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Transgene method for obtaining blue flowers through indigo blue synthesis via glutamine catalysis

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 display blue, and achieve easy decolorization. Effect

Active Publication Date: 2018-07-27
TIANJIN UNIV +1
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  • Summary
  • 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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  • Transgene method for obtaining blue flowers through indigo blue synthesis via glutamine catalysis
  • Transgene method for obtaining blue flowers through indigo blue synthesis via glutamine catalysis
  • Transgene method for obtaining blue flowers through indigo blue synthesis via glutamine catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Gene selection

[0036] Phosphopantetheinyl Transferases activate the sulfhydrylation domain T of non-ribosomal polypeptide synthase, the reaction mechanism is shown in the schematic diagram 2-1 , The activated T domain performs the function of fixing the substrate, which is necessary for the non-ribosomal polypeptide synthase to exert 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 to obtain nucleotides Sequence SEQ ID NO.1. The amino acid sequence encoded by the Sfp gene is shown in SEQ ID NO.2. The selection of Sfp gene should not limit the scope of protection for phosphopantetheinyl transferase in the present invention, and use any gene encoding phosphopantetheinyl transferase from other species, or encoding phosphopantetheinyl ...

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

[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, using 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, purified and recovered to obtain fragment CHS-RhAG and plasmid pBI121 ...

Embodiment 3

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

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

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

[0050] 2. Monoclonal colonies were inserted into 5mL LB liquid culture medium (10μg / mL rifampicin and 50μg / mL gentamicin) containing the same antibiotics and incubated overnight at 28°C on a shaker at a rotation speed of 150 revolutions per minute.

[0051] 3.2mL of Agrobacterium culture broth cultured overnight, diluted to 200mL LB broth, cultured to cell density OD in a shaker at 28°C (rotating speed 250 revolutions per minute) 600 From 0.3 to 0.5.

[0052] 4. The Agrobacterium culture solution was put into a 50mL centrifuge tube, cooled on ice and cent...

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Abstract

The invention discloses a transgene method for obtaining blue flowers through indigo blue synthesis via glutamine catalysis. The method comprises the following steps of (1) selecting a gene Sfp for coding phosphopantetheine transferase and a gene bpsA for coding indigo blue synthetase; respectively cloning the genes into a plant promoter downstream of plasmids containing plant promoters; (2) amplifying the obtained plasmids in escherichia coli; transferring the plasmids into soil agrobacterium; (3) transferring DNA containing Sfp and bpsA into plants. The produced blue flowers have various characteristics of natural flowers, are fresh, have flower fragrance, realize color fastness and is nontoxic. The transgenic enzymes and generated indigo blue are not in vacuoles, are not influenced by low pH of the plant vacuole, and can form the pure blue color. The precursor materials of blue substances are generated; the enzyme substances are glutamine richly contained in the plant bodies; the related enzymatic reaction only has one step; the transgenic transformation can be performed from white flowers existing in the nature.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a genetically modified method for catalyzing the synthesis of indigo from glutamine to obtain blue flowers. Background technique [0002] Flower color is an important ornamental feature of ornamental plants. The improvement of flower color has always been one of the important goals of gardeners. The flower color of plants not only plays an important role in pollination and reproduction of plants, but also provides humans with colorful viewing experience and 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 for flower species, modern rose (Rosa hybrida) has a history of 5,000 years of cultivation. So far, humans have bred more than 2500 varieties, but among them there is no true blue rose. [0003] The existing blue roses on the market, such as "Blue Enchantress"...

Claims

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Application Information

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Patent Type & Authority Applications(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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