Plant pollen specific expression promoter POsPTD1 and application

A plant pollen and promoter technology, applied in the field of genetic engineering, can solve the problem of difficulty in reproducing completely sterile populations, and achieve the effect of blocking pollen-mediated gene drift and reducing exogenous transgenic components

Active Publication Date: 2021-01-22
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sporophyte genic male sterile has a greater heterosis utilization potential than the sporophytic male sterile, but its inherent genetic characteristics make it difficult to breed a total sterile population, and it is necessary to develop a corresponding sporophytic genic male sterile total sterile population. method

Method used

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  • Plant pollen specific expression promoter POsPTD1 and application
  • Plant pollen specific expression promoter POsPTD1 and application
  • Plant pollen specific expression promoter POsPTD1 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the cloning of rice male gametophyte-specific promoter POsPTD1

[0034] Genes related to male gametophyte development are often expressed in middle and late flower / pollen. In order to find some genes related to gametophyte development, the BAR rice gene expression database (http: / / bar.utoronto.ca / efprice / cgi-bin / efpWeb.cgi) was analyzed, and many genes expressed in the middle and late flowers were found. qPCR verification to select a group of genes that may be related to the development of male gametophytes. Among them, the rice Loc_Os05g40740 gene was specifically highly expressed in late flowers, and its Arabidopsis homologous gene was specifically expressed in pollen. The Loc_Os05g40740 gene was initially listed as a gene related to male gametophyte development, and its promoter was listed as a pollen-specific promoter. Subsequently, the temporal and spatial expression pattern of the gene was analyzed by qPCR. The qPCR primers of the Loc_Os05g40740 gen...

Embodiment 2

[0037] Example 2, Pollen-specific GUS staining identification of the rice pollen-specific promoter POsPTD1

[0038] Transform POsPTD1-GUS into Agrobacterium LBA4404, and then transfect the positive Agrobacterium into Zhonghua 11. GUS staining was carried out on the roots, leaves and flowers of the transgenic positive offspring at the flowering stage. The results showed that the roots, leaves, glumes, filaments and pistils were not stained, while the anthers were stained blue. The anthers were further crushed, and observed under a microscope: only about 50% of the GUS stained pollen of the transgenic heterozygous plant was found, and almost all of the pollen of the homozygous plant could be stained by GUS ( figure 1 ). The above results indicated that the promoter POsPTD1 was indeed a pollen-specific promoter.

Embodiment 3

[0039] Example 3. Preparation and identification of male gametophytic sterility by RNA interference driven by POsPTD1

[0040] In order to determine whether the POsPTD1 promoter can drive the target gene or DNA fragment to destroy pollen fertility, male gametophytic sterile materials were prepared and identified. Taking Loc_Os05g40740 as the object, the POsPTD1 promoter was used to drive the interference fragment of the Loc_Os05g40740 gene, and the creation and identification of male gametophytic sterility were implemented:

[0041] 1. In order to facilitate the loading of relevant target genes or DNA fragments, and in order to accurately and directly identify transgenic positive plants and male gametophyte sterility, we replaced the GUS gene with the modified gene OsMYB76R of rice anthocyanin synthesis-related genes, and modified the vector pCAMBIA1301 The multi-cloning site has been transformed, and the specific operation is as follows:

[0042] (1) OsMYB76 gene modificatio...

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Abstract

The invention discloses a plant pollen specific expression promoter POsPTD1 and application. The nucleotide of the promoter POsPTD1 is shown as SEQ ID NO.1; the pollen specific promoter POsPTD1 disclosed by the invention is from a promoter of a rice endogenous gene, and can specifically drive a target gene or a DNA fragment to be expressed in pollen; and when the promoter disclosed by the invention is used for driving a gene or a DNA fragment with a pollen fertility control function to be specifically expressed in the pollen, gametophyte male sterility can be prepared, and an important role isplayed in aspects of preventing transgenic ingredients from drifting along with the pollen, breeding sporophyte nuclear sterility and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a plant pollen-specific promoter POsPTD1 and its application. The promoter can drive the specific expression of target genes or DNA fragments in pollen, thereby changing pollen fertility, controlling transgene drift, and preparing male gametophytes. Fertility and reproduction sporophyte nuclear male sterility, etc. Background technique [0002] The promoter is generally located upstream of the coding region of the gene. The promoter contains cis-acting elements, which are some special DNA sequences that determine the combination of special transcription factors, thereby realizing the transcriptional regulation of the gene. The promoter also provides the positioning information of RNA polymerase, and RNA polymerase recognizes and binds to the relevant region of the promoter to initiate the transcription of RNA. Therefore, the promoter is necessary for gene transcription, and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84C12N5/10A01H5/00A01H6/46
CPCC07K14/415C12N15/8231C12N15/8289
Inventor 张毅陈云杜双林
Owner YUNNAN UNIV
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