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Isolation, identification and application of pollen-specific promoter

A pollen-specific and promoter technology, applied in the application field of landscaping plants, can solve the problems that the sequence and function have not been reported yet

Inactive Publication Date: 2011-12-21
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Brassica napusΔ 8 -The sequence and function of the sphingolipid dehydrogenase gene BrD8B promoter have not been reported

Method used

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  • Isolation, identification and application of pollen-specific promoter
  • Isolation, identification and application of pollen-specific promoter
  • Isolation, identification and application of pollen-specific promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1.Δ 8 Acquisition of BrD8B Promoter of Sphingolipid Dehydrogenase Gene

[0028] 1. Preparation of Brassica napus Genomic DNA

[0029] Using the live cabbage-type rape plant planted in this group as the material, take its young leaves (about 100 mg), put them in a mortar and add liquid nitrogen and grind them thoroughly until the materials are completely broken. Then transfer the fully ground material into a 1.5ml centrifuge tube, add 600 μl of preheated CTAB extract (see "Refined Molecular Biology Experiment Guide" 2001, Science Press, translated by Yan Ziying, Wang Hailin), and mix well. 65°C water bath for 30min. Add an equal volume of chloroform, and gently extract for about 5 minutes. Centrifuge at 12000rpm room temperature for 10min. Take the supernatant, add 1 / 2 volume of isopropanol to mix well, and place at room temperature for 10 min to precipitate DNA. Pick out the precipitated DNA with a pipette tip, add 70% ethanol and wash twice. Remove 70% et...

Embodiment 2

[0042] Example 2.Δ 8 Construction of plant expression vector of sphingolipid dehydrogenase gene BrD8B promoter

[0043] From the pEASY-Blunt vector, use restriction endonucleases Hind III and BamH I (purchased from Takara Company) to carry out double enzyme digestion, and connect with the pBI121 (purchased from Clontech Company) vector of the same double enzyme digestion, and construct the CaMV35S on pBI121 The plant expression vector with the promoter replaced by the BrD8B promoter was sequenced and verified, and it was named pBI-proBrD8B. The vector map is shown in figure 2 . The pBI121 vector contains the GUS gene, which turns blue after staining, and is used to detect the specific expression of the promoter.

Embodiment 3

[0044] Example 3. Obtaining of Arabidopsis thaliana transformed with pBI-proBrD8B

[0045] 1. Transformation of plant expression vector into Agrobacterium

[0046] Pick a single colony of Agrobacterium GV3101 (Biovector, Inc) and inoculate it in 5ml YEP liquid medium (10g / L yeast extract, 10g / L peptone, 5g / L NaCl), shake overnight at 28°C and 200rpm. Add 2ml of the overnight cultured bacterial solution to 50ml of YEP medium, 28°C, 220rpm, shaking culture to OD 600 Between 0.5-1.0. Centrifuge the bacterial liquid at 5000rpm, discard the supernatant, suspend the pellet in 10ml 0.5M NaCl, centrifuge at 5000rpm for 5min at 4°C, discard the supernatant, and replace with 1ml pre-cooled 20mM CaCl 2 Resuspend cells. Take 0.2ml and add about 0.5-1μg pBI-proBrD8B, mix gently, freeze in liquid nitrogen for 1min, heat shock at 37°C for 5min, add 1ml of YEP solution, and incubate at 28°C for 2-4h with shaking. Centrifuge the bacterial solution at 5000rpm, discard the supernatant, resus...

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Abstract

The invention provides a pollen-specific promoter and application thereof. The promoter is derived from Brassica rapa and is an upstream sequence of a Delta8 sphingolipid desaturase gene. The promoter provided by the invention is a stable pollen-specific promoter having high specificity. The promoter can be used to promote the expression of various genes in pollen, and can be used for cultivatingmale sterile crop varieties and cultivating landscaping plants and the like with pollen eliminated or greatly reduced.

Description

technical field [0001] The invention relates to a plant pollen-specific promoter and its application. Specifically, involving a Δ 8 The acquisition of the promoter of the sphingolipid dehydrogenase gene and the construction of the plant expression vector, as well as its application in the cultivation of male sterile lines of plants, pollen elimination or greatly reduced landscaping plants. Background technique [0002] In the development of higher plants, the development of male gametes is a very complex process, and its regulatory mechanism is very complicated. It involves a series of organ differentiation and tightly controlled cellular and biochemical changes, accompanied by the coordinated expression of a large number of genes. During pollen development, about 20,000 genes are involved in the expression, of which about 10% are pollen-specific expression [1] , and the pollen-specific promoters play a very important role in the specific expression of these genes in poll...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/10C12N5/10A01H5/00
Inventor 胡赞民李书粉宋丽英尹维波陈宇红
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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