Identification and application of plant anther specific expression promoter pTaASG005

An anther-specific and promoter technology, applied in the application of the DNA, the field of isolated DNA, can solve the problems of long waiting time, insignificant effect of time and space improvement of target gene expression, and influence on plant growth and development, etc.

Active Publication Date: 2016-02-10
BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some constitutive strong promoters are widely used in the field of agricultural biotechnology, such as the CaMV35S promoter and the corn Ubiquitin-1 promoter. , the improvement effect is often not obvious because the time (developmental stage-specific) or space (tissue-organ-specific) of the target gene expression is not well controlled, or the gene expression induced by these constitutive promoters is too high. The growth and development of plants are affected, these are the obstacles encountered in the current use of constitutive strong promoters combined with functional genes to improve crop quality
[0004] In addition, when studying certain metabolic processes or regulatory pathways, it is often necessary to transform two or more genes on the same pathway into the same line, transforming one of the genes to obtain a transgenic plant and then transforming another gene, or It takes a long time to wait for the hybridization after the two genes have been transformed separately. In order to improve the efficiency and shorten the time for transforming multiple genes, it has recently been reported that a new vector can be used to transform multiple genes at the same time, but in multi-gene If the same promoter is used repeatedly during transformation, gene silencing may also occur due to the high homology of the promoter sequence

Method used

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  • Identification and application of plant anther specific expression promoter pTaASG005
  • Identification and application of plant anther specific expression promoter pTaASG005
  • Identification and application of plant anther specific expression promoter pTaASG005

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Whole-genome expression profile analysis of wheat anthers at different developmental stages and the acquisition of anther expression contigs at later stages of pollen development

[0043] The wheat anthers with pollen in the meiotic, mononuclear, binuclear, and trinuclear stages were taken, and the total RNA was extracted with Trizol (Invitrogen), and treated with DNaseI (Promega) to purify mRNA (Ambion). The purified mRNA is subjected to reverse transcription (Invitrogen), ultrasonic interruption (Fisher), library preparation (illumina) and amplification (illumina), and finally a sequencing reaction is performed on the illumina machine.

[0044] The results of high-throughput sequencing of the wheat transcriptome are first used for sequence splicing through Trinity software, and the resulting spliced ​​sequence further removes redundancy and similarity clustering. For the expression change analysis of the spliced ​​transcript contig, the sequence of high-throughp...

Embodiment 2

[0046] Example 2. RT-PCR verification of the tissue expression specificity of TaASG005 gene

[0047] Wheat is a heterohexaploid consisting of three sets of genomes A, B, and D. The average number of copies of genes is 2.8, of which nearly half of the genes (46%) have 3-4 copies, and 12% of genes have 1 -2 copies, 42% of gene copies are ≥5. Starting from the sequence of comp153941_c0_seq4 (shown in SEQ ID NO: 1), using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and the wheat ancestor Urartu (Triticumurartu, A genome donor) published on Nature in 2013 and The sequencing information of Aegilopstauschii (Aegilopstauschii, D genome donor) was electronically cloned, and three TaASG005 genes were obtained, named TaASG005-1, TaASG005-2 and TaASG005-3, respectively, of which comp153941_c0_seq4 corresponds to TaASG005-1. 3 The cDNA sequences of the TaASG005 gene are shown in SEQ ID NO: 2, SEQ ID NO: 3 and...

Embodiment 3

[0057] Embodiment 3. TaASG005-1, TaASG005-2 and TaASG005-3 gene promoter sequence acquisition and cis-elements analysis

[0058] Starting from the cDNA sequences of TaASG005-1, TaASG005-2 and TaASG005-3 genes, using the sequencing information of common wheat published by CerealsDB and IWGSC (International WheatGenomeSequencingConsortium), and the wheat ancestor Triticumurartu (A genome) published on Nature in 2013 The sequence information of Aegilopstauschii (donor) and Aegilopstauschii (D genome donor) were cloned electronically, and the promoters of TaASG005-1, TaASG005-2 and TaASG005-3 genes were obtained, named as TaASG005-1 promoter and TaASG005, respectively. -2 promoter and TaASG005-3 promoter, the lengths of which are 2032bp, 2047bp and 2009bp, respectively, and the sequences are as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0059] Using PlantCARE database and PLACE database, the TaASG005-1 promoter, TaASG005-2 promoter and TaASG005-3 promoter wer...

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Abstract

The invention belongs to the technical field of plant biology, discloses identification and application of a plant anther specific expression promoter pTaASG005 and particularly relates to separation, function identification and application of the plant anther specific expression promoter. The promoter is specifically expressed in plant anthers, particularly in anthers of pollen in a uninucleate stage, a binucleate stage and a trinucleate stage. The plant anther specific expression promoter pTaASG005 has a promising application prospect in the field of plant transgene.

Description

Technical field [0001] The present invention belongs to the field of plant biotechnology. Specifically, the present invention relates to isolated DNA, which can direct the specific transcription and / or expression of nucleic acid operably linked downstream thereof in plant anthers. In addition, the present invention also relates to expression cassettes and plants containing the DNA, and to the application of the DNA. Background technique [0002] Plant gene regulation is mainly carried out at the transcriptional level, coordinated by a variety of cis-acting elements and trans-acting factors. Promoter is an important cis-acting element. It is a DNA sequence located in the upstream region of the 5′ end of the structural gene to regulate gene transcription. It can activate RNA polymerase to accurately bind to the template DNA and ensure accurate and effective transcription. It plays a key role in transcriptional regulation. According to the different characteristics of its driving ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/00
Inventor 李健马力耕邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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