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Gene combination expressing and producing betacyanin in rice, and application thereof

A technology of betatin and gene combination, applied in the field of genetic engineering, can solve problems affecting the production of betalain

Inactive Publication Date: 2019-12-20
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main source of beta vulgaris is beet (Beta vulgaris), but many factors can obviously affect the yield of beta vulgaris in beet. According to literature reports, the beet red from beet can only meet 10% of the global food additive demand at present ( Manchali et al., Stability of betalain pigments of red beet. In RedBeet Biotechnology; Neelwarne, B., Ed.; Springer US: Boston, MA, 55-74.2013)

Method used

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  • Gene combination expressing and producing betacyanin in rice, and application thereof
  • Gene combination expressing and producing betacyanin in rice, and application thereof
  • Gene combination expressing and producing betacyanin in rice, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of multi-gene plant expression vector

[0047] 1. Optimized synthesis of three genes

[0048] The optimization is carried out in accordance with the following principles: (1) Optimize gene codons according to rice codon preference to improve gene translation efficiency; (2) Eliminate the recognition sites of commonly used restriction enzymes in genes to facilitate the construction of expression cassettes; (3) Eliminate the reverse repeat sequence, stem-loop structure and transcription termination signal, balance the GC / AT within the gene, and improve the stability of RNA; (4) Make the gene encoded protein conform to the N-terminal principle to improve the stability of the translated protein; (5) ) Optimize the free energy of mRNA secondary structure to improve gene expression efficiency.

[0049] Using the melo gene (GenBank No.D37929.1) of Aspergillus oryzae as a template, according to the codon preference of rice, the DNA sequence meloS shown in SEQ I...

Embodiment 2

[0091] Example 2 Transformation of rice

[0092] 1. Acquisition and identification of genetically modified rice

[0093] 1.1 Preparation of Agrobacterium and electric shock

[0094] 1) Pick a single Agrobacterium and inoculate it in 5mL LB liquid medium (rifampicin 50μg / mL, chloramphenicol 100μg / mL), culture at 28°C, 250 rpm for 20h.

[0095] 2) Transfer 1 mL of bacterial solution to 20-30 mL of LB liquid medium (rifampicin 50μg / mL, chloramphenicol 100μg / mL), incubate at 28°C, 250 revolutions / min for about 12 hours, and measure OD600≈1.5.

[0096] 3) Collect the bacteria by centrifugation at 8000 rpm, 4°C for 10 min, resuspend in Agrobacterium transformation permeate (5wt% sucrose, 0.05wt% Silwet L-77) and dilute to OD600≈0.8.

[0097] 4) Resuspend the cells in 0.5 times the volume of sterile water pre-cooled at 4°C, centrifuge at 3500g for 10 minutes, and pour out the supernatant carefully. Resuspend the bacteria in 1-2 mL of sterile 10% glycerol pre-cooled at 4°C. The bacterial liquid...

Embodiment 3

[0131] Example 3 Observation of the appearance of transgenic rice plants and detection of betaine content

[0132] The rice seeds harvested from the T1 generation in Example 2 were germinated normally. When the plants grow to about 20 cm, it can be clearly seen that the transgenic rice plants have a deep red color ( Figure 5 ), which preliminarily showed that the introduction of the above 3 genes allowed the synthesis of betaine in the transgenic rice plants.

[0133] Mass spectrometry analysis and content determination of betaine in transgenic rice plants: 0.1g of rice plants were ground into powder on ice, and 4.5ml of 32% ethanol with pH 5.3 was added, and extracted with low temperature shaking (100rpm) protected from light for 30min; 4℃ The supernatant was collected by centrifugation at 8000 rpm for 5 min; the supernatant was centrifuged at 4° C. at 8000 rpm for 5 min, and the collected supernatant was concentrated and dried at low temperature and protected from light; finally,...

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Abstract

The present invention discloses a gene combination expressing and producing betacyanin in rice, and an application thereof. The gene combination comprises a melo gene, a BvDODA1 gene and a BvCYP76AD1gene. Based on codon preferences of rice, the melo gene is optimized as a meloS gene and a nucleotide sequence is shown as SEQ ID NO.1; the BvDODA1 gene is optimized as a BvDODA1S gene and a nucleotide sequence is shown as SEQ ID NO.2; the BvCYP76AD1 gene is optimized to a BvCYP76AD1S gene and a nucleotide sequence is shown as SEQ ID NO.3; the three genes are respectively fused with a 35S promoterof a cauliflower mosaic virus and a NOS terminator of agrobacterium rhizogenes, corresponding gene expression cassettes are respectively constructed, then the gene expression cassettes are ligated into plant expression vectors to obtain polygenic plant transforming vectors containing the three gene expression cassettes, and the vectors are transformed into rice to obtain betacyanin-enriched transgenic rice which has a betacyanin content about 2.0-2.6 mg / g FW.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a gene combination for expression and production of betarubin in rice and its application. Background technique [0002] The main component of betanin is betanin, which is a natural pigment extracted from sugar beet. It has no toxic side effects, bright color and contains nutrients necessary for human metabolism and growth and development, so it is widely used in various beverages and fruit flavors. The coloring of powder, fruit juice, soda, candy, pastry, ice cream, canned food, concentrated fruit juice, ice cream, jelly, sausage food not only increases the beautiful appearance of food, but also improves the nutritional value of food. [0003] Tesoriere et al. (Am.J.Clin.Nutr. (2004) 80, 391-395, 2005) found that eating prickly pear fruit containing betaine can significantly reduce lipid damage caused by peroxidation stress, and improve the body’s resistance. Oxidation le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82A01H5/00A01H6/46
CPCC07K14/38C07K14/415C12N15/825C12N2800/22C07K2319/00
Inventor 田永生姚泉洪彭日荷付晓燕许晶高建杰韩红娟王波王丽娟李振军张福建黄悠楠张文慧
Owner SHANGHAI ACAD OF AGRI SCI
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