Transcription factor capable of regulating rubber tree anthocyanin synthesis and encoding gene and application thereof

A technology encoding genes and transcription factors, applied in the field of molecular biology, can solve the problems of low screening efficiency and achieve good application prospects

Active Publication Date: 2017-09-19
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] As early as 1994, Arokiaraj et al. in Malaysia obtained the world's first rubber tree transgenic plant by using the gene gun method. However, so far, nptII is used

Method used

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  • Transcription factor capable of regulating rubber tree anthocyanin synthesis and encoding gene and application thereof
  • Transcription factor capable of regulating rubber tree anthocyanin synthesis and encoding gene and application thereof
  • Transcription factor capable of regulating rubber tree anthocyanin synthesis and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Controlling Rubber Tree Anthocyanin Synthesis R2R3-MYB Bioinformatics Discovery

[0030] Using the R2R3-MYB conserved domain as a probe, the Hevea R2R3-MYB transcription factors were retrieved from the Hevea Genome Database, and a total of 177 R2R3-MYBs of Hevea were obtained. Then, Arabidopsis AtPAP2 (AT1G66390.1), dwarf Morning glory PhAN2 (AB982128.1), Tobacco NtAN2 (FJ472647.1), Antirrhinum AmROSEA1 (DQ275529.1), Antirrhinum AmROSEA2 (DQ275530.1), Antirrhinum AmVENOSA (DQ275531.1), Apple MdMYB10 (DQ267896.1 ), potato StAN1, grape VvMYBA1 (AB097923.1), grape VvMYBA2 (AB097924.1) and other 10 R2R3-MYB related to anthocyanin synthesis, and the evolutionary analysis found that the rubber tree scaffold0374_923317 and Scaffold0598_393375 were identified as anthocyanin synthesis Related transcriptional regulators are in the same evolutionary branch ( figure 1 ), it is speculated that the two may be related to the regulation of rubber tree anthocyanin synthesis. ...

Embodiment 2

[0031] Example 2 Cloning and structural domain analysis of R2R3-MYB related to the regulation of rubber tree anthocyanin synthesis

[0032] The scaffold0374_923317 sequence was retrieved from the rubber tree genome database, and the sequence was used as a search sequence to retrieve the leaf transcriptome data of the author's unit, and a gene sequence with a complete ORF was retrieved, and then, specific amplification PCR primers (such as SEQ ID NO.3-4) to amplify its ORF and perform sequencing analysis. As a result, a gene sequence identical to the leaf transcriptome sequence was obtained, with a full length of 618bp, which was named HbAn1. The expression level of the other sequence was very low and was not amplified.

[0033]With Arabidopsis AtPAP2 (AT1G66390.1), Petunia PhAN2 (AB982128.1), Tobacco NtAN2 (FJ472647.1), Antirrhinum AmROSEA1 (DQ275529.1), Antirrhinum AmROSEA2 (DQ275530.1), Antirrhinum AmVENOSA (DQ275531.1), apple MdMYB10 (DQ267896.1), potato StAn1 (AY841127.1)...

Embodiment 3

[0034] Example 3 HbAn1 tissue expression analysis

[0035] Using the cDNA of leaves, dark culture and light culture somatic embryo plant tender stems in four developmental stages of bronze stage, discoloration stage, light green stage and stable stage as template, Premix Ex Taq as reaction enzyme, fluorescent quantitative PCR reaction was performed using LightCycler of Roche Company @2.0 fluorescent quantitative PCR instrument. Fluorescent quantitative primers are HbAn1 primers in Table 1. The reaction program was 95°C for 1min, 95°C for 10s, 58°C for 10s, 72°C for 20s, and 72°C for 1min. The reaction system was 10 μl, including 5 μl 2×Premix Ex Taq, 0.25 μl primer (10 μmol), and 0.5 μl cDNA template. The reaction results were analyzed using LightCycler Software4.05 software. RH8 was used as the internal reference gene, and the standard cDNA and the test samples were set to repeat 3 times. Quantitative analysis found that the expression level of HbAn1 gradually decreased w...

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Abstract

The invention relates to molecular biology and specifically discloses an R2R3-MYB transcription factor HbAn1 capable of positively regulating rubber tree anthocyanin synthesis and an encoding gene and application thereof. An amino acid sequence of the transcription factor HbAn1 is shown as SEQ ID NO.1, and a nucleotide sequence of the encoding gene is shown as SEQ ID NO.2. According to the invention, an encoding gene sequence of the R2R3-MYB transcription factor HbAn1 capable of regulating anthocyanin synthesis is separated from rubber trees for the first time, and functions and an application range of the R2R3-MYB transcription factor HbAn1 are determined. Researches and analysis find that the expression of HbAn1 is gradually reduced along with leaf senescence and anthocyanin fading; meanwhile, HbAn1 expression in dark-culture tender stems is higher than that in light-culture tender stems, and the HbAn1 expression has positive correlation with the anthocyanin content. The transcription factor can positively regulate rubber tree anthocyanin synthesis and achieves the purpose of increasing an anthocyanin accumulation amount through self overexpression.

Description

technical field [0001] The invention relates to molecular biology, in particular to the R2R3-MYB transcription factor HbAn1 positively regulating the synthesis of rubber tree anthocyanins, its coding gene and its application. Background technique [0002] As early as 1994, Arokiaraj et al. in Malaysia obtained the world's first rubber tree transgenic plant by using the gene gun method. However, so far, nptII is used as a screening marker, and gus or gfp is used as a reporter gene. The screening efficiency is low, and it is necessary to develop new Hevea transgenic screening genes. Anthocyanins are usually red or purple, and because they are directly visible to the naked eye, they have been applied to visual screening markers, virus infection tracers, and real-time monitoring of boron deficiency in corn, tobacco, tomato, sweet potato, grape, apple and other transgenic systems. It is mainly regulated by the MBW (MYB-bHLH-WD40) complex composed of three types of transcription ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/825
Inventor 黄天带华玉伟方永军畅姣陈涛辛士超黄华孙
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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