Fig. 4 is ZMAA1 expression cassette electrophoresis detection described in the embodiment of the present invention.

Specific embodiment

The present invention is described in detail below with reference to embodiment.To keep the objectives, technical solutions, and advantages of the present invention clearer, bright Really, the present invention is described in more detail below, but the invention is not limited to these embodiments.

Hygromycin gene and red fluorescent gene (DsRed) gene are carried out chain rear rice transformation maturation by the present invention The callus of embryonal induction is added hygromycin in callus cell culture screening stage, detects in combination with red fluorescence, fastly Fast, effective screening.

In one embodiment, the present invention provides the construction method of required carrier in a kind of initiative of third generation sterile line, packets Include following steps: three linked genes are contained in building double T-DNA carrier, one of area T-DNA, another area T-DNA contains anti- Property gene, for callus conversion and the plant regeneration stage screening.

Three linked gene is respectively as follows: DsRed gene, EAT1 gene, ZmAA gene.

The resistant gene is the resistant gene of hygromycin and/or kanamycins.

Building the area T-DNA containing three linked genes method include:

A, the DNA of rice is extracted with CTAB method；

B, three kinds of linked gene espression boxes are obtained respectively；

C, the acquisition of the pCAMBIA1300 plasmid of Hpt gene is removed；

D, the connection of each expression cassette；

E, the connection of segment and pCAMBIA1300-hpt- plasmid is merged.

Building the area T-DNA containing EAT1 gene method include: using step A obtain DNA as template, with For primer amplification rice EAT1 expression casette.

Building the area T-DNA containing DsRed gene method include: using the pGDR plasmid containing DsRED segment as template, It utilizes Primer amplification DsRED expression casette is obtained,

Building the area T-DNA containing ZmAA gene method include: using maize dna as template, with Gene ZmAA1 expression casette is obtained for primer amplification.

The step A extracts the DNA of rice with CTAB method, further includes steps of

1.1. liquid nitrogen grinding 0.8-1.2g plant tissue is used, the powder after grinding is transferred in the centrifuge tube of pre-cooling；

1.2. 500 μ 2 × CTAB of L are added into centrifuge tube, place 30~60min after being mixed by inversion in 65 DEG C of water-baths, Period will turn upside down mixing one to repeatedly；

1.3. centrifuge tube is taken out, chloroform: isoamyl alcohol, mixing of turning upside down is added；

1.4. 12000rpm is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6. 12000rpm is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, turns upside down for several times, washes Wash precipitating；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7. 12000rpm is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and blows It is dry；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placements make for 4-12 hours It is sufficiently dissolved.

In another embodiment, the present invention also provides required in a kind of aforementioned any one third generation sterile line initiative The carrier of the construction method building of carrier.

In another embodiment, the present invention is achieved by the following scheme:

A, the acquisition of DsRed expression casette；

B, the acquisition of EAT1 expression casette

C, the acquisition of ZmAA gene expression and box

D, the transformation of double carrier Ts

E, the connection of each expression cassette

Experimental procedure:

1, the DNA of rice is extracted with CTAB method, concrete operations are as follows:

1.1. the powder after grinding is transferred in the centrifuge tube of pre-cooling with liquid nitrogen grinding 1g or so plant tissue；

1.2. 500 μ L2 × CTAB (being pre-heated to 65 DEG C) is added into centrifuge tube, is put in 65 DEG C of water-baths after being mixed by inversion 30~60min is set, during which to turn upside down mixing for several times；

1.3. centrifuge tube is taken out, 24: 1 isometric (chloroforms: isoamyl alcohol), mixing of turning upside down is added；

1.4. 12000rpm is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6. 12000rpm is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, turns upside down for several times, washes Wash precipitating；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7. 12000rpm is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and blows It is dry；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placement a few hours make it Sufficiently dissolution.

2, the acquisition of each expression cassette

(1) using the DNA in 1 as template, with For primer amplification rice EAT1 expression casette, expand Increasing system is as follows:

PCR amplification program: 94 DEG C of initial denaturation 5min；94 DEG C, it is denaturalized 1min；56 DEG C (depending on different primers pair), annealing 1min；72 DEG C, extend 1min (depending on fragment length, 1kb/min), 30~35cycle；72 DEG C, extend 10min；4 DEG C of guarantors It deposits.

(2) it using the pGDR plasmid containing DsRED segment as template, utilizes Primer amplification obtains DsRED expression casette, and amplification system is as follows:

Distilled water constant volume is to 20 μ L

Amplification program:

(3) using maize dna as template, with To draw Object expands to obtain gene ZmAA1 expression casette.

3, the acquisition of the pCAMBIA1300 plasmid of Hpt gene is removed.

, from concatemerization, the plasmid of removal Hpt gene will be obtained after pCAMBIA1300 plasmid enzyme restriction with AscI first PCAMBIA1300-hpt-, specific digestion system and digestion program are as follows:

Then it is added 2 μ L and 10xT4 buffer of T4 ligase, 3 μ L in system, distilled water constant volume to 30 μ L, at 16 DEG C 12hour is kept, pCAMBIA1300-hpt- cyclic plasmid is obtained

4, the connection of each expression cassette

1.EAT1 expression cassette, three segments of DsRed expression cassette and ZmAA1 expression cassette template added with 1: 1: 1 number ratio Enter, using KOD archaeal dna polymerase, idle loop 5 times in the case where not adding primer；Then using DsRed-F-EcoR I F and EAT1-R-Hind III primer carries out amplification cycles 35 times, and specific reaction system is as follows:

2.PCR response procedures are as follows:

5, the connection of segment and pCAMBIA1300-hpt- plasmid is merged

First to the plasmid double digestion of pCAMBIA1300-hpt- cyclisation, digestion system is as follows:

37 DEG C of heat preservation 3hour

By on the carrier that is connected to that treated of the connection product in step 4, linked system

6, it takes 10 μ L to imported into Agrobacterium above-mentioned reaction solution, forms the engineering that can be used for mediated transformation plant cell Bacterium agrobacterium strains EHA105.

The preparation of Agrobacterium competent cell

Take the Agrobacterium of -70 DEG C of preservations in containing 50 μ g/ml rifampin plate streakings, 28 DEG C of cultures.

Picking single colonie is inoculated in 5ml YM fluid nutrient medium, 28 DEG C of shaken cultivation 12-16hr of 220rpm.

Take the switching of 2ml bacterium solution in 100ml YM fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5.

It is transferred to sterile centrifugation tube, 5000rpm is centrifuged 5min, removes supernatant.

The CaCl2 solution of the 0.1M of 10ml pre-cooling is added, gently suspension cell, places 20min on ice.4 DEG C of 5000rpm from Heart 5min, removes supernatant.

The CaCl2 solution of the 0.1M containing 15% glycerol of 4ml pre-cooling is added, gently suspends.

Agrobacterium suspension is sub-packed in sterile Eppendorf pipe, and every 200 μ l of pipe freezes in -70 DEG C.

Convert Agrobacterium

The above-mentioned Plasmid DNA built of 1 μ g or so is taken to be added in 200ml Agrobacterium competent cell, after mixing, ice Bathe 30min, -70 DEG C of placement 10min.Again in 37 DEG C of water-bath 5min or 42 DEG C of water-bath 1min, then ice bath 2min, is added 28 DEG C of 800mlYM fluid nutrient medium, 175rpm is coated on the YM plate containing 50 μ g/ml Kanamycin after shaking training 3hr.28 DEG C of trainings It supports to form single colonie,

It is reporter gene without hygromycin gene that the present invention provides a kind of based on DsRED fluorescence protein gene Engineered strain.

Segment is merged the present invention also provides a kind of, then a kind of experiment new approaches of conversion carrier.

Other explanations

1, primer needed for each expression cassette expands

The above is only several embodiments of the present invention, not any type of limitation is done to the present invention, although this hair It is bright to be disclosed as above with preferred embodiment, however be not intended to limit the invention, any person skilled in the art, it is not taking off In the range of technical solution of the present invention, a little variation or modification are made using the technology contents of the disclosure above and is equal to Case study on implementation is imitated, is belonged in technical solution of the present invention protection scope.

Sequence table

<110>Qingdao Yuan Ce Group Co., Ltd

<120>in a kind of initiative of third generation sterile line required carrier construction method

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atggagggca ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 1800

tacgagggcc acaacaccgt gaagctgaag gtgaccaagg gcggccccct gcccttcgcc 1860

tgggacatcc tgtcccccca gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc 1920

gacatccccg actacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1980

aacttcgagg acggcggcgt ggcgaccgtg acccaggact cctccctgca ggacggctgc 2040

ttcatctaca aggtgaagtt catcggcgtg aacttcccct ccgacggccc cgtgatgcag 2100

aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 2160

aagggcgaga cccacaaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 2220

aagtccatct acatggccaa gaagcccgtg cagctgcccg gctactacta cgtggacgcc 2280

aagctggaca tcacctccca caacgaggac tacaccatcg tggagcagta cgagcgcacc 2340

gagggccgcc accacctgtt cctgagatct cgagcaccac caccaccacc actaacctag 2400

gtagctgagc gcatgcgatc tcggcttcaa aacggtactg gattttggat tcaaacgaaa 2460

gccatcgcta caacagaaca aataaaagaa cattaatcaa aacgcataaa agatgggtta 2520

attgtattca ataaggagaa aagtaattcc tactagatag tttactatca cgcgaaagga 2580

tggccagtct tcactacggg aagacaacct cgctgggaat cgaaactctg tcaacgctgg 2640

aggtgccaac acatcttcgt aataaacatt tttacattta tccaggcgta aagaaacacg 2700

atttagttat caatttgtat ttttggttct tatgaagaat aaacttcttc aaattcactt 2760

ccacgaatat tccgtcccgt tccgccagtt ccattcgagc tc 2802

Claims (10)

1. the construction method of required carrier in a kind of third generation sterile line initiative, which comprises the following steps: building is double Three linked genes are contained in T-DNA carrier, one of area T-DNA, and resistant gene is contained in another area T-DNA, turn for callus Change and the screening in plant regeneration stage.

2. according to claim 1 the third generation sterile line initiative in required carrier construction method, which is characterized in that described three Linked gene is respectively as follows: DsRed gene, EAT1 gene, ZmAA gene.

3. according to claim 1 in the initiative of third generation sterile line required carrier construction method, which is characterized in that it is described anti- Property gene be hygromycin and/or kanamycins resistant gene.

4. according to claim 2 the third generation sterile line initiative in required carrier construction method, which is characterized in that building contain The method for having the area T-DNA of three linked genes includes:

A, the DNA of rice is extracted with CTAB method；

B, three kinds of linked gene espression boxes are obtained respectively；

C, the acquisition of the pCAMBIA1300 plasmid of Hpt gene is removed；

D, the connection of each expression cassette；

E, the connection of segment and pCAMBIA1300-hpt- plasmid is merged.

5. according to claim 4 the third generation sterile line initiative in required carrier construction method, which is characterized in that building contain The method for having the area T-DNA of EAT1 gene include: using step A obtain DNA as template, with For primer amplification rice EAT1 expression casette.

6. according to claim 4 the third generation sterile line initiative in required carrier construction method, which is characterized in that building contain The method for having the area T-DNA of DsRed gene includes: to utilize using the pGDR plasmid containing DsRED segment as template Primer amplification Obtain DsRED expression casette.

7. according to claim 4 the third generation sterile line initiative in required carrier construction method, which is characterized in that building contain The method for having the area T-DNA of ZmAA gene include: using maize dna as template, with Gene ZmAA1 expression casette is obtained for primer amplification.

8. according to claim 4 the third generation sterile line initiative in required carrier construction method, which is characterized in that the step Rapid A extracts the DNA of rice with CTAB method, further includes steps of

1.1. liquid nitrogen grinding 0.8-1.2g plant tissue is used, the powder after grinding is transferred in the centrifuge tube of pre-cooling；

1.2. 500 μ 2 × CTAB of L are added into centrifuge tube, place 30~60min after being mixed by inversion in 65 DEG C of water-baths, during which Mixing one turn upside down to repeatedly；

1.3. centrifuge tube is taken out, chloroform: isoamyl alcohol, mixing of turning upside down is added；

1.4.12000rpm, it is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6.12000rpm it is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, is turned upside down for several times, washing is heavy It forms sediment；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7.12000rpm it is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and dries up；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placements fill it in 4-12 hours Divide dissolution.

9. in a kind of initiative of the third generation sterile line as described in any one of claim 1-8 the construction method building of required carrier Carrier.

10. a kind of method for extracting paddy DNA with CTAB method, which comprises the following steps:

1.1. liquid nitrogen grinding 0.8-1.2g plant tissue is used, the powder after grinding is transferred in the centrifuge tube of pre-cooling；

1.2. 500 μ 2 × CTAB of L are added into centrifuge tube, place 30~60min after being mixed by inversion in 65 DEG C of water-baths, during which Mixing one turn upside down to repeatedly；

1.3. centrifuge tube is taken out, chloroform: isoamyl alcohol, mixing of turning upside down is added；

1.4.12000rpm, it is centrifuged 10min, upper strata aqueous phase is transferred in a new centrifuge tube after taking-up；

1.5. the dehydrated alcohol of 2 times of volumes pre-cooling is added into centrifuge tube, -20 DEG C of precipitating 15min are to overnight；

1.6.12000rpm it is centrifuged 10min, removes supernatant, the ethyl alcohol of 700 μ L70% is then added, is turned upside down for several times, washing is heavy It forms sediment；12000rpm is centrifuged 5min, removes supernatant, repeats the process 1 time；

1.7.12000rpm it is centrifuged 10min, removes supernatant, is inverted on blotting paper several minutes, is finally placed in super-clean bench and dries up；

1.8. the sterile deionized water or TE solution of 30~50 μ L are added in centrifuge tube, 4 DEG C of placements fill it in 4-12 hours Divide dissolution.