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Identification and applications of plant anther-specific expression promoter

An anther-specific, promoter-based technology, which is applied to isolated DNA. The application field of this DNA can solve the problems of plant growth and development, gene silencing with high promoter sequence homology, and long waiting time, etc.

Active Publication Date: 2015-10-14
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 applications of plant anther-specific expression promoter
  • Identification and applications of plant anther-specific expression promoter
  • Identification and applications of plant anther-specific expression promoter

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] Wheat anthers whose pollen was in meiosis, mononucleate, binucleate and trinucleate were collected, total RNA was extracted with Trizol (Invitrogen), treated with DNaseI (Promega), and then mRNA was purified (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 sampl...

Embodiment 2

[0046] Embodiment 2.RT-PCR verifies the tissue expression specificity of TaASG046 gene

[0047] Wheat is an allohexaploid composed of three sets of genomes A, B, and D. The average copy number of genes is 2.8, of which nearly half of the genes (46%) have 3-4 copies, and 12% of the genes have 1 -2 copies, 42% of genes had ≥5 copies. Starting from the sequence of comp178993_c2_seq1 (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 three TaASG046 genes were obtained, named TaASG046-1, TaASG046-2 and TaASG046-3, among which comp178993_c2_seq1 Corresponding to TaASG046-1. The cDNA sequences of the three TaASG046 genes are respectively shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, ...

Embodiment 3

[0057] Example 3. Obtaining of TaASG046-1 and TaASG046-2 Gene Promoter Sequences and Analysis of Cis Elements

[0058] Starting from the cDNA sequences of TaASG046-1 and TaASG046-2 genes, using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), as well as the wheat ancestor Triticum urartu (Triticum urartu, A. Genome donor) and the sequencing information of Aegilops tauschii (D genome donor) were electronically cloned, and the promoters of TaASG046-1 and TaASG046-2 genes were obtained, and the lengths were 2210bp and 2078bp respectively, and the sequences were as SEQ ID Shown in NO:5 and SEQ ID NO:6.

[0059] Using PlantCARE database and PLACE database, the promoters of TaASG046-1 and TaASG046-2 genes were analyzed in cis elements. Such as image 3 As shown, the translation initiation site ATG is indicated by a bold underline, and the A of the translation initiation site ATG is defined as "+1", the ...

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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 TaASG046. 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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IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 李健马力耕邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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