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Male tissue-perferred regulatory region and method of using same

A technology for regulating regions and tissues, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as lack of teaching

Inactive Publication Date: 2000-09-27
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent does not teach the use of the male tissue-preferred regulatory region or the male tissue-preferred regulatory region and foreign genes as a method of controlling male sterility

Method used

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  • Male tissue-perferred regulatory region and method of using same
  • Male tissue-perferred regulatory region and method of using same
  • Male tissue-perferred regulatory region and method of using same

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Embodiment approach

[0025] According to one embodiment of the present invention, said object and other objects are achieved by providing an isolated DNA molecule, wherein said DNA molecule comprises the nucleotide sequence shown in sequence 1.

[0026] According to another embodiment of the present invention, an expression vector containing an exogenous gene is provided, wherein the expression of the exogenous gene is controlled by the preferred regulatory region of the male tissue, and the product of the exogenous gene affects male fertility.

[0027] According to another embodiment of the present invention, there is provided a method for producing male sterile plants using the expression vector, comprising the following steps: introducing an expression vector into plant cells, wherein the exogenous gene of the expression vector Can be a complementary nucleotide unit, such as antisense molecules (callase antisense RNA, barnase antisense RNA and chalcone synthase antisense RNA, Ms45 antisense RNA...

example 1

[0097] Genome Cloning and Sequencing of the Ms45 Promoter

[0098] Ac labeling and identification of Ms45 cDNA, and Northern analysis are disclosed in US5,478,369.

[0099] The partial cDNA of Ms45 was used to screen the B73 maize genomic library. This library was made by cloning SAU3A1 into a BAMHI digested genomic cloning vector (λ DashII, Stratagene, La Jolla, CA). Escherichia coli strain (ER1647, New England Laboratory, MA) suitable for genomic DNA was used as host selection for approximately 1 × 10 6 plaque. After three rounds of selection, clone AC4.1 was purified to homozygosity. Restriction mapping of AC4.1 confirmed that the clone was approximately 13 kb long and contained 2 internal BAMHI sites (Figure 1). One of the sites is also present on the Ms45 partial cDNA. The two BamHI fragments were subcloned into the cloning vector (Bluscript SK+, La Jolla, CA). The 5' end clone was approximately 3.5 kb in length, corresponding to the sequence upstream (5...

example 2

[0103] Primer extension analysis

[0104] Total RNA was extracted from maize spike male flowers containing early anthers at the tetrad to uninucleate stages. The total RNA was precipitated with ethanol and magnesium chloride. 1 mg of total RNA was extracted and polyA+ mRNA was purified with oligo-dT cellulose. PolyA+ RNA was directly extracted from 6-day-old maize seedling leaves and maize anthers by methods known to those skilled in the art.

[0105] Using methods well known to those skilled in the art, single-stranded Ms45 oligonucleotides were used to prepare sequencing ladders and 35S-dATP was used in standard sequencing methods. Primer extension was performed as follows: 1. 5'-end labeled synthetic oligonucleotide primers. Mix: 5 pmol primer N11916 (PHL11916) in a volume of 1.0 μl

[0106] 5μl (50μCi) γ32P-ATP (>5000Ci / mmole)

[0107] 0.7 μl 10X Kinase Buffer

[0108] 0.7 μl T4 polynucleotide kinase

[0109] Incubate at 37°C for 45 min to inactiv...

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Abstract

The present invention relates to an isolated nucleic acid sequence encoding the Ms45 male tissue-preferred regulatory region. In one aspect this invention relates the use of this male tissue-preferred regulatory region in mediating fertility. An example of such use is the production of hybrid seed such as in a male sterility system. The Ms45 male tissue-preferred regulatory region can be operably linked with exogenous genes, such as those encoding cytotoxins, complementary nucleotidic units and inhibitory molecules. This invention also relates to plant cells, plant tissue and differentiated plants which contain the regulatory region in this invention.

Description

field of invention [0001] The present invention relates to isolated DNA sequences as regulatory regions in eukaryotic cells. More specifically, the present invention relates to isolated DNA sequences derived from maize that serve as male tissue-preferred regulatory regions and function in gene expression in maize tissue. The present invention also relates to a method for conferring the ability of a gene that can or cannot be expressed normally in maize tissue to be expressed in a male-tissue-preferred manner. Background of the invention [0002] Tissue- and timing-specific gene expression and regulation are particularly present during sexual reproduction in eukaryotes. In plant gametogenesis, important cytological and biochemical changes occur during pollen development, when asymmetric mitosis of haploid microspores leads to the formation of two cells; each with a different developmental fate. Vegetative cells support the growth of pollen, while germ cells undergo mitosis ...

Claims

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Application Information

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IPC IPC(8): A01H5/00C07K14/34C12N5/10C12N9/10C12N9/22C12N9/24C12N15/09C12N15/29C12N15/82
CPCC12N15/8289C12Y602/01003C12N9/93C12N9/2402C07K14/34C12Y302/01031C12Y302/01039C12N9/1037C12N15/8231C12N9/22
Inventor M·C·阿尔伯特森T·W·福克斯C·W·加纳特G·A·胡夫曼T·L·肯达尔
Owner PIONEER HI BRED INT INC
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