DNA comprising rice anther-specific gene and transgenic plant transformed therewith

An anther, specific technology, applied in the field of plant genetic engineering, can solve a lot of time and energy problems

Inactive Publication Date: 2002-02-06
SYNGENTA PARTICIPATIONS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem is that this method requires a lot of time and effort

Method used

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Examples

Experimental program
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Embodiment 1

[0071] The present invention will be described in more detail with reference to examples, which should not be construed as limiting the scope of the invention. Example 1: Isolation of rice anther-specific gene RA8 Step 1: Isolation of RA8 gene

[0072] Rice (Oryza sativa L. Nakdong) flowers in the late vacuolar stage were cross-cut into 3 parts. The middle part containing intact anthers and part of palea and lemma was taken as an anther enriched sample. Anther-enriched mRNA was isolated from this part using the poly(A) Rapid mRNA Isolation Kit (Stratagene, USA). Using this mRNA as a template, an anther cDNA library was constructed using the ZAP-cDNA Gigapack 2 Gold Cloning Kit (Stratagene).

[0073] The same method was applied to leaves from 6-day-old rice seedlings to construct a leaf cDNA library.

[0074] From the constructed anther cDNA library, 33 cDNA clones were randomly selected, and the corresponding clone DNA was cut with EcoRI to obtain the cDNA region. 32 It wa...

Embodiment 2

[0084] To investigate the genomic complexity of RA8 clones, genomic DNA isolated from leaves of 2-week-old rice seedlings was cut with EcoRI, HindIII, or PstI, followed by Southern blotting with RA8 cDNA. The RA8 cDNA probe hybridized to a single band of rice genomic DNA, whereby the RA8 gene exists in a single copy in the rice genome. Example 2: Production of Plant Expression Vectors

[0085] The construction of the binary vector comprising the reporter gene GUS and the selection gene hph (hygromycin phosphotransferase) is as follows:

[0086]Plasmid pGA748 (G. An, "Binary Ti Plasmid Vectors: Agrobacterium Protocol", Humana Press, pp. 47-58) was cut with BamHI and HindIII and inserted into the CaMV35S promoter (Benfey et al., Science 250: 959-966, 1993). The hygromycin resistance gene (hygromycin phosphotransferase, hph) was inserted at the BglII site of the plasmid thus obtained (Gritz et al., 1983). The plasmid was cut with HindIII and ClaI, blunt-ended, and ligated. Th...

Embodiment 3

[0091] The fragment was connected to the β-glucuronidase coding sequence in pGA1633 to construct the plant expression vector pGA1647. In the vector pGA1647, the above-mentioned RA8 gene-derived sequence was fused to the GUS gene at the newly introduced BamHI site in exon 2 without any change in its reading frame. Thus, a translational fusion was created between part of the RA8 gene and the β-glucuronidase coding sequence. Embodiment 3: the production of transgenic rice

[0092] Use the freeze-thaw method (G.An et al. (edited), "Handbook of Plant Molecular Biology", page A3 / 1-A3 / 19, Kluwer Academic Publishers, Dordrecht, 1988) to construct the expression vector pGA1647 in Example 2 Transformation into Agrobacterium tumefaciens LBA4404 carrying the binary Ti plasmid pAL4404 (Hoekema et al., Nature 303:179-181, 1983).

[0093] Transformed Agrobacterium tumefaciens LBA4404 was cultured for 3 days in the AB liquid medium that added 30 mg / L hygromycin B and 3 mg / L tetracycline, an...

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Abstract

This invention describes novel DNA sequences which function as promoters of anther-specific transcription of coding DNA sequences in recombinant or chimeric DNA sequences. The invention also describes recombinant or chimeric DNA sequences, which are expressed specifically in the anther of a plant. The said recombinant or chimeric DNA sequences may be used to create transgenic plants, but especially transgenic male-sterile plants.

Description

field of invention [0001] The invention belongs to the field of plant genetic engineering. [0002] The present invention mainly relates to novel DNA sequences that function as promoters of anther-specific transcription of coding DNA sequences in recombinant or chimeric DNA sequences. The invention also relates to recombinant or chimeric DNA sequences specifically expressed in plant anthers. The recombinant or chimeric DNA sequences can be used to generate transgenic plants, especially transgenic male sterile plants. Background of the invention [0003] The production of male sterile plants is an economical hotspot in hybrid seed production. Male sterility prevents self-pollination that occurs in many plant species and hinders breeding and hybrid seed production. Anthers are male reproductive organs of flowering plants, are composed of various tissues, such as tapetum, endothecium, connective tissue, vascular tissue, etc., and are responsible for produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/09C12N15/11C12N15/82A01H5/00
CPCC12N15/8289C07K14/415C12N15/8222C12N15/8231C12N15/11
Inventor 安镇兴田钟声郑镛伦李是哲
Owner SYNGENTA PARTICIPATIONS AG
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