Identification and Application of Plant Anther-Specific Expression Promoter ptaasg042

An anther-specific, promoter-based technology, applied in the field of DNA applications and isolated DNA, can solve the problems of gene silencing with high homology of promoter sequences, ineffective improvement of target gene expression in time and space, and long waiting times, etc.

Active Publication Date: 2018-11-30
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 CaMV 35S promoter and the corn Ubiquitin-1 promoter. Sometimes, the improvement effect is not obvious because the time (developmental stage-specific) or space (tissue-organ-specific) of the expression of the target gene cannot be well controlled, or the gene expression level induced by these constitutive promoters is too high. These are the obstacles encountered when using 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 ptaasg042
  • Identification and Application of Plant Anther-Specific Expression Promoter ptaasg042
  • Identification and Application of Plant Anther-Specific Expression Promoter ptaasg042

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Genome-wide expression profiling analysis of wheat anthers at different developmental stages and acquisition of anther expression contig at later stages of pollen development

[0043] The present invention takes wheat anthers whose pollen is in meiosis, mononucleate, binucleate and trinucleate, extracts total RNA with Trizol (Invitrogen), and treats with DNaseI (Promega) to further purify mRNA (Ambion). The purified mRNA was subjected to reverse transcription (Invitrogen), ultrasonic fragmentation (Fisher), library preparation (illumina) and amplification (illumina), and finally a sequencing reaction on an illumina machine.

[0044] The results of high-throughput sequencing of the wheat transcriptome were first assembled by Trinity software, and the resulting spliced ​​sequences were further removed from redundancy and similarity clustering. For the expression change analysis of the spliced ​​transcript contig, the high-throughput sequencing sequence in each ...

Embodiment 2

[0046] Embodiment 2.RT-PCR verifies the tissue expression specificity of TaASG042 gene

[0047] Starting from the sequence of comp177540_c2_seq11 (as 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 Triticum urartu published on Nature in 2013 , A genome donor) and the sequencing information of Aegilops tauschii (D genome donor) were electronically cloned, and the CDS sequence of the TaASG042 gene was obtained, as shown in SEQ ID NO:2. Design specific primers, and use RT-PCR method to analyze the expression specificity of the gene in various tissue materials such as wheat roots, stems, leaves, anthers at different development stages and other floral organs except anthers. The results are as follows figure 2 As shown, the TaASG042 gene is only specifically expressed in anthers whose pollen is in the uninucleate, binucleate and trinucleate stages, a...

Embodiment 3

[0051] Example 3. Obtaining of TaASG042 promoter sequence and analysis of cis-elements

[0052] Starting from the CDS sequence of the TaASG042 gene, using the sequencing information of common wheat published by CerealsDB and IWGSC (international wheatgenome sequencing consortium), as well as the wheat ancestor Triticum urartu (Triticum urartu, A genome donor) and rough goat published in Nature in 2013 The sequencing information of grass (Aegilopstauschii, D genome donor) was electronically cloned, and the promoter of the TaASG042 gene was obtained, named TaASG042 promoter, and its length was 2000bp. The sequence of the promoter and part of the coding region is shown in SEQ ID NO:3 .

[0053] The cis-element analysis of the TaASG042 promoter was performed using the PlantCARE database and the PLACE database. Such as image 3 As shown, the translation start site ATG is indicated by a bold underline, and the A of the translation start site ATG is defined as "+1", the AGAAA motif...

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Abstract

The present invention belongs to the field of plant biotechnology, and particularly relates to separation, functional identification and applications of a plant anther-specific expression promoter. According to the present invention, the promoter is specifically expressed in the plant anther, and provides good application prospects in the plant transgene field.

Description

technical field [0001] The present invention belongs to the field of plant biotechnology, in particular, the present invention relates to isolated DNA, which can guide the specific transcription and / or expression of nucleic acid operably linked downstream of it in plant anthers. In addition, the present invention also relates to expression cassettes and plants containing the DNA, and to applications of the DNA. Background technique [0002] Plant gene regulation is mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. The 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 combine with the template DNA to ensure accurate and efficient transcription. play a key role in transcriptional regulation. According to the different characteristics of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82
Inventor 马力耕李健邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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