JP2010119373A - Method for evaluating laticifer specificity of promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating laticifer specificity of a promoter derived from gum tree (Hevea brasiliensis) therefor using a plant having the laticifer. <P>SOLUTION: The method for evaluates the laticifer specificity of the promoter. The method for evaluating the laticifer specificity of the promoter includes preparing a transformant of Periploca using a vector in which a gene encoding a fluorescent protein is integrated into the downstream side of the promoter that is the object of evaluation, and investigating the expression site of the fluorescent protein in the transformant grown even to a shoot. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for evaluating the specificity of a milk duct of a promoter, which is suitable for identifying a milk duct specific promoter.

  Natural rubber is obtained from an emulsion produced in an organ called a latex tube of a specific plant. For example, Hevea brasiliensis is known as a main production species of natural rubber. Further, due to its importance as a resource, natural rubber is desired to further improve productivity and functionality. Therefore, the development of genetic recombination technology is being studied for the purpose of enhancing milk productivity and enhancing functionality, and searching for promoters that promote gene expression specifically in the milk ducts (milk duct-specific). . Breast duct specificity is required because gene expression is promoted at sites other than the breast ducts, which may place a burden on plant metabolism and milk production, and may have adverse effects.

  However, it is known that rubber tree is low in efficiency of redifferentiation and transformation and is not suitable for achieving the above object. Therefore, if a promoter derived from rubber tree is introduced by genetic recombination technology to a plant having a milk duct similar to rubber tree other than rubber tree, and the presence or absence of milk duct specificity in the plant can be confirmed, the milk duct of rubber tree It is thought that it can evaluate whether it is a specific promoter, It is very useful.

As described above, as a technique for introducing a rubber tree-derived promoter into a plant having a milk duct and specifying a gene expression site, a technique using periploca has been disclosed so far (non-patent document). 1). Periproca is a semi-woody plant of the genus Pleurotus that has a milk duct similar to rubber tree and produces an emulsion that is a raw material for natural rubber. In Non-Patent Document 1, specifically, the REF promoter of Para rubber tree is cloned, a vector using the GUS gene as a marker gene is prepared, and this is introduced into periproca to confirm GUS activity.
Nagai et al., 117th Annual Forest Society Conference Proceedings (2007), p520

  However, in the method described in Non-Patent Document 1, GUS activity is confirmed by staining, and the expression of GUS activity can be confirmed in the periprocica marrow and phloem, but the staining is unclear and the stained area is other than marrow and phloem. The GUS gene expression site is difficult to specify precisely, and it is difficult to evaluate the milk duct specificity of the promoter.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for evaluating the specificity of the ducts of a rubber tree-derived promoter using a plant having a duct.

To solve the above problem,
The present invention is a method for evaluating the specificity of a breast duct of a promoter, and a transformant of Periploca using a vector in which a gene encoding a fluorescent protein is incorporated downstream of the promoter to be evaluated Is provided, and the expression site of the fluorescent protein in the transformant grown up to the shoot is examined.
In the present invention, it is preferable to confirm the fluorescent protein expression site in the shoot having a main stem length of 5 cm or more.
In the present invention, it is preferable to confirm the expression site of the fluorescent protein in the cross section between the shoots.
In the present invention, the fluorescent protein is preferably a green fluorescent protein.
In the present invention, the transformant is preferably produced by the Agrobacterium method.

  According to the present invention, a plant having a milk duct can be used to evaluate the duct specificity of a promoter derived from rubber tree. Since it is not necessary to apply genetic recombination technology to rubber trees with low efficiency of redifferentiation and transformation, a milk duct-specific promoter can be identified efficiently and with high accuracy.

The present invention will be described in detail below.
The method for evaluating the milk duct specificity of a promoter of the present invention is a method for evaluating the milk duct specificity of a promoter, using a vector in which a gene encoding a fluorescent protein is incorporated downstream of the promoter to be evaluated. A transformant of Periploca is prepared, and the expression site of the fluorescent protein in the transformant grown to the shoot is examined.
Rubber tree has low efficiency of redifferentiation and transformation, and is not suitable for specifying a milk duct-specific promoter to which genetic recombination technology is applied. The present invention uses a periproca as a plant having a milk duct similar to rubber tree, calculating an emulsion of the same component, and having a high efficiency of redifferentiation and transformation. If it can be confirmed that the promoter is milk duct-specific in periproca, it can be determined that rubber tree having a similar structure is milk duct-specific.
Thus, this invention evaluates efficiently and highly accurately whether a promoter is milk duct specific in rubber tree by using a periploca.

  In the present invention, the promoter to be evaluated is not particularly limited as long as it is a promoter that can function in rubber tree. Examples of rubber tree include Hevea brasiliensis, sea rubber tree, Indian rubber tree, Panama rubber tree, guayule rubber tree, rubber dandelion, Lagos rubber tree, gutta pergano tree, parata rubber tree, cis-chickle and the like.

  Periproca is a vine shrub belonging to the genus Periproca genus, which grows naturally in Africa, Europe, North America, Asia, etc., and produces milk by damaging plants. Specific examples include Periproca sepium, Periproca graeca, Periproca laevigata, and the like.

A vector for producing a periproca transformant is a vector in which a promoter to be evaluated is incorporated upstream of a gene encoding the fluorescent protein so as to function as a promoter of the fluorescent protein. In the present invention, it is preferable to infect callus or the like with an Agrobacterium genus into which the vector has been introduced.
The Agrobacterium used in the present invention is not particularly limited as long as it is an Agrobacterium that can introduce a vector such as a plasmid into a plant cell. Agrobacterium tumefaciens (Agrobacterium tumefaciens) is preferred. This is because the infection efficiency is good and it is widely used in the Agrobacterium method.

  An Agrobacterium genus containing a vector containing a promoter or the like to be evaluated may be prepared using any conventionally known technique. For example, a gene recombination intermediate vector in which a promoter or the like to be evaluated is incorporated into a plasmid capable of homologous recombination with the T-DNA region of a Ti plasmid possessed by Agrobacterium is produced. It may be introduced into the fungus. In addition, a target gene binary vector in which a gene encoding a fluorescent protein as a gene to be expressed is incorporated into a binary vector widely used in the Agrobacterium method and a promoter to be evaluated is introduced into the Agrobacterium genus May be.

  By culturing and growing the Agrobacterium genus introduced with the vector thus prepared by a conventional method, an amount necessary for infecting callus or the like can be prepared.

For example, a transformant of periproca is made by contacting tissue such as roots, stems, leaves or flowers of periproca or cells such as callus with an infectious solution containing the vector, followed by tissue culture and further containing an antibiotic. It can be produced by culturing in a selective medium.
For example, it is preferable to apply the Agrobacterium method in which a desired vector is introduced into an Agrobacterium genus and inoculated into periproca tissue or cells.

  Tissues and cells are sterilized or sterilized before being used for tissue culture. Sterilization or sterilization may be performed using a known disinfectant / sterilant, and for example, ethanol, benzaconium hydrochloride, sodium hypochlorite aqueous solution, or the like is preferably used.

The tissue culture may be performed on a medium having the same composition under light-shielded conditions, preferably at 18 to 30 ° C., more preferably at 23 to 28 ° C., and preferably for about 2 to 4 days.
The culture with the selective medium is preferably performed for 10 to 30 days, more preferably for 15 to 20 days. The culture temperature may be the same as in the case of the tissue culture.

  The produced transformant may be cultured on a regeneration medium and grown until it becomes a shoot.

  The fluorescent protein may be a known one, and any fluorescent protein normally incorporated into a protein expression vector, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), ZsGreen1, DsRed, etc. However, GFP is particularly suitable.

When the promoter to be evaluated is a milk duct-specific promoter, the fluorescent protein is not expressed unless a periproca transformant grows to such an extent that a milk duct is formed. Therefore, after growing to the shoot where the milk duct is formed, the expression site of the fluorescent protein is examined.
In addition, when the size of the expression site is small, it may be difficult to detect a fluorescent signal. Therefore, a shoot having a main stem length of preferably 5 cm or more, more preferably 10 cm or more, may be used for confirmation of the fluorescent protein expression site so that the fluorescent protein expression site can be more clearly identified.

  The fluorescent protein expression site can be examined anywhere as long as the shoot milk ducts are present, but the amount of fluorescent protein expression is large and the fluorescent signal is easy to detect. It is preferable to carry out, and it is more preferable to carry out in the cross section by making a round slice.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[Example 1]
<Preparation of periproca transformants and shoots>
As shown in FIG. 1, a GFP expression cassette in which a GFP gene (sGFP) is connected downstream of a REF promoter (REF pro) and a nopaline synthase gene terminator (NOS ter) is connected, and a nopaline synthase gene promoter ( A kanamycin resistance gene (NPT II) downstream of NOS pro) and a nopaline synthase gene terminator connected to the kanamycin resistance gene expression cassette, and a cauliflower mosaic virus 35S promoter (35S pro) downstream of the hygromycin gene (35S pro) An expression vector (PBIsGFP (S65T)) having a cassette for hygromycin resistance gene expression linked to HPT) and further linked to a terminator of the nopaline synthase gene was constructed. In Agrobacterium LBA4404, infected, to prepare an expression vector introduced Agrobacterium. In FIG. 1, “I” means intron.
Next, a periprocassepium stalk was contacted with the infectious solution containing the expression vector-introduced Agrobacterium for 2 minutes, and this was cultured in a medium at 25 ° C. for 3 days under light-shielding conditions.
Subsequently, the cells were further cultured for 16 hours at 25 ° C. in a kanamycin resistant selection medium to prepare 22 transformants.
Then, DNA was extracted from the transformant according to a known method, and the GFP gene was amplified by the polymerase chain reaction (PCR) method. PCR conditions are as follows. Then, the amplification product was detected by agarose gel electrophoresis. As a result, it was confirmed that in all the produced transformants, a gene in which the REF promoter and the GFP gene were combined was introduced into the periproca tissue and transformed.
(PCR conditions)
95 ° C / 5 minutes → (95 ° C / 1 minute → 60 ° C / 1 minute → 74 ° C / 2 minutes) x 30 cycles → 4 ° C

<Confirmation of expression level>
The transformant was grown to a shoot having a growth length of about 10 cm in a regeneration medium to prepare a specimen.
Using the obtained specimen (below (1)), together with the following specimens (2) and (3), total RNA was extracted according to a known method, and cDNA was obtained by reverse transcription reaction. Amplified.
(1) 22 transformed periprocica (REF-GFP-1 to 22)
(2) One sample of periprocca (35S-GFP) transformed by introducing a gene in which the cauliflower mosaic virus 35S promoter and the GFP gene are combined.
(3) As a control, one non-transformed sample of periproca (Wildtype)
The corrected expression level was calculated by dividing the expression level of the GFP gene by the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. The result is shown in FIG. The conditions for real-time PCR are as follows.
(apparatus)
7300 FAST Real-time PCR System
(Analysis software)
7300 System SDS Software
(Detection reagent)
SYBER green PCR Master Mix
(PCR conditions)
50 ° C / 2 minutes → 95 ° C / 10 minutes → (95 ° C / 15 seconds → 60 ° C / 1 minute) x 40 cycles → 95 ° C / 15 seconds → 60 ° C / 1 minute → 95 ° C / 15 seconds → 95 ° C / 15 seconds → 60 ℃ / 15 seconds

<Tissue observation>
For specimens (REF-GFP-5, 11, 12, 15) with a large corrected expression level, the internodes of the shoots were cut off and the cross section was observed with a microscope. Similarly, 35S-GFP and Wildtype were also observed. An image of the cross section at this time is shown in FIG. (A) is a transmission light observation image of REF-GFP-5, (b) is a fluorescence observation image of REF-GFP-5, (c) is a transmission light observation image of 35S-GFP, (d) is a transmission light observation image of 35S-GFP. Fluorescence observation image, (e) is a Wildtype transmitted light observation image, and (f) is a Wildtype fluorescence observation image. The observation conditions are as follows.
(microscope)
Nikon ECLIPSE E600
(filter)
EX460-500
DM505
BA 510-560
(Light)
Nikon INTERSLIGHT C-HGFI

As a result, in Wildtype, no fluorescence signal was detected other than autofluorescence, and it was confirmed that GFP was not expressed. In 35S-GFP, a fluorescence signal was detected over a wide range of the cross section, and it was confirmed that GFP was expressed in the entire tissue. On the other hand, in REF-GFP-5, the fluorescence signal was clearly detected in a part of the periphery of the marrow and the formation layer. The detection of a fluorescent signal in the medulla is considered to indicate that GFP is expressed in the breast duct. In addition, it is known that REF protein exists in cells that produce lipid, and that the fluorescence signal was detected in a part of the periphery of the formation layer, the expressed REF protein is a lipid in the phloem. It is thought that it shows that it has moved to parenchyma to make.
From the above, the REF promoter is considered to be milk duct-specific, and it was confirmed that the evaluation method of the present invention using periproca is effective for specifying a milk duct-specific promoter.

[Comparative Example 1]
Similar to Example 1, except that PBIsGFP (S65T) was used as an expression vector, an expression vector (pIG121-Hm) in which a GUS gene encoding a GUS protein was incorporated downstream of the REF promoter was used. A converter was produced. About the obtained specimen, the stem is cut in the same manner as in Example 1, the substrate 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) is added and stained by a conventional method, and the cross section is stained. The site was confirmed with a microscope. An image of the cross section at this time is shown in FIG. As a result, the expression of GUS activity was confirmed in the medulla and phloem of Periproca, but the staining was unclear and the stained area spread over a wide area other than the medulla and phloem, and the GUS protein expression site could not be identified. .

  The present invention can be used to increase the productivity and functionality of natural rubber.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the expression vector used for the transformation of periproca in Example 1. 2 is a graph showing the corrected expression level of the GFP gene in Example 1. It is a cross-sectional image of the stem of the specimen in Example 1, (a) is a transmitted light observation image of REF-GFP-5, (b) is a fluorescence observation image of REF-GFP-5, (c) is 35S. -GFP transmitted light observation image, (d) is a 35S-GFP fluorescence observation image, (e) is a Wildtype transmitted light observation image, and (f) is a Wildtype fluorescence observation image. It is a dyeing | staining image of the cross section of the stem of the sample by the microscope in the comparative example 1.

Claims (5)

A method for assessing the milk duct specificity of a promoter comprising:
Using a vector incorporating a gene encoding a fluorescent protein downstream of the promoter to be evaluated, a periploca transformant is prepared,
A method for evaluating milk duct specificity of a promoter, comprising confirming an expression site of a fluorescent protein in the transformant grown to a shoot.   The method for evaluating the specificity of a milk duct of a promoter according to claim 1, wherein an expression site of the fluorescent protein in the shoot having a main stem length of 5 cm or more is confirmed.   3. The method of evaluating the milk duct specificity of a promoter according to claim 1 or 2, wherein an expression site of the fluorescent protein is confirmed in a cross section between the shoot nodes.   The method for evaluating the specificity of a milk duct of a promoter according to any one of claims 1 to 3, wherein the fluorescent protein is green fluorescent protein (GFP).   The method for evaluating the specificity of a milk duct of a promoter according to any one of claims 1 to 4, wherein the transformant is produced by the Agrobacterium method.

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