Wheat male sterility genes WMS and application of anther specific promoter thereof

A technology of transgenic cells and amino acids, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of controlling plant fertility, etc., and achieve the effect of creating male sterile characteristics of plants

Active Publication Date: 2015-09-16
泉脉农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of artificially controlling plant fertility, the invention provides an application of wheat male sterility gene WMS and its anther-specific promoter

Method used

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  • Wheat male sterility genes WMS and application of anther specific promoter thereof
  • Wheat male sterility genes WMS and application of anther specific promoter thereof
  • Wheat male sterility genes WMS and application of anther specific promoter thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0082] Example 1: Using transcriptomics to identify 'Lumai 15 Ms2 ’ Anther-specific expression gene

[0083] RNA sequencing (RNA-seq) utilizes high-throughput sequencing technology to directly determine all or part of cDNA molecules derived from a sample. The present invention compares wheat near-isogenic lines 'Lumai 15' and 'Lumai 15' by RNA sequencing Ms2 ’ transcriptome of the respective anther tissues. When the leaf ring distance between the flag leaf and the second leaf of the wheat main stem or tiller reaches 4 cm, the anther is roughly in the early stage of meiosis I (referred to herein as the initial stage of meiosis), and the young ears of the main stem / tiller are collected, The anthers of the first and second florets of the five spikelets in the middle of the young panicle were collected. For 'Lumai 15' and 'Lumai 15 Ms2 ’, 3 biological replicates were collected, each with approximately 150 anthers. Total RNA was extracted using TRIzol reagent and related metho...

Embodiment 2

[0085] Embodiment 2: Verification of wheat WMS gene full-length cDNA

[0086] In order to verify the full-length (full-length) cDNA of the WMS gene, the 'Lumai 15 Ms2’ total RNA from the young panicle, and then the cDNA template was prepared using the RevertAid Frist Strand cDNA Synthesis kit (Thermo Scientific, Waltham, MA, USA). The 5' and 3' ends of the full-length cDNA of the WMS gene (SEQ ID NO: 1) were isolated using rapid amplification of cDNA ends (RACE), using the SMARTer RACE cDNA Amplification kit (Clontech Laboratories, Mountain View , CA, USA), the operation method refers to the kit instructions. The nested primers of 5' end RACE PCR are WMS-RP1 and WMS-RP2, wherein WMS-RP2 is the nested primer of WMS-RP1; the nested primers of 3' end RACE PCR are WMS-FP1 and WMS - FP2, wherein WMS-FP2 is the nested primer of WMS-FP1. Sequencing of the RACE PCR product confirmed the integrity of both ends of the full-length cDNA sequence (SEQ ID NO:1) of the WMS gene, indicatin...

Embodiment 3

[0087] Embodiment 3: 'Lumai 15 Ms2 ’Construction of Genomic BAC Library

[0088] To facilitate the cloning of genomic DNA, the 'Lumai 15' was created Ms2 'The bacterial artificial chromosome (BAC) library of genomic DNA, the construction method refers to the published literature (Grotewold / ed.2003.Plant Functional Genomics / Humana / Totowa:3-19; Shi et al.2011.Plant Methods 7:33). In short, from 'Lumai 15 Ms2 'Extract high-molecular weight genomic DNA (high-molecular weight, HMW) from leaves, partially digest with restriction endonuclease HindIII, use 1% agarose gel and pulsed-field gel electrophoresis (pulsed-field gel electrophoresis, PFGE) to separate enzyme Cut the product, purify DNA with a fragment size between 100-300kb, repeat the pulse electrophoresis and fragment purification steps, and insert the DNA fragment obtained through two purifications into the BAC vector pIndigoBAC536-S (completely digested by HindIII and dephosphorylated at the end ), and then transform E...

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Abstract

The invention relates to wheat male sterility genes WMS and an application of an anther specific promoter thereof. The anther transcriptomes of the wheat near-isogenic line 'Lu wheat 15' and 'Lu wheat 15+Ms2' ('Lu wheat 15Ms2' for short) during the meiosis early period are compared by means of the RNA sequencing technique, and the genes WMS only expressed in 'Lu wheat 15Ms2' anther tissue are found. The full-length cDNA sequence of the WMS genes is shown in SEQ ID NO:1. The amino acid sequence of WMS gene coding protein, namely WMS protein, is shown in SEQ ID NO:2. The WMS gene promoter has the activity of triggering anther tissue specificities, the fertility of the stamen of wheat and slender false brome grass can be changed by manually operating the WMS genes. Therefore, the wheat male sterility genes WMS and the application of the anther specific promoter thereof can be used for realizing anther specificity expression of target genes, establishment of plant male sterility, establishment of a plant recurrent selection system and development of the plant hybrid seed production technique.

Description

1. Technical field [0001] The invention relates to the application of a wheat male sterility gene WMS and an anther-specific promoter thereof. 2. Technical background [0002] Plant male sterility is essential for selective breeding and hybrid seed production. On many plants, a large number of male sterile lines have been found, and their collection and preservation provide valuable genetic and germplasm resources for modern agriculture. In recent years, artificially created plant male sterile lines, especially for important food crops rice (Oryza sativa L.) and corn (Zea mays L.), have begun to play a role in the production. [0003] American Pioneer Hi-Bred International has created a new seed production technology (Seed Production Technology, SPT) (Waltz.2012.Nature Biotechnology 30:215-217). In maize, SPT technology is applied to three key genes: dominant male fertility gene Ms45, recessive male sterile gene ms45 and red fluorescent (visible) marker gene DsRed2. Pione...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N5/10A01H5/00
CPCC07K14/415C12N15/8218C12N15/8289Y02A40/146C12N15/113C12N15/8205C12N15/8231
Inventor 付道林倪飞齐娟吕波罗明成王树芸
Owner 泉脉农业科技有限公司
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