Rhododendron hybridum petal RhCHS gene promoter and flower color breeding application

A rhododendron petal and promoter technology, applied in the field of genetic engineering, can solve problems such as insufficient research on CHS gene promoters

Active Publication Date: 2021-09-17
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is: provide a RhCHS gene promoter of red Belgian Rhododendron petals to solve the shortage of CHS gen...

Method used

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  • Rhododendron hybridum petal RhCHS gene promoter and flower color breeding application
  • Rhododendron hybridum petal RhCHS gene promoter and flower color breeding application
  • Rhododendron hybridum petal RhCHS gene promoter and flower color breeding application

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Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1, rhododendron RsCHS gene promoter cloning

[0023] (1) DNA Extraction of Rhododendron Rhododendron

[0024] The petals of the red Belgian rhododendron used in the experiment were collected from Ningbo Beilun Yirun Flower Co., Ltd. The sampling plants are required to be healthy, with luxuriant flowers and bright colors, and the experimental petals are required to be clean and well-proportioned, with uniform color and no insect spots. The petals of living plants are first cleaned with a washing bottle to clean the floating dust on the surface, and the petals are collected and quickly frozen in liquid nitrogen. Frozen petal samples were extracted in the laboratory using CTAB method to extract Rhododendron genomic DNA for future use.

[0025] (2) Tail-PCR primer design and verification

[0026] Referring to the instructions of the Genome Walking Kit (CodeNo.6108) kit, design 3 R-terminal primers SP1, SP2, and SP3 (Table 1), pair with 4 random primers in the ...

Embodiment 2

[0041] Example 2, analysis of cis-acting elements of the RhCHS gene promoter of Rhododendron Rhododendron Rhododendron Rhododendron

[0042] With the help of PlantCARE, cis-acting element analysis was performed on the obtained 1477 promoter sequences. The name, quantity and position of the cis-element are shown in Table 2, figure 1 shown.

[0043] Table 2: Types, positions and quantities of cis-acting elements in the RhCHS gene promoter of the red Belgian cuckoo

[0044]

example 3

[0045] Example 3, construction of recombinant vector

[0046]The pCambia1301 vector was digested with PstI and NcoI, the 35S Promoter in front of the GUS gene was excised, and the vector backbone was recovered for recombination reaction with the CHSP fragment. The reaction product was transformed into Escherichia coli DH5α, coated with a Kana resistant plate, cultured overnight at 37°C, and 8 resistant colonies were picked. After positive colonies were identified by PCR using the above amplification primers, clone No. 15 was picked for sequencing. Bidirectional sequencing primers were M13-R:CAGGAAACAGCTATGAC, Gus-R:GAAAAGGGTCCTAACCAAGA.

[0047] The results showed that the promoter sequence obtained in the recombinant vector was completely consistent with the promoter sequence obtained in the Tail-PCR experiment, which proved that the recombinant vector was constructed successfully. For the sequencing results and analysis results, see figure 2 .

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Abstract

The invention relates to a rhododendron hybridum petal RhCHS gene promoter and flower color breeding application. According to the invention, a promoter with relatively high promoter activity is obtained from petal tissue of rhododendron hybridum. The full length of the promoter is 1477bp, the expression of a Chalcone synthase (CHS) gene can be efficiently started, the promoter disclosed by the invention can replace a promoter of the CHS gene with a low expression level in other species by virtue of a gene recombination technology, the enzyme activity of a CHS is improved, and the synthesis of anthocyanidin and anthocyanin in a downstream pathway of an anthocyanin metabolism pathway is increased, so that the color performance of petals is influenced. The promoter plays an important role in promoting breeding research on a plurality of horticultural flowers and plants including rhododendron and has good economic and social values.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the acquisition of a chalcone synthase (CHS) gene promoter derived from the petal tissue of the red Belgian rhododendron and its application in flower color breeding research. Background technique [0002] The promoter is a DNA sequence located in the upstream region of the 5' end of the structural gene, with a length ranging from 1000-2000bp. This DNA sequence can activate RNA polymerase to accurately combine with template DNA and start transcription. The specific transcription of a gene depends on whether the enzyme can effectively form a binary complex with the promoter, and the sequence of the promoter will affect its affinity with RNA polymerase, thereby affecting the level of gene expression. Near the transcription start site of the core region of the promoter usually contains a TATA box structure with the sequence TATAAA; upstream of the 5' end of ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84C12N1/21A01H5/02A01H6/36C12N15/10C12R1/19C12R1/01
CPCC12N15/825C12N15/8222C12N9/1037C12Y203/01074C12Q1/6848C12Q2531/113C12Q2549/119
Inventor 贾永红吴月燕
Owner ZHEJIANG WANLI UNIV
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