Anther specificity expression promoter and purpose thereof

An anther-specific and promoter technology, applied in the field of plant genetic engineering, can solve the problem of lack of RA8 gene promoter

Inactive Publication Date: 2009-12-02
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few RA8 gene promoters reported so far, and strengthening the cloning and application of RA8 gene promoters will play an important role in creating new male sterile line materials of monocotyledonous gramineous crops such as rice and wheat by genetic engineering

Method used

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  • Anther specificity expression promoter and purpose thereof
  • Anther specificity expression promoter and purpose thereof
  • Anther specificity expression promoter and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Cloning of Tsp2 promoter

[0020] 1. Genomic DNA extraction: Genomic DNA of rice variety Nipponbare was extracted by CTAB method.

[0021] 2. Amplify the Tsp2 promoter by polymerase chain reaction (PCR):

[0022] About 100 ng of genomic DNA was taken as a template, and primers raup (5'-AGCAATGGCGTGCAGGGAGTTTGATA-3') and radn (5'-GGGAGGAGCTGGAAGGAGAAGATGGA-3') were used as primers for conventional polymerase chain reaction (PCR). Concrete system and reaction condition are as follows:

[0023] 10× Reaction Buffer 5 μL

[0024] 25mM MgCl 2 4μL

[0025] 10mM deoxynucleotide mixture (dNTP) 1μL

[0026] Raup (10μM) 2μL

[0027] radn (10μM) 2μL

[0028] Template 2 μL

[0029] Taq DNA polymerase 0.5 μL

[0030] Total volume 50μL

[0031] The PCR reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycle: 94°C denaturation for 1 minute, 50°C renaturation for 1 minute, 72°C extension for 1 minu...

Embodiment 2

[0035] Embodiment 2: Construction of expression vector

[0036] 1. According to the above sequence, design the primers for constructing the plant expression vector:

[0037] Forward primer ragup: 5′-GCC GGTACC GCTACTAGGCGTTCG-3′

[0038] Reverse primer ragdn: 5′-GCC AGATCT GGGAGGAGCTGGAA-3' has a KpnI restriction site upstream and a BglII restriction site downstream (underlined part).

[0039] The polymerase chain reaction was performed using the plasmid DNA containing the Tsp2 promoter as a template. The reaction system and procedure are the same as in Example 1.

[0040]2. Take 1 μL of the purified PCR product and connect it to the pMD18-T vector. The operation steps are carried out according to the instructions of the pMD18-T vector kit produced by TaKaRa Company. Then the ligation product was transformed into Escherichia coli JM109 strain, grown overnight on an LB medium plate containing ampicillin (100 μg / μL), picked resistant colonies, and cultured overnight in LB...

Embodiment 3

[0042] Embodiment 3: Transformation of tobacco with expression vector biolistic method containing Tsp2 promoter

[0043] 1. Collect tobacco leaves and flower buds, peel off the anthers, sterilize the surface of the receptor with 70% ethanol, cut the leaves into small pieces, cut the anthers into 2 to 3 sections, and place the samples in a place without Hormone MS solid medium, samples centered at 3cm 2 within the area.

[0044] 2. The preparation of the microprojectile carrier, the plasmid coating and the specific operation of the gene gun were carried out according to the instructions of BioRad's Biolistic ParticalDelivery System (PDS-1000 / He). The samples were transformed with 8 μL (plasmid and gold powder) mixture per gun, and bombarded twice per dish.

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Abstract

The invention belongs to the technical field of plant genetic engineering and provides a promoter sequence for the specificity expression of plant anthers, a recombined nucleic acid sequence containing a promoter, a construction body and an expression system, wherein the promoter can drive functional genes to express the specificity in the plant anthers.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a promoter sequence specifically expressed in plant anthers, a recombinant nucleic acid sequence, a construct and an expression system containing the promoter, and a method for specifically expressing a functional gene in plant anthers. use. technical background [0002] The utilization of heterosis is one of the important ways to increase crop yield and improve crop quality. In recent years, there have been many successful reports on the cultivation of plant male sterile lines by means of genetic engineering. The anther-specific expression promoter is an important element for constructing male sterility. Most of the male organ-specific expression promoters reported for the construction of male sterility expression vectors are promoters from dicotyledons such as tobacco or Arabidopsis. These Most promoters are difficult to express in monocot crops....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63A01H1/00A01H5/00
Inventor 余懋群张金辉潘志芬邓光兵龙海
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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