JP2015081252A - Growth promoter for animals and plants - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a growth promoter for animals and plants having better effect than straight chain polyamine.SOLUTION: A growth promoter for animals and plants comprises branched chain polyamine or long chain polyamine.

Description

  The present invention relates to a growth promoter for animals and plants.

  The polyamine is an aliphatic hydrocarbon having three or more primary amino groups (monovalent functional group obtained by removing hydrogen from the primary amine (-NHR (R represents an aliphatic hydrocarbon group))). It is a generic name.

  Polyamines are low molecular weight basic substances that are involved in various life phenomena. Polyamines produced by many organisms are linear aliphatic hydrocarbons (referred to herein as “linear polyamines”).

  With respect to linear polyamines, effects of promoting plant growth (Patent Document 1), extending the life of animals (Patent Document 2), RNA stabilizing action (Patent Document 3), and the like have been reported so far.

JP 2002-29905 A International Publication No. 2009/093454 JP 2006-20575 A

  This invention makes it a subject to provide the growth promoter for animals and plants which has the effect superior to the linear polyamine.

The present inventors have paid attention to special polyamines produced by thermophilic bacteria. That is, polyamines having a branched structure (referred to herein as “branched polyamines”) and longer-chain polyamines (referred to herein as “long-chain polyamines”) than those produced by other organisms. )). The inventors have found that these special polyamines have higher nucleic acid stabilization ability than linear polyamines, and are superior in growth promoting effects on animals and plants. The present invention has been completed through further studies based on such findings, and includes the following embodiments.
Item 1.
A growth promoter for animals and plants containing a branched-chain polyamine or a long-chain polyamine.
Item 2.
Item 2. The growth promoter for animals and plants according to Item 1, wherein the branched polyamine is a tertiary polyamine represented by the following formula (I) or a quaternary polyamine represented by the following formula (II).

(In the formula, R ^{1 to} R ⁶ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ² , R ^3, and R ⁵ are respectively The repeating units may be the same or different, and n ^{1 to} n ³ are the same or different and are 0 or an integer of 3 or less.)

^(Wherein, R 7 ^{to R 15} are the same or different, having 2 to 6 carbon atoms, which may be substituted, an alkyl group or an alkenyl group, and ^{R 8, R 9, R 12} and ^{R 14} May be the same or different for each repeating unit, and n ^{4 to} n ⁷ are the same or different and are 0 or an integer of 3 or less.)
Item 3.
The growth promoter for animals and plants according to claim 1, wherein the long-chain polyamine is a long-chain polyamine represented by the following formula (III):

(Wherein R ^{16 to} R ¹⁹ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ¹⁷ and R ¹⁸ are each a repeating unit. Each may be the same or different, and n ⁸ and n ⁹ are the same or different and are integers of 3 or less.)
Item 4.
Item 2. The growth promoter for animals and plants according to Item 1, wherein the branched-chain polyamine and the long-chain polyamine can be produced by thermophilic bacteria.
Item 5.
A growth promoter for animals and plants comprising a thermophilic bacterium or a cell extract thereof.
Item 6.
An animal or plant growth promoter containing an aminoalkyltransferase or an aminoalkenyltransferase (provided that the alkyl group and the alkenyl group have 2 to 6 carbon atoms and may be substituted, an alkyl group or an alkenyl group) Group).
Item 7.
A growth promoter for animals and plants containing a gene encoding an aminoalkyltransferase or an aminoalkenyltransferase, or an expression vector containing the gene (however, the alkyl group and the alkenyl group have 2 to 6 carbon atoms) An alkyl group or an alkenyl group which may be substituted.

  According to the present invention, it is possible to provide an agent that is superior in a growth promoting action on animals and plants.

It is drawing which shows the phylogenetic tree of TK1691 ortholog. It is drawing which shows the area | region like S-adenosylmethionine-dependent methyltransferase preserve | saved in the ortholog of TK1691. It is drawing which shows the polyamine biosynthetic pathway of T. kodakarensis. A shows the polyamine analysis of BL21 (DE3) codonplus-RIL strain / pET21a, and B shows the polyamine analysis of BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691 (the arrow indicates a peak derived from bisaminopropyl spermidine). Represent). It is a graph which shows the result of having investigated the length extension in the nematode which gave each diet (Control is a nematode which grew on BL21 (DE3) codonplus-RIL strain / pET21a, TK1691 is BL21 (DE3) codonplus- Represents nematodes grown on RIL strain / pET21a-Tk1691). It is a graph showing the results of examining pharyngeal elongation in nematodes fed each diet (Control: nematodes grown on BL21 (DE3) codonplus-RIL strain / pET21a, TK1691: BL21 (DE3) codonplus-RIL strain / Represents nematodes grown on pET21a-Tk1691). It is a graph which shows the result of having investigated the change of the body length and the body weight in the beetle larva which gave each diet. It is the graph which investigated the activation effect of translation about various polyamines.

1. Branched-chain polyamine In the present invention, the branched-chain polyamine means one having a branched structure in part, unlike a linear polyamine produced by organisms other than thermophilic bacteria. In the present invention, the branched polyamines are not limited to those that can be produced by thermophilic bacteria, but include all those having a partial branched structure.

  Although not particularly limited, a tertiary polyamine represented by the following formula (I) can be given as a representative example of the branched polyamine.

(In the formula, R ^{1 to} R ⁶ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ² , R ^3, and R ⁵ are respectively The repeating units may be the same or different, and n ^{1 to} n ³ are the same or different and are 0 or an integer of 3 or less.)
In the above, Preferably, R < ¹ > -R < ⁶ > is a C3-C4 alkyl group or alkenyl group which may be substituted. R ^{1 to} R ⁶ are more preferably an alkyl group.

In the above, n ^{1 to} n ³ are preferably 0 or an integer of 2 or less, more preferably 0 or 1, and still more preferably 0.

Although not particularly limited, as representative examples of branched polyamines that can be produced by thermophilic bacteria, N ⁴ -aminopropyl spermidine represented by the following formula (IV) and N ⁴ -aminopropyl spermine represented by the following formula (V): Etc.

  Although it does not specifically limit, The quaternary polyamine represented by following formula (II) is mentioned as another typical example of a branched chain polyamine.

^(Wherein, R 7 ^{to R 15} are the same or different, having 2 to 6 carbon atoms, which may be substituted, an alkyl group or an alkenyl group, and ^{R 8, R 9, R 12} and ^{R 14} May be the same or different for each repeating unit, and n ^{4 to} n ⁷ are the same or different and are 0 or an integer of 3 or less.)

In the above, Preferably, R < ⁷ > -R < ¹⁵ > is a C3-C4 alkyl group or alkenyl group which may be substituted. R ^{7 to} R ¹⁵ are more preferably an alkyl group.

In the ^above, n 4 ~n ⁷ is preferably 0 or 2 an integer, more preferably, 0 or 1, more preferably 0.

Is not particularly limited, as a typical example of branched polyamines may be produced by thermophilic bacteria, N ⁴ represented by the following formula (VI) - include bis (aminopropyl) spermidine.

2. Long-chain polyamine In the present invention, a long-chain polyamine means one having a longer chain length than a linear polyamine produced by an organism other than thermophilic bacteria. In the present invention, the long-chain polyamine is not limited to those that can be produced by thermophilic bacteria.

  Although not particularly limited, a representative example of the long-chain polyamine is a secondary polyamine represented by the following formula (III).

(Wherein R ^{16 to} R ¹⁹ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ¹⁷ and R ¹⁸ are each a repeating unit. Each may be the same or different, and n ⁸ and n ⁹ are the same or different and are integers of 3 or less.)

In the above, Preferably, R < ¹⁶ > -R < ¹⁹ > is a C3-C4 alkyl group or alkenyl group which may be substituted. R ^{16 to} R ¹⁹ are more preferably an alkyl group.

In the above, n ⁸ and n ⁹ are preferably 0 or 2 an integer, more preferably, 0 or 1, more preferably 0.

  Although not particularly limited, as typical examples of long-chain polyamines that can be produced by thermophilic bacteria, thermopentamine represented by the following formula (VII), cardohexamine represented by the following formula (VIII), etc. Is mentioned.

  These branched-chain polyamines and long-chain polyamines are part of a primary amino group (a monovalent functional group obtained by removing hydrogen from a primary amine (-NHR (R represents an aliphatic hydrocarbon group))). It can be bent relatively flexibly. These branched polyamines and long-chain polyamines are considered to be able to stabilize tRNA by entering the loop structure of tRNA while being bent at the primary amino group. Although not bound by this theory, in consideration of the size of the tRNA loop structure, the branched chain polyamine and the long chain polyamine have a chain length of the aliphatic hydrocarbon group part sandwiched between the primary amino groups, the number of carbon atoms. If it is 3-4, it is preferable at the point of tRNA stabilization ability.

  Although not bound by theory, in terms of tRNA stabilization ability, branched polyamines and long-chain polyamines are more preferable because the greater the number of amino groups, the higher the basicity.

  Branched-chain polyamines and long-chain polyamines are considered to promote translation efficiency through tRNA stabilizing ability, and thus exhibit growth promoting ability.

2. Thermophilic bacteria In the present invention, the branched-chain polyamine and the long-chain polyamine may be those that can be produced by thermophilic bacteria.

  In the present invention, the thermophile means a microorganism having an optimum growth temperature of 45 ° C. or higher or a growth limit temperature of 55 ° C. or higher.

  The thermophile is not particularly limited as long as it produces branched-chain polyamines and long-chain polyamines. The thermophilic bacterium may belong to any one of archaea, eubacteria and eukaryotes.

  The thermophilic bacterium is not particularly limited, but is preferably a hyperthermophilic bacterium having an optimum growth temperature exceeding 80 ° C.

  Although it does not specifically limit as a hyperthermophile, For example, Thermococcus genus, Pyrococcus genus, Methanocardococcus genus, Archaeoglobus genus, Methanococcus genus, Thermotoga genus, Aquifex genus, Thermoanater etc. Although it does not specifically limit as what belongs to Thermococcus genus, Thermococcus kodakarensis, Thermococcus profundus, Thermococcus litralis, etc. are mentioned.

  In the present invention, a thermophilic bacterium that is not a hyperthermophilic bacterium can also be used, and such a thermophilic bacterium is not particularly limited. , Desulfurobacterium genus, Thermovirga genus, Caldicelloulosirptor genus, Bacillus genus, Rhodopseudomonas genus, Clostridium genus and the like. Although it does not specifically limit as what belongs to Thermus genus, For example, Thermus thermophilus, Thermus aquaticus, etc. are mentioned.

  The growth promoter for animals and plants of the present invention may contain a thermophilic bacterium that produces a branched-chain polyamine or a long-chain polyamine or a cell extract thereof.

  The cell extract is not particularly limited as long as it contains a branched-chain polyamine or a long-chain polyamine, and those extracted by a method of crushing with an ultrasonic wave or a surfactant can be used.

3. Aminoalkyltransferases and aminoalkenyltransferases Even if animals and plants that do not produce branched-chain polyamines, the aminoamine transferase or aminoalkenyl group from the outside against the polyamine biosynthetic system it originally has By introducing a transferase, a branched polyamine can be synthesized. Therefore, the growth promoter for animals and plants of the present invention may contain an aminoalkyltransferase or an aminoalkenyltransferase as an active ingredient.

  Examples of aminoalkyltransferase or aminoalkenyltransferase include TK1691 derived from Thermococcus kodakarensis that catalyzes a reaction of adding an aminopropyl group to spermidine. The orthologue of TK1691 is widely found in thermophilic bacteria (FIG. 1). Furthermore, the TK1691 ortholog contains a wide range of S-adenomethyline-dependent methyltransferase-like regions (FIG. 2).

  The amino acid sequence of TK1691 is shown in SEQ ID NO: 1. The base sequence encoding it is shown in SEQ ID NO: 2.

  In the present invention, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having aminopropyl group transfer activity can also be used. In the left column, the deletion, substitution, or addition is preferably performed in a region different from the above-mentioned S-adenoylmethineine-dependent methyltransferase-like region.

  TK1691, including T.K. The Kodakarensis polyamine biosynthetic pathway is shown in FIG.

  The growth promoter for animals and plants of the present invention may contain a gene encoding an aminoalkyltransferase or an aminoalkenyltransferase, or an expression vector containing the gene as an active ingredient.

  Although it does not specifically limit as an expression vector, According to an application object, what has the said gene under control of a suitable promoter can be used.

  Even animals and plants that do not produce long-chain polyamines can synthesize long-chain polyamines by introducing long-chain polyamine synthase from the outside into the polyamine biosynthesis system that they originally possess. . Therefore, the growth promoter for animals and plants of the present invention may contain a long-chain polyamine synthase as an active ingredient.

4). Growth promoter for animals and plants In the present invention, animals and plants widely include animals and plants. Note that animals include insects.

Although not particularly limited, examples of animals include so-called domestic animals in general, specifically dogs, cats, horses, cows, pigs, sheep, goats, donkeys, mules, camels, llamas, Asian elephants,
Examples include alpaca, reindeer (caribou), cob cow, water buffalo, yak, guinea pig, rabbit (rabbit), mink, chicken, duck, goose, turkey, barricane, quail, ostrich, dove, pheasant and sea urchin; and fish.

  As other specific examples of animals, there are so-called breeding insects in general, specifically, bees, silkworms, beetles, stag beetles and the like: locusts and other edible insects.

  Although not particularly limited, examples of plants include cabbage, Chinese cabbage, spinach, sweet potato, potato, radish, carrot, apple, pear, grape, peach, strawberry, mandarin orange, cypress, cedar, pine, rose, cherry, ume, Examples include rice, wheat, shiitake mushrooms and eringi.

  The content ratio of the above-mentioned active ingredient in the growth promoter for animals and plants of the present invention can be appropriately set. Although it does not specifically limit, For example, you may contain 0.05-2 weight%.

  Although it does not specifically limit as a dosage form of the growth promoter for animals and plants of this invention, A liquid agent, a spray agent, a solid agent, a powder agent, etc. are mentioned. The dosage form may be such that the above-mentioned active ingredient is continuously released for a necessary period.

  The growth promoter for animals and plants of the present invention may further contain other active ingredients or other additives (stabilizers, excipients, etc.) in addition to the above-mentioned active ingredients, if necessary.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

1. Example: Effect of branched polyamines on nematode body length
1.1 Genetic manipulation General genetic manipulation was based on the previous report (Green and Sambrook). For cloning of DNA fragments, Escherichia coli DH5α strain was used, and transformants were selected by culturing in LB medium supplemented with 50 μg ml ^-1 ampicillin. Plasmid mini Kit (Qiagen) was used for extraction of plasmid DNA. Primers used in the construction of the gene disruption vector described below are genomic data of Thermococcus kodakarensis KOD1 strain (Morikawa M., et al, Appl. Environ. Microbiol., 1994, 60: 4559-4566) (http: // Designed based on www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=69014). As the DNA polymerase, KOD plus (Toyobo) was used. Restriction enzymes and other nucleic acid modifying enzymes were purchased from Takara Bio Inc. and Nippon Gene. For DNA sequencing, BigDye Terminator Cycle Sequencing kit, ver. 3.1 and 3130 Genetic Analyzer (both Applied Biosystems) were used.

1.2 Construction of Tk1691 gene expression vector
After extracting the genomic DNA of T. kodakarensis KOD1 strain (wild type strain), Tk1691-Fw (5'- AAAAAA CATATG ATGAGGGAGATAATTGAGAG-3 (SEQ ID NO: 3) '; underline, NdeI recognition site) -Tk1691-Rv (5′-A GAATTC TCAGGTAGTCGAGCTCTCCT-3 ′ (SEQ ID NO: 4); underlined, EcoRI recognition site) was used for PCR amplification. The obtained DNA fragment was digested with a restriction enzyme and cloned into the corresponding position of pET21a (Novagen) to obtain a Tk1691 expression vector (pET21a-Tk1691).

1.3 Analysis of polyamines in E. coli expressing Tk1691
pET21a-Tk1691 was introduced into Escherichia coli BL21 (DE3) codonplus-RIL strain (Agilent Technology) by the TSS method (Chung CT et al., Proc. Natl. Acad. Sci., 1989, 86: 2172-2175). After culturing E. coli BL21 (DE3) codonplus strain (hereinafter referred to as BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691) having pET21a-Tk1691 in LB liquid medium at 37 ° C. for 12 hours, 50 μg ml ⁻¹ ampicillin and 1 5 ml of M9 medium containing mM isopropyl-β-thiogalactopyranoside was inoculated and cultured at 37 ° C. for 12 hours. The cells were collected from the culture solution by centrifugation at 6000 rpm for 10 minutes, and a crude polyamine fraction was obtained by trichloroacetic acid precipitation. The amount of branched polyamine (bisaminopropylspermidine, [3 (3) (3) 4]) present in the bacterial body was analyzed by high performance liquid chromatography (CCPM, Tosoh Corp.) The column was CK-10S (6.0 mm × 50 mm) (GL Science Co., Ltd.) 100 mM potassium citrate, 2.0 M potassium chloride, 650 mM 2-propanol, 2.4 mM polyethylene lauryl ether, 0.195 N HCl in developing solvent with a flow rate of 1.0 mL min ^The polyamine was fractionated at -1.400 mL boric acid, 400 mM sodium chloride, 4.9 mM polyethylene lauryl ether, 7.5 mM O-phthalaldehyde, 171 mM ethanol, 28 mM 2 at a flow rate of 0.5 mL min ^-1 -A polyamine derivatized with a fluorescence detector (GL-7453A, GL Sciences Inc.) with an excitation wavelength of 325 nm and a fluorescence wavelength of 450 nm was detected by reacting with a reaction solvent with the composition of mercaptoethanol. As pET21a E. coli BL21 (DE3) codonplus-RIL strain (hereinafter referred to as BL21 (DE3) codonplus-RIL strain / pET21a) transformed with the above is cultured in the same manner, crude polyamine is extracted in the same manner, and the extract bis Aminopropylspermidine was analyzed.

1.4 Measurement of length and throat of nematodes feeding on E. coli expressing Tk1691 E. coli BL21 (DE3) codonplus-RIL strain / pET21a and BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691 in 50 μg ml ^-1 Pre-cultured at 37 ° C. for 12 hours in 5 ml of LB liquid medium containing ampicillin. Inoculated into 200 ml of M9 medium containing 50 μg ml ⁻¹ ampicillin and 1 mM isopropyl-β-thiogalactopyranoside and cultured at 37 ° C. for 12 hours. The culture was collected by centrifugation at 6000 rpm for 10 minutes, and then washed twice with 0.8% (wt / v) NaCl water. Next, E. coli was suspended in 100 μl of 0.8% (wt / v) NaCl water, 100 μl of the bacterial solution was added to M9 solid medium, and cultured at 42 ° C. for 12 hours. Five adults with eggs were placed on M9 solid medium coated with the fungus solution and reared at 20 ° C. for 3 days. Thirteen young adults were relaxed with 2% Sodium Azide (Sigma) dissolved in M9 liquid medium, photographed at a microscope magnification of 10 × 10, and body length and throat length were measured. ImageJ (http://rsbweb.nih.gov/ij/) was used for the measurement of body length. Three independent length measurement experiments were performed. Independent pharyngeal length measurement experiments were conducted twice.

1.5 Results
Spermidine was produced by polyamine analysis of BL21 (DE3) codonplus-RIL strain / pET21a grown in M9 liquid medium, but bisaminopropyl spermidine was not produced. On the other hand, bisaminopropylspermidine was produced by polyamine analysis in BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691 grown in M9 liquid medium (FIG. 4). The body length and throat length of nematodes fed BL21 (DE3) codonplus-RIL strain / pET21a or BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691 on M9 agar medium were measured, and BL21 (DE3) codonplus It was found that the body length was significantly increased in nematodes fed with -RIL strain / pET21a-Tk1691 (FIG. 5). However, the length of the pharynx did not change in nematodes fed BL21 (DE3) codonplus-RIL strain / pET21a-Tk1691 and BL21 (DE3) codonplus-RIL strain / pET21a (FIG. 6). This indicates that the elongation of tissues other than the pharynx was promoted by bisaminopropyl spermidine.

2. Example: Effect of an extract of the hyperthermophilic bacterium T. kodakarensis on the growth of beetle larvae
2.1. Preparation of cell-free extract of T. kodakarensis and administration to beetle larvae
T. kodakarensis KOD1 strain (Morikawa M., et al, Appl. Environ. Microbiol., 1994, 60: 4559-4566) produces bisaminopropyl spermidine. T. kodakarensis KOD1 strain 1.5 L ASW-YT-Pyruvate medium (1.25-fold artificial seawater (0.8 × ASW) (Robb and Place 1995), 5 gl ^-1 yeast extract (Nacalai Tesque), 5 gl ^-1 Polypeptone (Nacalai Tesque), 5 gl ⁻¹ pyruvate] was cultured at 60 ° C. for 48 hours. A Bacillus subtilis 168 trpC strain that does not produce bisaminopropylspermidine was cultured in LB medium at 37 ° C. for 12 hours. After collecting each culture by centrifugation at 6000 rpm for 10 minutes, T. kodakarensis KOD1 strain was 0.8 × ASW, Bacillus subtilis 168 trpC strain was suspended twice in 0.8% (wt / v) NaCl water, Washing was performed by repeating centrifugation at 6000 rpm for 10 minutes. Each microbial cell having a wet weight of 0.6 g was suspended in 200 ml of ultrapure water, and the microbial cells were sufficiently crushed with an ultrasonic crusher (Sonifier 25, Branson). 200 ml of ultrapure water or each cell disruption solution was mixed with 200 g of dry sawdust. Twenty-four first-instar larvae beetles of approximately the same length and weight were divided into eight groups, and the groups were put into sawdust mixed with each microbial cell disruption solution. The larvae were weighed after standing at room temperature for 1 month.

2.2 Results
Beetle larvae grown on sawdust containing extract of T. kodakarensis KOD1 grown at 60 ° C have significantly increased body length and weight than beetle larvae grown on sawdust containing Bacillus subtilis 168trpC or ultrapure water (Figure 7).
3. Example: Effect of activation of cell-free translation system of hyperthermophilic bacterium T. kodakarensis given by branched-chain polyamines
3.1 Preparation of S30 fraction of hyperthermophile T. kodakarensis T. kodakarensis KC1 strain (Endoh et al., J. Biotechnol., 2006, 126: 186-195) deficient in chitinase activity -YT-Pyruvate medium [1.25-fold diluted artificial seawater (0.8 × ASW) (Robb and Place 1995), 5 gl -1 yeast extract (Nacalai Tesque), 5 gl ^-1 polypeptone (Nacalai Tesque), 5 gl ^{- 1} pyruvic acid] was cultured at 60 ° C. for 48 hours. The culture broth was collected by centrifugation, and then washed by repeating suspension and centrifugation twice with 0.8 × ASW. The cells were suspended in S30 protease inhibitor buffer (10 mM Tris-acetic acid pH 7.4, 1 mM dithiothreitol, 1.4 mM magnesium acetate, 6.0 mM potassium acetate, protease inhibitor (Roch) 1 tablet / 10 ml). This cell suspension was disrupted at 7,500 psi for 1 minute using a homogenizer (EmulsiFlex C-5, AVESTIN). The obtained cell lysate was centrifuged at 30,000 × g, 4 ° C. for 30 minutes. The supernatant was centrifuged again at 30,000 xg, 4 ° C for 30 minutes. The supernatant is collected and placed in a dialysis membrane with a size exclusion limit of 6-8 kDa (100 mM S30 buffer (10 mM Tris-acetate pH 7.4, 1 mM dithiothreitol, 1.4 mM magnesium acetate). , 6.0 mM potassium acetate) for 45 minutes, the dialysis operation was repeated twice more, and the supernatant was diluted 1 million times with S30 buffer, then centrifuged at 4,000 xg, 4 ° C for 10 minutes, S30 fraction was obtained.

3.2 Preparation of chiA Δ4 mRNA from T. kodakarensis
Using the plasmid pTRC1 (Endoh et al., J. Biotechnol., 2006, 126: 186-195) having the chiAΔ4 gene region encoding chitinase derived from T. kodakarensis, T7 RiboMax® Express RNA system (Promega) ) To prepare mRNA encoding chitinase.

3.3 Measurement of cell- free translation system and chitinase activity using the S30 fraction of the hyperthermophilic bacterium T. kodakarensis Cell- free translation reaction was performed using 11 mM magnesium acetate, 2 mM dithiothreitol, 80 mM ammonium acetate, 275 mM Potassium acetate, 1.5 mM guanosine triphosphate, 1.5 mM cytidine triphosphate, 1.5 mM uridine triphosphate, 10 mM phosphoenolpyruvate, 45 mM bicine (pH 8.5), 3 mM adenosine triphosphate, 2 mM L- Glycine, 2 mM L-cysteine, 2 mM L-glutamic acid, 2 mM L-arginine, 2 mM L-proline, 2 mM L-asparagine, 2 mM L-histidine, 2 mM L-isoleucine, 2 mM L-leucine, 2 mM L-lysine, 2 mM L-threonine, 2 mM L-tyrosine, 2 mM L-alanine, 2 mM L-methionine, 2 mM L-phenylalanine, 2 mM L-serine, 2 mM L-tryptophan, 2 mM L-aspartic acid, 2 mM L-glutamine, 2 mM L-valine, 4% (v / v) RNAsecure (registered trademark) Nology), 2% (v / v) polyethylene glycol, 0.3 mg / ml ChiAΔ4 mRNA, 6.5 mg / ml S30 fraction, 0.2 mM Each polyamine was used in a total volume of 30 μl. The absence of polyamine was used as a control experiment for cell-free translation reaction. The polyamines added to the cell-free translation reaction were 1,3-propanediamine [3], 1,4-butanediamine [4], N ^1- (3-aminopropyl) butane-1,4-diamine [34], respectively. , N ¹ , N ¹ -bis (2-aminoethyl) ethane-1,2-diamine [2 (2) 2], N ¹ , N ¹ -bis (2-aminopropyl) propane-1,2-diamine [ 3 (3) 3], N , N 1 - bis (3-aminopropyl) butane-1,4-diamine ^{[343], N 1 - (} 3- aminopropyl) -N ³ - (3- (3-amino Propylamino) propyl) propane-1,3-diamine [3333], N ^1- (3- (3-aminopropylamino) propyl) butane-1,4-diamine [334], 1,3-bis [3- (3-Aminopropylamino) propylamino] propane [33333], N ¹ , N ¹ -bis (3-aminopropyl) butane-1,4-diamine [3 (3) 4], N ^1- (3-amino propyl) -N ¹ - (3- (3- aminopropyl amino) propyl) butane-1,4-diamine [3 (4) 33], N 1 - (3- aminopropyl) -N ⁴ - (3- ( 3-aminopropylamino) Propyl) butane-1,4-diamine ^{[3343], N 1, N} 1, N 4 - tris (3-aminopropyl) butane-1,4-diamine [3 (3) 43], N 1 - (3- (3- (3-Aminopropylamino) propylamino) propyl) butane-1,4-diamine [3334], 4-amino-N, N, N-tris (3-aminopropyl) butane-1-aminium [bis Aminopropylspermidine] [3 (3) (3) 4], 4-amino-N- (4-aminobutyl) -N, N-bis (3-aminopropyl) butane-1-aminium [3 (3) ( 4) 4] was used (the numbers in square brackets represent the number of carbon chains through the amino group, and the numbers in parentheses in the brackets represent the number of carbon chains branched through the amino group. Represent). The cell-free translation reaction solution having these compositions was reacted at 80 ° C. for 1 hour and cooled to 4 ° C. to stop the reaction. Add 3 μl of the cell-free translation reaction solution after the cell-free translation reaction to 33 mM 4-methylumbelliferyl-β-DN, N'-diacetyl chitobioside (Sigma), and allow it to react at 90 ° C for 30 minutes. It was. The chitinase reaction solution was added to 50 mM Glycine (pH 11.0) to stop the chitinase reaction. Thereafter, the mixture was centrifuged at 4 ° C., 13,200 rpm for 5 minutes, and 200 μl of the supernatant was dispensed into a 96-well fluorescent plate (Perkielmer). The fluorescence of the compound produced by the chitinase reaction was measured with a fluorescence plate reader (ARVOMx / Light, 1420 Multilabel / Lumescence Counter, Perkin Elmer Japan) at an excitation wavelength of 365 nm and a detection wavelength of 455 nm.

3.4 Results
In the cell-free translation reaction at 80 ° C., it was found that polyamines having a valence of 3 (3) 3 or 343 or more have a translational activation effect as compared with the system not added with polyamine. On the other hand, spermidine and putrescine inhibited translation. In addition, as the number of branched aminopropyl groups increased, a translational activation effect was observed (FIG. 8; translation activity of without polyamine was set to 1).

SEQ ID NO: 3 Tk1691-Fw primer
SEQ ID NO: 4 Tk1691-Rv primer

Claims (7)

A growth promoter for animals and plants containing a branched-chain polyamine or a long-chain polyamine. The growth promoter for animals and plants according to claim 1, wherein the branched polyamine is a tertiary polyamine represented by the following formula (I) or a quaternary polyamine represented by the following formula (II).
(In the formula, R ^{1 to} R ⁶ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ² , R ^3, and R ⁵ are respectively The repeating units may be the same or different, and n ^{1 to} n ³ are the same or different and are 0 or an integer of 3 or less.)
^(Wherein, R 7 ^{to R 15} are the same or different, having 2 to 6 carbon atoms, which may be substituted, an alkyl group or an alkenyl group, and ^{R 8, R 9, R 12} and ^{R 14} May be the same or different for each repeating unit, and n ^{4 to} n ⁷ are the same or different and are 0 or an integer of 3 or less.) The growth promoter for animals and plants according to claim 1, wherein the long-chain polyamine is a long-chain polyamine represented by the following formula (III):
(Wherein R ^{16 to} R ¹⁹ are the same or different and each represents an optionally substituted alkyl group or alkenyl group having 2 to 6 carbon atoms, and R ¹⁷ and R ¹⁸ are each a repeating unit. Each may be the same or different, and n ⁸ and n ⁹ are the same or different and are integers of 3 or less.) The growth promoter for animals and plants according to claim 1, wherein the branched-chain polyamine and the long-chain polyamine can be produced by thermophilic bacteria. A growth promoter for animals and plants comprising a thermophilic bacterium or a cell extract thereof. An animal or plant growth promoter containing an aminoalkyltransferase or an aminoalkenyltransferase (provided that the alkyl group and the alkenyl group have 2 to 6 carbon atoms and may be substituted, an alkyl group or an alkenyl group) Group). A growth promoter for animals and plants containing a gene encoding an aminoalkyltransferase or an aminoalkenyltransferase, or an expression vector containing the gene (however, the alkyl group and the alkenyl group have 2 to 6 carbon atoms) An alkyl group or an alkenyl group which may be substituted.