阅读说明：本技术 一种与胡萝卜类胡萝卜素合成相关的DcLCYB1基因序列及其应用 (DcLCYB1 gene sequence related to synthesis of carrot carotenoid and application thereof ) 是由 熊爱生 王雅慧 徐志胜 于 2018-08-09 设计创作，主要内容包括：番茄红素β-环化酶(Lycopeneβ-cyclase,LCYB)是类胡萝卜素合成途径中的一个关键酶。本发明从胡萝卜品种‘山西黄胡萝卜’中克隆得到一个DcLCYB1基因,该基因含有一个1527bp的开放阅读框(ORF),编码508个氨基酸,属于亲水性蛋白。二级结构预测显示,胡萝卜DcLCYB1蛋白由38.98％的α-螺旋、4.13％的β-折叠、16.14％的延伸主链和40.75％的随机卷曲组成。荧光定量结果显示,DcLCYB1基因在黄色和紫色品种胡萝卜的叶片中表达水平较高；而红色胡萝卜品种中仅在叶柄中少量表达。本发明利用RT-PCR技术从胡萝卜中克隆得到DcLCYB1基因,该基因能够调控胡萝卜中类胡萝卜素的积累,从而影响胡萝卜的营养品质。(The invention clones a DcLCYB1 gene from a carrot variety 'Shanxi yellow carrot', the gene comprises an Open Reading Frame (ORF) of 1527bp, codes 508 amino acids and belongs to hydrophilic protein.)

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.

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a carotenoid synthesis related gene of carrots and application thereof. Specifically, a carotenoid synthesis related gene DcLCYB1 is obtained by cloning a carrot variety 'Shanxi yellow carrot', and the gene can be used for the synthesis research and application of the carrot carotenoid.

Background

Carrot (Daucus carota L.) belongs to the family Umbelliferae (Apiaceae) and genus Daucus, and is an important root vegetable crop (Liu Li Feng, Shanghai vegetable, 2006, 2: 4-6). Carrots originate in the southwest asia and have a cultivation history of 2000 years. The carrot cultivation area in China is wide, accounts for 37 percent of the total area of the world, and is the first carrot producing country in the world (food and agricultural organization of the United nations, 2016). The fleshy root of carrot contains many kinds of nutrients such as carotenoid, vitamins, minerals and special pectin etc. (ruanwanzhen, chinese food and nutrition, 2007, 6: 51-53). Carotenoids are an important class of natural terpenoid pigments. As a precursor of vitamin A, carotenoid plays a crucial role in the growth and development process of plants, and has the effects of resisting oxidation, resisting cancer, delaying senescence and the like (Zhu Yunzhi et al, molecular plant breeding, 2016, 2: 471-.

Under the catalytic action of Lycopene β -cyclase (LCYB), the two ends of Lycopene are cyclized to generate α -carotene and β -carotene (Zhuchanfu, et al, Proc. phytophysiology and molecular biology, 2004, 30 (6): 609. 618), the types and contents of carotenoids in crops can be changed by regulating the expression of LCYB gene, and then the quality and commodity value of crops are improved, the Lycopene 2-cyclase LCYB gene is originally found in Arabidopsis thaliana, at present, plants such as tomato (Solanum lycopersicum mL.), pepper (Capsicum annum L.,), European plum (Cerasius (Bge) Sok.), calendula officinalis (Calendaflor L., 2015.), plant (Calendaflor L., et al) are cloned to obtain the gene (YB, 2088), and the like, but the variety of Lycopene is still found in the national cultivars, 20811. although the variety is still lack of YB, 2088. the genes of Lycopene is researched.

Disclosure of Invention

The invention provides a preparation method and application of a gene DcLCYB1 related to synthesis of carotinoid. The obtained DcLCYB1 gene is beneficial to further understanding the synthesis mechanism of the carotenoid of the carrot, and can be used for improving the nutritional quality of the carrot.

Drawings

FIG. 1 conserved domain prediction of carrot DcLCYB1 protein

FIG. 2 evolution analysis of carrot and other species LCYB proteins

FIG. 3 analysis of amino acid sequence of carrot DcLCYB1 protein for hydrophobicity (A) and hydrophilicity (B)

FIG. 4 Secondary Structure prediction of carrot DcLCYB1 protein

FIG. 5 expression analysis of carrot DcLCYB1 gene in carrots of different colors

Detailed Description

1. Extraction of carrot total RNA and cDNA synthesis: the carrot material is yellow carrot variety 'Shanxi yellow carrot', red carrot variety 'Shaanxi red carrot' and purple carrot variety 'Yunnan purple carrot', the carrot variety is stored in an Umbelliferae vegetable crop inheritance and germplasm innovation laboratory of Nanjing agriculture university, and the plant is planted in a national key laboratory phytotron of Nanjing agriculture university crop inheritance and germplasm innovation. Total RNA of carrot samples was extracted using a 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 wrapper) (Biotech Co., Ltd. Nuo Zan, Nanjing).

2. Cloning of carrot DcLCYB1 Gene, carrot DcLCYB1 gene is obtained by searching according to the carrot transcriptome Database (Xu et al, Database, 2014, bau096) of the subject group, a pair of cloning primers is designed according to the full-length sequence of the gene, wherein the forward primer sequence is 5'-ATGAAAGTGATGGATACTCTACT-3', the reverse primer sequence is 5'-CTATTCTCTATCTTTGATCAGAT-3', the obtained 'Shanxi yellow carrot' cDNA is taken as a template, a 20 mu L system is adopted for PCR amplification, the amplification procedure is that 94 ℃ is used for pre-denaturation for 5min, 94 ℃ is used for denaturation for 30s, 54 ℃ is used for annealing for 30s, 72 ℃ is used for extension for 90s, the total cycle is 35, finally 72 ℃ is used for extension for 10min, 1.2% agarose gel electrophoresis is used for separating PCR amplification products, the PCR amplification products are recovered and then connected to a pMD19-T vector (Kyoto TaRa Biotechnology Co., Ltd.), and the PCR amplification products are transformed in Escherichia coli DH5 α and are sequenced by Nanjin Stry Biotechnology.

3. Sequence analysis: completing the search of nucleotide and amino acid sequences and the prediction of conserved domains in NCBI related websites (http:// blast. NCBI. nlm. nih. gov); multiple sequence comparison of LCYB proteins among different species and the hydrophobicity/hydrophilicity analysis of carrot LCYB1 protein are completed by adopting DNAMAN 6.0; constructing a phylogenetic tree by using MEGA 5.0 software (Tumara et al, Mol biolEvol, 2011, 28 (10): 2731-; protein secondary structure prediction was done with the aid of the SOPMA website (http:// pbil. ibcp. fr /).

4. Real-time fluorescent quantitative PCR reaction: primer Premier 6.0 software (Singh et al, Biotechniques, 1998, 24 (2): 318-; the reverse primer sequence is 5'-GATGTTCTTCTACTTCTGCCACTAT-3'. The expression quantity of the DcLCYB1 gene in leaves, petioles and roots of three varieties of carrots with different colors is detected by using a ChamQ SYBR qPCR Master Mix (Nanjing Nodezan Biotechnology Co., Ltd.), amplification is completed by using a CFX96 real-time fluorescence quantitative PCR instrument (American Bio-Rad Co., Ltd.) and matched software, and the amplification program is as follows: 5min at 95 ℃; at 95 ℃ for 10s, at 60 ℃ for 10s and at 72 ℃ for 30s for 40 cycles; the melting curve was then obtained by stepwise amplification from 60 ℃ to 95 ℃. Carrot DcAnn gene is selected as an internal reference gene (Wang et al J Hot Sci Biotechnol, 2016, 91 (3): 264-) -270), and the expression level of the DcLCYB1 gene in different tissues of different varieties of carrots is calculated by a relative quantitative method (Pfafll, Nucleic Acid Res, 2001, 29 (9): e 45).

5. The test result is 1). the sequence determination result shows that the DcLCYB1 gene contains an Open Reading Frame (ORF) of 1527bp and codes 509 amino acids, the conservative domain prediction shows that the DcLCYB1 protein contains 1 highly conservative structural domain (figure 1). 2). the amino acid sequence coded by the DcLCYB1 gene is compared with the sequences of other species of LCYB proteins to construct an evolutionary tree, and the result shows that the carrot DcYB 1 protein has a close relationship with medlar, tomato and pepper of solanaceae (figure 2). 3). the result of the secondary structure prediction of the protein of LCYB1 shows that the main component of the protein of DcLCYB1 includes 38.98% of helix, 4.13% of fold, 16.14% of extension and 40.14% of main chain extension, the protein of the DcYB 4835 includes 38.98% of helix, 4.13% of helix, the yellow main chain extension and the leaf extension of 40.14% of the carrot yellow leaf, and the leaf of the carrot is expressed in yellow leaves of the southern yellow leaf variety, and the yellow leaf of carrot 3627' yellow leaf, and the yellow leaf of carrot variety is expressed in the yellow leaf map of the yellow.