CN102492719B - Method for preparing broad-spectrum plant virus-resistant transgenic plant and application - Google Patents
Method for preparing broad-spectrum plant virus-resistant transgenic plant and application Download PDFInfo
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- CN102492719B CN102492719B CN 201110402593 CN201110402593A CN102492719B CN 102492719 B CN102492719 B CN 102492719B CN 201110402593 CN201110402593 CN 201110402593 CN 201110402593 A CN201110402593 A CN 201110402593A CN 102492719 B CN102492719 B CN 102492719B
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Abstract
The invention discloses a method for preparing a broad-spectrum plant virus-resistant transgenic plant and application. The method comprises the following steps of: a, constructing a transformation vector, namely performing restriction enzyme digestion on an aiiA gene of Bacillus thuringiensis by using restriction enzyme, connecting the gene to a plant binary transformation vector pA8, and transferring the connected plant binary transformation vector pA8-aiiA into a strain EHA105 of Agrobacterium tumefaciens by a freeze-thawing method; and b, performing genetic transformation of plants, namely transforming selenium tobacco and Nicotiana benthamiana respectively by a leaf disc transformation method, screening on a Murashige and Skoog (MS) culture medium containing hygromycin to obtain transformed seedlings, and obtaining intact regenerated plants through a rooting culture medium, wherein the regenerated plants comprise a pA8 regenerated plant for transforming an empty vector and a regenerated plant for transforming the pA8-aiiA. The method is feasible and easy to operate, and can be widely applied to the control of viral diseases of economical crops because the broad-spectrum resistance to different plant viruses is strengthened.
Description
Technical field
The present invention relates to biological technical field, more specifically relate to the preparation method of a kind of transgenic plant of wide spectrum Antiphytoviral.The purposes that also relates to simultaneously a kind of transgenic plant of wide spectrum Antiphytoviral, specifically, the present invention relates to that colony induction signaling molecule protein degradation encoding gene aiiA passes through Agrobacterium conversion tobacco in the Tribactur, obtain the transgene tobacco of wide spectrum Antiphytoviral, different types of important plant virus such as tobacco mosaic virus (TMV), potato virus X, marmor upsilon etc. are had preferably antiviral effect.Technological method of the present invention can be widely used in the virus disease control of cash crop.
Background technology
Occurring in nature, a lot of plants all can be subject to virus infection and cause virus disease.Though plant virus is strict cytozoon, specialization is not strong, and often a kind of virus can colonize on the plant not of the same race, that genus is even not equal, and Combined Infection usually occurs.Almost every kind of farm crop and cash crop all are subject to the harm of several even tens kinds of viruses and the underproduction or total crop failure.The viroses of plant are a kind of global diseases, and the crop loss that the annual whole world is caused by the viroses of plant is difficult to counting.China is large agricultural country, and along with the change of ecological condition and the introduction of plant germplasm resource, viral species has gradually and increases the trend that harm increases the weight of gradually.
Virus obligate parasitism in vegetable cell, it copies required energy, material, place and is provided by host cell fully, and plant does not have complete immune metabolic system in addition, and this is so that the control of the viroses of plant is very difficult.What mostly use for the control of viral diseases of plants is aggregate measures, but all there is limitation in these traditional methods.As can killing with agricultural chemicals the insect of virus spread for some arbovirus, but the used chemical pesticide of this method easily causes environmental pollution, and not remarkable to the disease drug effect behind the virus infection plant.Can organize detoxicity method to obtain the planting material of anosis viral disease for being applicable to vegetative farm crop, but that shortcoming is cost is high, workload is large, particularly because infecting again of field kept nontoxic validity period not long.The in recent years birth of genetic engineering for plant virus resistance has brought hope for people prevent and treat virus disease.
Dong in 2000 etc. have been cloned into the protein gene aiiA of coding degraded quorum sensing (Quorum sensing) signaling molecule N-acyl homoserine lactones (N-acyl-homoserine lactones, AHLs) from a strain bacterial strain of bacillus 240B 1.After using afterwards the agrobacterium tumefaciens method this gene transformation tobacco, potato, potato, tobacco have been strengthened to the resistance of Chinese cabbage soft rot bacteria (Erwinia carotovora SCGl).Find that in addition AiiA albumen can not only improve transgenic plant to the resistance against diseases of pathogenetic bacteria, can also improve the immunizing power of animal.But also there is not relevant report about this gene in the function aspect the Antiphytoviral.
Summary of the invention
The objective of the invention is to be to provide the preparation method of a kind of transgenic plant of wide spectrum Antiphytoviral, easy to implement the method, easy and simple to handle, enhancing is to the resistance of wide spectrum of different plant viruses, the transgenic plant that obtain can strengthen the resistance to different plant viruses, can be widely used in the virus disease control of cash crop.
Another object of the present invention is the application of transgenic plant in tobacco that has been to provide a kind of wide spectrum Antiphytoviral, the major advantage of this invention is the disease resisting effect that important plant virus is had wide spectrum, and the transgenosis that can be applied to staple crop is antiviral.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The preparation method of a kind of transgenic plant of wide spectrum Antiphytoviral the steps include:
A. the structure of conversion carrier:
With Tribactur (Bacillus thuringiensis, the Yuan Zhiming researcher of our unit is so kind as to give, ask for an interview the genetic resources table) the aiiA gene to be connected to plant binary conversion carrier pA8 after with digestion with restriction enzyme upper (available from pCAMBIA 1305.1 carriers of CAMBIA company, referred to as the pA8 carrier, ask for an interview the genetic resources table), by freeze-thaw method the plant binary conversion carrier pA8-aiiA that connects is changed among agrobacterium strains (Agrobacterium tumefaciens) EHA105 (professor S.Gelvin of Purdue Univ-West Lafayette USA is so kind as to give, and asks for an interview the genetic resources table).
B. Genetic Transformation in Higher Plants:
By leaf dish method for transformation (with reference to Horsch et al., 1985) transform respectively coral selenium tobacco (Nicotiana tabacum cv.Xanthi) and this uncured tobacco (N.benthamiana), containing the MS substratum of Totomycin (MS+1.0mg/L BA+0.1mg/L NAA+30mg/L Hygromycin+100mg/LTimentin) screening acquisition transformation seedlings, obtain complete aftergrowth by root media (MS+0.1mg/LNAA+30mg/LHygromycin+100mg/L Timentin), comprise the pA8 aftergrowth that transforms empty carrier and the aftergrowth that transforms pA8-aiiA.
The present invention finds that the aiiA gene with bacillus thuringiensis changes in the tobacco by Agrobacterium and expresses, can obtain the transgene tobacco of wide spectrum Antiphytoviral.
The application of a kind of transgenic plant of wide spectrum Antiphytoviral in tobacco, its application process is as follows:
When reconstituted tobacco plant length has 4-5 sheet leaf, select PCR and RT-PCR to detect the blade of the transfer-gen plant of the same size that all is positive as material.Inoculate different plant viruses by the method for frictional inoculation, observe anaphylaxis Lesion size and fluorescence size and the power of inoculation position, relatively incidence.
The transgenic plant that the present invention relates to can show the resistance of wide spectrum to plant virus.May have resistance of wide spectrum to different pathogens (virus, bacterium, fungi etc.), can be widely used in the virus disease control of cash crop.
The present invention compared with prior art has the following advantages and effect:
The control of plant virus does not have effective means at present, and prior art is just carried out for a certain plant virus, often just plays certain preventive effect, occurs then to be difficult to control after the plant virus infection.Existing transgenic technology is also just for a certain specific plant virus.The transgenic plant that the present invention obtains have the effect of wide spectrum Antiphytoviral, and the main virus of present serious harm crop is all shown comparatively ideal disease resisting effect.
Description of drawings
Fig. 1 is PCR and the RT-PCR detected result figure that turns the aiiA genetic tobacco.
The reconstituted tobacco plant that obtains is carried out PCR and RT-PCR detection, and upper row is the PCR detected result, and lower row is the RT-PCR detected result, and M is dna molecular amount mark, the difference regeneration strain of 1-8 for obtaining ,-negative contrast.
Fig. 2 turns the aiiA genetic tobacco to the resistance of tobacco mosaic virus (TMV) TMV figure as a result.
Detect transgene tobacco to the resistance of TMV virus, A figure is the pA8 coral selenium tobacco that transforms the empty carrier contrast, and B figure is the coral selenium tobacco that transforms pA8-aiiA.The anaphylaxis scab that the tobacco of conversion pA8-aiiA gene forms is less and less than the pA8 tobacco that transforms the empty carrier contrast.C figure is this uncured tobacco of pA8 that transforms the empty carrier contrast, and D is for transforming this uncured tobacco of pA8-aiiA.E figure is the TMV that detects the GFP mark under ultra-violet lamp, is this uncured tobacco that transforms pA8-aiiA above wherein, and is lower to transforming this uncured tobacco of pA8 of empty carrier contrast.This uncured tobacco of transgenosis GFP fluorescence intensity is starkly lower than and transforms the empty carrier contrast.
Fig. 3 turns the aiiA genetic tobacco to the resistance of potato virus X PVX figure as a result.
Detect transgene tobacco to the resistance of PVX virus, A figure is the pA8 tobacco that transforms the empty carrier contrast, and B figure is the tobacco that transforms pA8-aiiA.The line shape anaphylaxis scab that the tobacco of conversion pA8-aiiA gene forms is thinner and more weak than the pA8 tobacco that transforms the empty carrier contrast.
Fig. 4 turns the aiiA genetic tobacco to the resistance of marmor upsilon PVY figure as a result.
Detect transgene tobacco to the resistance of PVY virus, A figure is the pA8 tobacco leaf that transforms the empty carrier contrast, and B figure is the tobacco leaf that transforms pA8-aiiA.Transform the anaphylaxis scab that the tobacco of pA8-aiiA gene forms more weak and less than the pA8 tobacco medicine that transforms the empty carrier contrast.
Fig. 5 turns the aiiA genetic tobacco to the resistance of PPV virus figure as a result.
Detect the resistance of this uncured tobacco of transgenosis PPV virus.A figure is this uncured tobacco that transforms pA8, and B figure is this uncured tobacco that transforms pA8-aiiA, and C is the PPV that detects the GFP mark under ultra-violet lamp.
Embodiment
The invention will be further described below in conjunction with embodiment.Experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment 1:
The preparation method of a kind of transgenic plant of wide spectrum Antiphytoviral the steps include:
. the structure of conversion carrier and Plant Transformation:
The structure of conversion carrier (. the structure of conversion carrier and Plant Transformation) according to bacillus thuringiensis (B.thuringiensis; the Yuan Zhiming researcher of our unit is so kind as to give; ask for an interview the genetic resources table) aiiA gene order design primer; 5 ' end at primer adds respectively Bgl II and BstE II restriction enzyme site and protection base; extract total DNA of bacillus thuringiensis (acquisition is separated in this laboratory voluntarily); go out aiiA gene (Wang etc. by pcr clone; Science in China Series C:Life Sciences; 2007; 50 (3): 385-39); be connected to behind the double digestion with the expression vector pA8 behind Bgl II and the BstE II double digestion (available from pCAMBIA 1305.1 carriers of CAMBIA company; referred to as the pA8 carrier) on, called after pA8-aiiA plasmid.Empty carrier pA8 is changed among Agrobacterium (A.tumefaciens) the bacterial strain EHA105 (preserve in this laboratory, and professor S.Gelvin of Purdue Univ-West Lafayette USA is so kind as to give, and asks for an interview the genetic resources table) by freeze-thaw method respectively with the pA8K-aiiA plasmid that builds.
B, Plant Transformation:
With above-mentioned conversion Agrobacterium (A.tumefaciens EHA105) difference incubated overnight, centrifugal, thalline suspends with liquid MS1/10 substratum.Coral selenium tobacco (N.tabacum cv.Xanthi) and this uncured tobacco (N.benthamiana) tobacco leaf are cut into the fritter of 0.5cm * 0.5cm, a part is put into pA8 plasmid bacterium liquid and was soaked 15 minutes, the bacterium liquid that a part is put into the pA8-aiiA plasmid soaked 15 minutes, a part is put into liquid MS medium and was soaked 15 minutes, after blotting unnecessary bacterium liquid or liquid with filter paper, be placed on and cultivated altogether on the MS substratum three days, then forward on the screening culture medium (MS+1.0mg/L BA+0.1mg/L NAA+30mg/L Hygromycin+100mg/L Timentin), simultaneously, a blade part of soaking with liquid MS medium is forwarded on the normal substratum (MS+1.0mg/L BA+0.1mg/L NAA), a part is transferred on the screening culture medium as the contrast that transforms blade, 26 ℃, the 16h illumination cultivation is carried out preliminary screening, and per two weeks are changed a subculture.The seedling of regeneration goes to root media (MS+0.1mg/LNAA+30mg/L Hygromycin+100mg/L Timentin) and takes root.The result shows that the leaf dish after the conversion selecting substratum growth 3-4 can form callus after week and differentiation is sprouted, and the bud that directly differentiates changes that root media can be taken root and continued growth over to, forms regeneration plant.
Embodiment 2:
The application of a kind of transgenic plant of wide spectrum Antiphytoviral in tobacco, its application process is as follows:
Transformation of tobacco is to the resistance of different plant viruses:
When reconstituted tobacco plant length had 4-5 sheet leaf, selection PCR and RT-PCR detected the transfer-gen plant of the same size of be positive (Fig. 1) as supplying the examination plant.Different plant virus toxogens are added appropriate amount of buffer solution (K
2HPO
41g/100mL, Na
2SO
30.1g/100mL) and appropriate amount of quartz sand grind to form together homogenate, dip viral juice with writing brush and pass through the method for frictional inoculation the different plant viruses of Plant Leaf surface seeding, comprise tobacco mosaic virus (TMV) (TMV), potato X and Y virus (PVX and PVY), the TMV of GFP mark virus (being called for short GFP-TMV) and Plum pox potyvirus virus (abbreviation GFP-PPV) (TMV wherein, PVX, PVY virus is provided by applicant unit one belongs to-Wuhan Virology Institute,Chinan academy of Sciences street virus preservation center, GFP-TMV and GFP-PPV are so kind as to give by the Guo Huishan researcher of Institute of Microorganism, Academia Sinica, ask for an interview the genetic resources table).Use the residual juice on the clear water flush away inoculation blade after the inoculation, postvaccinal tobacco is placed in the greenhouse cultivates, observe anaphylaxis Lesion size power and the fluorescence area discrepancy of the formation of inoculation position in 7-14 days, more susceptible situation.
Resistance detection to different plant viruses describes to transformation of tobacco below in conjunction with different virus:
1. transformation of tobacco is to the antiviral effect of tobacco mosaic virus (TMV).
With the coral selenium tobacco of above-mentioned conversion and the seed germination of this uncured tobacco acquisition, in the sand of plantation after sterilization.When the transformation of tobacco plant grows to 4-5 sheet leaf, detect by PCR and RT-PCR, conclusive evidence transforms succeed (Fig. 1).Select transfer-gen plant of the same size as being used for the Antiphytoviral effect for the examination plant.Tobacco mosaic virus (TMV) is added appropriate amount of buffer solution (K
2HPO
41g/100mL, Na
2SO
30.1g/100mL) and appropriate amount of quartz sand grind to form together homogenate, dip the method for viral juice by frictional inoculation in Plant Leaf surface seeding tobacco mosaic virus (TMV) (TMV) with writing brush, and the TMV of GFP mark virus.Use the residual juice on the clear water flush away inoculation blade after the inoculation, postvaccinal tobacco is placed in the greenhouse cultivates, observe anaphylaxis Lesion size power and the fluorescence area discrepancy of the formation of inoculation position in 7-14 days, relatively antiviral effect the results are shown in Figure 2.
The coral selenium tobacco that as can be seen from the figure transforms the pA8-aiiA gene obviously is eager to excel to the resistance of tobacco mosaic virus (TMV) than the pA8 tobacco that transforms the empty carrier contrast, and scab is little and few, and susceptible symptom is light.This uncured tobacco that transforms the pA8-aiiA gene also obviously is eager to excel to the resistance of TMV virus than this uncured tobacco of pA8 that transforms the empty carrier contrast, and the area of the green fluorescence of formation is wanted much less.
2. transformation of tobacco is to the antiviral effect of potato virus X.
Adopt above-mentioned same method to detect the coral selenium tobacco of conversion to the antiviral effect of potato X, the results are shown in Figure 3.The line shape anaphylaxis scab that the tobacco of conversion pA8-aiiA gene forms is thinner and more weak than the pA8 tobacco that transforms the empty carrier contrast.
3. transformation of tobacco is to the antiviral effect of marmor upsilon.
Adopt above-mentioned same method to detect the coral selenium tobacco of conversion to the antiviral effect of potato Y, the results are shown in Figure 4.Transform anaphylaxis scab that the tobacco of pA8-aiiA gene forms than the pA8 tobacco that transforms the empty carrier contrast a little less than and lack.
4. transformation of tobacco is to the antiviral effect of Plum pox potyvirus (PPV) virus.
Adopt above-mentioned same method to detect this uncured tobacco of conversion pA8-aiiA gene to the antiviral effect of GFP-PPV, the results are shown in Figure 5.This uncured tobacco that transforms the pA8-aiiA gene obviously is eager to excel to the resistance of PPV virus than this uncured tobacco of pA8 that transforms the empty carrier contrast, and the area of the green fluorescence of formation is wanted much less.
Claims (2)
1. the preparation method of the transgenic plant of a wide spectrum Antiphytoviral the steps include:
A. the structure of conversion carrier:
AiiA gene order design primer according to bacillus thuringiensis, 5 ' end at primer adds respectively Bgl II and BstE II restriction enzyme site and protection base, extract total DNA of bacillus thuringiensis, go out the aiiA gene by pcr clone, be connected to behind the double digestion with on the expression vector pA8 behind Bgl II and the BstE II double digestion, by freeze-thaw method the plant binary conversion carrier pA8-aiiA that connects changed among the agrobacterium strains EHA105;
B. Genetic Transformation in Higher Plants:
Transform respectively coral selenium tobacco and this uncured tobacco by leaf dish method for transformation, obtain transformation seedlings in the MS substratum screening that contains Totomycin, obtain complete aftergrowth by root media MS, comprise the pA8 aftergrowth that transforms empty carrier and the aftergrowth that transforms pA8-aiiA;
Described MS substratum is: the special U.S.A of MS+1.0mg/L 6-benzyl aminoadenine+0.1mg/L naphthylacetic acid+30mg/L Totomycin+100mg/L stings;
Described root media is that the special U.S.A of MS+0.1mg/L naphthylacetic acid+30mg/L Totomycin+100mg/L stings;
Described pA8 carrier is pCAMBIA 1305.1 carriers available from CAMBIA company.
2. the application of the preparation method of the transgenic plant of a kind of wide spectrum Antiphytoviral claimed in claim 1 in the transgene tobacco of preparation Antiphytoviral.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1594583A (en) * | 2004-06-25 | 2005-03-16 | 中国科学院武汉病毒研究所 | Transgenic plant and biological repair applied to polluted environment thereof |
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| CN1594583A (en) * | 2004-06-25 | 2005-03-16 | 中国科学院武汉病毒研究所 | Transgenic plant and biological repair applied to polluted environment thereof |
Non-Patent Citations (3)
| Title |
|---|
| Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase;Yi-Hu Dong et al.;《NATURE》;20010614;第411卷;第813-817页 * |
| Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease;Huifang Ban et al.;《Plant Cell Rep》;20091231;第28卷(第12期);第1847-1855页 * |
| 苏云金芽孢杆菌口i诅基因载体构建及石斛兰转化;潘丽晶等;《中山大学学报(自然科学版)》;20090131;第48卷(第1期);第67-71页 * |
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