CN101914540A

Movatterモバイル変換

Info

Publication number: CN101914540A
Application number: CN2010102482138A
Authority: CN
Inventors: 侯义龙
Original assignee: Dalian University
Current assignee: Dalian University
Priority date: 2010-08-09
Filing date: 2010-08-09
Publication date: 2010-12-15

Abstract

The invention discloses a method for introducing RNA interference into plants. The method comprises the following steps of: 1, designing and synthesizing siRNA according to a target gene sequence; 2, preparing 1 to 10 percent solution of siRNA by using double distilled water; and 3, spraying the solution of siRNA on the surface of plant leaves. According to the method, the RNA interference is introduced into the plants from the surface of the leaves, and stomatal cells and epidermic cells of the plants can absorb the siRNA. After the siRNA fluorescently-labeled enters the cells, the siRNA can also be detected by a flow cytometer. The method for introducing the RNA interference into the plants of the invention has the advantages of simple and convenient operation, high speed, low cost, quick response, high timeliness, stability, high efficiency, low using cost, easy popularization and the like.

Description

A kind of method of RNA being disturbed introduced plant

Technical field

The present invention relates to a kind of RNA interferential introducing method, more particularly, relate to the method that RNA disturbs introduced plant.

Background technology

The application of RNAi in nematode, fruit bat and plant at present obtained a lot of achievements, and research in Mammals and application have also obtained many progress.The gene silencing of RNA mediation is the self-protective mechanism of plant on the gene regulating level; play a significant role at the intrusion of antagonism foreign DNA, virus infection, DNA swivel base with in resetting; having ubiquity, also is the result of gene expression regulation in the vegetable cell simultaneously.At present, RNAi has become one of popular research field, and its potential using value is very huge.Can predict, be applied to the parsing of large-scale plant function genome, plant disease-resistant and the aspects such as regulation and control of growing have broad application prospects based on the technical tactic of RNAi.Design a kind of working method is easy, effect is obvious, use cost is low introducing RNA and disturb method in plant, application prospect must be very wide.

Summary of the invention

The present invention is based on modern biotechnology, and a kind of easy and simple to handle, speed is fast, cost is low, rapid-action, real-time novel method of RNA being disturbed introduced plant is provided, for the plant gene function analysis, enhancing disease resistance provides advanced means.

In order to achieve the above object, a kind of method that RNA is disturbed introduced plant of the present invention comprises the steps:

S1, according to target-gene sequence design, synthetic siRNA;

S2, utilize distilledwater preparation 1%-10%siRNA solution;

S3, described siRNA solution is sprayed on the plant leaf surface.

Under the optimal way, spraying of siRNA solution is chosen in the enforcement of plant-growth animated period.

It is a kind of FA Protocols in Molecular Biology that grew up in recent years that RNA disturbs, and is widely used in that large-scale functional genome resolves, the disease-resistant and aspects such as regulation and control of growing.As realizing a kind of means of RNA interferential, siRNA has the effect of the RNA of bringing out interferential in vivo.The present invention adopts the foliage applying method that RNA is disturbed introduced plant, and plant stomata cell and epidermic cell thereof can absorb siRNA.Carried out to detect by flow cytometer again after fluorescently-labeled siRNA enters cell.The present invention is as a kind of method of RNA being disturbed introduced plant, have easy and simple to handle, speed is fast, cost is low, rapid-action, real-time, stable, efficient, use cost is low, is easy to advantages such as popularization.Although there is false positive to exist sometimes, can solve by contrast reasonably is set.

Description of drawings

Fig. 1 is the detection design sketch after an embodiment of the present invention RNA disturbs introduced plant.

Embodiment

The present invention disturbs the method for introduced plant with RNA, and the core scheme comprises the steps:

1. also synthesize it according to target-gene sequence design siRNA sequence, carry out fluorescent mark simultaneously.

Wherein, the process of design, synthetic siRNA sequence adopts prior art, becomes the cDNA sequence by the mRNA reverse transcription with target gene, then the cDNA sequence is input to design software as getting final product in " siRNA designer ".Fluorescent mark can entrust biotech company to carry out.

2. this fluorescently-labeled siRNA is mixed with 1%-10%siRNA solution.

Wherein, siRNA solution is preferentially selected the distilled water preparation for use.

3. in plant-growth vigorous period, certain density fluorescently-labeled siRNA solution is sprayed on the plant leaf surface.

For example,, on the blade of cherry, apple, peach,spray 5% siRNA solution, every spray in 3 days once, spray continuously 3 times in the mid-June in every year.

4. get blade, solution is made on the flushing surface.

5. this solution is carried out flow cytometry analysis, confirm the existence of siRNA.

Embodiment:

Be used for the treatment of plant virus with the inventive method, be specially the treatment apple chlorotic leaf spot virus (Apple chlorotic leaf spot virus, ACLSV).Step is as follows:

1. the RNA reverse transcription with the ACLSV coat protein gene becomes the cDNA sequence, (sequence is: 5 '-CAG then the cDNA sequence to be input to the middle design of design software " siRNA designer " siRNA sequence and synthetic this sequence, ACC, CUU, AUU, GAA, GUC, GAA-3 '), carry out fluorescent mark (entrusting biotech company to carry out fluorescent mark) simultaneously.

2. this fluorescently-labeled siRNA is mixed with 1%, 5% and 10%siRNA solution (distilled water preparation).

3.6spray 1%, 5% and 10% siRNA solution the middle of the month on the blade of peach, every spray in 3 days once, spray continuously 3 times.

4. get blade, solution is made on the flushing surface.

5. this solution is carried out flow cytometry analysis, can confirm the existence of siRNA.

6. last, the blade that sprays siRNA solution is extracted total RNA, carry out the reverse transcription-pcr analysis, to detect result of treatment to ACLSV.

7. from detected result, as shown in Figure 1,1 represents 10%siRNA solution effect; 2,3expression 5%siRNA solution effects; 4expression 1%siRNA solution effects; 5 expression DNA marker.Among the figure, concentration is that 1% siRNA solution result of treatment siRNA solution result of treatment the poorest in the three, 5% is better, and 10% siRNA solution result of treatment is best.

The above; only be the preferable embodiment of the present invention; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, all should be encompassed within protection scope of the present invention.

Claims

1. one kind is disturbed the method for introduced plant with RNA, it is characterized in that, comprises the steps:

S1, according to target-gene sequence design, synthetic siRNA;

S2, utilize distilled water preparation 1%-10%siRNA solution;

S3, described siRNA solution is sprayed on the plant leaf surface.

2. according to claim 1 is described RNA is disturbed the method for introduced plant, it is characterized in that, described step S3 sprays described siRNA solution at the plant-growth animated period.

3. according to claim 2 is described RNA is disturbed the method for introduced plant, it is characterized in that, spray 5% siRNA solution, every spray in 3 days once, spray continuously 3 times.

4. according to claim 3 is described RNA is disturbed the method for introduced plant, it is characterized in that,, siRNA solution is sprayed on the blade of cherry, apple or peach in the mid-June in every year.