JP4331335B2 - Cotton plant with improved cotton fiber characteristics, method for producing the same, and method for producing cotton fiber from the cotton plant - Google Patents

Description

【００５５】
（２）隔離圃場での栽培試験（１９９８年５月〜１２月に実施）
表１に記載したセルラインの種子と遺伝子組換えを行っていないコーカー３１２を供試材料として用いた。１９９８年５月２０日に、テキサス工科大学付属の隔離圃場に播種した。１９９８年７月５日から、これらのワタ植物は開花を始め、受粉は自家受粉を行った。１９９８年７月〜９月にかけて栽培している全ての形質転換体から健全な若い葉を採取し、常法に従って導入した目的遺伝子をプローブにしてノザン解析を行い、目的遺伝子の発現を調べた。１９９８年９月から朔果が開じょし、乾燥後、胚珠を収穫した。Texas A & M Agricultural Experiment Station でマシンジニングを行い, ワタ繊維を種子から分離した。得られたワタ繊維について、（１）９００ＨＶＩシステム（Ｓｐｉｎｌａｂ社製）を用いて、繊維繊度、繊維長均斉度、繊維長および繊維強度を測定した。その結果を表２に示す。
【００５６】
【表２】

【００５７】
表２より明らかなように、エンドウ由来のカタラーゼ遺伝子であるＣＡＴが導入された形質転換体は、全てのセルラインで顕著に繊維繊度、繊維長均斉度および繊維強度が増大した。
以上の結果から、ワタ植物にエンドウ由来のカタラーゼ遺伝子であるＣＡＴを導入することによって、繊維繊度および繊維長均斉度等の繊維特性が有意に増大したワタ繊維を生産するワタ植物が得られることが明らかとなった。
【００５８】
【発明の効果】
上述したように、本発明により、ワタ繊維における繊度、繊維長均斉度等の繊維特性の改良を行うことができる、例えば、繊度が改良されることにより糸の染色性が高まり、また、繊維長均斉度が改良されることにより糸の強度が高まる。したがって、本発明のワタ植物から採取されるワタ繊維により、織物品質の向上が可能となる。さらに本発明のワタ植物は、より優れたワタ繊維特性および／またはワタ繊維生産性を有する、さらに新規なワタ品種の作出のための植物材料としても利用することができる。
【００５９】
【配列表】

【図面の簡単な説明】
【図１】エンドウ由来カタラーゼ遺伝子の発現カセットを含むバイナリーベクターｐＢＩＮ３５Ｓ−Ｓ．Ｓ−ＣＡＴの構築手順を示す図である。なお、図中の略語の意味は以下の通りである。Ｓ．Ｓ：ワタペルオキシダーゼ由来細胞壁移行シグナル配列，Ｆｒ．１：フラグメント１，Ｆｒ．２：フラグメント２，Ｆｒ．３：フラグメント３，３５Ｓ ｐｒｏ：カリフラワーモザイクウイルス由来３５Ｓプロモーター，ｔｅｒ：ターミネーター，ＮＯＳ ｐｒｏ：ノパリン合成酵素プロモーター，ｎｐｔII：ネオマイシンホスホトランスフェラーゼIIコード配列，Ｓ．Ｓ−ＣＡＴ：ワタペルオキシダーゼ由来細胞壁移行シグナル配列とエンドウ由来カタラーゼｃＤＮＡの融合遺伝子，ＲＢ：ライトボーダー配列，ＬＢ：レフトボーダー配列，Ｘｈ：ＸｈｏＩ制限サイト，Ｂ：ＢａｍＨＩ制限サイト，Ｈ：ＨｉｎｄIII 制限サイト，Ｅ：ＥｃｏＲＩ制限サイト，Ｘｂ：ＸｂａＩ制限サイト，Ｐ：ＰｓｔＩ制限サイト[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cotton plant that produces cotton fibers with improved fiber properties compared to wild strains, and in particular, fiber properties such as increased fiber fineness (thickness) and fiber length uniformity (short fiber content). The present invention relates to a cotton plant that produces cotton fiber having the following. The present invention also relates to a method for producing the cotton plant and a method for producing cotton fiber having improved fiber characteristics from the cotton plant.
[0002]
[Prior art]
Cotton fiber is usually produced by cultivating a cotton plant belonging to the genus Gossypium and collecting it from the obtained fruit (cotton ball). Cotton fiber is a single cell in which the epidermal cells of the seed coat are differentiated, initiation, elongation, secondary cell wall thickening, and maturation ) Through 4 stages. More specifically, cotton fiber elongation starts fiber elongation immediately after ovule epidermis flowering, and then the fiber grows rapidly and completes elongation about 25 days after flowering. Thereafter, the elongation of the fiber stops and a secondary cell wall is generated, and after maturing, it becomes one mature cotton fiber.
[0003]
There are many varieties of cotton plants, cotton fibers having different fiber characteristics are obtained, and they are used properly according to various uses. There are various characteristic parameters indicating fiber characteristics, and particularly important ones include fiber length, fineness, strength, and fiber length uniformity. Conventionally, great efforts have been made to improve the properties of cotton fibers, but the main points of improving the fiber properties are fiber length and fineness. Among them, longer and thinner fibers have been desired. Umijima cotton is famous as a variety having such fiber characteristics, but its productivity is low and it is very expensive. If cotton fibers having fiber characteristics equivalent to or better than Umishima cotton can be obtained with higher productivity, it is very beneficial in the industry.
[0004]
Means for improving fiber characteristics and / or productivity are roughly divided into (1) breed improvement by crossing, (2) treatment with plant hormones, and (3) breed improvement using gene recombination technology. There are three.
[0005]
Varietal improvement by crossing has been the most widely used hitherto, and the current cotton plant varieties have been bred by this method. However, since this method can introduce useful characters only from closely related species, it takes a lot of time, and the degree of change is low. Especially, in terms of cotton fiber characteristics or cotton fiber yield, there is no significant improvement. I can't expect much.
[0006]
Plant hormones such as auxin, gibberellin, cytokinin, and ethylene are widely used for regulating the growth of agricultural crops and horticultural plants. The effects of plant hormones on the fiber properties of cotton plants, especially on the fiber elongation mechanism, are known for gibberellins, auxins, brassinosteroids, etc., but in fact, sufficient effects are available for industrial use. At present, it has not been put into practical use.
[0007]
On the other hand, in recent years, there has been remarkable progress in gene recombination technology in the field of plant breeding, and introduction and expression of specific useful foreign genes in various plants (for example, cotton, soybean, corn, tomato, etc.). Thus, many examples have been reported that succeeded in imparting desired useful traits to the plant.
[0008]
In cotton plants, insect-resistant recombinant cotton introduced with BT toxin [insecticidal protein toxin produced by Bacillus thuringiensis] gene and 5-enolpyruvylshikimate-3-phosphate synthase gene Herbicide (glyphosate) -tolerant recombinant cotton and the like that have been introduced have been developed and commercialized (it is said that the share of planted area of recombinant cotton in the US has already reached a majority).
[0009]
Similarly, instead of pest resistance genes and herbicide resistance genes, genes related to cotton fiber formation and elongation are introduced into cotton plants and expressed in large quantities, dramatically increasing their fiber properties or productivity. It is expected to improve. Conversely, it is theoretically possible to change the fiber characteristics by incorporating an antisense gene and suppressing the function of the endogenous gene. A method using such a genetic engineering technique is expected to be able to more reliably control fiber elongation and formation within a short period of time than breeding by conventional mating and selection.
[0010]
However, in order to obtain cotton fibers with the desired fiber characteristics by genetic recombination technology, it is essential to clarify the mechanisms of fiber elongation and formation at the gene level and to determine genes that are closely involved in those mechanisms. However, at present, molecular biological knowledge about such genes is very poor. Although much research has been conducted on plant cell elongation so far, there are still many unclear points about the control factors, and the control mechanism has not been elucidated until now.
[0011]
However, there are no reports of genes involved in cotton fiber elongation and formation. For example, John et al., E6 gene (Maliyakal E. John and Laura J. Crow, Proc.) Expressed preferentially in the fiber tissue on the 15th and 24th day of flowering by the differential screening method (Differential Screening). Natl. Acad. Sci. USA, 89, 5769-5773 (1992)) and the H6 gene encoding a proline-rich protein that works actively during secondary cell wall formation (Maliyakal E. John and Greg Keller, Plant physiol., 108, 669-676 (1995)). In addition, John introduced the E6 gene into a cotton plant in an antisense form to suppress the expression level of the endogenous E6 gene and examined the effect of the E6 gene on cotton fiber properties (Maliyakal E. Jhon, Plant Molecular Biology, 30, 297-306 (1996)). However, although the expression level of E6 RNA in the fiber tissue decreased, it was reported that the fiber length, strength, and fineness did not change significantly, and concluded that the E6 gene is not important for cotton fiber development.
Meanwhile, Song et al. Identified a cDNA encoding an acyl carrier protein (ACP) specifically expressed in cotton fiber tissue from a differential plant (Ping Song, Randy D. Allen, Biochimica et Biophysica). Acta, 1351, 305-312 (1997)). In addition, the cDNA of the catalytic subunit of cellulose synthase has been isolated as a gene related to cellulose synthesis of cotton fibers (Julie R. Pear, Yasushi Kawagoe, et al, Proc. Natl. Acad. Sci. USA, 93, 12637). -12642 (1996)).
[0012]
In addition, several examples of the production of recombinant cotton plants introduced with genes implicated in fiber properties have been reported. For example, Agracetus in the United States has improved fiber strength by introducing a peroxidase gene, which is thought to be related to ovule and fiber development, downstream of the promoter of a fiber-specific expression gene into cotton plants. It has been reported that a recombinant cotton plant producing cotton fiber has been developed (International Publication WO95 / 08914). In addition, the present inventors isolated and identified five genes that are specifically expressed during cotton fiber elongation by a differential screening method and a differential display method (Japanese Patent Laid-Open No. 9-75093), of which By introducing the KC22 gene, which is one of the above, into a cotton plant, a recombinant cotton plant producing cotton fiber with an improved fiber length has been obtained.
[0013]
However, only a few genes that are specifically expressed during cotton fiber formation / elongation have been isolated as described above, and moreover, which of these fiber-specific genes is the fiber length, fiber At present, little has been clarified as to whether it plays an important role in determining the parameters of cotton fiber characteristics such as strength, fineness, and fiber length uniformity. Furthermore, a recombinant cotton plant that produces cotton fibers with improved characteristic parameters such as fiber fineness and fiber length uniformity has not yet been reported.
[0014]
[Problems to be solved by the invention]
Therefore, the object of the present invention is important for the determination of cotton fiber properties such as genes involved in cotton fiber elongation and formation, particularly fiber fineness (fiber thickness) and fiber length uniformity (short fiber content). By screening for genes that play a role and artificially controlling the expression of the genes, the above-mentioned recombinant cotton plants with improved cotton fiber properties are produced. Moreover, the objective of this invention is providing the manufacturing method of the cotton fiber by which the fiber characteristic from this cotton plant was improved.
[0015]
[Means for Solving the Problems]
As a result of diligent efforts to achieve the above object, the present inventors have introduced a catalase gene into cotton plants and overexpressed in cotton fibers, thereby allowing characteristic parameters such as fineness and fiber length uniformity of cotton fibers. In addition, by harvesting and isolating cotton fiber from this recombinant cotton plant, cotton fiber with improved fiber properties was successfully produced and the present invention was completed. It was.
[0016]
That is, the present invention is as follows.
1. Cotton plant belonging to the genus Gospium, which stably retains an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber, and produces cotton fiber having improved fiber characteristics compared to the wild strain, particularly The cotton plant, which is a transgenic plant obtained by transforming the wild strain with an expression vector containing an exogenous catalase gene under the control of a promoter that can function in cotton fiber.
2. The said cotton plant which is a homozygote about this catalase gene, especially the said cotton plant which is the form of a seed.
3. Cotton fiber obtained from the above cotton plant and having improved fiber characteristics compared to the wild strain.
4). An expression vector containing an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber, and comprising an improved step compared to the wild strain, comprising the step of transforming a cotton plant wild strain belonging to the genus Gospium A method for producing a cotton plant producing cotton fibers having fiber characteristics, in particular, a method for regenerating a plant from the transformed cell.
5. The following steps:
(1) An expression vector containing an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber, transforming a cotton plant cell line belonging to the genus Gospidium,
(2) Regenerating a cotton plant (R0) that produces cotton fibers having improved fiber characteristics compared to the wild type strain from the transformed cells;
(3) collecting seeds from the plant body by self-pollination; and
(4) Test the separation ratio of the catalase gene in seeds obtained by self-pollination from cotton plants (R1) obtained by cultivating the seeds
A method for producing a cotton plant having a fixed trait that produces a cotton fiber that is homozygous for the catalase gene and has improved fiber characteristics as compared to the wild type.
6). A method for producing cotton fiber having improved fiber characteristics compared to a wild strain, comprising a step of collecting seed obtained by self-pollination of any of the above cotton plants and separating cotton fiber from the seed coat of the seed.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
The cotton plant of the present invention is not particularly limited as long as it belongs to the genus Gossypium, and is derived from a plant that produces cotton fiber. For example, G. hirsutum, G. hirsutum, barbadense), Gosypium arboreum, G. anomalum, G. armourianum, G. klotzchianum, G. klotzchianum, G. herbaceum (G. herbaceum) Examples include those derived from cotton plants such as G. raimondii.
[0018]
Here, the “cotton plant” finally produces cotton fiber equivalent to the plant body even during or before the formation of the cotton fiber, in addition to the plant body containing the matured cotton fiber in the fruits. Obtaining growing cotton plant, cotton plant cell or tissue capable of differentiating into immature cotton plant, and seed collected from the cotton plant (provided by cultivating the seed) All produce cotton fiber equivalent to the parent).
[0019]
In the present invention, the “wild strain” means any cotton plant that does not have an exogenous catalase gene on its genome. Therefore, in addition to so-called wild species, all cultivars established by normal mating, natural or artificial mutants thereof, and transgenic cotton plants introduced with exogenous genes other than catalase are included.
[0020]
In the present invention, "cotton fiber having improved fiber characteristics" means, for example, at least one of various parameters usually used for evaluation of fiber characteristics such as fiber length, fineness, fiber strength, fiber length uniformity, It means improved cotton fiber compared to the wild type. Preferably, the cotton plant of the present invention produces cotton fibers in which at least one characteristic parameter of fiber fineness and fiber length uniformity is improved compared to the wild type. More preferably, the cotton plant of the present invention produces cotton fibers having improved fiber fineness and fiber length uniformity as compared to the wild type.
[0021]
The cotton plant of the present invention is one in which an exogenous catalase gene has been introduced and stably maintained by a genetic engineering technique in these cotton plants belonging to the genus Gospium. Here, “stablely maintained” means that the catalase gene is expressed in cotton fiber produced by a plant of the present generation into which at least the catalase gene has been introduced, and is sufficient to improve the fiber characteristics of cotton fiber. It means being retained in the cotton plant cell for a period of time. Therefore, in reality, the catalase gene needs to be integrated on the chromosome of the host cotton plant. More preferably, the catalase gene is stably inherited in the next generation.
[0022]
Many plant catalases are localized in microbodies and are produced by metabolically toxic H ₂ O ₂ Is a detoxifying enzyme that breaks down water into oxygen. However, its function in plants is not fully understood. Catalase is a plant's cold adaptability and pathogen resistance (Sanchez-Casas, P. and Klessing, DF Plant Physiol., 106, 1675-1679 (1994), Prasad, TK, Anderson, MD, et al, Plant Cell, 6 , 65-74 (1994)), but there has been no report on the relationship between catalase and the fiber properties of cotton plants.
[0023]
In the present invention, “catalase gene” refers to a decomposition reaction of hydrogen peroxide (2H ₂ O ₂ → O ₂ + 2H ₂ DNA having a base sequence encoding the amino acid sequence of an enzyme that catalyzes O). The origin of the catalase gene is not particularly limited, and a catalase gene that has already been cloned and is readily available can be used. The catalase gene may be a cDNA obtained from mRNA (or a cDNA library), a genomic DNA obtained from a genomic DNA library, or by chemical synthesis. Preferably, the catalase gene is a plant-derived gene, more preferably a pea-derived gene (Reference Example 1 described below shows a cloning procedure for pea-derived catalase cDNA).
[0024]
As a preferable example of the catalase gene used in the present invention, DNA having the base sequence shown by base numbers 57 to 1541 in the base sequence shown in SEQ ID NO: 1 in the sequence listing, or hybridizes with the sequence under stringent conditions And a DNA encoding a polypeptide having a base sequence that can be converted and having a catalase activity equivalent to that sequence. The term “stringent conditions” as used herein means that only a base sequence encoding a polypeptide having a catalase activity equivalent to a catalase encoded by a specific catalase gene sequence forms a hybrid (so-called specific hybrid) with the specific sequence. A base sequence encoding a polypeptide having no equivalent activity means a condition that does not form a hybrid (so-called non-specific hybrid) with the specific sequence. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0.2 M NaH) ₂ PO _Four , 20 mM EDTA · 2Na, pH 7.4) at 42 ° C., and further washed with 0.5 × SSC at 42 ° C. is one example of the stringent conditions of the present invention. It is not limited.
[0025]
The “base sequence capable of hybridizing under stringent conditions” of the present invention has the base sequence of SEQ ID NO: 1 with respect to an arbitrary DNA pool (for example, a cDNA derived from catalase-producing cells / tissues or a genomic DNA library). It can be obtained by performing a hybridization reaction under stringent conditions using DNA as a probe.
[0026]
The term “exogenous” as used herein means that the cotton plant does not naturally exist and is introduced from the outside. Therefore, the “exogenous catalase gene” of the present invention may be a catalase gene of the same kind as the host cotton plant (ie, derived from the host cotton plant) introduced from the outside by genetic manipulation. Considering the identity of codon usage, the use of a host-derived catalase gene is also preferred.
[0027]
The exogenous catalase gene may be introduced into cotton plants by any genetic engineering technique, for example, a protoplast fusion with a heterologous plant cell having the catalase gene, a plant having a viral genome genetically engineered to express the catalase gene Examples thereof include infection by virus, or transformation of host cotton plant cells by an expression vector containing a catalase gene.
[0028]
Preferably, however, the cotton plant of the present invention is obtained by transforming a cotton plant cell line with an expression vector comprising an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber. It is a transgenic plant.
[0029]
Promoters that can function in cotton fibers include promoters of genes that are constitutively expressed in plant cells, preferably constitutive promoters derived from plants or plant viruses (eg, cauliflower mosaic virus (CaMV) 35S promoter, CaMV19S promoter, NOS Promoters, etc.), and cotton plant endogenous promoters that are specifically expressed during cotton fiber formation and elongation. Examples of such cotton fiber specific promoters include various promoters described in International Publication No. WO95 / 08914.
[0030]
In the expression vector of the present invention, the exogenous catalase gene is placed downstream of the promoter so that its transcription is controlled by a promoter that can function in cotton fiber. It is preferable that a transcription termination signal (terminator region) that can function in cotton fibers is further added downstream of the catalase gene. Examples of such terminator include NOS (nopaline synthase) gene terminator.
[0031]
Since cotton fiber elongation is accompanied by cell wall development, it is expected that genes involved in fiber elongation preferably express activity in and around the cell wall. Therefore, when the exogenous catalase gene to be introduced does not have a coding sequence for a cell wall transition signal recognized by cotton plant cells, the coding sequence of the signal peptide is inserted between the promoter and the mature catalase coding sequence. It is desirable to do. As such a signal sequence, a signal sequence of cotton plant-derived peroxidase is preferably exemplified.
[0032]
The expression vector of the present invention may further contain a cis-regulatory element such as an enhancer sequence. The expression vector is a marker gene for selecting a transformant such as a drug resistance gene marker, such as a neomycin phosphotransferase II (NPTII) gene, a phosphinothricin acetyltransferase (PAT) gene, a glyphosate resistance gene, etc. It is desirable to further include. Under the condition where selective pressure is not applied, the incorporated gene may drop off. Therefore, if the herbicide tolerance gene is allowed to coexist on the vector, the herbicide is always used during cultivation. There is also an advantage that a condition under selective pressure can be realized.
Furthermore, in order to facilitate mass preparation and purification, the expression vector contains an origin of replication that allows autonomous replication in E. coli and a selectable marker gene in E. coli (eg, ampicillin resistance gene, tetracycline resistance gene, etc.). Is desirable. The expression vector of the present invention can be conveniently constructed by inserting the above-mentioned catalase gene expression cassette and, if necessary, a selection marker gene into the cloning site of a pUC or pBR E. coli vector.
[0033]
When an exogenous catalase gene is introduced by utilizing infection by Agrobacterium tumefaciens or Agrobacterium rhizogenes, on the Ti or Ri plasmid carried by the bacterium The catalase gene expression cassette can be inserted into a T-DNA region (region that is transferred to a plant chromosome). Currently, the standard method for transformation by the Agrobacterium method uses a binary vector system. The functions required for T-DNA transfer are supplied independently from both the T-DNA itself and the Ti (or Ri) plasmid, and each component can be divided on separate vectors. The binary plasmid has 25 bp border sequences at both ends necessary for excision and integration of T-DNA, and the plant hormone gene causing crown gall (or hairy root) is removed, and at the same time, room for insertion of a foreign gene is provided. ing. As such binary vectors, for example, pBI101 and pBI121 (both CLONTECH) are commercially available. The vir region acting on T-DNA integration is located on another Ti (or Ri) plasmid called a helper plasmid and acts on trans.
[0034]
Various conventionally known methods can be used for transformation of cotton plants. For example, protoplasts are isolated from cotton plant cells by treatment with cell wall degrading enzymes such as cellulase and hemicellulase, and polyethylene glycol is added to a suspension of the protoplasts and an expression vector containing the above-mentioned catalase gene expression cassette to obtain endocytosis. Incorporation of the expression vector into the protoplast during the tosis-like process (PEG method), phosphophatidylcholine and other lipid membrane vesicles are placed by sonication, etc., and the vesicle and protoplast are fused in the presence of PEG Method (liposome method), fusion method using minicells in the same process, method of applying electric pulse to suspension of protoplast and expression vector and incorporating vector in external solution into protoplast (electroporation) Law). However, these methods are complicated in that they require a culture technique for redifferentiation from protoplasts to plants. As a means for gene introduction into intact cells with cell walls, a micropipette is inserted into the cell, and vector DNA in the pipette is injected into the cell by hydraulic pressure or gas pressure, and micro metal particles coated with DNA There are a direct introduction method such as a particle gun method that accelerates the explosives using explosives and gas pressure, and introduces them into cells, and a method that uses infection by Agrobacterium. Microinjection requires the skill of operation and has the disadvantage that the number of cells that can be handled is small. Therefore, considering the ease of operation, it is preferable to transform cotton plants by the Agrobacterium method and the particle gun method. The particle gun method is further useful in that a gene can be directly introduced into the apical meristem of a plant under cultivation. Further, in the Agrobacterium method, a plant vector, for example, a genomic DNA of a gemini virus such as tomato golden mosaic virus (TGMV) is simultaneously inserted into a binary vector between border sequences, so that an arbitrary site of a plant being cultivated can be detected. By simply inoculating cells with a bacterial suspension using a syringe or the like, viral infection spreads throughout the plant body, and the target gene is also introduced into the entire plant body.
[0035]
However, in the particle gun method and the Agrobacterium method, gene transfer is often a chimera, so a sample cell in which a catalase gene is frequently introduced into germ line cells is used for transformation. Need to be used for. Examples include embryos, hypocotyl slices, embryogenic callus, isolated growth points, and the like. On the other hand, since each of the above transformation methods and microinjection methods using protoplasts can select a redifferentiated plant from a single cell, it is said that a homogeneous transgenic plant having a catalase gene introduced into all cells is obtained. It can be said that it is an excellent transformation means from the viewpoint.
[0036]
As a specific example, a method for introducing a target gene by Agrobacterium and regenerating a transformed cell into a plant body in a cotton plant is shown below.
The seeds of cotton plants were prepared according to conventional methods, Stewart seed germination medium (Stewart concentrate, 0.75 g / L MgCl ₂ , 2.0 g / L fighter gel, pH 6.8) and cultivated aseptically. On the other hand, Agrobacterium transformed with a plasmid having a target gene connected to a promoter and having a kanamycin resistance gene is cultured and diluted with MSNH (MS inorganic salts, 30 g / L glucose, pH 5.8). Dispense into. The hypocotyl is cut out from the germinated cotton seedling, and the hypocotyl section is immersed in the above-mentioned bacterial dilution to infect it. Agrobacterium diluted solution is removed on a filter paper sterilized from a hypocotyl section infected with Agrobacterium, and T2 plate (MS inorganic salt, 0.75 g / L MgCl ₂ , 0.1 mg / L 2,4-D, 0.5 mg / L kinetin, 30 g / L glucose, 2.0 g / L fighter gel, pH 5.8) for 3 days. Co-cultured hypocotyls were placed on MS2NKKCL plates (MS inorganic salts, 0.75 g / L MgCl ₂ , 30 g / L glucose, 2 mg / L naphthalene acetic acid, 0.1 mg / L kinetin, 2.0 g / L fighter gel, 50 μg / ml kanamycin, 500 μg / ml cefotaxime, pH 5.8) for 3-4 weeks Incubate. When callus of about 4 mm size is observed, the callus is removed from the hypocotyl and transferred to a fresh MS2NKKKCL plate for further growth. After sufficient growth until the callus is about 1 cm in size, it is transferred to a kanamycin-containing MSNH liquid medium for embryo induction, and liquid suspension culture is started. Liquid suspension culture is performed until slightly spherical and light green cells can be observed with the naked eye. Suspension cultures containing fully grown cells are diluted with MKNH and MSK50K plates (MS inorganic salts, 0.75 g / L MgCl ₂ 1.9g / L KNO _Three , 30 g / L glucose, 2.0 g / L fighter gel, 50 μg / ml kanamycin, pH 5.8) and spread on a plate for embryo formation. Cultivation is continued until embryos of at least about 1 mm size are formed and transferred to SA plates (Stewart concentrate, 20 g / L sucrose, 20 g / L agar, pH 6.8) as germination medium. After several weeks of culture and rooting is observed, the roots are removed and the embryos are placed in SGA plates (Stewart concentrate, 5 g / L sucrose, 0.75 g / L MgCl ₂ 5 g / L agar, 1.5 g / L fighter gel, pH 6.8) and continue culturing until true leaves are observed. Once the first true leaf is observed, cotton seedlings can be transplanted into pint-sized canned bottles containing SGA medium. If the height of the cotton seedling becomes 10 cm and several true leaves are observed, it can be planted in a suitable pot and cultivated in a greenhouse. Seeds and cotton fibers can be obtained from the cotton transformant thus obtained.
[0037]
Genomic DNA is extracted from this transformant according to a conventional method, this DNA is cleaved with an appropriate restriction enzyme, Southern hybridization is performed using the introduced target gene as a probe, and the presence or absence of transformation can be confirmed. it can. Alternatively, a primer that specifically amplifies the target gene can be synthesized and the presence or absence of transformation can be confirmed by PCR.
In addition, RNA is extracted from transformants and non-transformants according to a conventional method, probes having sense or antisense sequences of the introduced target gene are prepared, and Northern hybridization is performed using these probes. The state of expression of the target gene can be examined.
[0038]
Cotton fibers differentiated from seeds obtained by self-pollination of transgenic cotton plants obtained in this way have improved fiber characteristics compared to cotton fibers obtained from wild strains. It is included in a statistically constant ratio depending on the style. Preferably, the cotton fibers have fiber characteristics with improved parameters of at least either fiber fineness or fiber length uniformity, more preferably both parameters. More preferably, the cotton fiber further has fiber characteristics in which the fiber length and fiber strength are improved as compared with the wild type.
[0039]
The appearance ratio of the improved fiber properties in cotton fibers differentiated from R1 seeds obtained by self-pollination of regenerated generation (R0) plants usually follows Mendel's law. For example, when the catalase gene is heterozygically integrated at one locus, improved cotton fiber properties separate at a ratio of 3: 1 in R1 seeds. In R2 seeds obtained by cultivating R1 seeds differentiated from cotton fibers having improved fiber characteristics and self-pollinating, if the improved cotton fiber characteristics are retained in all seeds, the R1 plant is If the introduced catalase gene is homozygous for the introduced catalase gene and the cotton fiber property segregates 3: 1, then the R1 plant can be determined to be heterozygous for the introduced catalase gene.
[0040]
Cotton plants homozygous for the introduced catalase gene selected in this way are extremely useful in the field of the seed industry as lines with improved cotton fiber properties fixed.
[0041]
The present invention also provides a method for producing cotton fibers from a cotton plant that produces cotton fibers having improved fiber properties obtained as described above.
Using the seeds of the selected lines, a cultivation test can be performed in a closed greenhouse or an isolated field. In the cultivation test, selected cotton seeds are sown according to a conventional method, and after the cotton plants are grown and flowered, they are self-pollinated to obtain the fruit, and the seeds can be collected from the opened fruit.
[0042]
Cotton fiber can be isolated by machine-ginning the collected seeds according to a conventional method. About the obtained cotton fiber, cotton fiber characteristics can be evaluated using, for example, a sorter method, a pressley method, a sterometer method, an HVI (mass high speed inspection machine) system method, and the like.
[0043]
【Example】
EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.
[0044]
Reference Example 1 Isolation of pea-derived catalase cDNA
The cDNA cloning was performed according to the method described in Plant Mol. Biol., 17, 1263-1265 (1991), page 1263, left column, line 18 to right column, line 15.
(1) Construction of cDNA library
Leaves were collected from pea (Pisum sativum) seedlings on the 10th day of germination, and poly (A) was collected according to a conventional method. ⁺ RNA was extracted. Isolated poly (A) ⁺ Using RNA as a template, cDNA is synthesized using an oligo (dT) primer and reverse transcriptase, and then double-stranded by a polymerase reaction is inserted into a vector, and E. coli is transformed to create a cDNA library. did. poly (A) ⁺ RNA isolation and cDNA synthesis were performed using a commercially available cDNA cloning kit according to the attached protocol.
[0045]
(2) Screening of target genes by plaque hybridization
A catalase gene derived from cotton plants (Ni W., Turley RB, Trelease RN, Biochim. Biophys. Acta., 1049, 219-222 (1990)) was applied to the filter replicated from the phage plaque of the cDNA library prepared by the above method. cDNA is a radioisotope ³² The target gene cDNA was selected by hybridizing with P as a probe and detecting a positive signal. The base sequence of the cloned cDNA was determined by the dideoxy method using an automatic sequencer. The obtained base sequence is shown in Sequence Listing SEQ ID NO: 1. An ORF encoding 494 amino acids (base numbers 57-1541) was found. This pea-derived catalase gene was named CAT.
[0046]
Example 1 Production of transformant of cotton plant
(1) Construction of catalase gene expression plasmid
A cell wall transition signal sequence of cotton plant-derived peroxidase was inserted between the CaMV35S promoter and the CAT gene shown in SEQ ID NO: 1. The vector construction procedure is shown in FIG. The signal sequence (SS) was amplified from a cotton plant-derived cDNA library by PCR according to a conventional method using specific primers to which XhoI and BamHI restriction enzyme sites were added, and subcloned into a pRTL2 vector. Next, the CAT gene was divided into fragment 1 (Fr.1) cleaved with BamHI and HindIII, fragment 2 (Fr.2) cleaved with HindIII and EcoRI, and fragment 3 (Fr.3) cleaved with EcoRI and XbaI. . Fragment 1 was ligated to the signal sequence, and fragment 2 was further ligated. Fragment 3 was inserted between the 35S promoter sequence (35S pro) and the terminator sequence (ter) of the pRTL2 vector. The fragment in which the signal sequence and fragments 1 and 2 were linked was cleaved with XhoI and EcoRI and subcloned between the 35S promoter and fragment 3. Next, this clone was cleaved with PstI and subcloned into a binary vector pBIN19 containing an expression cassette (Nos pro-nptII-ter) of a kanamycin resistance gene (nptII) using a BamHI adapter. This plasmid was transformed into pBIN35S-S. It was named S-CAT. In addition, Escherichia coli JM109 transformed with the plasmid was transformed into Escherichia coli JM109 / pBIN35S-S. It was named S-CAT.
[0047]
(2) Introduction of plasmid into Agrobacterium
Escherichia coli JM109 / pBIN35S-S. S-CAT and the Escherichia coli HB101 strain having the helper plasmid pRK2013 were each 37 ° C. overnight in an LB medium containing 50 μg / ml kanamycin, and 37 mg of an Agrobacterium EHA101 strain in an LB medium containing 50 μg / ml kanamycin. The culture was performed at 0 ° C. for 2 nights. 1.5 ml of each culture solution was collected in an Eppendorf tube by centrifugation and then washed with LB medium. After suspending these cells in 1 ml of LB medium, put 100 μl of each suspension in a separate tube, mix the three bacteria, spread this on LB agar medium, and culture at 28 ° C. The plasmid was ligated and transferred to Agrobacterium. One to two days later, a part of the colony was scraped off with a platinum loop and coated on an LB agar medium containing 50 μg / ml kanamycin. After culturing at 28 ° C. for 2 days, a single colony was selected. The resulting transformant was EHA101 / pBIN35S-S. It was named S-CAT.
[0048]
(3) Seed disinfection and germination
The seeds of the cotton plant (G. hirsutum cultivar Coke 312) were left in 70% ethanol for 30 seconds. Seeds were collected and washed with 100 ml of sterile water. Subsequently, it was immersed in a sterilizing solution (Clorox / Tween 20) and shaken (110 rpm) for 20 minutes. The seeds were collected and washed with 100 ml of sterilized water, and then the seeds were sufficiently washed with sterilized water for about 30 to 60 minutes. Sterilized seeds on seed germination medium plate (Stewart concentrate, 0.75 g / L MgCl ₂ , 2.0 g / L fighter gel, pH 6.8). The plate was cultured at 30 ° C. for 7-10 days.
[0049]
(4) Agrobacterium infection
The cotton seedlings obtained in (3) were taken out, the cotyledons and roots were removed with a scalpel, and 6 to 8 mm size hypocotyls were cut out. The transformed Agrobacterium obtained in (2) above was cultured at 30 ° C. for 24 hours, and the culture solution was diluted 19-fold with MSNH (MS inorganic salts, 30 g / L glucose, pH 5.8). Hypocotyl sections were immersed in the diluted culture for 30 seconds. The hypocotyls were arranged on two sterilized filter papers, excess water was removed, and T2 plate (MS inorganic salts, 0.75 g / L MgCl ₂ 0.1 mg / L 2,4-D, 0.5 mg / L kinetin, 30 g / L glucose, 2.0 g / L fighter gel, pH 5.8). T2 plates were co-cultured for 3 days at room temperature in the dark. Co-cultured hypocotyls were placed on MS2NKKCL plates (MS inorganic salts, 0.75 g / L MgCl ₂ , 30 g / L glucose, 2 mg / L naphthalene acetic acid, 0.1 mg / L kinetin, 2.0 g / L fighter gel, 50 μg / ml kanamycin, 500 μg / ml cefotaxime, pH 5.8) and parafilm to prevent drying And cultured at 30 ° C. under bright conditions.
[0050]
(5) Callus formation
Hypocotyls were cultured on MS2NKKCL plates and transferred to new plates every month. Transformed callus appeared after about 3-4 weeks. The cells were cultured until the callus was about 4 mm in size, cut from the hypocotyl with a scalpel and transferred to a fresh MS2NKKCL plate. Sufficient growth (necessary for 3 to 5 months) was made until the callus was about 1 cm in size.
[0051]
(6) Embryogenesis
The sufficiently grown callus was transferred to a 50 ml culture flask to which 10 ml of MSNH liquid medium containing kanamycin (250 μg / ml) was added, and liquid suspension culture was performed. Liquid suspension culture was performed for about 2-8 weeks by shaking (110 rpm) culture at 30 ° C. under light conditions. After spherical and light green cells were observed in the culture, the culture was transferred to a 50 ml sterile tube and the cells were collected at the bottom of the tube. The supernatant MSNH liquid medium was removed and approximately 20-30 ml of fresh MSNH liquid medium was added to wash the cells. This operation was performed twice. The cells were collected at the bottom of the tube, and 9.5 ml of MSNH liquid medium was added to 0.5 ml of cells to prepare a cell dilution. Aliquots of cell dilutions were taken in 2 ml increments, each with MSK50 plates (MS inorganic salts, 0.75 g / L MgCl ₂ 1.9g / L KNO _Three , 30 g / L glucose, 2.0 g / L fighter gel, 50 μg / ml kanamycin, pH 5.8) and spread evenly on the plate. Plates were sealed with parafilm to prevent drying and incubated at 30 ° C. under light conditions. When the development state of the somatic embryo was confirmed, some somatic embryos were observed on the plate after about 3 weeks. Appearing somatic embryos were developed to at least 1 mm size and transferred to SA plates (Stewart concentrate, 20 g / L sucrose, 20 g / L agar, pH 6.8).
[0052]
(7) Regeneration of transformed plants
SA plates were sealed with parafilm and incubated in the dark at 30 ° C. for about 2 weeks. Two weeks later, the roots were cut off with a scalpel and the embryos were SGA plates (Stewart concentrate, 5 g / L sucrose, 0.75 g / L MgCl ₂ 5 g / L agar, 1.5 g / L fighter gel, pH 6.8). SGA plates were incubated at 30 ° C. under bright conditions without being sealed with parafilm. Germinated embryos were incubated on SGA plates until the true leaves appeared. Seedlings with observed true leaves were transplanted into pint-sized canned bottles. Transformants in which the height was about 10 cm and several true leaves were observed were transferred to a pot and grown in a greenhouse. Southern hybridization and Northern hybridization analysis are performed on the regenerated transformant and the R1 plant grown from the R1 seed obtained from the transformant, and the target catalase gene is stably integrated and expressed. Transformants were confirmed.
[0053]
Example 2 Field test of transformant
(1) Selection of lines (cell lines) into which the target gene has been introduced
The transformant produced in Example 1 was subjected to a cultivation test in a closed greenhouse attached to Texas Institute of Technology from 1996 to 1997, and confirmed introduction of the target gene by Northern analysis and evaluation of the properties of the obtained cotton fiber. It was. From the results, cell lines in which the target catalase gene was reliably introduced and the gene was expressed were selected (Table 1).
[0054]
[Table 1]

[0055]
(2) Cultivation tests in isolated fields (implemented from May to December 1998)
Cell line seeds listed in Table 1 and a coker 312 that was not genetically modified were used as test materials. On May 20, 1998, seeded in an isolated field attached to Texas Tech University. From July 5, 1998, these cotton plants began to blossom and pollination was self-pollinated. Healthy young leaves were collected from all the transformants cultivated from July to September 1998, and Northern analysis was performed using the target gene introduced according to a conventional method as a probe to examine the expression of the target gene. From September 1998, the fruit was opened, and after drying, the ovule was harvested. Machine fibering was performed at Texas A & M Agricultural Experiment Station to separate cotton fibers from seeds. About the obtained cotton fiber, (1) Fiber fineness, fiber length uniformity, fiber length, and fiber strength were measured using 900HVI system (made by Spinlab). The results are shown in Table 2.
[0056]
[Table 2]

[0057]
As is apparent from Table 2, the transformant introduced with CAT, which is a pea-derived catalase gene, significantly increased fiber fineness, fiber length uniformity and fiber strength in all cell lines.
From the above results, by introducing CAT, which is a catalase gene derived from peas, to a cotton plant, it is possible to obtain a cotton plant that produces cotton fibers with significantly increased fiber properties such as fiber fineness and fiber length uniformity. It became clear.
[0058]
【The invention's effect】
As described above, according to the present invention, it is possible to improve fiber properties such as fineness and fiber length uniformity in cotton fiber, for example, by improving the fineness, the dyeability of the yarn is increased, and the fiber length The strength of the yarn is increased by improving the uniformity. Therefore, the cotton fiber collected from the cotton plant of the present invention can improve the quality of the fabric. Furthermore, the cotton plant of the present invention can be used as a plant material for producing a new cotton variety having superior cotton fiber characteristics and / or cotton fiber productivity.
[0059]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a binary vector pBIN35S-S containing an expression cassette for a pea-derived catalase gene. It is a figure which shows the construction procedure of S-CAT. In addition, the meaning of the abbreviation in a figure is as follows. S. S: Cotton peroxidase-derived cell wall transition signal sequence, Fr. 1: Fragment 1, Fr. 2: Fragment 2, Fr. 3: Fragment 3, 35S pro: Cauliflower mosaic virus-derived 35S promoter, ter: terminator, NOS pro: nopaline synthase promoter, nptII: neomycin phosphotransferase II coding sequence, S-CAT: fusion gene of cotton peroxidase-derived cell wall transition signal sequence and pea-derived catalase cDNA, RB: right border sequence, LB: left border sequence, Xh: XhoI restriction site, B: BamHI restriction site, H: HindIII restriction site, E: EcoRI restriction site, Xb: XbaI restriction site, P: PstI restriction site

Claims (17)

The exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber and the coding sequence of the cell wall transition signal are stably retained and selected from the group consisting of the following (a) to (c) as compared to the wild type at least one fiber characteristics produce cotton fibers with improved that, cotton plants belonging to the Gossypium genus:
(A) fiber strength;
(B) Fiber fineness;
(C) Fiber length uniformity .   The cotton plant according to claim 1, wherein the cotton plant is a transgenic plant obtained by transforming the wild strain with an expression vector containing an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber. .   The cotton plant according to claim 1 or 2, wherein the catalase gene is derived from a plant.   The cotton plant according to claim 3, wherein the catalase gene is derived from peas. The cotton plant according to claim 1 or 2, wherein the catalase gene has the following base sequence (a) or (b).
(A) the base sequence represented by base numbers 57 to 1541 in the base sequence shown in the sequence listing SEQ ID NO: 1 (b) hybridizes with the base sequence of (a) above under stringent conditions , and (a) above nucleotide sequence encoding a polypeptide having a polypeptide equivalent catalase activity encoded by the nucleic acids having the sequence of   The cotton plant is derived from a plant selected from the group consisting of Gospium hirstum, Gospium barbadense, Gospium alborium, Gospium anomalum, Gospium alumianum, Gospium clotzkianoum, Gospium herbaqueum and Gospium lemonday The cotton plant according to any one of claims 1 to 5. The cotton plant according to any one of claims 1 to 6, wherein the cell wall transition signal is a signal sequence of a peroxidase derived from a cotton plant. The cotton plant according to any one of claims 1 to 7 , which is homozygous for the catalase gene. The cotton plant according to claim 8, which is in the form of a seed. An expression vector containing an exogenous catalase gene under the control of a promoter capable of functioning in cotton fibers, and a step of transforming a cotton plant wild strain belonging to the genus Gosypium, the following ( Method for producing cotton plant producing cotton fiber having at least one improved fiber property selected from the group consisting of a) to (c) :
(A) fiber strength;
(B) Fiber fineness;
(C) Fiber length uniformity .   The method according to claim 10, further comprising the step of regenerating a plant from the transformed cell.   The method according to claim 10 or 11, wherein the catalase gene is derived from a plant.   The method according to claim 12, wherein the catalase gene is derived from peas. The method according to claim 10 or 11, wherein the catalase gene has the following base sequence (a) or (b).
(A) the base sequence represented by base numbers 57 to 1541 in the base sequence shown in the sequence listing SEQ ID NO: 1 (b) hybridizes with the base sequence of (a) above under stringent conditions , and (a) above nucleotide sequence encoding a polypeptide having a polypeptide equivalent catalase activity encoded by the nucleic acids having the sequence of   The cotton plant wild strain is selected from the group consisting of Gospium hirstum, Gosypium barbadense, Gospium alborium, Gospium anomalum, Gospium alumianum, Gospium clotzkianoum, Gospium herbaqueum and Gospium lemonday The method in any one of 10-14. The following steps:
(1) An expression vector containing an exogenous catalase gene under the control of a promoter capable of functioning in cotton fiber, transforming a cotton plant wild-type cell belonging to the genus Gosypium,
(2) A cotton plant (RO) that produces cotton fibers having at least one improved fiber characteristic selected from the group consisting of the following (a) to (c) from the transformed cells, compared to the wild type: )
(3) collecting seeds from the plant body by self-pollination; and
(4) The seed comprises assaying the separation ratio of the catalase gene in seeds obtained by self-pollination from cultivation to cotton plants obtained (R1), and homozygous for the catalase gene, wild A method for producing a cotton plant having a trait that produces cotton fiber having at least one improved fiber property selected from the group consisting of the following (a) to (c) as compared to a strain :
(A) fiber strength;
(B) Fiber fineness;
(C) Fiber length uniformity . The seeds obtained by self-pollination of the cotton plant according to any one of claims 1 to 8 are collected, and the following (a) to (a) A method for producing cotton fibers having at least one improved fiber property selected from the group consisting of c) :
(A) fiber strength;
(B) Fiber fineness;
(C) Fiber length uniformity .

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