Tea tree MYB transcription factor CsAN1 and application thereof in regulation of anthocyanin metabolism

A technology of transcription factor and tea tree, applied in the field of plant genetic engineering, can solve the problem that the regulation mechanism of anthocyanin synthesis-related transcription factors is unclear and other problems.

Inactive Publication Date: 2017-03-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for identification of specific genes involved in plant growth or development through their ability to regulate certain chemical reactions within plants called enzyme pathways. These genes are located close together along DNA sequences known as chromatin structures like nucleosomal histones which control how much different parts of an organism' s own cells produce each other during cell division. By analyzing this structure, researchers have been able to identify these genes and determine whether they may be useful for agricultural purposes such as increasing yield potential or improving resistance against disease agents.

Problems solved by technology

This patents describes how different types of vegetables like rice, barley, citrus, lime, carrot seed pods or rootlets contain significant amounts of certain compounds called phloretoxylindones ("Phenomenochlaquinosis") and chelates therapy agents - including zinc ionophyllum berbons, lycopene, quercitol, rhodamines, diknoferisminerals, dibenzyluximides, α-,β-unsaturable phenone gamma coumarns, xanthanoidenans, and gallaminage bacteria. They contribute towards improving nutritional values during ferry culture processes and reducing negative side effect associated with traditional methods involving pesants.

Method used

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  • Tea tree MYB transcription factor CsAN1 and application thereof in regulation of anthocyanin metabolism
  • Tea tree MYB transcription factor CsAN1 and application thereof in regulation of anthocyanin metabolism
  • Tea tree MYB transcription factor CsAN1 and application thereof in regulation of anthocyanin metabolism

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Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Tea tree MYB transcription factor CsAN1 Isolation and cloning

[0040] 1. Primer design: In the early stage of the present invention, transcriptome sequencing (Anoroad Gene Technology (Beijing) Co., Ltd.) was performed on the leaves of the tea tree "Zijuan" at different developmental stages, and the transcriptome database obtained CsAN1 The full-length, according to the full-length known sequence design amplification CsAN1 Specific primers:

[0041] F: 5'-TACTTATTCAGACAAACGCACAC-3';

[0042] R: 5'-ATTTCAACTCTAATTTCAACCCA-3';

[0043] 2. RNA extraction: Hipure Plant RNA Mini Kit (Meiji, Guangzhou) was used to extract the total RNA of the terminal bud, the second leaf, the third and fourth leaves of the tea tree "Zijuan" and the old leaves, and measured it with a nucleic acid protein analyzer OD260 / OD280 and OD260 / OD230 values ​​were used to judge the quality and yield of RNA, and 1.0% agarose gel electrophoresis was used to detect the integrity of RNA.

[...

Embodiment 2

[0059] Example 2 Tea tree MYB transcription factor CsAN1 Construction and transformation of overexpression vector

[0060] 1. Schematic diagram of the construction of the overexpression vector figure 2 As shown, the specific steps are: the positive clone PMD19T- CsAN1 The vector is used as a template, and specific primers are designed: pBI121- CsAN1 -F:5'-GGACTCTAGAGGATCCATGGACATTGTTTGTTGTTGTGT-3', pBI121- CsAN1 -R: 5'-GACCACCCGGGGATCCTCATCATTCATCACCTAACA-3', for PCR amplification. Use at the same time Bam H The pBI121 overexpression vector was digested with I (TaKaRa) to linearize it, and then ligated using the ClonExpress® II One Step Cloning Kit (Nanjing Nuoweizan Biotechnology Co., Ltd.). Enzyme cutting system: Bam H I 1μL+Buffer 1μL+pBI121 plasmid 3μL+H 2 O to make up to 10 μL. Digest in a water bath at 37°C for 4 hours, then transfer to a water bath at 85°C for inactivation for 20 minutes.

[0061] Table 1 Connection system of ClonExpress® II One Step Cloning...

Embodiment 3

[0086] Example 3 Tea tree MYB transcription factor CsAN1 Anthocyanin measurement in overexpressing plants

[0087] 1. Take and transform separately CsAN1 , CsAN1 +CsGL3 and CsAN1 +CsGL3+CsTTG1 tobacco leaves, using a microscope to observe the microstructure of various transgenic tobacco leaves, the results are shown in image 3 .

[0088] 2. Anthocyanin extraction: Grind 3 g of fresh leaf samples in a mortar with liquid nitrogen, then transfer them to test tubes, add 3 mL of extraction buffer (made of 18% n-propanol, 1 % hydrochloric acid: prepared from 81% water), in a boiling water bath for 3 min, and left at room temperature overnight. For extraction results, see Figure 4 .

[0089] 3. Measurement of anthocyanins: use a UV spectrophotometer to measure the concentration of the extract in A 535 and A 650 The absorbance value, the final anthocyanin content with (A 535 ~A 650 ) g -1 (FW) representation. See the test results Figure 5 .

[0090] 4. Determination o...

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Abstract

The invention specifically discloses a tea tree MYB transcription factor CsAN1 and an application thereof in regulation of anthocyanin metabolism. A nucleotide sequence of the tea tree MYB transcription factor CsAN1 is represented as SEQ ID NO:1. CsAN1 is connected with a 35S promoter and then is connected to a pBI121 carrier, meditated transformation of tobaccos is realized by agrobacteria GV3101, meanwhile, the gene as well as genes CsGL3 and CsTTG1 capable of forming compounds with the gene is used for transforming the tobaccos respectively, leaves of overexpressed tobacco plants turn red, anthocyanin content detected by a spectrophotometer is remarkably increased, and two main pigments including cyanidine and delphinidin are detected through HPLC (high performance liquid chromatography).

Description

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Claims

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Application Information

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Owner SOUTH CHINA AGRI UNIV
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