Function and application of SiLCYE in regulating and controlling anabolism of millet carotenoids such as zeaxanthin

A technology of zeaxanthin and millet, applied in the direction of application, cells and enzymes modified by the introduction of foreign genetic material

Active Publication Date: 2021-12-17
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are relatively few studies on LCYE, and there are few reports on LCYE in millet

Method used

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  • Function and application of SiLCYE in regulating and controlling anabolism of millet carotenoids such as zeaxanthin
  • Function and application of SiLCYE in regulating and controlling anabolism of millet carotenoids such as zeaxanthin
  • Function and application of SiLCYE in regulating and controlling anabolism of millet carotenoids such as zeaxanthin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1, SiLCYE gene editing target design

[0081] 1.1 Amino acid sequence, coding sequence and genome sequence of SiLCYE

[0082] The amino acid sequence of SiLCYE is shown in sequence 1 in the sequence listing, the coding gene of SiLCYE is shown in sequence 2 in the sequence listing, and the genome sequence of SiLCYE is shown in sequence 3 in the sequence listing. Through bioinformatics analysis, the SiLCYE genome has a total of 10 exons and 9 introns, and the full-length CDS is 1623bp.

[0083] 1.2. SiLCYE gene editing target design

[0084] According to the millet SiLCYE genome sequence, through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, and finally select the nucleotides 171-190 of sequence 3 in the sequence list, that is, 5'-GAAGCACGATGAGACGGCGG-3' as the target sequence.

Embodiment 2

[0085] Example 2, Construction of pYLCRISPR / Cas9Pubi-H-SiLCYE recombinant vector

[0086] 2.1. Construction of LacZ-U6a-sgRNA expression cassette

[0087] according to figure 1 The flowchart for constructing the sgRNA expression cassette shown in the figure shows the construction of the LacZ-U6a-sgRNA expression cassette containing the 20bp target sequence of the SiLCYE gene. (4): e0154027) and LacZ-U6a promoter adapter primer sequence Cas9U6aF (+), LacZ-U6a promoter sequence, 20bpSiLCYE target sequence, sgRNA sequence, sgRNA and the adapter primer sequence gR- of pYLCRISPR / Cas9Pubi-H binary vector Rcas9(-). The nucleotide sequence (862bp) of the expression cassette is shown as sequence 4 in the sequence listing.

[0088] The build process is as follows:

[0089] first as figure 1 Design 4 primers at the indicated position: Cas9U6aF(+), U6aSiLCYET1, gRSiLCYET1, gR-Rcas9(-), wherein the Cas9U6aF(+) primer contains the 6 nucleotide sequence of the Bsa I restriction site o...

Embodiment 3

[0106] Example 3. Obtainment of SiLCYE-positive transgenic millet and identification of edited strains

[0107] 3.1. Ci846 mature embryo callus culture and callus transformation mediated by pYLCRISPR / Cas9Pubi-H-SiLCYE Agrobacterium strain EHA105

[0108] The millet variety Ci846, which is intended to be transformed as a receptor material, was provided by the research group of Researcher Diao Xianmin of the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0109] According to Biomed EHA105 Agrobacterium Competent Cell Reagent Manual (Product No. BC303-01), the recombinant vector pYLCRISPR / Cas9Pubi-H-SiLCYE was introduced into EHA105 competent cells to obtain pYLCRISPR / Cas9Pubi- Agrobacterium strains with H-SiLCYE plasmid.

[0110] The mature embryos of millet Ci846 were used as explants for callus induction culture, and the infection and transformation experiments of Agrobacterium strains containing pYLCRISPR / Cas9Pubi-H-SiLCYE plasmid were commissioned by th...

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Abstract

The invention discloses a method for increasing the content of zeaxanthin or/and lutein stearate or/and antheraxanthin in millet. The method comprises the following steps of inhibiting or reducing the expression quantity of a protein coding gene in the millet, and/or inhibiting or reducing the activity and/or content of the protein in the millet, so as to increase the content of the zeaxanthin or/and the lutein stearate or/and the antheraxanthin in the millet, wherein the protein is SiLCYE protein. SiLCYE is cloned through a homologous cloning technology. An amino acid sequence of the SiLCYE is as shown in SEQ ID NO. 1, and a coding sequence of the SiLCYE is as shown in SEQ ID NO. 2. An edited strain of SiLCYE genes is obtained through a CRISPR-Cas9 editing technology, and the regulation and control effects of the two genes in the metabolic reaction of the millet carotenoids are determined through metabonomics analysis. An experimental basis and gene resources are provided for revealing a metabolic pathway regulation and control mechanism of the millet carotenoids and cultivating new germplasm of the millet which is rich in carotenoid and high in nutritional quality.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the function and application of SiLCYE protein regulating the synthesis and metabolism of zeaxanthin and other millet carotenoids. Background technique [0002] Carotenoids are essential nutrients for the human body. They have unique nutritional and health effects such as anti-oxidation, immune regulation, anti-cancer, anti-aging, and vision protection. Therefore, it is very important to strengthen the content of carotenoids in grains and promote their metabolism and absorption. There is necessary research work. There are many kinds of carotenoids, more than 700 kinds of natural carotenoids have been found, which can be divided into two categories according to the different chemical structures: one is carotene (only containing two elements of carbon and hydrogen, such as β-carotene The other is lutein (in addition to carbon and hydrogen elements, there are also oxygen-containing funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/84C12N15/55C12N1/21C12N5/10A01H5/10A01H6/46
CPCC12N9/90C12N15/8218C12N15/8247C12N15/825C12N15/8205C12N9/22C12Y505/01018
Inventor 刘君高英王欢石添添王竞馨王芮程溪柳朱梦圆魏钰李莎刘远航
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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