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A transgenic breeding method for producing astaxanthin in crop seed endosperm
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A technology of astaxanthin and seed embryo, applied in the field of genetic engineering, can solve the problems of decreased nutritional value, lack of synthetic astaxanthin, etc.
Active Publication Date: 2020-02-21
SOUTH CHINA AGRI UNIV
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[0003] Rice is an important food crop and model plant, and its seed is also an excellent bioreactor. The protein and active nutrients of rice mainly exist in the seed coat and embryo, while the polished rice for daily consumption is removed from the seed coat and The endosperm of the embryo (the main component is starch), its nutritional value is greatly reduced
However, there is no report on the production of astaxanthin in rice endosperm, because rice lacks key enzymes for synthesizing astaxanthin, and it is also a very difficult problem to specifically need those enzymes to synthesize astaxanthin in endosperm. Subject research
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Embodiment 1
[0036] The present invention finds through research that PSY, CrtI, BHY and BKT are four essential key enzymes in order to synthesize astaxanthin in crop endosperm. Based on the above research basis, the present embodiment establishes a transgenic breeding method for producing astaxanthin in rice endosperm, which specifically includes the following steps:
[0037] S1. Construction of four essential gene expression cassettes:
[0038] S11. Synthesis of the coding regions of four essential genes (PSY-r, CrtI-p, BHY-p and BKT-p): Using the PSY gene (GenBank No.U32636.1) of maize as a template, according to monocotyledonous plants and rice Codon preference, using the CodonOptimization Tool program to optimize the codon, synthetically obtain the PSY-r of the DNA sequence shown in SEQ ID NO:1, clone it into a plasmid vector, and determine its sequence by sequencing; ) as a template, according to the codon preference of monocotyledonous plants and rice, utilize the Codon Optimizatio...
Embodiment 2
[0080] Appearance and HPLC detection of astaxanthin in transgenic rice endosperm:
[0081] Observation of astaxanthin in the endosperm of transgenic rice: After the transgenic rice seeds introduced with the above four genes were processed into polished rice (removing the seed coat) and the rice grains were chopped and observed, it can be seen that the inside and outside of the whole polished rice are orange-red ( Image 6 ), indicating that astaxanthin was synthesized in the endosperm.
[0082] Extraction and high-performance liquid chromatography (HPLC) identification of astaxanthin from transgenic rice seeds: Take 0.1 g of rice seeds and grind them into powder on ice, add 2 mL of methanol and continue to grind uniformly for 5 min; 100rpm) extraction for 10min; centrifuge at 4°C 8000rpm for 5min to collect the supernatant; repeat the extraction of the precipitate until the precipitate is white; centrifuge the supernatant at 4°C 8000rpm for 5min and then combine the supernatan...
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Abstract
The invention discloses a transgenic breeding method producing astaxanthin in crop seed endosperm. The transgenic breeding method specifically includes the steps of S1, optimizing the sequence of a PSY gene, a CrtI gene, a BHY gene and a BKT gene according to the codon preference of a breeding crop, and respectively fusing the four genes with the optimized sequences with breeding crop endosperm expression promoters and transit peptides to build gene expression boxes; S2, building the four gene expression boxes obtained in the S1 into plant transformation vectors to obtain the plant transformation vectors of the PSY, the CrtI, the BHY and the BKT; S3, using the plant transformation vectors obtained in the S2 to transform the breeding crop to obtain a transgenic crop with the salmon-colored endosperm and synthesized astaxanthin. The transgenic breeding method has the advantages that the astaxanthin can be synthesized in the seed astaxanthin of crops (especially paddy rice), the crops can be directly used for eating and also be used as the raw materials for producing the astaxanthin, and the method has important guiding significance and production application value to the novel functional crop breeding.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a transgenic breeding method for producing astaxanthin in the endosperm of crop seeds. Background technique [0002] Astaxanthin, chemical name 3,3'-dihydroxy-4,4'-diketone-ketone, β'-carotene, also known as super vitamin E, is a kind of orange-red carotenoid pigment, in Organisms have super antioxidant properties, and their antioxidant activity is 550 times that of vitamin E. It has the functions of anti-radiation, preventing tumors, preventing cardiovascular diseases, preventing diabetes, delaying aging and improving immunity. It has broad application value in food, health care products, medical supplies, cosmetics, food additives, aquaculture and other fields. In nature, astaxanthin is mainly produced by some algae, bacteria, and phytoplankton, and higher plants cannot synthesize it due to the lack of key β-carotenoid ketolase (BKT). At present, the sources of astaxanthin ...
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