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Phalaenopsis miR172 coding sequence and application thereof

A phalaenopsis, sequence technology, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2011-08-31
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few reports on higher ornamental plants with economic value

Method used

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  • Phalaenopsis miR172 coding sequence and application thereof
  • Phalaenopsis miR172 coding sequence and application thereof
  • Phalaenopsis miR172 coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Phalaenopsis miRNA172 clone

[0031] 1. According to known miRNA172 Conserved region of gene sequence, designer Phalaenopsis miRNA172 Primers such as SEQ ID NO.2 and SEQ ID NO.3. Specific PCR amplification of the Phalaenopsis genome extracted from the tissue;

[0032] 2. The product was separated by 13V gel electrophoresis at room temperature, and a band with a size of about 250bp was detected;

[0033] 3. The bands were recovered and purified, amplified again with the same primer and then sequenced. The result is shown in SEQ ID NO.1.

Embodiment 2

[0034] Embodiment 2 Phalaenopsis miRNA172 Gene sequence information and homology analysis

[0035] 1. The sequence obtained in Example 1 and the known sequences of Arabidopsis and rice miR172 Homology analysis and comparison of conserved regions were carried out on the gene, and it was found that it was related to two genes from Arabidopsis thaliana miR172 Family sequences clustered together ( figure 1 A) and has the above miR172 The consensus length of about 20bp homologous conserved region sequence ( figure 1 B), initially determined as miRNA172 Gene;

[0036] 2. Using the obtained sequence to predict the miRNA secondary structure with RNA-fold software, the results are as follows figure 2 , the RNA can form a typical neck loop structure.

Embodiment 3

[0037] Example 3 Phalaenopsis in different tissues PhmiR172 and target genes TOE1 The expression of (mRNA semi-quantitative PCR)

[0038] 1. According to miR172 homologous target gene TOE1 (Sequence such as SEQ ID NO.6) Design primers (SEQ ID NO.4 and SEQ ID NO.5);

[0039] 2. Using the above primers, and PhmiR172 Primers SEQ ID NO.2 and SEQ ID NO.3 respectively amplified in the cDNA of each tissue of Phalaenopsis PhmiR172 and target genes PhTOE1-LIKE ;

[0040] The result is as image 3 shown. In some organizations, PhmiR172 There is a complementary effect between the expression of the target gene and the expression of the target gene. as in roots and stems PhmiR172 transcripts of TOE1 expression. Not detected in leaves and sepals PhmiR172 , the expression of the target gene was detected. also PhmiR172 High expression in pedicels and buds, indicating that it may be involved in the regulation of flowering.

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Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, in particular to a Phalaenopsis miRNA172 sequence and application thereof. A miR172 gene coding sequence cloned from the Phalaenopsis genome, gene expression mode analysis, construction of a transgenic expression vector in Arabidopsis, a primer sequence, a transgenic pure plant and a corresponding phenotype are included. The expression of Phalaenopsis miRNA172 in Arabidopsis can promote apical meristem to be active for presenting premature flowering.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and specifically relates to Phalaenopsis miR172 and its applications, including Phalaenopsis miR172 Phalaenopsis transgene expression vector, primer sequence, transgenic pure line plant, and the functional application of the gene verified by the pure line plant. Background technique [0002] Phalaenopsis( Phalaenopsis amabilis ) belongs to the angiosperm monocotyledon Phalaenopsis Phalaenopsis. It is an aerial orchid with aerial roots that can absorb nutrients in the air to survive. It mostly grows in tropical and humid regions of Asia, adapting to high temperature, high humidity and ventilated environment. The single stem is short, the leaves are fleshy and fleshy, and the flowers are shaped like butterflies with wings. The colors are bright and colorful. It is an important economic flower and has become more and more popular among consumers in recent years...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/82C12Q1/68A01H5/00
Inventor 明凤严钦骅韩颖颖
Owner FUDAN UNIV
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