Identification and applications of plant anther-specific expression promoter pTaASG033

An anther-specific, promoter-based technology, which is applied to isolated DNA, can solve problems such as long waiting times, effects on plant growth and development, and gene silencing with high homology of promoter sequences.

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 pTaASG033
  • Identification and applications of plant anther-specific expression promoter pTaASG033
  • Identification and applications of plant anther-specific expression promoter pTaASG033

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 contigs at later stages of pollen development

[0043] Wheat anthers with pollen at meiotic, mononucleate, binucleate and trinucleate stages were taken, and total RNA was extracted with Trizol (Invitrogen) and treated with DNaseI (Promega) to purify mRNA (Ambion). The purified mRNA was reverse transcribed (Invitrogen), interrupted by ultrasound (Fisher), library preparation (illumina) and amplified (illumina), and finally the sequencing reaction was performed on the illumina machine.

[0044] The results of high-throughput sequencing of the wheat transcriptome were first spliced ​​by Trinity software, and the obtained spliced ​​sequences were further removed from redundancy and similarity clustering. For the analysis of expression changes of transcript contigs obtained by splicing, the sequences of high-throughput sequencing in eac...

Embodiment 2

[0046] Example 2. RT-PCR to verify the tissue expression specificity of TaASG033 gene

[0047] Wheat is an allohexaploid 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 the genes have 1 -2 copies, 42% of genes have ≥5 copies. Starting from the sequence of comp163920_c0_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 in Nature in 2013 , A genome donor) and Aegilops tauschii (Aegilops tauschii, D genome donor) were electronically cloned, and three TaASG033 genes were obtained, named TaASG033-1, TaASG033-2 and TaASG033-3, among which comp163920_c0_seq1 Corresponding to TaASG033-1. The cDNA sequences of the three TaASG033 genes are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and ...

Embodiment 3

[0057] Example 3. Acquisition of TaASG033-1, TaASG033-2 and TaASG033-3 gene promoter sequences and analysis of cis-elements

[0058] Starting from the cDNA sequences of TaASG033-1, TaASG033-2 and TaASG033-3 genes, using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and the wheat ancestor Urartu wheat published in Nature in 2013 ( The sequencing information of Triticum urartu, A genome donor) and Aegilops tauschii (D genome donor) were electronically cloned, and the promoters of TaASG033-1, TaASG033-2 and TaASG033-3 genes were obtained and named as TaASG033, respectively. -1 promoter, TaASG033-2 promoter and TaASG033-3 promoter, their lengths are 2092bp, 1997bp and 2000bp respectively, and their sequences are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 respectively.

[0059] Using the PlantCARE database and the PLACE database, cis-element analysis was performed on the TaASG033-1 promoter, th...

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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 an isolated DNA capable of directing the specific transcription and / or expression of a nucleic acid operably linked downstream thereof in plant anthers. In addition, the present invention also relates to expression cassettes, plants, etc. 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 that regulates gene transcription. It can activate RNA polymerase and make it accurately combine with the template DNA to ensure accurate and efficient transcription. plays a key role in transcriptional regulation. According to their...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N15/82A01H5/00
Inventor 马力耕李健邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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