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Cymbidium sinense chlorophyll catabolism regulatory protein as well as coding gene and application thereof

A technology for chlorophyll degradation and metabolism regulation, which is applied in orchid chlorophyll degradation metabolism regulation protein and its coding gene and application field, which can solve the problems of long cycle and unpredictable traits, and achieve the effect of chlorophyll reduction and trait improvement

Active Publication Date: 2017-12-19
ENVIRONMENTAL HORTICULTURE RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the directional improvement of leaf art traits is particularly important, while traditional hybrid breeding generally has a long cycle and unpredictable traits.

Method used

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  • Cymbidium sinense chlorophyll catabolism regulatory protein as well as coding gene and application thereof
  • Cymbidium sinense chlorophyll catabolism regulatory protein as well as coding gene and application thereof
  • Cymbidium sinense chlorophyll catabolism regulatory protein as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Cloning and sequence analysis of CsCLH2 gene

[0020] 1. Extraction of RNA

[0021] Take 2 g of the young leaf tissue of Molan cultivar Dharma, extract its total RNA with plant Trizol (Invitrogen) reagent, and reverse transcribe it into cDNA (Thermo Scientific RevertAid First Strand cDNASynthesis Kit).

[0022] 2. Acquisition of the target gene CsCLH2

[0023] Using CsCLH2-F1 (SEQ ID NO: 3) and CsCLH2-R1 (SEQ ID NO: 4) as primers and the cDNA obtained in the above step as a template, PCR was performed using Ex-Taq enzyme (TaKaRa Biotechnology Co.) according to the following conditions: 94°C for 4min, then 34cycles (94°C for 40s, 59.5°C for 40s, 72°C for 1.5min, 72°C for 10min). The PCR product was recovered from the agarose gel, then connected to the pMD19-T vector (TaKaRa Biotechnology Co.) and sent to Huada Gene Research Institute for sequencing. The analysis of the sequencing results found that the amplified fragment contained the complete CDs sequence o...

Embodiment 2

[0026] Example 2 Prokaryotic expression of CsCLH2 protein

[0027] 1. Protein prokaryotic expression vector construction

[0028] Using CsCLH2-F2 (SEQ ID NO: 5) and CsCLH2-R2 (SEQ ID NO: 6) as primers, using the cDNA of Molan leaves as a template, use Ex-Taq enzyme (TaKaRa Biotechnology Co.) to perform PCR according to the following conditions : 94°C for 4min, then 34cycles (94°C for 40s, 59.5°C for 40s, 72°C for 1.5min, 72°C for 10min). The PCR product (CsCLH2 gene) was recovered from the agarose gel, then connected to the pGEX6P-1 vector (TaKaRa Biotechnology Co.) and sequenced to verify its accuracy, confirming that the CsCLH2 gene was inserted into the pGEX6P-1 vector, thus obtaining pGEX6P- 1-CsCLH2 expression vector. The Escherichia coli containing pGEX6P-1-CsCLH2 is currently preserved in the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences.

[0029] 2. Protein induction expression and enzyme activity analysis

[0030] 1) Inoculate...

Embodiment 3

[0036] Example 3 Expression pattern of CsCLH2 in orchids

[0037] 1. Extraction of RNA

[0038] Take 2 g of different tissues of the young leaves, old leaves, roots, pseudobulbs, and flowers of Molan cultivar Dharma, use the plant Trizol (Invitrogen) reagent to extract its total RNA, and take 2 μl of it using the reverse transcription kit Thermo Scientific RevertAid First Strand cDNASynthesis Kit reverse transcription into cDNA.

[0039] 2. Quantitative PCR

[0040] Using primers CsCLH2QRT-F (SEQ ID NO: 7) and CsCLH2QRT-R (SEQ ID NO: 8), the expression of CsCLH2 gene in different tissues of orchids was detected by real-time quantitative PCR. Actin QRT-F (SEQ ID NO:9) and ActinQRT-R (SEQ ID NO:10) were used as primers to amplify Actin as an internal reference. The following procedure was followed: pre-denaturation at 95°C for 30 s, followed by 40 cycles (10 s at 95°C, 10 s at 59.5°C, 30 s at 72°C), and extension at 72°C for 10 min. The iCycler IQ Real-timePCR Detection Syst...

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Abstract

The invention discloses cymbidium sinense chlorophyll catabolism regulatory protein as well as a coding gene and application thereof. The amino acid sequence of the cymbidium sinense chlorophyll catabolism regulatory protein disclosed by the invention comprises 307 amino acid residues and is shown in SEQ ID NO:2; the nucleotide sequence of a cymbidium sinense chlorophyll catabolism regulatory gene for coding the cymbidium sinense chlorophyll catabolism regulatory protein comprises 924 basic groups and is shown in SEQ ID NO:1. The cymbidium sinense chlorophyll catabolism regulatory protein disclosed by the invention has the activity of chlorophyll degrading enzyme; after the expression quantity of the cymbidium sinense chlorophyll catabolism regulatory protein is increased, the content of chlorophyll in arabidopsis thaliana leaf blades can be decreased; after the cymbidium sinense chlorophyll catabolism regulatory protein is highly expressed in cymbidium sinense, the contents of chlorophyll in leaf blades and flowering branches of cymbidium sinense are obviously decreased. The cymbidium sinense chlorophyll catabolism regulatory gene and the cymbidium sinense chlorophyll catabolism regulatory protein coded by the same can be used for research on a chlorophyll degradation molecular mechanism and character improvement of leaf color or flower color of plants.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to an orchid chlorophyll degradation metabolism regulation protein and its coding gene and application. Background technique [0002] The color of plant leaves depends on the content of chlorophyll in them. Therefore, the regulation of chlorophyll synthesis metabolism not only affects the efficiency of plants absorbing light energy for photosynthesis, but also directly determines the traits of leaf color variation, which is an important value embodiment of ornamental plants. Coleus plants with colorful leaves caused by chlorotic leaves have long attracted the attention of breeders. [0003] The study found that in the process of leaf chlorosis, chlorophyll is degraded into colorless metabolites need four key enzymes. First, chlorophyll is catalyzed by chlorophyllase (Chlorophyllase, CLH) to remove phytol groups to form phytochlorophyll (Chlide), and then, a metal ...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/82A01H5/00
CPCC12N9/18C12N15/825C12Y301/01014
Inventor 杨凤玺朱根发许庆全梁迪魏永路
Owner ENVIRONMENTAL HORTICULTURE RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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