CN103233029A - Construct and application thereof - Google Patents
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- CN103233029A CN103233029A CN2013101630672A CN201310163067A CN103233029A CN 103233029 A CN103233029 A CN 103233029A CN 2013101630672 A CN2013101630672 A CN 2013101630672A CN 201310163067 A CN201310163067 A CN 201310163067A CN 103233029 A CN103233029 A CN 103233029A
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Abstract
The invention discloses a construct and an application thereof. The construct comprises a nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence shown in SEQ ID NO: 1, namely a blue-green algae ictB gene can be successfully introduced into a receiver plant by using the construct, thus the photosynthetic efficiency of the receiver plant can be remarkably reinforced, and the yield of plants is increased.
Description
Technical Field
The invention relates to the technical field of biological engineering. In particular, the invention relates to constructs and uses thereof. More specifically, the invention relates to constructs, recombinant cells and their use in the preparation of transgenic plant cells, tissues, organs or cultures thereof, transgenic plants, transgenic plant cells, tissues, organs or cultures thereof, and methods of making transgenic plants.
Background
The substance accumulation way of crops is mainly through photosynthesis, and the high-light-efficiency breeding at the present stage is a new way for cultivating high-yield plant varieties. However, conventional breeding has limited available genetic resources, and to obtain better high photosynthetic efficiency strains or varieties, excellent gene resources need to be introduced through modern biotechnology, particularly molecular breeding technology, so as to innovate high photosynthetic efficiency germplasm.
With the continuous maturity and perfection of gene sequencing, molecular breeding technology and transgenic technology, the screening and discovery of excellent photosynthetic efficiency and yield related genes and the genetic transformation by utilizing the genes to obtain high photosynthetic efficiency plants and germplasm are realized, however, the research still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a means for improving the photosynthesis efficiency and yield of plants.
It should be noted that the present invention has been completed based on the following findings of the inventors:
the inventor successfully introduces the plant high-photosynthetic-efficiency gene-blue-green algae ictB gene into receptor rice and expresses the gene by adopting a method combining conventional breeding and biotechnology, and verifies that the photosynthesis efficiency of the receptor plant can be improved by the transformation of the blue-green algae ictB gene through multiple experiments, so that the yield of the receptor plant is improved.
Specifically, the inventor artificially synthesizes the cyanobacteria ictB gene, prepares a construct (namely a recombinant expression vector P6+ ictB) containing the gene, transforms Agrobacterium tumefaciens (Agrobacterium tumefaciens) LBA4404 (namely EHA105-P6+ ictB) by a direct method, transforms rice callus by using the Agrobacterium, calli GUS staining, regenerated plant GUS staining, transplants the transgenic plantlet into a field for character observation, and measures the photosynthetic rate. As a result, the inventors found that:
1. the cyanobacteria ictB gene is transferred into the rice callus by an agrobacterium tumefaciens mediated method, and the result of the callus and the GUS staining of a regeneration plant shows that the cyanobacteria ictB gene is successfully transferred into a receptor rice genome;
2. the field character survey result shows that the basic agronomic characters and economic characters of the rice with the transgenic ictB are obviously superior to those of a wild control group. The concrete expression is as follows: in the growth period, the transgenic rice grows vigorously, tillering is fast and much, plants are robust, the characters such as effective spike, sword leaf width and spike length are obviously superior to those of a control group, and the photosynthesis efficiency of the transgenic rice with the ictB gene is preliminarily improved; in addition, the photosynthetic rate measurement result shows that the photosynthetic rate of the rice with the transgenic ictB is obviously higher than that of the wild control group. In the mature period, the single plant full grain number character of the transgenic rice is obviously superior to that of a wild control group, and the single plant full grain number character is directly and positively correlated with the yield, so that the transgenic ictB gene can improve the yield of the rice.
Thus, the invention proposes constructs comprising the cyanobacteria ictB gene and their use in increasing the efficiency of photosynthesis in plants.
According to one aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises SEQ ID NO: 1. The inventors have surprisingly found that the use of this construct enables the expression of SEQ ID NO: the nucleotide sequence shown in 1, namely the cyanobacteria ictB gene (sometimes referred to as the 'ictB gene' for short) is successfully introduced into a receptor plant, so that the photosynthesis efficiency of the receptor plant can be obviously enhanced through the expression of the ictB gene, and the yield of the receptor plant can be further effectively improved, thereby providing a theoretical basis for high photosynthetic efficiency breeding, and providing a new way for cultivating high-yield plant varieties with high photosynthetic capacity, low respiratory consumption, lasting photosynthetic function, and ideal plant types and growth vigor, such as rice varieties.
According to some embodiments of the invention, the construct of the invention is in the form of at least one selected from the group consisting of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus, preferably a plasmid. This can effectively improve the efficiency of genetic transformation using the construct.
According to one embodiment of the invention, the construct of the invention further comprises: the maize Ubi promoter; and a NOS terminator. Thus, the efficiency of genetic transformation can be effectively improved.
According to yet another aspect of the present invention, there is also provided a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises the construct described above. The recombinant cell is used for transforming plants, so that the blue-green algae ictB gene can be successfully introduced into receptor plants, and further the photosynthesis efficiency of the receptor plants can be remarkably enhanced, so that the yield of the receptor plants can be effectively improved.
According to some specific examples of the present invention, preferably, the recombinant cell of the present invention is a recombinant agrobacterium tumefaciens EHA105-p6+ ictB cell. The inventor finds that the transgenic plant obtained by transforming the receptor plant by using the recombinant cell has very high positive rate.
According to a further aspect of the invention, the invention also provides the use of the constructs and recombinant cells described above in the preparation of transgenic plant cells, tissues, organs or cultures thereof and transgenic plants.
According to another aspect of the present invention, there is also provided a transgenic plant cell, tissue, organ or culture thereof. According to an embodiment of the invention, the transgenic plant cell, tissue, organ or culture thereof is obtained by transforming a recipient plant cell, tissue or organ with the previously described construct or recombinant cell. According to the embodiment of the invention, the transgenic plant cell, tissue, organ or culture thereof can effectively express the cyanobacteria ictB gene, and after the transgenic plant cell, tissue, organ or culture thereof is cultured under a proper condition, a high-yield transgenic plant with high photosynthetic capacity, low respiratory consumption and lasting photosynthetic performance can be effectively obtained.
According to a further aspect of the present invention, there is also provided a method of producing a transgenic plant, according to an embodiment of the present invention, the method comprising: transforming a recipient plant cell, tissue or organ with the construct or recombinant cell described above; and isolating the transgenic plant cell, tissue or organ and culturing under suitable conditions to obtain the transgenic plant. The inventor surprisingly finds that the method can be used for efficiently preparing the transgenic plant capable of effectively expressing the cyanobacteria ictB gene, and the transgenic plant has the advantages of high photosynthetic capacity, low respiratory consumption, lasting photosynthetic performance and the like and has very high photosynthetic efficiency.
According to some embodiments of the invention, in the method of producing a transgenic plant of the invention, the transformation is performed using agrobacterium-mediated methods, and the recipient plant is in the form of callus. Thus, the genetic transformation efficiency is very high.
According to a further aspect of the present invention, there is also provided a transgenic plant or progeny thereof. According to an embodiment of the present invention, the transgenic plant or its progeny is obtained by the method of preparing a transgenic plant as described in the preceding. The inventor finds that the transgenic plant or the progeny thereof has the advantages of high photosynthetic capacity, low respiratory consumption, durable photosynthetic performance and the like, and the photosynthetic efficiency is very high.
According to yet another aspect of the present invention, there is provided a method for enhancing photosynthesis and yield in a plant. According to an embodiment of the invention, the method is: the method of claim 7 or 8, preparing a transgenic plant, causing the transgenic plant to express the cyanobacteria ictB gene. Therefore, the photosynthesis of the transgenic plant can be effectively enhanced, and the yield is obviously improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a diagram of electrophoretic validation of recombinant Agrobacterium tumefaciens comprising the recombinant vector p6+ ictB according to one embodiment of the present invention;
FIG. 2 shows GUS staining results of rice calli transformed with Agrobacterium tumefaciens containing p6+ ictB recombinant vector and a control according to one embodiment of the present invention;
FIG. 3 shows the GUS staining results of roots and leaves of rice seedlings transformed with Agrobacterium tumefaciens containing p6+ ictB recombinant vector and controls according to one embodiment of the present invention;
FIG. 4 shows the results of a fertility phase trait survey after field planting of rice transgenic for the ictB gene according to one embodiment of the present invention;
FIG. 5 shows the measurement results of photosynthetic rate in the filling stage of each line after the field planting of the ictB transgenic rice T1 generation according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The nucleotide sequence of the cyanobacteria ictB gene is shown as follows:
ACCATGGCTGAGGCTTCAGAACCGTCTCCATGGTTGCTGCGCTGGCAAGGATGTCTCCCCAGCTCGGCAGCCCAACAAAAGCGCCTCGCCAATCTGGCCGGCATTGTTTTGATCCTCCTGCTTGCAGGACTGCCCCTGGTGACGCGCACGGGGCTGGGACTGATCGTGCTGGCGTGTGGTGCGCTCTGGCTGCTGTGGTCGCTCAGCAAGCCTCCCGAGCGGCTGGGTGAAATCAGCGGTTGGCTGCTGCTGTTCCTGGCTGTCGCCGTGCTGGCCACAGGCTTCTCGCCAGTTCCCGCCGCGGCCCTCAAGGGGCTGGTGAAGCTGCTCAGCTATCTGGGGGTTTATGCACTGATGCGCCAGCTTCTCGCCGTTCGCCCTGCATGGTGGGACCGGCTGCTGGCGGCTCTGCTCGGGGGATCGTTGCTGACCGATGTGCTCGCCCTGCGCCAGCTCTATGCCCCCACCGAAGAGCTGGCCCGCTGGGCTGATCCGAATTCAGTGGCCGCAGGAACGATCCGGATCTATGGGCCGCTCGGCAACCCCAATCTGTTGGCGGGCTACCTGGTGCCGATCCTGCCGCTGGCGCTTGTGGCCTGCATCCGCTGGCGCGGATGGGGCAGCCGGGCTTTTGCCGCAACGGCCTTCAGCCTCGGCTGTGCATCGGTGCTCTTCAGCTACAGCCGGGGGGGATGGCTCGCCCTTGTGGCTGCGATCGGCAGCCTGGTGCTGCTGCTGGTGCTTCGCGCCATTCGCCACTGGCCCCCACTCTGGAAGCGATTGGTGCCCCTGGCCCTGCTGGGAGCCGGGGGGCTTCTGCTGGCCCTGGCCGTCACCCAGGTCGACCCGATCCGCACCCGGGTGATGAGCCTGCTTGCCGGTCGCGGTGACAGCTCCAACAACTTCCGGATCAATGTGTGGCTGGCTGCGATCGACATGATCCAAGACCGCCCCTGGCTGGGGATCGGTCCGGGGAACGCAGCCTTCAACAGCGTCTACCCCCTCTATCAACAACCGAAGTTCAATGCCCTGAGCGCCTACTCCGTGCCGCTCGAACTTCTGGTGGAAACAGGCATTCCCGGCCTGATCGCCTGCATCGGCCTCGCCCTTGCCAGCCTGCGCAGAGGCTTGAGAGCGTTGGCGTCCGATGCGGATCTGGCCTTGCCCTGCATCGGCTGCCTTGCCGCGATCACCGGGCTTCTGGTGCATGGCGCTGCCGACACGATCTTCTTCCGCCCCGAAGTGCAGATCACTGGTTGGTTCTGTTTGGCCACCCTCAGCCAATGCCGGCAGAGGGCATGA（SEQ ID NO：1）
the inventor finds and proves that the cyanobacteria ictB gene is a functional gene related to the plant photosynthesis efficiency, and further provides the following means for improving the plant photosynthesis efficiency and yield.
Vector constructs, recombinant cells and uses thereof
According to one aspect of the invention, the invention provides a construct. According to an embodiment of the invention, the construct comprises SEQ ID NO: 1. The inventors have surprisingly found that the use of this construct enables the expression of SEQ ID NO: the nucleotide sequence shown in 1, namely the cyanobacteria ictB gene (sometimes referred to as the 'ictB gene' for short) is successfully introduced into a receptor plant, so that the photosynthesis efficiency of the receptor plant can be obviously enhanced through the expression of the ictB gene, and the yield of the receptor plant can be further effectively improved, thereby providing a theoretical basis for high photosynthetic efficiency breeding, and providing a new way for cultivating high-yield plant varieties with high photosynthetic capacity, low respiratory consumption, lasting photosynthetic function, and ideal plant types and growth vigor, such as rice varieties.
The term "construct" as used in the present invention refers to a genetic vector which comprises a specific nucleic acid sequence and is capable of transferring the nucleic acid sequence of interest into a host cell, such that the nucleic acid sequence of interest is integrated into the genome of the host cell to obtain a recombinant cell. According to an embodiment of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a Cosmid (Cosmid), and a virus. According to a particular example of the invention, the construct is in the form of a plasmid. The plasmid is used as a genetic carrier, has the characteristics of simple operation, capability of carrying larger fragments and convenience for operation and treatment. The form of the plasmid is not particularly limited, and may be a circular plasmid or a linear plasmid, and may be either single-stranded or double-stranded. The skilled person can select as desired. The term "nucleic acid" used in the present invention may be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, the length of which is not subject to any particular limitation. For constructs used to construct recombinant cells, it is preferred that the nucleic acid be DNA, as DNA is more stable and easier to manipulate than RNA.
Additional elements may also be included in the constructs according to embodiments of the present invention to confer additional beneficial effects on the constructs. According to one embodiment of the invention, the construct may further comprise a promoter sequence and a terminator sequence. The term "promoter" as used in the present invention refers to a nucleic acid sequence capable of directing the transcription of a nucleic acid molecule to which it is operably linked. The term "operably" as used in the present invention refers to a functional linkage between a nucleic acid expression control sequence, e.g., a promoter, signal sequence, enhancer, etc., and a nucleic acid sequence of interest, wherein the expression control sequence affects the transcription and/or translation of the nucleic acid corresponding to the nucleic acid sequence of interest when a suitable molecule, e.g., a transcription activating molecule, is associated with the expression control sequence. Thus, a particular promoter can be introduced directly into a plant cell via a construct, and can be used to drive the promoter of SEQ ID NO: 1, namely the transcription and expression of the cyanobacteria ictB gene, thereby improving the expression efficiency of the ictB gene in the obtained recombinant cell.
According to some embodiments of the invention, the construct of the invention further comprises a maize ubiquitin (ubi) promoter and a NOS terminator. Thus, the integration efficiency of the target nucleic acid sequence ictB gene can be significantly improved.
According to one embodiment of the invention, the construct may further comprise a selectable marker gene. The term "selectable marker gene" as used herein refers to a gene that encodes a product that confers on a cell receiving the gene in conjunction with a construct a specific property that allows the cell receiving the gene to be readily distinguished from a cell not receiving the gene. Thereby, it is convenient to screen plant cells for receiving the construct. According to embodiments of the present invention, the type of the selection marker gene is not particularly limited, and according to some specific examples of the present invention, the selection marker gene is a drug resistance gene. Thus, selection can be readily made by resistance of recombinant cells receiving the exogenous construct, for example by addition of a bactericidal agent to the culture medium, and cells which correspondingly receive and express a bactericidal agent resistance gene in conjunction with the construct will survive on the culture medium. According to a specific example of the present invention, the selection marker gene may be at least one selected from the group consisting of a neomycin resistance gene, a hygromycin resistance gene and a carbenicillin resistance gene, preferably a mycin resistance gene and a carbenicillin resistance gene. This can further improve the efficiency of screening for recombinant cells that receive the exogenous construct.
According to one embodiment of the invention, the construct may further comprise a sequence encoding a reporter protein. The term "reporter protein" as used in the present invention refers to a protein which, upon expression, produces a signal which can be detected directly or indirectly and which in turn reflects whether the foreign nucleic acid sequence carried by the construct has been successfully expressed in the cell. According to the embodiment of the present invention, the kind of the reporter protein is not particularly limited as long as it has a detectable activity. According to embodiments of the invention, the reporter protein may be a protein capable of generating an optical signal, such as a luminescent protein or a fluorescent protein, such as green fluorescent protein or the like. Thus, the reporter protein may be monitored according to conventional methods. For example, there may be employed: the reporter protein is detected by a colorimetric method, a fluorescent method, a bioluminescence method, a chemiluminescence method, an enzyme-linked immunosorbent assay (ELISA) method and an in-situ staining method. Thus, by the above method, it is possible to easily and efficiently determine whether or not the foreign nucleic acid sequence carried by the construct has been successfully expressed in the cell.
In the present invention, according to a specific example of the present invention, the construct of the present invention is a p6+ ictB recombinant vector.
The inventors have surprisingly found that genetic transformation using this construct is very efficient.
Constructs according to embodiments of the invention are described above. Of course, as will be appreciated by those skilled in the art, the construct may also contain other conventional elements to facilitate the transfer of the construct into a host cell and integration into the genome of the host cell for normal function, such as an origin of replication, a multiple cloning site, and the like. These elements will not be described in detail herein.
According to yet another aspect of the present invention, there is also provided a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises the construct described above. The recombinant cell is used for transforming plants, so that the blue-green algae ictB gene can be successfully introduced into receptor plants, and further the photosynthesis efficiency of the receptor plants can be remarkably enhanced, so that the yield of the receptor plants can be effectively improved.
According to some specific examples of the present invention, preferably, the recombinant cell of the present invention is a recombinant agrobacterium tumefaciens EHA105-p6+ ictB cell. Therefore, the transgenic plant obtained by transforming the receptor plant by using the recombinant cell has very high positive rate.
According to a further aspect of the invention, the invention also provides the use of the constructs and recombinant cells described above in the preparation of transgenic plant cells, tissues, organs or cultures thereof and transgenic plants. According to an embodiment of the present invention, the plant is a monocotyledonous plant, preferably rice, millet, wheat, sorghum and corn, more preferably rice, and further preferably nipponlily.
Transgenic plant cell, tissue, organ or culture thereof, transgenic plant and preparation method thereof
According to another aspect of the present invention, there is also provided a transgenic plant cell, tissue, organ or culture thereof. According to an embodiment of the invention, the transgenic plant cell, tissue, organ or culture thereof is obtained by transforming a recipient plant cell, tissue or organ with the previously described construct or recombinant cell. According to the embodiment of the invention, the transgenic plant cell, tissue, organ or culture thereof can effectively express the cyanobacteria ictB gene, and after the transgenic plant cell, tissue, organ or culture thereof is cultured under a proper condition, a high-yield transgenic plant with high photosynthetic capacity, low respiratory consumption and lasting photosynthetic performance can be effectively obtained. The term "culture" as used herein refers to derivatives obtained by culturing transgenic plant cells, tissues or organs under conditions suitable for growth, which derivatives have the same genome as the original transgenic plant cells, tissues or organs.
Among them, according to some embodiments of the present invention, the method for transforming a recipient plant cell, tissue or organ using the aforementioned construct or recombinant cell is not particularly limited as long as the ictB gene carried in the construct or recombinant cell can be efficiently introduced into the recipient plant cell, tissue or organ. According to some embodiments of the invention, the transformation may be performed by agrobacterium-mediated transformation. Thus, the efficiency of genetic transformation can be effectively improved.
According to an embodiment of the present invention, the transgenic plant cell, tissue, organ of the present invention may be derived from a monocotyledonous plant, preferably rice, millet, wheat, sorghum, and maize, more preferably rice, and further preferably nipponlily.
According to a further aspect of the present invention, there is also provided a method of producing a transgenic plant, according to an embodiment of the present invention, the method comprising: transforming a recipient plant cell, tissue or organ with the construct or recombinant cell described above; and isolating the transgenic plant cell, tissue or organ and culturing under suitable conditions to obtain the transgenic plant. It should be noted that the expression "culturing under suitable conditions" as used herein is to be understood in a broad sense and encompasses various operations for supplying energy to cells/cell aggregates which are experienced in obtaining individual plants from cells, tissues or organs of transgenic plants, and the conditions for culturing cells, tissues or organs of transgenic plants are not particularly limited, and any of various operating conditions can be used for achieving "culturing". The inventor surprisingly finds that the method can be used for efficiently preparing the transgenic plant capable of effectively expressing the cyanobacteria ictB gene, and compared with a wild type, the transgenic plant has the advantages of high photosynthetic capacity, low respiration consumption, lasting photosynthetic capacity and the like, and the photosynthetic efficiency is very high.
According to the embodiments of the present invention, the plant to which the method for producing a transgenic plant cell of the present invention is applied is not particularly limited, i.e., the source of the recipient plant cell, tissue or organ is not particularly limited. According to some embodiments of the invention, the recipient plant cell, tissue or organ may be derived from a monocot. According to further embodiments of the present invention, the monocotyledonous plant includes, but is not limited to, rice, millet, wheat, sorghum, corn, preferably rice, including, but not limited to, medium flower 9, medium flower 10, medium flower 11, taibei 309, danjiang 8, yunnao 2, shanyou 63, shanyou 608, fengyou 22, qiayou 88, qiyou 416, iiyou 107, iiyou 128, iiyou 718, siliangyou 527, chuan nong 1, hetero 0152, wan rice 88, wan rice 90, wan rice 92, wan rice 94, wan rice 96, wan rice 185, wan rice 187, wan rice 189, wan rice 191, wan rice 193, wan rice 195, wan rice 197, rice 199, kawa 201, oryza 203, oryza 205, oryza 207, jin Yuan 101, and Ningqing (wherein the above rice varieties are commercially available, for example, from Anjia rice, Anhui Hui). According to a preferred embodiment of the invention, the recipient plant cell, tissue or organ is derived from Oryza sativa.
According to an embodiment of the present invention, a method for transforming a recipient plant cell, tissue or organ using the construct or recombinant cell of the present invention is not particularly limited. According to some embodiments of the invention, the transformation may be performed by agrobacterium-mediated transformation. Thus, the efficiency of introducing the construct into the host plant cell can be improved, and transgenic cells, tissues or organs in which the nucleic acid sequence of interest is successfully integrated with the chromosome of the host cell can be conveniently selected, further enabling the efficiency of producing transgenic plants to be improved. It is noted that the terms "transformation" and "transfection" are used interchangeably herein and refer to the process of introducing an exogenous nucleic acid sequence into a host cell.
According to one embodiment of the present invention, in the method for producing a transgenic plant of the present invention, the form of the transformation receptor is not particularly limited and may be a cell, a tissue or an organ as long as it can efficiently receive the foreign nucleic acid sequence carried in the construct or the recombinant cell. According to a specific example of the invention, the recipient plant is in the form of callus. Thus, the integration efficiency of the foreign nucleic acid sequence is very high.
According to an embodiment of the present invention, in the method of producing a transgenic plant of the present invention, the kind of the recipient plant is not particularly limited. According to some embodiments of the invention, the recipient plant may be a monocot or a dicot, preferably a monocot, more preferably rice, millet, wheat and maize, most preferably rice.
It should be noted that the expression "isolating a transgenic plant cell, tissue or organ" as used herein refers to selecting a corresponding screening method based on the nucleic acid sequence carried by the construct, such as a screening marker gene or a reporter gene, and the genetic transformation method used, so as to distinguish an individual receiving the gene of interest from an individual not receiving the gene of interest, and isolating the plant cell, tissue or organ selected to receive the construct, i.e., the transgenic plant cell, tissue or organ. For example, when the construct carries a selectable marker gene such as a drug resistance gene, selection can be made by resistance of the transgenic cells that receive the exogenous construct, e.g., by adding a bactericide to the culture medium, cells, tissues or organs that correspondingly receive and express the bactericide resistance gene along with the construct will survive on the culture medium; when the construct carries a reporter protein, such as a luminescent or fluorescent protein, one may employ: the reporter protein is detected by colorimetry, fluorescence, bioluminescence, chemiluminescence, enzyme-linked immunosorbent assay (ELISA) and in-situ staining, and then the reporter protein gene is received by the construct and expressed, and cells, tissues or organs emit fluorescence. Therefore, by the method, the receptor plant cells, tissues or organs which receive the target genes and do not receive the target genes can be distinguished conveniently and efficiently, and then the transgenic plant cells, tissues or organs are screened and separated.
According to a further aspect of the present invention, there is also provided a transgenic plant or progeny thereof. According to an embodiment of the present invention, the transgenic plant or its progeny is obtained by the method of preparing a transgenic plant as described in the preceding. The inventor finds that the transgenic plant or the progeny thereof has the advantages of high photosynthetic capacity, low respiratory consumption, durable photosynthetic performance and the like, and the photosynthetic efficiency is very high. According to an embodiment of the present invention, the transgenic plant is a monocotyledonous plant, preferably rice, millet, wheat, sorghum and maize, more preferably rice, and further preferably nipponica.
According to yet another aspect of the present invention, there is provided a method for enhancing photosynthesis and yield in a plant. According to an embodiment of the invention, the method is: according to the method for preparing the transgenic plant, the transgenic plant is prepared, and the transgenic plant expresses the cyanobacteria ictB gene. Therefore, the photosynthesis of the transgenic plant can be effectively enhanced, and the yield is obviously improved. Wherein, according to some embodiments of the invention, the plant is rice. Therefore, compared with the wild type, the obtained transgenic rice has obviously enhanced photosynthesis and obviously improved yield.
It should be noted that the construct of the present invention and the use thereof are discovered and completed by the inventors of the present application through hard creative efforts and optimization works.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the third edition of the molecular cloning, laboratory Manual, or related products, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.
Example 1 construct preparation
Synthesis of cyanobacteria ictB gene and construction of vector pUC57-ictB
The inventors of the present invention synthesized the cyanobacteria ictB gene (SEQ ID NO: 1) by the Tokyo Biotechnology, Ltd, and added restriction enzyme sites BamHI and XbaI to the upstream and downstream of the synthesized gene, respectively, to obtain the followingSEQ ID NO: 2 is shown inThe nucleic acid sequence was ligated to vector pUC57 to obtain pUC57-ictB vector.
GGATCCACCATGGCTGAGGCTTCAGAACCGTCTCCATGGTTGCTGCGCTGGCAAGGATGTCTCCCCAGCTCGGCAGCCCAACAAAAGCGCCTCGCCAATCTGGCCGGCATTGTTTTGATCCTCCTGCTTGCAGGACTGCCCCTGGTGACGCGCACGGGGCTGGGACTGATCGTGCTGGCGTGTGGTGCGCTCTGGCTGCTGTGGTCGCTCAGCAAGCCTCCCGAGCGGCTGGGTGAAATCAGCGGTTGGCTGCTGCTGTTCCTGGCTGTCGCCGTGCTGGCCACAGGCTTCTCGCCAGTTCCCGCCGCGGCCCTCAAGGGGCTGGTGAAGCTGCTCAGCTATCTGGGGGTTTATGCACTGATGCGCCAGCTTCTCGCCGTTCGCCCTGCATGGTGGGACCGGCTGCTGGCGGCTCTGCTCGGGGGATCGTTGCTGACCGATGTGCTCGCCCTGCGCCAGCTCTATGCCCCCACCGAAGAGCTGGCCCGCTGGGCTGATCCGAATTCAGTGGCCGCAGGAACGATCCGGATCTATGGGCCGCTCGGCAACCCCAATCTGTTGGCGGGCTACCTGGTGCCGATCCTGCCGCTGGCGCTTGTGGCCTGCATCCGCTGGCGCGGATGGGGCAGCCGGGCTTTTGCCGCAACGGCCTTCAGCCTCGGCTGTGCATCGGTGCTCTTCAGCTACAGCCGGGGGGGATGGCTCGCCCTTGTGGCTGCGATCGGCAGCCTGGTGCTGCTGCTGGTGCTTCGCGCCATTCGCCACTGGCCCCCACTCTGGAAGCGATTGGTGCCCCTGGCCCTGCTGGGAGCCGGGGGGCTTCTGCTGGCCCTGGCCGTCACCCAGGTCGACCCGATCCGCACCCGGGTGATGAGCCTGCTTGCCGGTCGCGGTGACAGCTCCAACAACTTCCGGATCAATGTGTGGCTGGCTGCGATCGACATGATCCAAGACCGCCCCTGGCTGGGGATCGGTCCGGGGAACGCAGCCTTCAACAGCGTCTACCCCCTCTATCAACAACCGAAGTTCAATGCCCTGAGCGCCTACTCCGTGCCGCTCGAACTTCTGGTGGAAACAGGCATTCCCGGCCTGATCGCCTGCATCGGCCTCGCCCTTGCCAGCCTGCGCAGAGGCTTGAGAGCGTTGGCGTCCGATGCGGATCTGGCCTTGCCCTGCATCGGCTGCCTTGCCGCGATCACCGGGCTTCTGGTGCATGGCGCTGCCGACACGATCTTCTTCCGCCCCGAAGTGCAGATCACTGGTTGGTTCTGTTTGGCCACCCTCAGCCAATGCCGGCAGAGGGCATGATCTAGA（SEQ ID NO：2）
In the above sequence, the restriction sites (BamHI/XbaI) are underlined.
II, construction of p6 recombinant vector
1. PCR amplification of maize ubiquitin (Ubi) promoter fragment and construction of pMD18-T + Ubi recombinant vector
(1) PCR amplification of the Ubi promoter
Genomic DNA of maize variety B73 (Zea mays mays cv. B73) was extracted using a plant genomic DNA extraction kit (TIANGEN novel plant genomic DNA extraction kit, catalog number: DP 320-02). According to the sequence of the promoter in the corn B73gDNA, a pair of PCR specific amplification primers are respectively designed at the head and the tail: the upstream primer F1: GG (GG)CTGCAGTGCAGCGTGACCCGGTCGT (SEQ ID NO: 3), plus a restriction site Pst I and a protecting base; the downstream primer R1: GG (GG)CTGCAGAAGTAACACCAAAC (SEQ ID NO: 4), plus a restriction enzyme site Pst I and a protecting base.
Then, the gDNA of the corn B73 extracted above is used as a template, and an upstream primer F1, a downstream primer R1 and high fidelity Ex Taq are utilizedTM(TaKaRa, DRR 100B) POLYMERThe synthase was subjected to PCR amplification. Among them, the PCR amplification system is shown in Table 1.
TABLE 1
| Composition of | Volume (μ l) |
| Genomic DNA | 0.2 |
| dNTPs（2.5mM） | 2 |
| 10 XEx Buffer (containing magnesium ions) | 2.5 |
| Primer F1 (50. mu.M) | 1 |
| Primer R1 (50. mu.M) | 1 |
| Ex taq | 0.2 |
| ddH2O | Make up to 25 |
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5min, followed by denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 50s, extension at 72 ℃ for 90s, 35 reaction cycles, and final extension at 72 ℃ for 7 min.
The PCR amplification product was separated by 1.0% agarose gel electrophoresis, and then purified and recovered using a TIANGEN agarose gel DNA recovery kit (catalog No.: DP 209-03).
(2) Construction of pMD18-T + Ubi recombinant vector
The PCR amplification product obtained by the method is subjected to T/A cloning (pMD 18-T plasmid, TaKaRa, D103A), transformed into escherichia coli, and the positive clone is selected to be sequenced by Shenzhen Hua Dagen science and technology corporation, so that the result is accurate.
Wherein, the connection conditions of the T/A clone are as follows:
connecting in an energy-saving intelligent thermostatic bath (SDC-6) at 16 ℃ for more than 8h to obtain a pMD18-T + P604 recombinant vector. The product after the connection is transformed into escherichia coli according to the following method:
after thawing on ice, 100. mu.l of DH 5. alpha. (supplied by Town Populus, the institute for Kunming animals, national academy of sciences, China; or commercially available from, for example, Shanghai Producer) of competent cells prepared according to the calcium chloride method as shown in the molecular cloning laboratory Manual (third edition, scientific Press) were taken out of the ultra-low temperature refrigerator, 10. mu.l of the ligation product obtained as above, i.e., pMD18-T + P604 recombinant vector, gently stirred, ice-cooled for 30min, heat-shocked for 60s at 42 ℃ and ice-cooled for 5min, 600. mu.l of SOC medium precooled at 4 ℃ was added (detailed in the molecular cloning laboratory Manual, third edition, scientific Press, incorporated herein by reference in its entirety), thawed at 37 ℃ at 220rpm for 45min, centrifuged at 8000rpm for 30s, the supernatant was removed, 150. mu.l of the remaining mixture after precipitation with 150. mu.l of the remaining supernatant was gently blown, and LB (kanamycin) plates were spread with glass beads (detailed in the molecular cloning laboratory Manual, third edition, scientific press, incorporated herein by reference in its entirety), cultured for 16-24h at 37 ℃ in an inverted state. A recombinant E.coli containing pMD18-T + Ubi cloning vector was obtained and named DH5 α -Ubi.
2. Construction of pCAMBIA-1301+ Ubi recombinant vector
According to the manual of the TIANGEN ordinary plasmid miniprep kit (catalog number: DP 103-03), extracting a cloning vector pMD18-T + Ubi with a promoter sequence of the maize Ubi from the Escherichia coli DH5 alpha-Ubi transformed with the promoter Ubi obtained by the construction; after purification, the fragment was digested with the corresponding restriction enzyme Pst I (NEB), and the corresponding promoter fragment was recovered using a TIANGEN agarose gel DNA recovery kit (Cat. No.: DP 209-03).
The pCAMBIA-1301 plasmid (supplied by The Kingming animal research institute of Chinese academy of sciences; or commercially available from, for example, Shanghai Riegen Gene science Co., Ltd.) was digested with restriction enzyme Pst I, and The original source of The pCAMBIA-1301 plasmid was The Cambian Bios (biological open source) plasmid, Australia) and recovered.
Cloning vector pMD18-T + Ubi or pCAMBIA-1301 plasmid 10. mu.l (< 1000 ng)
The recovered promoter fragment Ubi and the recovered vector fragment were ligated according to the T4 ligase (TaKaRa, D2011A) protocol, as follows:
connecting in 16 deg.C energy-saving intelligent thermostatic bath (SDC-6) for more than 8 h.
Taking out 100 mul of competent cell DH5 alpha prepared by calcium chloride method from ultra-low temperature refrigerator, melting on ice, adding 10 mul of the above ligation product, stirring gently, ice-bath for 30min, heat shock at 42 ℃ for 60s, ice-bath for 5min, adding 600 mul of SOC precooled at 4 ℃, resuscitating at 37 ℃ for 45min at 220rpm, centrifuging at 8000rpm for 30s, removing supernatant, leaving 150 mul, blowing gently, coating glass beads with LB (Kan), and culturing at 37 ℃ for 16-24h in inversion. Thus, a recombinant vector pCAMBIA-1301+ Ubi was obtained.
The PCR detection of the obtained recombinant vector pCAMBIA-1301+ Ubi was carried out using F1 (SEQ ID NO: 3) and R1 (SEQ ID NO: 4) as primers, respectively, to confirm that the obtained recombinant vector pCAMBIA-1301+ Ubi contains the desired promoter Ubi.
3. PCR amplification of NOS terminator and construction of pMD18-T + NOS recombinant vector
According to the sequence of the NOS terminator in the pCAMBIA-1301 plasmid, a pair of PCR specific amplification primers are respectively designed at the head and the tail: the upstream primer F2: GG (GG)GAGCTCGAATTTCCCCGATCGTTCAA (SEQ ID NO: 5), adding a restriction enzyme cleavage site Sac I and a protecting base; the downstream primer R2: GG (GG)GAATTCCCGATCTAGTAACATAGAT (SEQ ID NO: 6), plus a restriction enzyme site EcoR I and a protecting base. Taking the pCAMBIA-1301 plasmid extracted above as a template, and utilizing an upstream primer F2, a downstream primer R2 and high fidelity Ex TaqTM(TaKaRa, DRR 100B) polymerase was used for PCR amplification. Wherein, the PCR amplification system is as follows:
| composition of | Volume (μ l) |
| Plasmid DNA | 0.1 |
| dNTPs（2.5mM） | 2 |
| 10 XEx Buffer (containing magnesium ions) | 2.5 |
| Primer F1 (50. mu.M) | 1 |
| Primer R1 (50. mu.M) | 1 |
| Ex taq | 0.2 |
| ddH2O | Make up to 25 |
The PCR amplification product obtained above was subjected to T/A cloning (pMD 18-T plasmid, TaKaRa, D103A) to transform E.coli, to obtain recombinant E.coli containing pMD18-T + NOS cloning vector, which was designated DH 5. alpha. -NOS. The positive clones were picked and sequenced by Shenzhen Hua Dagen science and technology Limited, which proved to be accurate.
4. Construction of pCAMBIA-1301+ Ubi + NOS, i.e., p6 recombinant vector
Extracting the constructed cloning vector pMD18-T + NOS according to the operation manual of a TIANGEN common plasmid miniextract kit (catalog number: DP 103-03); after purification, the DNA fragment was digested with the corresponding restriction enzymes Sac I (NEB) and EcoR I (NEB), and then the corresponding NOS terminator fragment was recovered using a TIANGEN agarose gel DNA recovery kit (catalog No.: DP 209-03). Meanwhile, the pCAMBIA-1301+ Ubi plasmid is cut by restriction enzymes Sac I and EcoR I and recovered.
Wherein,
vector pMD18-T + NOS or pCAMBIA-1301+ Ubi plasmid 10. mu.l (< 1000 ng)
The recovered terminator fragment NOS and the recovered vector fragment are connected by T4 ligase, and competent cells DH5 alpha are transformed to obtain a recombinant vector pCAMBIA-1301+ Ubi + NOS, namely p 6.
Construction of a III, p6+ ictB recombinant vector
The pUC57-ictB plasmid prepared previously was digested with restriction enzymes BamHI/XbaI, and the ictB gene fragment was recovered, and at the same time, the p6 plasmid was extracted, and the large fragment was recovered after digestion with the corresponding restriction enzymes BamHI/XbaI, and the recovered product was ligated, followed by transformation of competent cell DH 5. alpha. to obtain the recombinant vector p6+ ictB. Screening positive clones, performing PCR detection, and sequencing to determine the correct insertion of the target fragment. Thus, a p6+ ictB construct was prepared and ready for use.
EXAMPLE 2 preparation of recombinant Agrobacterium tumefaciens EHA105-p6+ ictB cells
The plasmid p6+ ictB recombinant construct prepared in example 1 was transformed into competent cells of Agrobacterium tumefaciens EHA105 (deposited at the preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Hubei, 2009, 12 and 24 days, China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M209315) according to the calcium chloride method described in molecular cloning Experimental Manual (third edition, scientific Press):
the Agrobacterium tumefaciens competent cell EHA105 was taken out in an ultra-low temperature refrigerator and thawed on ice. After thawing, 5. mu.l of p6+ ictB recombinant vector was added, mixed gently, ice-washed for 10min, frozen in liquid nitrogen for 5min, thawed at 37 ℃ for 5min, added to 800. mu.l of room-temperature LB liquid medium, thawed at 28 ℃ at 160rpm for 3h, centrifuged at 8000rpm for 30s, the supernatant was aspirated, 200. mu.l was left to be blown up, and spread on YM medium plates (50 mg/l Kan, 10mg/l Rif) to which kan-Rif (kanamycin-rifampin) double antibody was added. Culturing at 28 deg.C for 2-3 days.
PCR was performed using F1 (SEQ ID NO: 3) and R1 (SEQ ID NO: 4) as primers and transformants were selected by digestion with BamHI/XbaI.
The recombinant Agrobacterium tumefaciens containing the recombinant construct p6+ ictB, from which about 1310bp band was amplified by PCR and about 1302bp band was cleaved by enzyme (see FIG. 1). Wherein, as shown in figure 1, lane 1: molecular weight standard DL2000, lane 2: and (3) PCR products.
In the present invention, the recombinant Agrobacterium with the recombinant construct p6+ ictB obtained as described above was designated recombinant Agrobacterium tumefaciens EHA105-p6+ ictB.
Example 3 Induction and transformation of Rice calli
Rice calli were induced as follows and transformed with recombinant Agrobacterium tumefaciens EHA105-p6+ ictB prepared as described in example 2:
(1) removing hull of Japanese fine rice seeds (which are preserved in the preservation center of Wuchang Lojia mountain Wuhan university, Wuhan city, Wuhan, 2009, 12 and 18 days, namely China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC P200910), sterilizing the surface of 70% ethanol for 30s, then sterilizing with sodium hypochlorite with the effective chlorine of 1.5% for 30min, shaking violently during the period, and cleaning with sterilized water for 5 times after the sterilization; placing the sterilized seeds on an N6D culture medium, and sealing the seeds with a sealing film; dark culturing at 28 deg.C for 6-8 weeks;
(2) selecting actively growing callus (yellow white, dry, diameter 1-3 mm), and culturing in dark at 28 deg.C for 2-3 weeks on new N6D culture medium;
(3) single colonies of recombinant Agrobacterium tumefaciens (recombinant Agrobacterium tumefaciens EHA105-p6+ ictB) constructed as described in example 2 were picked up and streaked on YM medium supplemented with antibiotics (50 mg/l Kan, 10mg/l Rif) for 3 days at 28 ℃; the recombinant Agrobacterium tumefaciens cells were scraped off and placed in 30ml of AAM medium supplemented with 30. mu.l of 100mM AS (Acetosyringone ), and the recombinant Agrobacterium tumefaciens cells were gently resuspended (recombinant Agrobacterium tumefaciens EHA105-p6+ ictB prepared in example 2);
(4) placing the subcultured callus in a sterilized culture dish; pouring the recombinant agrobacterium tumefaciens suspension prepared in the step 3 into a culture dish, and immersing the callus in the culture dish for 15 min;
(5) pouring out the recombinant agrobacterium tumefaciens suspension, and sucking the redundant liquid of the callus by using sterilized absorbent paper; placing a piece of sterilized filter paper on the N6-AS culture medium, adding 1ml of the AAM culture medium containing AS, and transferring the callus onto the filter paper; sealing the culture dish, and performing dark culture at 22 ℃ for 48-60 h;
(6) placing the infected callus in a 50ml sterilizing tube, and washing with sterilized water by shaking until the supernatant becomes clear; soaking the callus in sterile water containing 500mg/l carbenicillin (Carb) for 30min to kill recombinant Agrobacterium tumefaciens; removing excess water from the callus with sterilized absorbent paper, and transferring to N6-AS medium containing 50mg/l hygromycin B (HmB) and 250mg/l Carb; sealing the culture dish with sealing film, and culturing at 28 deg.C under illumination for 2-3 weeks. After the resistant callus grew out, the callus was transferred to a new N6-AS medium containing 50mg/l hygromycin B (HmB) and 250mg/l Carb; sealing the culture dish with sealing film, and culturing at 28 deg.C under illumination for 2-3 weeks.
Example 4 detection of GUS expression in Rice callus
GUS staining was performed on the recombinant Agrobacterium tumefaciens EHA105-p6+ ictB-transformed rice calli obtained in example 3 according to the method described by Chen S Y et al (Journal of Integrated Plant Biology, 2008, 50 (6): 742-751), the entire contents of which are incorporated herein by reference, in order to detect the expression of GUS in the transformed rice calli.
Wherein, the formula (1 ml) of GUS staining solution is as follows: 610. mu.l 0.2M Na2HPO4Solution (pH = 7.0); 390. mu.l 0.2MNaH2PO4Solution and 10. mu.l of 0.1M X-gluc.
Specifically, the rice callus transformed with recombinant Agrobacterium tumefaciens EHA105-p6+ ictB was soaked in GUS staining solution, incubated at 37 ℃ for 2 hours or overnight until blue color appeared, and then photographed to record the staining results, as shown in FIG. 2. As shown in FIG. 2, the Agrobacterium tumefaciens-mediated transformation of rice calli containing the p6+ ictB recombinant construct prepared in example 3 appeared blue after staining (right panel in FIG. 2), whereas the untransformed calli, i.e., the control, did not change in color after GUS staining (left panel in FIG. 2). The results show that the p6+ ictB construct of the invention has been transferred into rice calli.
Example 5: detection of GUS expression in transgenic rice seedlings
The transformed callus prepared in example 3 was transferred to MS-R differentiation medium containing 50mg/l hygromycin B (HmB) to differentiate shoots; sealing the culture dish with a sealing film, and culturing at 28 deg.C for 4-6 weeks; when the seedlings grow to 3-4cm, the seedlings are transferred to 1/2MS rooting medium containing 50mg/l hygromycin B (HmB) for rooting screening.
Then, transgenic rice seedlings were subjected to GUS staining with reference to the GUS staining method for callus in example 4, and the staining results were recorded by photographing, and the results are shown in FIG. 3. As can be seen from FIG. 3, the roots (right side of upper panel in FIG. 3), leaves (right side of lower panel in FIG. 3) of rice seedlings transformed by Agrobacterium tumefaciens-mediated transformation with p6+ ictB recombinant construct appeared blue after staining, and the color of untransformed seedlings, i.e., the roots (left side of upper panel in FIG. 3), leaves (left side of lower panel in FIG. 3) were unchanged after GUS staining. The results show that the p6+ ictB recombinant construct of the invention has been transformed into rice seedlings.
Example 6: determination of agronomic characters and photosynthetic rate of transgenic rice seedlings
Transplanting the seedlings to a field after the seedlings grow for 20 days in an 1/2MS rooting culture medium, and measuring the field agronomic characters of the seedlings, such as the plant height, the effective spike, the length of the sword leaf, the width of the sword leaf, the spike length and the like of the seedlings in the growth period of the transgenic rice; measuring the photosynthetic rate of the water in the grouting period; after harvesting, the number of branches at one time, the number of full grains per plant, the total number of grains, the maturing rate, the thousand-grain weight and the like are measured. The agronomic trait survey results of rice transformed with the ictB gene are shown in Table 2 and FIG. 4.
Then, seeds of transgenic rice of T0 generation are planted in the field, and the photosynthetic rate of each line of T1 generation of transgenic rice is measured in the grouting period. The results are shown in FIG. 5.
Table 2 investigation results of basic agronomic traits and economic traits of rice transformed with ictB gene
Note: the representations had significant differences.
"K030" represents the ictB gene.
As can be seen from table 2:
1. compared with a control group, the rice with the transgenic ictB gene has the characteristics of plant height, effective panicle width, sword leaf width, panicle length and the like, and more than half of the lines show remarkable advantages, and the function of improving the photosynthesis of the rice of the ictB gene is preliminarily verified;
2. because the single plant full grain number character is directly and positively correlated with the yield, the character performance of the transgenic line is obviously superior to that of a control line, and the transgenic ictB gene can improve the rice yield.
Wherein, FIG. 4 shows a checking chart of the growth period traits of rice with the transgenic ictB gene, as shown in FIG. 4, wherein the left picture is a wild type control; the right panel shows rice with the transgenic ictB gene. As can be seen from the figure, the growth vigor of the rice with the transgenic ictB is superior to that of the control, the tillering speed is high, the number of plants is large, and the effective ears are obviously more than that of the control.
FIG. 5 shows the result of determination of photosynthetic rate of rice lines T1 generation transformed with ictB gene in the filling stage. As shown in FIG. 5, "K030" in the figure represents the ictB gene. As can be seen from the figure, the photosynthetic rate of the transgenic rice with the ictB gene of T1 generation is obviously superior to that of the wild type control.
From the results, it can be seen that SEQ ID NO: the nucleotide sequence shown in 1, namely the blue-green algae ictB gene, is successfully introduced into a receptor plant, so that the photosynthesis efficiency of the receptor plant can be obviously enhanced, and the plant yield is improved.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A construct comprising SEQ ID NO: 1.
2. The construct according to claim 1, wherein the construct is in the form of at least one selected from the group consisting of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus, preferably a plasmid.
3. The construct of claim 1, further comprising:
the maize Ubi promoter; and
the NOS terminator.
4. A recombinant cell comprising the construct of any one of claims 1 to 3,
preferably, the recombinant cell is recombinant Agrobacterium tumefaciens EHA105-p6+ ictB.
5. Use of the construct according to any one of claims 1 to 3 or the recombinant cell according to claim 4 for the preparation of transgenic plant cells, tissues, organs or cultures thereof and transgenic plants,
optionally, the plant is a monocotyledonous plant, preferably rice, millet, wheat, sorghum and maize, more preferably rice, further preferably nipponica.
6. A transgenic plant cell, tissue, organ or culture thereof obtained by transforming a recipient plant cell, tissue or organ with the construct of any one of claims 1-3, or the recombinant cell of claim 4,
optionally, the transgenic plant cell, tissue, organ is derived from a monocotyledonous plant, preferably rice, millet, wheat, sorghum and maize, more preferably rice, and further preferably nipponica.
7. A method of making a transgenic plant comprising:
transforming a recipient plant cell, tissue or organ with the construct of any one of claims 1-3, or the recombinant cell of claim 4; and
isolating the transgenic plant cell, tissue or organ and culturing under suitable conditions to obtain a transgenic plant,
optionally, the recipient plant cell, tissue or organ is derived from a monocotyledonous plant, preferably rice, millet, wheat, sorghum and maize, more preferably rice, and even more preferably nipponica.
8. The method according to claim 7, wherein said transformation is carried out using Agrobacterium-mediated transformation and said recipient plant is in the form of callus.
9. A method for enhancing photosynthesis and increasing yield of plants,
the method of claim 7 or 8, preparing a transgenic plant, causing the transgenic plant to express the cyanobacteria ictB gene.
10. The method of claim 9, wherein the plant is rice.
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