A phalaenopsis cyclophilin cyp gene and its application

A cyclophilin and Phalaenopsis technology, applied in the field of Phalaenopsis genetic engineering, can solve the problem that the stability detection of Phalaenopsis cyclophilin has not been reported.

Active Publication Date: 2021-07-09
ZHENGZHOU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] As far as the Phalaenopsis genome research is concerned, the actin gene ( ACTIN ) as an internal reference gene, while the Phalaenopsis cyclophilin CYP Gene cloning and stability testing of expression have not been reported yet

Method used

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  • A phalaenopsis cyclophilin cyp gene and its application
  • A phalaenopsis cyclophilin cyp gene and its application
  • A phalaenopsis cyclophilin cyp gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This embodiment is just Phalaenopsis CYP The cloning process of gene cDNA sequence is briefly introduced as follows. Phalaenopsis obtained from specific PCR clones CYP In the process of gene cDNA full-length sequence, first cloned separately CYP The conserved fragment, 3' end sequence and 5' end sequence in the cDNA sequence of the gene were further spliced ​​and verified. The relevant experimental process is briefly introduced as follows.

[0050] (1) Extract total RNA and reverse transcribe it into cDNA;

[0051] The leaves of the Phalaenopsis cultivar "Treasure Red Rose" were used as experimental materials to extract total RNA. The specific operation steps are as follows:

[0052] Take 1.0 g of leaves at the full flowering stage, grind them fully in liquid nitrogen, put the powder into a 1.5 mL centrifuge tube, add 1 mL of Trizol reagent from Invitrogen Company, shake and mix well, and place at room temperature for 5 to 10 minutes;

[0053] Centrifuge at 4°C and ...

Embodiment 2

[0104] On the basis of embodiment 1, the present embodiment is mainly about Phalaenopsis CYP The stability of gene transcription and expression in Phalaenopsis was studied and analyzed, and the relevant experiments are briefly introduced as follows.

[0105] (1) Total RNA extraction and cDNA preparation

[0106] Select the roots and leaves of Phalaenopsis seedlings, roots, leaves and pedicels in the early flowering stage, roots, leaves, pedicels, sepals, petals, lips, stamens, rudimentary ovary, and developing ovary after pollination 15 kinds of tissues such as seeds and fruits at the mature stage were used as experimental materials. Referring to the relevant operations in Example 1, total RNA was extracted respectively and transcribed into single-stranded cDNA. The cDNA was diluted 6 to 10 times and used directly, or stored at -20°C spare.

[0107] (2) Design primers

[0108] Phalaenopsis obtained according to embodiment 1 CYP Gene sequence, design a pair of specific prim...

Embodiment 3

[0142] Based on the results of Example 2, it can be judged that Phalaenopsis CYP The gene has the potential to be used as an internal reference gene. For this reason, inventor uses Phalaenopsis CYP gene as an internal reference gene, with MADS-box Gene DtpsMADS1 (NCBI accession number: JQ065097) as an example, the gene is used in different tissues of Phalaenopsis in full flowering stage (tissue materials include: leaves, roots, pedicels, sepals, petals, lips, stamens, rudimentary ovaries, etc. 11 materials) A preliminary study of the expression characteristics in

[0143] Fluorescent quantitative PCR detection DtpsMADS1 For genes, the primer sequences were designed as follows:

[0144] qMADS1-F: TGTCGCTCTCATCATTTTTTCG-3',

[0145] qMADS1-R: 5'-GCTTTCAACCTTTGCCTTCA-3';

[0146] The 25 μL reaction system was designed as follows:

[0147] 2X SYBR buffer, 12.5μL;

[0148] 10 μM forward primer (qMADS1-F), 1 μL;

[0149] 10 μM reverse primer (qMADS1-R), 1 μL;

[0150]c...

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Abstract

The invention belongs to the technical field of Phalaenopsis genetic engineering, in particular to a Phalaenopsis cyclophilin CYP Gene and its application patent application matters. The cDNA sequence length of this gene is 901bp, and the 277th~728th bases are its conservative sequences. Phalaenopsis cyclophilin CYP The gene encodes the cyclophilin CYP, including 173 amino acids. This application is the first to study the cyclophilin gene in Phalaenopsis CYP After cloning and analysis, using real-time fluorescent quantitative PCR technology, preliminary research shows that the gene can be stably expressed in different stages of Phalaenopsis growth and development and in different tissues. This result shows that this gene is suitable for the related genes in Phalaenopsis It was used as an internal reference gene for expression analysis. Generally speaking, this application enriches the types of internal reference genes used in the research on gene expression of Phalaenopsis, lays a good foundation for accurate analysis of gene expression detection, and thus has good application value.

Description

technical field [0001] The invention belongs to the technical field of Phalaenopsis genetic engineering, in particular to a Phalaenopsis cyclophilin CYP Gene and its application patent application matters. Background technique [0002] Phalaenopsis( Phalaenopsis spp. ) is an orchid plant with beautiful flower shape and bright color, and the flowering period is as long as 3-5 months. Therefore, it is known as the "Queen of Orchids" and has high ornamental value. It is also the first flagship product of Chinese New Year flowers in many areas In recent years, it has played an increasingly important role in the flower industry. [0003] With the widespread application of biotechnology in flower breeding and the research on the molecular regulation mechanism of Phalaenopsis physiological metabolism, the cloning and expression characteristics of various functional genes of Phalaenopsis have gradually become a research hotspot. Meanwhile, orchids are the most evolved group of m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/11C12Q1/6895
CPCC07K14/415C12Q1/6895C12Q2600/166
Inventor 许申平袁秀云岳媛张燕梁芳
Owner ZHENGZHOU NORMAL UNIV
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