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A transgenic method for obtaining blue flowers by catalyzing the synthesis of indigo from glutamine

A technology for synthesizing indigo and glutamine, which is applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the problems of high production cost, complex precursor substances, and inability to show blue, and achieve easy decolorization. Effect

Active Publication Date: 2020-06-02
TIANJIN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method involves the modification of multiple genes, the required precursor substances are complex, and the production cost is extremely high. This technology has been successful in carnations and chrysanthemums, and because the vacuole pH of roses is very low (pH is about 2.7), this Technology applied on roses only results in lavender flowers, unable to show true blue ( figure 1 )(Katsumoto Y, Fukuchimizutani M, Fukui Y, et al.Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-huedflowers accumulating delphinidin.[J].Plant&Cell Physiology,2007,48(11):1589.)

Method used

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  • A transgenic method for obtaining blue flowers by catalyzing the synthesis of indigo from glutamine
  • A transgenic method for obtaining blue flowers by catalyzing the synthesis of indigo from glutamine
  • A transgenic method for obtaining blue flowers by catalyzing the synthesis of indigo from glutamine

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Experimental program
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Effect test

Embodiment 1

[0035]Example 1 Gene Selection

[0036] Phosphopantetheinyl Transferases (Phosphopantetheinyl Transferases) activate the thiol domain T of non-ribosomal polypeptide synthetases. The reaction mechanism is shown in the schematic diagram Figure 2-1 , the activated T domain performs the function of immobilizing the substrate, which is necessary for the non-ribosomal polypeptide synthetase to perform its catalytic function.

[0037] The gene Sfp of the phosphopantetheinyl transferase used in the present invention is derived from Bacillus subtilis ATCC21332 (NCBI number: ALS83446). We adjusted and optimized the gene codon according to the gene codon preference of Rosaceae plants, and obtained the nucleotide Sequence SEQ ID NO.1. The amino acid sequence encoded by the Sfp gene is shown in SEQ ID NO.2. The selection of the Sfp gene should not limit the scope of protection of phosphopantetheinyl transferase in the present invention, use any other species-derived gene encoding phosph...

Embodiment 2

[0041] Example 2 Plasmid Cloning

[0042] The present invention selects the plant binary expression vector pBI121 as the starting plasmid vector.

[0043] Insert the artificially synthesized fragment (SEQ ID NO.7) between the restriction enzyme sites PmeI and SacI of the plasmid pBI121 to form the plasmid pBI121-CHS-RhAG, see image 3 . SEQ ID NO.7 includes the terminator sequence MASt, promoter sequence CHSp and promoter sequence RhAGp of Mannopine Synthase.

[0044] Using the plasmid containing the sequence of SEQ ID NO.3 as a template and using BpsA-FG (SEQ ID NO.8) and BpsA-RG (SEQ ID NO.9) as primers, the fragment bpsA was obtained by PCR.

[0045] Using the plasmid containing the sequence of SEQ ID NO.1 as a template and Sfp-FG (SEQ ID NO.10) and Sfp-RG (SEQ ID NO.11) as primers, the fragment Sfp was obtained by PCR.

[0046] Plasmid pBI121-CHS-RhAG was digested with restriction enzymes BamHI and SpeI, and the fragment CHS-RhAG and plasmid pBI121 backbone were obtaine...

Embodiment 3

[0047] Embodiment 3: Preparation of Competent Agrobacterium Agrobacterium Carrying Target Gene

[0048] The plasmid pBI121-GENES2 extracted from Escherichia coli was transformed into Agrobacterium agrobacterium. The preparation of soil Agrobacterium competent cells is a general method, specifically:

[0049] 1. Agrobacterium tumefaciens GV3101 was cultured on LB agar medium containing 10 μg / mL rifampicin and 50 μg / mL gentamicin at 28° C. for two days;

[0050] 2. The monoclonal colonies were inserted into 5 mL of LB liquid culture solution containing the same antibiotics (10 μg / mL rifampicin and 50 μg / mL gentamicin) and cultivated overnight in a shaker at 28° C., with a rotation speed of 150 rpm.

[0051] Dilute 3.2mL of the Agrobacterium culture solution cultivated overnight to 200ml of LB culture solution and cultivate it in a shaker at 28°C (250 rpm) until the cell density is OD 600 0.3 to 0.5.

[0052] 4. The soil Agrobacterium culture solution was put into a 50 mL cent...

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Abstract

The invention discloses a transgenic method for catalyzing glutamine to synthesize indigo to obtain blue flowers. The steps are as follows: 1) select the Sfp gene encoding phosphopantetheinyl transferase and the bpsA gene encoding indigo synthase, and clone them respectively in Downstream of the plant promoter of the plasmid of the plant promoter; 2) the plasmid obtained is amplified in Escherichia coli, and transformed into Agrobacterium agrobacterium; 3) the DNA containing Sfp and bpsA is transferred into the plant; the present invention produces Blue flowers, with various characteristics of natural flowers, fresh and floral, non-marking, non-toxic. The transgene-encoded enzyme and the resulting indigo are out of the vacuole and unaffected by the low pH of the plant vacuole to form a pure blue color. The precursor of the blue substance, that is, the substrate of the enzyme is glutamine, which is rich in plants. The enzymatic reaction involved is only one step, and it can be transgenic from white flowers in nature.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a transgenic method for synthesizing indigo from glutamine to obtain blue flowers. Background technique [0002] Flower color is an important ornamental feature of ornamental plants, and the improvement of flower color has always been one of the important goals of horticultural workers. The flower color of plants not only plays an important role in the pollination and reproduction of plants, but also provides humans with a colorful viewing experience, which has important aesthetic value (Grotewold, E. The genetics and biochemistry of floral pigments [J]. Annual Review of Plant Biology, 2006, 57(1):761.). As an important ornamental plant, the modern rose (Rosa hybrida) has been cultivated for 5,000 years. So far, humans have cultivated more than 2,500 varieties, but there is no real blue rose among them. [0003] The existing blue roses on the market, such as "Blue Enchantress" are a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/60C12N15/54A01H5/00A01H6/56A01H6/74A01H6/30A01H6/62
CPCC12N9/1288C12N9/88C12N15/825C12Y207/08C12Y207/08007C12N15/8205
Inventor 张雁陈义华南迦纳杰·阿瑟·安卡拉哈里胡逸灵李鹏伟
Owner TIANJIN UNIV
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