WO2018037635A1 - Mir398-targeted nucleic acid sequence-containing virus vector for gene transfer - Google Patents

Abstract

The present invention pertains to a virus vector for gene transfer, said virus vector containing a nucleic acid sequence targeted by miR398.

Description

Viral vector for gene transfer comprising miR398 target nucleic acid sequence

The present invention relates to a viral vector for gene transfer comprising a miR398 target nucleic acid sequence,
And a gene transfer method comprising the step of infecting a plant with the same.

There are several methods for expressing foreign genes in plants, but plant virus vectors have the advantage that they can be expressed easily and without high-level expression and insertion of foreign DNA into the plant genome. . Although genetically engineered plants produced by genome editing technology, which has been attracting attention in recent years, may not be regulated as recombinants, in order to produce genome-edited plants, genome editing enzymes are often expressed. Since it is necessary to produce a recombinant to be used once, there is a competition for development of alternative technologies (Non-Patent Document 1). The use of a viral vector is one promising alternative technique, but it has been necessary to remove the viral vector after completion of genome editing, which has been a technical obstacle.

On the other hand, miR398 is a small RNA that has been shown to be involved in stress response and copper metabolism, but it has been reported as an miRNA whose expression is induced by environmental stress in Arabidopsis and the like and suppresses the target gene. (Non-patent document 2). Examples of the stress that induces the expression of miR398 include sucrose contained in the dedifferentiation / redifferentiation medium of many plants.

Woo et al. Nature Biotechnology 33, 1162-1164 (2015) Dugas and Bartel, Plant Mol Biol, 67: 403-417 (2008)

The purpose of the present invention is to find a plant virus vector that can be easily removed after the completion of gene introduction and can minimize the adverse effects on the plant by the removal operation.

As a result of diligent research, the present inventors introduced a genome editing enzyme gene into a plant by using a viral vector for gene introduction containing a miR398 target nucleic acid sequence, which was not conventionally known, and genome editing. By removing viral vectors that became unnecessary after completion with minimal adverse effects on plants, we found that genome editing is possible without incorporating foreign DNA into plant chromosomes, and the present invention was completed. It was.

That is, the gist of the present invention is as follows.

[1] A viral vector for gene transfer comprising a miR398 target nucleic acid sequence.
[2] The virus vector according to [1], which has a miR398 target nucleic acid sequence targeted by miR398 having a sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequences of SEQ ID NOS: 5-26.
[3] The viral vector according to [1] or [2] above, wherein the miR398 target nucleic acid sequence is complementary to a nucleic acid sequence having at least 70% identity with miR398.
[4] The miR398 target nucleic acid sequence is complementary to a nucleic acid sequence having at least 80% identity in the region of positions 2 to 17 of the sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NOs: 5 to 26 The virus vector according to any one of [1] to [3], which is a target.
[5] Any of the above [1] to [4], wherein the miR398 target nucleic acid sequence is selected from the group consisting of the sequence of SEQ ID NO: 2, 3 or 4, or a sequence having at least 90% identity thereto The described viral vector.
[6] The viral vector according to any one of [1] to [5], wherein the miR398 target nucleic acid sequence is the sequence of SEQ ID NO: 2, 3 or 4.
[7] The viral vector according to any one of [1] to [6], which is a plant viral vector.
[8] The virus vector according to [7], which is a solanaceous plant virus vector.
[9] The viral vector according to any one of [1] to [8], which lacks a coat protein.
[10] A gene introduction method comprising a step of infecting a plant with the virus vector according to any one of [1] to [9].
[11] The gene introduction method according to [10], further comprising a step of dedifferentiating and redifferentiating the plant.

According to the present invention, it is not necessary to perform a special operation for removing the viral vector after completion of gene introduction, and the gene can be introduced into the plant with minimal adverse effects on the plant.

Figure 3 shows the design of a viral vector according to one embodiment of the present invention.

The present invention provides a viral vector for gene transfer containing a miR398 target nucleic acid sequence, and a gene transfer method comprising a step of infecting a plant with the virus vector.

MiR398 is widely preserved in plants regardless of monocotyledonous or dicotyledonous. The miR398 in the present invention may be any plant miR398 that has the same expression format as tobacco and can be regenerated from a virus-infected leaf. Examples of such miR398 include, but are not limited to, miR398 such as tobacco, Arabidopsis, rice, corn, soybean, wheat, potato, melon, apple apple, poplar, and oilseed rape. Preferably, miR398 in the present invention is tobacco miR398.

The sequence of tobacco miR398 is
5'-UGUGUUCUCAGGUCGCCCCUG-3 '(SEQ ID NO: 1)
However, additional miR398 sequences available in the present invention are illustrated in Table 1.

The miR398 target nucleic acid sequence in the viral vector of the present invention may be complementary to a nucleic acid sequence having at least 70%, preferably 80%, more preferably 90% identity with the miR398 sequence, preferably SEQ ID NO: 1. And at least 80%, preferably 85%, more preferably 90%, most preferably 100% identity in the region of positions 2-17 of a sequence selected from the group consisting of What is necessary is just to be complementary to the nucleic acid sequence to have.

The miR398 target nucleic acid sequence in the viral vector of the present invention is:
5′-CAGGGGCGACCCTGAGAACACA-3 ′ (SEQ ID NO: 2)
5′-TGCGGGTGACCCTGGGAAATACATA-3 ′ (SEQ ID NO: 3)
5′-ACTCGGTGACCTGGGACACACT-3 ′ (SEQ ID NO: 4);
And a sequence consisting of a sequence having at least 90% identity with them, preferably the sequence of SEQ ID NO: 2, 3 or 4.

All known plant viruses are affected by host RNA silencing. Then, the production of resistant plants against many viruses has been reported by expressing miRNAs artificially designed to target the virus (eg, Turnip yellow mosaic virus: Niuetetal., Nat Biotechnol. 2006 Nov; 24 (11): 1420-8; turnip mosaic virus: Niu et al., Nat Biotechnol. 2006 Nov; 24 (11): 1420-8; cucumber mosaic virus: Qu et al., J Virol. 2007 Jun ; 81 (12): 6690-9). Therefore, the virus vector of the present invention may be any plant virus vector, but preferably a solanaceous plant virus vector such as a tomato mosaic virus vector, a tomato bushy stunt virus vector, a tobacco mosaic virus vector, a tobacco stem virus. A vector, a potato X virus vector, a potato Y virus vector and the like can be mentioned, but not limited thereto.

The viral vector of the present invention is preferably deficient in the coat protein, which makes it easier to remove.

As a plant into which a gene can be introduced according to the present invention, any plant can be used as long as the expression pattern of miR398 is the same as that of tobacco and the plant can be regenerated from infected leaves. For example, tobacco, Arabidopsis thaliana, rice, corn, soybean, Examples include, but are not limited to, wheat, potato, melon, apple apple, poplar, oilseed rape. Preferably, the plant into which the gene can be introduced according to the present invention is tobacco.

The present invention provides a gene introduction method including a step of infecting a plant with the virus vector. The gene introduction method according to the present invention preferably further comprises a step of dedifferentiating and redifferentiating the plant.

The step of infecting a plant with a viral vector and the step of dedifferentiating and redifferentiating the plant may be any step commonly used in the art. An example thereof includes, but is not limited to, a step of inoculating tobacco with a virus, and then a step of forming callus from infected leaves and redifferentiating to obtain shoots.

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples unless it exceeds the gist.

1. Viral vector design To easily measure the efficiency of gene transfer, coat protein coding region of tomato mosaic virus (ToMV) vector pTLW3 (Kubota et al. J Virol., 2013, 77 (20): 11016-1026) Was replaced with the yellow fluorescent protein (YFP) gene or the meganuclease I-SceI gene. The YFP gene was used because it is convenient for visualizing the site of infection, but it does not affect the properties of the viral vector and can be replaced with any sequence. I-SceI is an endonuclease that specifically recognizes an 18-base sequence. Tobacco normally does not express fluorescence, but by using transformed tobacco with a gene cassette in the genome that expresses firefly luciferase when the I-SceI target site is cleaved and subsequently restored to frame when repaired, Visualization of genome editing sites is possible. Furthermore, the miR398 target nucleic acid sequence or Spacer sequence having SEQ ID NO: 2, 3 or 4 was placed immediately after the stop codon of the YFP gene or I-SceI gene. The design of the viral vector is shown in FIG.

2. Infection with viral vector In vitro transcription reaction was performed using AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript, USA) using linear DNA obtained by cutting each ToMV vector plasmid with MluI as a template. A 10-fold dilution was mechanically inoculated into tobacco leaves using carborundum.

Observation of the spread of YFP fluorescence showed that there was no significant difference in the presence or absence of the miR398 target sequence. Therefore, it is considered that the ToMV vector growth and the expression of the YFP gene in tobacco leaves are hardly affected by the insertion of the miR398 target nucleic acid sequence It was.

3. Sterilized tobacco leaf 14 days after plant dedifferentiation and redifferentiation virus inoculation with Kitchen Hiter (registered trademark) diluted 5 times, from about 1 cm square leaf disk including the site where expression of YFP or firefly luciferase was observed Callus was formed, and shoots were formed according to the known tobacco transformation method (Oshima et al., Plant Cell. 1990, 2: 95-106). However, the medium used for redifferentiation was one that did not contain antibiotics for selection.

4). Detection of virus by RT-PCR Shoots were cut from callus and transferred to fresh medium. Three weeks later, RNA was extracted from the first leaf of the shoot using ISOSPIN Plant RNA (Wako Pure Chemical Industries, Ltd.). Next, a primer (To86: 5′-TCAACAGAGTAACTGGGT-3 ′ (SEQ ID NO: 27); and To87: 5′-GCAGCTACAGACTTAGA-3 ′ (SEQ ID NO: 27), which uses 100 ng of RNA as a template and targets the ToMV replication protein coding region. 28)) and PrimeScript One Step RT-PCR Kit Ver. RT-PCR (40 cycles) was performed using 2 (Dye Plus) (Takara Bio Inc.). As positive control and negative control, viral RNA prepared by in vitro transcription and RNA extracted from virus-free tobacco leaves were used, respectively. The sample after RT-PCR was subjected to agarose gel electrophoresis, and an image was captured using LAS-3000 (Fuji Film Co., Ltd.).

In the negative control Spacer sequence, virus was detected in 100% (25/25) sample, whereas in shoots redifferentiated from virus vector-infected leaves having SEQ ID NO: 2, 60% (15/25) sample. Only virus was detected, and the remaining 40% of samples had viral genome accumulation below the detection limit.

5). Detection of virus by real-time PCR RNA was extracted from the third leaf of the redifferentiation shoot in the same manner as described above. Next, primers (511F: 5′-TATACCTTTCTAGGCTCGAGAGGGG-3 ′ (SEQ ID NO: 29); and 657R: 5′-TTCCCGTGTAACATTCTTGAGAATG-3 ′ (SEQ ID NO: 30)) and iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad, USA), and real-time PCR was performed using the iQ ^™ 5 real-time PCR analysis system.

Individuals in which the amount of viral genome accumulated in shoots redifferentiated from the viral vector having SEQ ID NO: 3 or 4 is reduced from 1 / 10,000 to 1 / 100,000 as compared to the negative control Spacer sequence There were many. Combined with the result of paragraph [0027], the effect of decreasing the amount of accumulated viral genome was higher in the order of SEQ ID NOs: 2, 3, and 4. From these results, it can be said that the higher the homology with miR398, the more effective the target sequence used.

6). Results By using a viral vector containing the miR398 target nucleic acid sequence, it was found that the amount of viral vector accumulated in the redifferentiated shoot decreased without any special treatment, and some individuals were removed below the detection limit. .

Since the gene transfer method according to the present invention does not require mating for removing foreign genes, it can be used for non-recombinant gene introduction into plants that want to avoid mating of vegetatively breeding crops, F1 varieties, fruit trees, etc. . Further, the present invention can be applied even to plants that are difficult to transform. In addition, the present invention provides virus removal after carrying out eradication treatment of the virus by suppressing the expression of endogenous genes using a virus vector or inducing RNA silencing for other viruses already infected. It can also be applied to.

Claims (11)

Virus vector for gene transfer containing miR398 target nucleic acid sequence. The viral vector according to claim 1, comprising a miR398 target nucleic acid sequence targeted by miR398 having a sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequences of SEQ ID NOS: 5-26. The viral vector according to claim 1 or 2, wherein the miR398 target nucleic acid sequence is complementary to a nucleic acid sequence having at least 70% identity with miR398. The miR398 target nucleic acid sequence is complementary to a nucleic acid sequence having at least 80% identity in a region from position 2 to 17 of a sequence selected from the group consisting of the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NOs: 5-26 The viral vector according to any one of claims 1 to 3. The viral vector according to any one of claims 1 to 4, wherein the miR398 target nucleic acid sequence is selected from the group consisting of the sequence of SEQ ID NO: 2, 3 or 4 or a sequence having at least 90% identity thereto. . The viral vector according to any one of claims 1 to 5, wherein the miR398 target nucleic acid sequence is the sequence of SEQ ID NO: 2, 3 or 4. The virus vector according to any one of claims 1 to 6, which is a plant virus vector. The virus vector according to claim 7, which is a solanaceous plant virus vector. The viral vector according to any one of claims 1 to 8, which lacks a coat protein. A gene transfer method comprising a step of infecting a plant with the virus vector according to any one of claims 1 to 9. The gene introduction method according to claim 10, further comprising a step of dedifferentiating and redifferentiating the plant.

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