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
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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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