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Pollen specific promoter and application thereof

A pollen-specific and promoter technology, applied in the biological field, can solve the problems of single source of sterility genes, long cycle of male sterility, etc., and achieve the effect of great application prospects

Inactive Publication Date: 2017-08-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the naturally occurring male sterility types rarely have problems such as long cycle and single source of sterility genes, and there are also certain deficiencies in the cultivation of restorer lines and the selection of offspring.

Method used

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  • Pollen specific promoter and application thereof
  • Pollen specific promoter and application thereof
  • Pollen specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning of Maize Pollen Specific Promoter Sequence pZm908

[0041]A pollen-specific cDNA clone (682bp fragment), Zm401 (Li et al., 2001), was isolated from the mature pollen cDNA library of maize, and was used as a probe to screen the maize genome library (Cat.#FL1032D, Lot#5443, Clontech, Palo Alto, CA, USA) obtained the genomic clone pGZM908 of the Zm908 gene.

Embodiment 2

[0042] Example 2 Isolation of pollen-specific promoter sequence pZm908

[0043] The following two primers were designed and synthesized, and a restriction endonuclease SphI site and a restriction endonuclease BamHI site were respectively added to the 5' ends of the two primers for the needs of subsequent isolation and construction:

[0044] Primer 1 5'CgcGCATGCGGTGTGCGTAATGACTCGA 3'SphI

[0045] Primer 2 5'Cgcggatccgaaacatatgaatagtac3'BamHI

[0046] Using the plasmid DNA pGZM908 as a template, carry out PCR reaction, the reaction system is as follows:

[0047]

[0048]

[0049] Amplification conditions: 95°C for 10 min; 95°C for 1 min, 58°C for 1 min, 72°C for 2 min, a total of 30 cycles; 72°C for 10 min, a 2126bp DNA fragment (SEQ ID No.1) was amplified. Recover with DNA gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), subclone the recovered fragment into the sequencing vector pM19-T, and transform the ligated product into 2 Escherichia coli DH5α competen...

Embodiment 3

[0050] Example 3 Construction of Plant Expression Vector pZm908::GUS

[0051] Plasmid p19T-pZm908 was extracted by alkaline lysis, digested with SphI, filled in with Klenow, and digested with BamHI to obtain the pZm908 promoter fragment, which was digested with HindⅢ, filled in with Klenow, and digested with BamHI. The large fragments of pBI121 were connected ( figure 1 ), and then convert the ligation product to CaCl 2 In E. coli DH5α competent cells treated by the method, cultivate overnight on LB solid medium containing kanamycin (final concentration 100 μg / ml); pick the white colony grown on the plate, insert into containing kanamycin ( The LB liquid medium with a final concentration of 100 μg / ml) was cultured overnight, and the bacteria were collected by centrifugation to extract the plasmid by alkaline lysis. The sequencing results showed that the vector pZm908::GUS was constructed correctly.

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PUM

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Abstract

The invention discloses a pollen specific promoter sequence separated from corns and application of the pollen specific promoter sequence. The pollen specific promoter sequence is obtained by screening corn genome libraries and carrying out cloning in corn genomes. A pollen specific promoter disclosed by the invention comprises a nucleotide sequence represented by SEQ ID NO:1 or a specific fragment of the nucleotide sequence. The invention discloses a genetic engineering method for creating male sterile plants by virtue of the promoter sequence. More particularly, the method comprises the steps of linking a heterogenous or homologous gene to the downstream side of the promoter, constructing a plant expression vector, and transforming a host plant. By utilizing the promoter sequence, downstream genes can be driven to specifically express target proteins in pollen, so as to realize the creation of male sterile plants or the transformation safety of transgenic plants.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pollen-specific promoter sequence isolated from maize and its application. The pollen-specific promoter is cloned from the maize genome DNA, and connected with different homologous and heterologous genes to construct a plant expression vector, transforming a host plant, and making the transgenic plant pollen specifically express the method and application of its downstream genes. Background technique [0002] In the process of research and development of transgenic plants, it is found that the time, space and expression amount of exogenous gene expression are often important factors that make it impossible to obtain ideal transgenic plants and effectively study the mechanism of gene action. Regulation at the transcriptional level is the most important regulatory mode of plant gene expression regulation. The driving activity and characteristics of the promoter directly determine th...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00
CPCC07K14/415C12N15/829
Inventor 于静娟陈希阳齐鑫彭静刘鹏王冬雪
Owner CHINA AGRI UNIV
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