Auxin Signaling Transcription Regulatory Protein aux/iaa2, Its Encoding Gene and Probe
A coding and protein technology, applied in DNA/RNA fragments, genetic engineering, plant genetic improvement, etc., can solve problems such as uncoded gene sequences
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Embodiment 1
[0040] Embodiment 1, the cloning of Agapanthus agapanthus Aux / IAA2 gene
[0041] 1. Acquisition of plant material
[0042] Take the leaf tissue of Agapanthus adult seedlings for RNA extraction;
[0043] 2. Extraction of RNA
[0044] Total RNA (Trizol: Invitrogen) was extracted with "RNA prep pure Plant Total RNA Extraction Kit", and the integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration;
[0045] 3. Full-length cloning of genes
[0046] According to the protein function annotation results of Agapanthus japonica transcriptome sequencing (RNA-seq), the core fragment of Agapanthus japonica Aux / IAA2 gene was obtained. Using the RACE method (SMARTer TM RACE cDNA Amplification Kit: Clonetech) for full-length cDNA cloning in three stages:
[0047] (1) RT-PCR to obtain the middle fragment of the gene
[0048] The extracted RNA was reverse-transcribed (Prime Script II 1st Strand cDNA Synthesis Kit: Treasure Bi...
Embodiment 2
[0059] Example 2, Sequence Information and Homology Analysis of Agapanthus Agapanthus Aux / IAA2 Gene
[0060] The full-length open reading frame sequence of the new Agapanthus agapanthus Aux / IAA2 gene of the present invention is 480 bp, and the detailed sequence is shown in the sequence shown in SEQ ID NO.3. According to the sequence of the open reading frame, the amino acid sequence of Agapanthia Auxiliae Aux / IAA2 protein is deduced, with a total of 159 amino acid residues, a molecular weight of 18.07kDa, and an isoelectric point (pI) of 6.15. For the detailed sequence, see the sequence shown in SEQ ID NO.4 ;
[0061] The open reading frame sequence of agapanthus Aux / IAA2 and the amino acid sequence of its encoded protein were stored in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology searches were carried out, and it was found that it has 96% identity at ...
Embodiment 3
[0062] Embodiment 3, agapanthus Aux / IAA2 gene transformation model plant Arabidopsis thaliana
[0063] 1. Construction of the expression vector containing the gene of interest (Agapanthus agapanthus Aux / IAA2 gene)
[0064] According to the full-length coding sequence of Agapanthus Aux / IAA2 gene (SEQ ID NO.3), design amplifying primers in the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be selected depending on the vector) in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the sequence of the coding region of the Agapanthus Agapanthus Aux / IAA2 gene was connected to an intermediate vector (such as pMD19-T) for sequencing, and then the sequenced correct Agapanthus The coding region sequence of lotus Aux / IAA2 gene was further cloned into an expression vector (such as pHB), and it was transferred into Agrob...
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