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Two Anther Development Specific Promoters and Their Applications

A promoter and anther technology applied in the field of plant genetic engineering

Inactive Publication Date: 2020-01-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are few promoters that are specifically expressed in anther development and induced by high temperature. Using such promoters to drive the expression of specific genes is also one of the ways of genetic engineering improvement

Method used

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  • Two Anther Development Specific Promoters and Their Applications
  • Two Anther Development Specific Promoters and Their Applications
  • Two Anther Development Specific Promoters and Their Applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Cloning of promoters PAtCKL2 and PAtCKL7

[0036] The cloning of promoter PAtCKL2 and PAtCKL7 of the present invention is based on the report of 13 caseinkinase I-like gene members in existing Arabidopsis (Lee, Jung-Youn, Taoka, Ken-ichiro, Yoo, Byung-Chun, Ben -Nissan, Gili, Kim, Dong-Jin, & Lucas, William J.(2005).Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.The Plant Cell,17(10),2817- 2831). The promoter sequences of genes AtCKL2 (Accession No. AT1G72710) and AtCKL7 (Accession No. AT5G44100) were retrieved from the TAIR database (The Arabidopsis Information Resource, http: / / www.arabidopsis.org / ).

[0037] According to the promoter sequences of AtCKL2 and AtCKL7 genes, the forward and reverse sequences of the promoters of AtCKL2 and AtCKL7 were designed respectively, and the primers PAtCKL2-TA-F, PAtCKL2-TA-R, PAtCKL7-TA-F, PAtCKL7-TA-R Sequences are described below and PCR amplified...

Embodiment 2

[0046] Embodiment 2: The expression vector construction of promoter PAtCKL2 and PAtCKL7 driving reporter gene

[0047] Primers were designed according to the promoters PAtCKL2 and PAtCKL7, and attB site sequences and protective bases for recombination were added to both ends of the primers, and the primers were named primers JPAtCKL2-BP-F, JPAtCKL2-BP-R, and JPAtCKL7, respectively. -BP-F and JPAtCKL7-BP-R, and then use the promoters PAtCKL2 and PAtCKL7 obtained in Example 1 as templates to carry out PCR amplification with high-fidelity DNA polymerase, and the products obtained are constructed by BP and LR recombination reactions into pGWB433 (see Tsuyoshi et al., Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem, 2007, 71 (8): 2095-2100 for the pGWB433 vector), so as to obtain the driving report Expression vectors PAtCKL2::GUS and PAtCKL7::GUS for the gene GUS.

[0048] The...

Embodiment 3

[0057] Example 3: Transformation of Arabidopsis thaliana using promoter expression vectors PAtCKL2::GUS and PAtCKL7::GUS

[0058] The vector plasmid constructed in the verified embodiment 2 was transferred into the Agrobacterium strain GV3101 (R Hellens, P Mullineaux. (2000) A guide to Agrobacterium binary Ti vectors. Trendsin Plant Science, 5 (10). 446-451 .). The acceptor material used in the transformation of Arabidopsis involved in the present invention is Colombian wild type, and the transformation method and program of the Agrobacterium-mediated flower organ soaking method to transform Arabidopsis thaliana refer to (Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols. 1:641-646), the specific steps are as follows. The activated Agrobacterium (GV3101) containing the target vector was inoculated into 200 mL liquid LB medium, and cultured overnight on a shaker at 28...

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Abstract

The invention belongs to the field of plant gene engineering, and more specifically relates to two anther growth specific promoters, and applications thereof. The two promoters PAtCKL2 and PAtCKL7 areobtained from arabidopsis thaliana Columbia wild type via anther later period high efficiency specific expression and high temperature induced expression; the nucleic acid sequences of PAtCKL2 and PAtCKL7 are represented by SEQ ID NO:1 and SEQ ID NO:2. It is shown that under high temperature stress, PAtCKL2 and PAtCKL7 are capable of driving expression of GUS gene in arabidopsis thaliana anther early tissues via high temperature treatment of PAtCKL2 and PAtCKL7 transgenetic arabidopsis thaliana. The invention also discloses an expression cassette structure constructed by PAtCKL2 and PAtCKL7,and gene engineering modification of the expression cassette structure in arabidopsis thaliana and other crops, and applications of PAtCKL2 and PAtCKL7 in construction of male sterile lines or improvement of regulation on male reproductive organ growth under high temperature stress.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to two anther development-specific promoters (PAtCKL2 and PAtCKL7) and their application. The promoter is isolated from the late anther of wild type Arabidopsis thaliana. These two promoters drive the target gene to be efficiently and specifically expressed in the late anther period, and the expression level reaches the highest in the anther on the day of flowering, and can be expressed in the early tissue of the anther after being induced by high temperature. [0002] The present invention also relates to the preparation of an Arabidopsis anther development-specific and high-efficiency expression promoter, a plant expression vector and a transformant containing the promoter, and the application of the promoter in Arabidopsis transformation and cotton genetic improvement. The invented promoter can be used to regulate male development and create male steri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/02A01H6/20A01H6/60
Inventor 朱龙付闵玲李耀耀张献龙
Owner HUAZHONG AGRI UNIV
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