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Lilium chalcone synthase genes (chs) promoter as well as preparation method and use thereof

A lily chalcone synthase technology, applied in the field of genetic engineering, can solve problems such as the lack of a chalcone synthase gene promoter

Inactive Publication Date: 2008-07-30
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, so far, there have been no reports of obtaining the chalcone synthase gene promoter from lily and applying it in transgenic plants

Method used

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  • Lilium chalcone synthase genes (chs) promoter as well as preparation method and use thereof
  • Lilium chalcone synthase genes (chs) promoter as well as preparation method and use thereof
  • Lilium chalcone synthase genes (chs) promoter as well as preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 100

[0068] Example 1 Extraction of Lily Leaf Genomic DNA

[0069] Grind 0.5-2.0g young leaves of Oriental Lily "Sorbonne" in liquid nitrogen, put them into a 2ml centrifuge tube, and add 1×CTAB buffer (760ul), PVP 20% (140ul) and β-mercaptoethanol (20ul), Water bath at 65°C for 20-40min, then add an equal volume of chloroform-isoamyl alcohol, centrifuge at 12000rpm for 20-40min, and repeat once; take the supernatant and put it in a 5ml centrifuge tube, slowly add 1 / 10 volume of absolute ethanol (-20°C pre-cooled), 1 / 5 volume CTAB buffer, NaCl (25ul 5M), and 1 / 10 volume KA C (5M pre-cooling) mix well, freeze on ice for 20-40min; centrifuge at -4°C, 7000rpm for 10-15min, add 1×CTAB buffer (1000ul), 65°C, water bath for 20-40min; add 25ul saturated NaCl and 800ul Chloroform-isoamyl alcohol (24:1) 12000rpm, centrifuge for 10-20min; take the precipitate, add 550ul TE and RNase, at 37°C, add 2 times absolute ethanol (pre-cooled at -20°C) after 30-40min in water bath Invert and mix wel...

Embodiment 2

[0111] Embodiment 2 contains the plant expression vector of chalcone synthase gene promoter

[0112] The technical procedures for the construction and identification of expression vectors are as follows:

[0113] Digest the T vector with Hind III and Xba I double enzymes, recover the target large fragment containing CHSA, in T 4 Under the action of ligase, ligate with the corresponding double-enzyme-digested plasmid pBI121 containing the Lc gene at 16°C for 5 h. Take 10 μL of the ligated product to transform Escherichia coli DH5α, spread it on an LB plate containing kanamycin (50 mg / L), Cultivate overnight at 37°C, select a single colony and shake, extract the plasmid, and perform PCR identification and double enzyme digestion identification.

[0114] In order to identify the correctness of the clone obtained by replacing the CaMV35S promoter on pBI1210 with the PCHS promoter, 9 clones were selected to extract plasmids, and PCR analysis was performed first, and then the posit...

Embodiment 3

[0116] Example 3 Application of Chalcone Synthase Gene Promoter in Transgenic Plants

[0117] 1. Source and treatment of lily explants

[0118] Use well-growing red and white lily bulbs and leaves as materials, rinse with running water overnight, rinse with distilled water once, soak in 70% alcohol for 1 minute, rinse with sterile water for 3 times, disinfect with 0.15% mercury liter for 25-30 minutes, and then rinse with sterile water Rinse 3 times, 5min each time. Cut the sterile bulbs into small pieces of 0.5cm X 0.5cm, cut the leaves into small pieces of 0.8cm in length, and inoculate them in M 2 On the culture medium, culture at 25°C, with 16 hours of light every day, and the light intensity is 2000-10000Lx.

[0119] 2. Direct differentiation and emergence of explants

[0120] Sterile material was inoculated in M 2 After 4-5 weeks on the culture medium, they began to differentiate and sprout. After about 3 weeks of cultivation, they can be used as transformation mate...

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Abstract

The invention discloses a newly found DNA sequence of the promoter of the chalcone synthase (chs) gene and the manufacturing method and use for the same. The promoter is used for PCR with a joint from sorbonne, and clones 899bp of the upstream regulation sequence of chs gene by combining the characteristics of the nested PCR, TD PCR, hot-started PCR and two-step PCR. The analysis in the promoter database Plant CARE shows that, the sequence has a basically conservative region TACPyAT which expresses specifically in flowers, and has the main cis-acting elements with which the primary promote is provided such as a transcription initiation related TATA box, a transcription auxiliary CAAT box, a G box and CCAAT etc. The promoter can regulate the specific expression of the target gene in the plant, and can provide an effective promoter element for the constructing the plant expression vector, increase the expression of foreign gene, and minimum the biological energy consumption.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a newly discovered DNA sequence of a plant gene tissue-specific expression promoter, in particular to a DNA sequence of a lily chalcone synthase gene (chs) promoter and a preparation method thereof and a method containing the same Plant expression vectors for promoters and their use in transgenic plants. Background technique [0002] Lily is a perennial bulbous flower with huge flowers, rich colors and beautiful flower posture. It can be used not only as cut flowers, potted flowers, but also in gardens and green spaces. With the continuous improvement of human economy and development, people have higher and higher requirements for the ornamental and cultivation characters of lily, which makes the process of commercial breeding of lily continue to accelerate. Thousands of new varieties of lilies have been bred all over the world, centered on the Netherlands and the Unit...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/52C12N15/82
Inventor 刘雅莉张宗勤杨丽常小丽解燕祁银燕
Owner NORTHWEST A & F UNIV
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