Plant pollen-specific promoter PSP1 and application thereof

A pollen-specific, plant pollen technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as affecting plant growth and development, poor control of the spatiotemporal specificity of target gene expression, and insignificant improvement effects, and achieves broad application prospects and agricultural Economic value, effect of avoiding biosecurity problems

Active Publication Date: 2017-11-21
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, some constitutive strong promoters are widely used in the field of agricultural biotechnology, such as Ubiquitin-type promoters and CaMV35S promoters. However, when using these promoters to induce the target gene to transform plants such as rice for plant impr...

Method used

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  • Plant pollen-specific promoter PSP1 and application thereof
  • Plant pollen-specific promoter PSP1 and application thereof
  • Plant pollen-specific promoter PSP1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Cloning of rice pollen-specific promoter PSP1

[0039] According to the whole genome sequence of rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided by NCBI, design specific amplification primers for PSP1 sequence, and add Specific enzyme cutting site ( figure 1 ). The specifically designed primers are: a KpnI restriction site (GGTACC) is added to the 5' end of the forward primer (P1F), and a NcoI restriction site (CCATGG) is added to the 5' end of the reverse primer (P1R). The primer sequences are as follows:

[0040] P1F forward primer: 5'-CGGggtaccAACCAAACCTGTGGCTGC-3'KpnI

[0041] P1R reverse primer: 5'-CATGccatggCGCTCTGTGACGCCAT-3'NcoI

[0042] Then the genomic DNA of the rice variety Nipponbare was extracted, and using the Nipponbare genomic DNA as a template, the primers (P1F and P1R) designed above were used to amplify the promoter PSP1 sequence, and the following amplification procedures were used: pre-denaturation at 94°C for 4 minu...

Embodiment 2

[0043] Example 2. Construction of a plant expression vector driven by the promoter PSP1

[0044] The positive clone obtained in Implementation 1 and identified by sequencing was subjected to plasmid extraction, and the extracted plasmid was double digested with KpnI and NcoI, and the PSP1 promoter fragment was recovered. At the same time, pCambia1305 was linearized by double digestion with KpnI and NcoI, and the pCambia1305 backbone was recovered. The PSP1 promoter fragment recovered after digestion was ligated with the pCambia1305 backbone recovered after digestion with T4 ligase (purchased from NEB Company), Obtain the plant expression vector PSP1-GUS-pCambia1305 ( image 3 ), the PSP1-GUS-pCambia1305 expression vector was transformed into Agrobacterium tumefaciens EHA105 by electric shock transformation method.

Embodiment 3

[0045] Example 3. Transforming rice with the expression vector of the GUS gene driven by the promoter PSP1

[0046] Using the Agrobacterium-mediated method of rice mature embryo callus dissemination transformation, the recombinant expression vector PSP1-GUS-pCambia1305 was transferred into rice mature embryos, and the transformation method was as follows: (1) induction of rice mature embryo callus: mature The Nipponbare seeds were shelled, then surface-sterilized with 75% alcohol for 2 minutes, then soaked in 30% NaClO solution for 30 minutes, and repeated once, and then washed 4-5 times with sterilized water. The seeds were then placed on the induction medium and cultured, and cultured in the dark at 26 degrees to induce callus for transformation ( Figure 4 A). (2) Co-culture of rice callus and Agrobacterium: activate, enrich and resuspend the EHA105 strain identified in Example 2 containing the PSP1-GUS-pCambia1305 expression vector, and adjust OD600=0.3-0.5. Collect the ...

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Abstract

The invention discloses plant pollen-specific promoter PSP1 and application thereof and also discloses an expression kit containing the plant pollen-specific promoter PSP1, a recombinant expression vector and a host cell, as well as a primer pair for amplifying the plant pollen-specific promoter PSP1, and a method of using the plant pollen-specific promoter PSP1 to drive target gene expression in plant pollen. A transgenic rice plant is acquired herein by transforming via the expression vector constructed with the plant pollen-specific promoter PSP1; verification and histochemical staining analysis show that the promoter can drive efficient specific expression of a target gene in pollen, and cloning of the plant pollen-specific promoter PSP1 helps develop seed breeding.

Description

Technical field: [0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a plant pollen-specific promoter PSP1 and its application. Background technique: [0002] Genetically modified technology is considered to be one of the most effective ways to solve the contradiction between the sharp decrease of land resources and the rapid expansion of population. The use of transgenic technology to improve and breed new varieties can bring huge economic and social benefits and significant ecological benefits, so it has attracted the attention of governments and scientists from various countries. [0003] The exogenous genes carried by transgenic plants may drift to related species or wild species with pollen, and "gene drift" occurs, which may lead to internal genetic changes in nearby related species or wild species, resulting in new traits Or form new species, thereby causing changes in the ecosystem. In particular, the drift of gen...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/82A01H5/00
CPCC07K14/415C12N15/8231C12N15/8289
Inventor 胡时开钱前郭龙彪胡培松唐绍清曾大力魏祥进焦桂爱圣忠华邵高能谢黎红
Owner CHINA NAT RICE RES INST
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