CN103184226A - Novel cinnamomum hupehanum ribosome inactivated protein gene CcRIPII - Google Patents

Abstract

The invention designs a PCR (Polymerase Chain Reaction) primer according to a conserved sequence of a reported plant ribosome inactivated protein gene CcRIPII, and obtains a novel partial sequence of a novel cinnamomum hupehanum ribosome inactivated protein gene CcRIPII through homologous amplification; subsequently a full-length gene cDNA (Complementary Deoxyribonucleic Acid) sequence is obtained through RACE (Rapid-Amplification of Complementary Deoxyribonucleic Acid Ends); the CcRIPII gene does not contain internal functors but contains a complete ORF (Open Reading Frame) area; and the ORF contains full-length 1758 nucleotides, coding 585 amino acids and one termination codon (TGA). A plant expression carrier pCAMBUA2300-CcRIPII of the CcRIPII gene is established, and a transgenetic seedling is obtained by using pCAMBIA2300-CcRIPII transformed Ningyang Chinese-date. The CcRIPII is the first woody plant ribosome inactivated protein gene which is cloned and subjected to wood genetic transformation on the international.

Description

A kind of new fragrant camphor tree ribosome-inactivating protein gene CcRIPII

One, technical field

The present invention relates to a kind of new ribosome-inactivating protein gene CcRIPII of Cortex cinnamomi camphorae.The CcRIPII gene does not have interior functor, but contains the complete ORF zone, 1758 Nucleotide of its encoding sequence total length (29-1786), encode 585 amino acid and a terminator codon (TGA).

Made up the plant expression vector pCAMBIA2300-CcRIPII of CcRIPII gene, transformed Ningyang's date with pCAMBIA2300-CcRIPII and obtained transfer-gen plant.CcRIPII clones and finishes first xylophyta ribosome-inactivating protein gene that forest tree genetic is transformed in the world, and its clone and the success that forest tree genetic is transformed are significant to the antiviral research of xylophyta and genetic breeding.

Two, background technology

Ningyang's date in Shandong is the famous good local speciality of China, and the popular development that has a strong impact on Ningyang's date of virus disease has caused heavy losses in recent years.Virus disease not only threatens to Ningyang's date, but also endangers the production of multiple fruit tree.Some Cortex cinnamomi camphorae that the Feng Dian of Taian Taishan Academy of Forestry Sciences is neat and Liu Jing researcher finds and Ningyang's date is planted in areal have highly resistance to virus disease.

Ribosome-inactivating protein gene (RIP) is a class broad-spectrum disease resistance virus gene, avoids having vital role aspect the viral harm at protective plant.Existing many reports are about the report of RIP gene-transformed plant, acquisition antivirus plant new germ plasm both at home and abroad.But the genetic transformation of xylophyta is not seen successfully report so far as yet.A breach is opened in this research hope in this field.

Three, summary of the invention

The present invention is according to the plant ribosome inactivating protein gene conservative sequences Design PCR primer of having reported, and amplification has obtained the partial sequence of a kind of new Cortex cinnamomi camphorae ribosome-inactivating protein gene CcRIPII through homology.Obtained this full length gene cDNA sequence (Fig. 1) by the RACE amplification then.The CcRIPII gene does not contain interior functor, but contains the complete ORF zone, and 1758 Nucleotide of ORF total length are encoded 585 amino acid and a terminator codon (TGA) (Fig. 1).Made up the plant expression vector pCAMBIA2300-CcRIPII (Fig. 3) of CcRIPII gene subsequently, transformed Ningyang's date with pCAMBIA2300-CcRIPII and obtained transfer-gen plant.CcRIPII clones and finishes first xylophyta ribosome-inactivating protein gene that forest tree genetic is transformed in the world.

The double base cloning vector pCAMBIA2300 of our usefulness (available from company Australia Cambia company, its structure is seen Fig. 2) has made up the plant expression vector of CcRIPII gene.When making up the plant expression vector of goal gene CcRIPII, the CcRIPII gene fragment that molecular cloning is obtained is inserted among the carrier pCAMBIA2300 at SalI and KpnI double enzyme site, and the expression vector that builds up is named as pCAMBIA2300-CcRIPII (Fig. 3).Used marker gene is the neomycin phosphotransferase gene (NPTII) of widespread use, and the product of its coding has resistance to glucosamine glycoside microbiotic such as kantlex etc.When making up this expression vector, consider too conference influence expression of fusion gene, therefore do not comprise reporter gene GUS.

Because the structure of herbaceous plant and xylophyta differs widely, the gene that past RIP gene genetic Study on Transformation is used all is from herbaceous plant, and plant transformed also all is herbaceous plant.We use the RIP gene transformation xylophyta from xylophyta in this research, and goal gene is easier expression in the xylophyta host.

In addition, our goal gene used in transforming is grown in the plant of areal from the locality with recipient plant, plant transformed to environment without any influence.

According to CcRIPII gene order and used carrier design a pair of specific PCR primer, the forward primer design is on 5 ' end carrier sequence, the reverse primer design is at 3 of goal gene ' end, can guarantee that like this pcr amplification product can only be from the CcRIPII gene that imports, and can not improve the specificity and the accuracy that detect from the native gene sequence.In addition, be 550bp with this PCR product that primer amplified is gone out, size to fit, easy to detect, the efficient height.

Four, embodiment

(1) with the Regeneration in Vitro system of xylophyta as converting material, utilize the During Agrobacterium method to carry out genetic transformation.

(2) conversion operation step

1. Agrobacterium-mediated Transformation method

(1) by the triparental cross method pCAMBIA2300-CcRIPII expression vector is transformed in the agrobacterium tumefaciens lba4404, is used for the During Agrobacterium transformation experiment.

(2) bacterium liquid preparation

1) scrapes the culture surface that the agrobacterium strains that contains recombinant plasmid freezes with transfering loop, line and contain on the corresponding antibiotic above-mentioned YEP flat board, place 28 ℃ of constant incubators to cultivate 2 days, when treating to have on the flat board bacterium colony to grow, namely can be used for liquid culture.The flat board that grows bacterium colony places 4 ℃ of refrigerators to preserve.Be transferred to after one month on the new YEP flat board, to keep the activity of bacterium.

2) picking list bacterium colony from the flat board, be inoculated in contain the 10ml additional phase should antibiotic YEP liquid nutrient medium (pH7.0) in, on the constant temperature shaking table, in 28 ℃, the 180-200rpm shaking culture is spent the night.

3) morning next day is as bacterium liquid OD ₆₀₀During=0.5-0.6, can be used for transforming.(be generally on the experience and carry out shaking culture about 5 of afternoons on the previous day, next day, morning, 8 left and right sides bacterial concentrations can reach standard)

Remarks: add in the substratum antibiotic the time, must wait until the substratum cooling but (the endurable temperature of staff) adding when not solidifying, otherwise microbiotic can lose efficacy at high temperature.

(3) Agrobacterium-mediated Transformation

1) the pre-cultivation: aseptic Regeneration in Vitro is organized a transverse cutting 2-3 otch, after be seeded in and cultivated in advance on the division culture medium 1-2 days.

2) cultivate altogether: with sterilized water with Agrobacterium bacterium liquid dilution 5-20 doubly, to be immersed in the diluent 10-20 minute through pre-incubated Regeneration in Vitro tissue, take out the stem section, after blotting with aseptic filter paper, change dark the cultivation 2-4 days on the division culture medium over to, control treatment is that the Regeneration in Vitro of will cut wound is organized in and soaks 10-20 minute in the sterilized water, and other processing is the same with experiment material.

3) select to cultivate: after Regeneration in Vitro tissue and Agrobacterium are cultivated altogether, can see that the Regeneration in Vitro organization edge has milky Agrobacterium bacterium colony to occur, and with aseptic water washing stem section, blots with aseptic filter paper then.The Regeneration in Vitro tissue transferred to be added with taking off on the bacterium division culture medium of select pressing, at illumination 4000-10000lx, select under 25 ℃ of conditions to cultivate.

4) the subculture selection is cultivated: select to cultivate 3-4 after week, the transformant of Regeneration in Vitro tissue will differentiate the resistance indefinite bud, change these resistant materials over to corresponding increasing and select the bacterium that takes off of pressure to select to cultivate in the substratum.

5) root culture: when treating more than the indefinite bud length to 2 centimetre, downcut also to insert and contain taking off of selecting to press and carry out root culture on the bacterium root media, grow adventive root about two weeks, obtain transformed plant.

(3) PCR Molecular Detection

1, the extraction of the total DNA of plant (adopting the CTAB method)

(1) takes by weighing the fresh blade of 2 gram left and right sides plant test-tube plantlets, put in the mortar, add the PVPP of 1/10 vegetable material volume, pour liquid nitrogen into vegetable material is ground to form smalls.

(2) this powder is put into big centrifuge tube, add 65 ℃ of preheatings with the isopyknic 2 * CTAB extracting solution of vegetable material, add 1% beta-mercaptoethanol again, put upside down mixing repeatedly, 65 ℃ of insulation 15-20min.

(3) take out, cool to room temperature adds isopyknic chloroform, gently put upside down about 10min, centrifugal 15 minutes of room temperature 12000rpm repeatedly.

(4) supernatant liquor is placed another centrifuge tube, repeat previous step 1-2 time.

(5) add 2 times of volume dehydrated alcohols, fully mixing is scratched out flocks with the glass hook, places the Eppendorf pipe of piling 70% ethanol in advance, washing DNA, and 12000rpm is centrifugal, outwells ethanol.

(6) add 2 times of volume 70% ethanol, wash DNA2 time.

(7) dry up, be dissolved in an amount of TE (pH8.0) damping fluid.

(8) add RNase solution, make final concentration reach 10mg/ml, handle 20min for 35 ℃.Use phenol then: chloroform: primary isoamyl alcohol (24: 24: 1), extracting once, upward please liquid use chloroform again: primary isoamyl alcohol (24: 1), extracting is once.Be settled out DNA with the two volumes dehydrated alcohol.Wash twice with 75% ethanol again, dry up, DNA be dissolved in the sterilized water be put in-20 ℃ standby.

2, polymerase chain reaction (PCR) detects

(1) reaction system

, as negative control, the transformation tissue culture seedling is carried out pcr amplification detect with the tissue cultured seedling of unconverted as positive control with plasmid DNA.

Design a pair of Auele Specific Primer according to the CcRIPII gene order.

35S-F (5 ' forward primer) AGG GAT GAC GCA CAATCC CAC TAT (be positioned at pCABIA2300-35S carrier cloning site-150bp place).

GSP3-403R (3 ' reverse primer) CAATTG CAG GGT CAG GAT TAT CTT CAC GT (positions of 403 bases in the middle of 5 of this primer ' be positioned at RIPII gene C cRIPII).

The PCR product is 550bp.

The forward primer design is on 5 ' end carrier sequence, the reverse primer design is at 3 of goal gene ' end, be 550bp with this PCR product that primer amplified is gone out, comprising is the complete sequence of goal gene, can guarantee that like this pcr amplification product can only be from the CcRIPII gene order that imports, and can not improve the specificity and the accuracy that detect from the native gene sequence.The used probe of Southern hybridization is the fragment that pcr amplification goes out, warp ³²As the probe of Southern hybridization, what PCR and Southern hybridization detected the dual positive then is considered to transfer-gen plant behind the P-dCTP mark.

1) reaction solution

2) response procedures

Pre-sex change: 94 ℃, 5min

35 circulations of increasing under the following conditions: sex change, 94 ℃ of 45sec; Renaturation, 55 ℃ of 45sec; Extend, 72 ℃, 1min;

Extend at last: 72 ℃, 5min:4 ℃, preserve standby.

Five, accompanying drawing and explanation thereof

The encoding sequence (A) of accompanying drawing 1.CcRIPII gene and the aminoacid sequence of deriving (B).

The encoding sequence of the fragrant camphor tree ribosome-inactivating protein gene of Fig. 1-A., initial numeral is ATG, terminator codon TGA.

The aminoacid sequence of the fragrant camphor tree ribosome inactivating protein of Fig. 1-B. ,-symbol illustrates the termination codon position

The configuration diagram of the binary vector pCAMBIA2300 that accompanying drawing 2. clone's goal gene are used.

The structure iron of accompanying drawing 3.CcRIPII gene plant expression vector pCAMBIA2300-CcRIPII.

Claims (5)

1. the present invention has cloned a kind of new ribosome-inactivating protein gene CcRIPII from the Cortex cinnamomi camphorae of China's viral diseases.The CcRIPII gene does not contain interior functor, but contains the complete ORF zone, and 1758 Nucleotide of its encoding sequence total length (29-1786) are encoded 585 amino acid and a terminator codon (TGA) (Fig. 1).

2. with the pCAMBIA2300 plant expression vector pCAMBIA2300-CcRIPII (Fig. 3) of CcRIPII that has been vector construction, CaMV35S promotor, goal gene, terminator OCS and marker gene NPTII combine and are built into mosaic gene in the expression vector that builds up, and are conducive to transform and express.

3. transform Ningyang's date with pCAMBIA2300-CcRIPII, obtained transfer-gen plant.

4.CcRIPII be to clone and finish first xylophyta ribosome-inactivating protein gene that forest tree genetic is transformed in the world, we have completely independent intellecture property.Therefore the CcRIPII gene source transforms the easier expression of xylophyta in xylophyta, and transfer-gen plant does not have detrimentally affect to environment.Its clone and significant to the antiviral research of xylophyta and genetic breeding to the success of forest tree genetic conversion.

5. design a pair of specific PCR primer according to the CcRIPII gene order.

35S-F (5 ' forward primer) AGG GAT GAC GCA CAA TCC CAC TAT (be positioned at pCABIA2300-35S carrier cloning site-150bp place).

GSP3-403R (3 ' reverse primer) CAA TTG CAG GGT CAG GAT TAT CTT CAC GT (positions of 403 bases in the middle of 5 of this primer ' be positioned at RIPII gene C cRIPII).

The PCR product is 550bp.

Because the forward primer design is on 5 ' end carrier sequence, the reverse primer design is at 3 of goal gene ' end, can guarantee that like this pcr amplification product can only be from the CcRIPII gene that imports, and can not improve the specificity and the accuracy that detect from the native gene sequence.In addition, be 550bp with this PCR product that primer amplified is gone out, size to fit, easy to detect, the efficient height.

1) reaction solution:

2) response procedures

Pre-sex change: 94 ℃, 5min

35 circulations of increasing under the following conditions: sex change, 94 ℃ of 45sec; Renaturation, 55 ℃ of 45sec; Extend, 72 ℃, 1min;

Extend at last: 72 ℃, 5min4 ℃, preserve standby.

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