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Screening and function verification of carotenoid cleavage dioxygenase encoding gene participating in synthesis of gardenia jasminoides ellis crocin

A carotene and dioxygenase technology, which is applied in the field of plant genetic engineering and can solve the problems of unknown genes that perform functions.

Inactive Publication Date: 2017-12-22
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, the research on the biosynthetic pathway of crocin has received extensive attention. Although the key enzymes involved in the biosynthetic pathway of crocin are basically clear, the genes of these key enzymes are multi-gene families, and the genes that perform specific functions are unknown.

Method used

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  • Screening and function verification of carotenoid cleavage dioxygenase encoding gene participating in synthesis of gardenia jasminoides ellis crocin
  • Screening and function verification of carotenoid cleavage dioxygenase encoding gene participating in synthesis of gardenia jasminoides ellis crocin
  • Screening and function verification of carotenoid cleavage dioxygenase encoding gene participating in synthesis of gardenia jasminoides ellis crocin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Detection of crocin content in gardenia green fruit, red fruit and leaves

[0024] 1.1 Experimental method

[0025] Leaves, green fruit and red fruit samples were vacuum freeze-dried, pulverized and sieved with a grinder, and 0.25 g of each sample was accurately weighed in a Erlenmeyer flask, and 25 mL of 50% chromatographic grade methanol was added for ultrasonic extraction for 45 minutes. After extraction, a small amount of 50% methanol was carefully added to compensate for the weight loss. The extract was filtered and passed through a 0.22 μm filter membrane to obtain the test sample. Standard product preparation: Accurately weigh the standard products crocin I and crocin II to make a mixed standard, and the respective concentrations are 0.035 mg / ml crocin I and 0.015 mg / ml crocin II. Chromatographic conditions: Waters BEH-C18 chromatographic column (100mm×2.1mm, 1.7μm), column temperature 35°C, gradient elution with acetonitrile (A) and water (B) as mobi...

Embodiment 2

[0028] Example 2 Transcriptome sequencing, assembly and annotation of gardenia green fruits, red fruits and leaves

[0029] 2.1 Experimental method

[0030] 2.1.1 Gardenia RNA extraction, cDNA library construction and computer

[0031] Gardenia plants were planted in the Botanical Garden of the Chongqing Branch of the Institute of Medicinal Botany, Chinese Academy of Medical Sciences. The leaves and fruits were collected in October, washed immediately after collection, and quickly frozen with liquid nitrogen and stored in a -80°C refrigerator. The QIAGEN RNeasyPlus Mini Kit (74134) was used for extraction, and the specific operation was referred to its instruction manual. The integrity of the extracted total RNA was detected by gel electrophoresis, and the RNA concentration was detected by a NanoDrop micro-volume fluorescence spectrophotometer. The total amount should be greater than 3.5 micrograms. Detected by Agilent2100 Bioanalyzer, the RIN (RNA integrity number) value sh...

Embodiment 3

[0044] Example 3 Identification and differential expression analysis of key enzyme genes in the crocin synthesis pathway

[0045] 3.1 Experimental method

[0046]Using the amino acid sequences of key enzymes in the crocin pathway of other species downloaded by NCBI as QUERY, perform BLASTP comparison in the transcriptome database with a threshold of 1E-10 to obtain the protein sequences of key enzymes, and then use the Pfam annotation results to confirm the protein domains . The obtained results were again compared with BLASTP in the NCBI database to verify the correctness of the sequences. Use RSEM software to estimate the expression levels of all genes in the transcriptome, and use DESeq Bioconductor package (1.14.0) software to analyze differentially expressed genes among different organs. The genes whose parameters meet log2fold changes>=1 (FDR value<0.001) belong to differential expression Gene.

[0047] 3.2 Results and analysis

[0048] According to gene expression t...

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Abstract

The present invention relates to a carotenoid cleavage dioxygenase CCD4a involved in the synthesis of gardenia jasminoides ellis crocin, an encoding gene and functions thereof, belongs to the technical field of gene engineering, and discloses the open reading frame sequence of a carotenoid cleavage dioxygenase CCD4a gene and the amino acid sequence encoded by the carotenoid cleavage dioxygenase CCD4a gene. According to the present invention, the CCD4a gene is screened based on transcriptomics, the functions of CCD4a are verified in vitro by using zeaxanthin as the substrate and constructing the pET28a-CCD4a prokaryotic expression vector, and the results prove that CCD4a has the functions of carotenoid cleavage dioxygenase so as to establish the foundation for the analysis and biosynthetic research of the crocin source pathway.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for screening and function verification of crocin biosynthesis genes. Background technique [0002] Crocin is a carotene compound, mainly accumulated in saffron stigma and gardenia fruit. Crocin has certain curative effects in inhibiting cancer, improving memory, anti-depression, and treating cardiovascular diseases. In view of the medicinal and economic value of crocin, the plant saffron to which it belongs is often given a lot of halo, known as the "red gold" in medicinal materials, and the retail price is as high as 2,000 to 7,000 ￡ / kg. [0003] Gardenia jasminoides Ellis, a traditional Chinese medicine in my country, is also rich in crocin. According to reports, the content of crocin I and crocin II in the pulp is as high as 27.54 mg g-1. At the same time, the chemical and pharmacological studies of Gardenia jasminoides are more in-dep...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12Q1/68
CPCC12N9/0069C12Q1/6869C12Y113/11071C12Q2535/122
Inventor 宋经元季爱加贾静徐志超
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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