CN110819646A - DcLCYB1 gene sequence related to synthesis of carrot carotenoid and application thereof - Google Patents

Abstract

Lycopene β-cyclase (LCYB) is a key enzyme in the carotenoid synthesis pathway. The invention clones a DcLCYB1 gene from the carrot variety 'Shanxi Yellow Carrot', the gene contains an open reading frame (ORF) of 1527bp, encodes 508 amino acids, and belongs to a hydrophilic protein. Secondary structure prediction revealed that carrot DcLCYB1 protein was composed of 38.98% α-helix, 4.13% β-sheet, 16.14% extended backbone and 40.75% random coil. Fluorescence quantitative results showed that the expression level of DcLCYB1 gene was higher in the leaves of yellow and purple varieties of carrots, while only a small amount of expression in petioles of red carrot varieties. The invention utilizes RT-PCR technology to clone the DcLCYB1 gene from carrots, and the gene can regulate the accumulation of carotenoids in carrots, thereby affecting the nutritional quality of carrots.

Description

A DcLCYB1 gene sequence related to carotenoid synthesis in carrots and its application

technical field

The invention belongs to the field of plant genetic engineering, and relates to a carotenoid synthesis-related gene of carrot and its application. Specifically, a carotenoid synthesis-related gene DcLCYB1 was cloned from the carrot variety 'Shanxi Yellow Carrot', which can be used for the research and application of carotenoid synthesis.

Background technique

Carrot (Daucus carota L.) is a biennial herb belonging to the family Apiaceae and is an important root vegetable crop (Liu Lifeng, Shanghai Vegetables, 2006, 2: 4-6). Carrots originated in southwestern Asia and have a cultivation history of more than 2,000 years. my country's carrot cultivation area is extensive, accounting for 37% of the world's total, making it the world's largest carrot producer (Food and Agriculture Organization of the United Nations, 2016). Carrot fleshy roots contain a variety of nutrients, such as carotenoids, vitamins, minerals and special pectin (Ruan Wanzhen, Chinese Food and Nutrition, 2007, 6: 51-53). Carotenoids are an important class of natural terpenoid pigments. As the precursor of vitamin A, carotenoids play a crucial role in the process of plant growth and development, and also have anti-oxidation, anti-cancer, anti-aging and other effects (Zhu Yunqin et al., Molecular Plant Breeding, 2016, 2: 471 -474).

Lycopene β-cyclase (LCYB) is an important regulatory enzyme in carotenoid synthesis pathway. Under the catalysis of lycopene β-cyclase (LCYB), both ends of lycopene are cyclized to generate α-carotene and β-carotene (Zhu Changfu et al., Chinese Journal of Plant Physiology and Molecular Biology, 2004, 30 (6): 609-618). By regulating the expression of LCYB gene, the types and contents of carotenoids in crops can be changed, and then the quality and commercial value of crops can be improved. The LCYB gene of lycopene β-cyclase was first discovered in Arabidopsis thaliana, and is currently found in tomato (Solanum lycopersicum L.), pepper (Capsicum annum L.), Cerasus humilis (Bge) Sok., calendula (Calendulaofficinalis L.) and other plants have cloned the lycopene β-cyclase LCYB gene (Zhang Jiancheng et al., Chinese Journal of Horticulture, 2017, 44(11): 2075-2088). very lacking.

SUMMARY OF THE INVENTION

The invention provides a preparation method and application of a carotenoid synthesis-related gene DcLCYB1 in carrots. The obtained DcLCYB1 gene is helpful to further understand the mechanism of carotenoid synthesis in carrots, and can also be used to improve the nutritional quality of carrots.

Description of drawings

Figure 1. Conserved domain prediction results of carrot DcLCYB1 protein

Figure 2. Evolutionary analysis of LCYB proteins in carrot and other species

Figure 3. Hydrophobicity (A) and hydrophilicity (B) analysis of the amino acid sequence of carrot DcLCYB1 protein

Figure 4. Secondary structure prediction of carrot DcLCYB1 protein

Figure 5. Expression analysis of carrot DcLCYB1 gene in carrots with different colors

Detailed ways

1. Extraction of carrot total RNA and synthesis of cDNA: carrot materials are yellow carrot variety 'Shanxi Yellow Carrot', red carrot variety 'Shaanxi Red Carrot' and purple carrot variety 'Yunnan Purple Carrot', the above carrot varieties were obtained from the umbrella of Nanjing Agricultural University. The plant is preserved in the Laboratory of Crop Genetics and Germplasm Innovation of Vegetable Crops, and the plants are planted in the Artificial Climate Room of the State Key Laboratory of Crop Genetics and Germplasm Innovation of Nanjing Agricultural University. The total RNA of carrot samples was extracted using the plant total RNA extraction kit RNA simple Total RNAKit (Beijing Tiangen Biochemical Technology Co., Ltd.). The RNA samples were reverse transcribed into cDNA according to the instructions of the reverse transcription kit HiScript IIQ RT SuperMix for qPCR (+gDNA wiper) (Nanjing Nova Biotechnology Co., Ltd.).

2. Cloning of carrot DcLCYB1 gene: The carrot DcLCYB1 gene was retrieved according to the carrot transcriptome database of our research group (Xu et al., Database, 2014, bau096), and a pair of cloning primers were designed according to the full-length sequence of the gene: forward primer sequence is 5'-ATGAAAGTGATGGATACTCTACT-3'; the reverse primer sequence is 5'-CTATTCTCTATCTTTGATCAGAT-3'. Using the obtained 'Shanxi yellow carrot' cDNA as a template, PCR amplification was carried out in a 20 μL system. The amplification program was: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 54 °C annealing for 30 s, and 72 °C extension for 90 s, a total of 35 cycles ; Final extension at 72°C for 10min. The PCR amplification products were separated by 1.2% agarose gel electrophoresis, recovered and connected to pMD19-T vector (Dalian TaKaRa Biotechnology Co., Ltd.), transformed into E. coli DH5α, and sequenced by Nanjing GenScript Biotechnology Co., Ltd.

3. Sequence analysis: Complete nucleotide and amino acid sequence searches and conserved domain predictions on the NCBI related website (http://blast.ncbi.nlm.nih.gov); LCYB protein multiple sequence alignment among different species and carrot LCYB 1 protein hydrophobicity/hydrophilicity analysis was completed by DNAMAN 6.0; phylogenetic tree was constructed by MEGA 5.0 software (Tumara et al., Mol Biol Evol, 2011, 28(10): 2731-2739); protein secondary structure was predicted with the help of SOPMA website (http://pbil.ibcp.fr/) completed.

4. Real-time fluorescence quantitative PCR reaction: use Primer Premier 6.0 software (Singh et al., Biotechniques, 1998, 24(2): 318-319) to design DcLCYB1 gene expression detection primers, the forward primer sequence is 5'-TTGCTAGATTGCCTTGACACTAC-3 '; the reverse primer sequence is 5'-GATGTTCTCTACTTCTGCCACTAT-3'. ChamQ SYBR qPCR Master Mix (Nanjing Nuoweizan Biotechnology Co., Ltd.) was used to detect the expression of DcLCYB1 gene in the leaves, petioles and roots of carrots with three different colors. Company) and supporting software, the amplification procedure is as follows: 95°C for 5 min; 95°C for 10s, 60°C for 10s, 72°C for 30s, a total of 40 cycles; then the melting curve is obtained by stepwise amplification from 60°C to 95°C. The carrot DcActin gene was selected as the internal reference gene (Wang et al. J Hortic Sci Biotechnol, 2016, 91(3): 264-270), and the relative quantification method was used to calculate the expression of DcLCYB1 gene in different tissues of different varieties of carrot (Pfaffl, Nucleic Acid Res, 2001, 29(9):e45).

5. Test results: 1). The sequence determination results show that the DcLCYB1 gene contains an open reading frame (ORF) of 1527 bp, encoding 509 amino acids; the conservative domain prediction shows that the DcLCYB1 protein contains a highly conserved structural domain (Figure 1). 2). Sequence alignment of the amino acid sequence encoded by the DcLCYB1 gene with LCYB proteins of other species to construct an evolutionary tree. The results showed that the DcLCYB1 protein of carrot was closely related to Lycium barbarum, tomato and pepper of Solanaceae (Fig. 2). 3). The hydrophobicity/hydrophilicity analysis of DcLCYB1 protein found that the number of hydrophilic amino acids in DcLCYB1 protein was more than that of hydrophobic amino acids, which belonged to hydrophilic protein (Fig. 3). 4). The secondary structure prediction results of DcLCYB1 protein showed that the main components of DcLCYB1 protein included 38.98% α-helix, 4.13% β-sheet, 16.14% extended backbone and 40.75% random coil (Fig. 4). . 5). The results of fluorescence quantitative expression analysis showed that the expression level of DcLCYB1 gene was higher in leaves of yellow variety 'Shanxi Huangruo' and purple variety 'Yunnan Ziruo', but lower in petiole and root; In Shaanxi Red Carrot', the DcLCYB1 gene was only slightly expressed in petioles, and hardly expressed in leaves and roots.

Claims (5)

1. A carotenoid synthesis related gene DcLCYB1 obtained from carrot variety 'Shanxi yellow carrot'.

2. The nucleotide sequence of the carotenoid synthesis-associated gene DcLCYB1 according to claim 1.

3. A process for producing the carotenoid synthesis-associated gene DcLCYB1 derived from carrot of claim 1, which comprises the steps of:

1) design of cloning primer, forward primer: 5'-ATGAAAGTGATGGATACTCTACT-3', reverse primer: 5'-CTATTCTCTATCTTTGATCAGAT-3', respectively;

2) the DcLCYB1 gene is cloned from the Shanxi yellow carrot by RT-PCR method.

4. Functional studies of the carotenoid synthesis-associated DcLCYB1 gene of claim 1: the transcription level of carrot carotenoid synthesis related gene DcLCYB1 in different tissues of carrot varieties with different colors is researched by adopting a real-time fluorescent quantitative PCR method.

5. The use of the DcLCYB1 gene associated with carotenoid synthesis according to claim 1.

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