<i>CiMYB15</i> from <i>Caragana Intermedia</i> Positively Regulates Flavonoids Metabolism of Arabidopsis

doi:10.13523/j.cb.20181002

China Biotechnology

2018, Vol. 38

Issue (10): 8-19 DOI: 10.13523/j.cb.20181002

Orginal Article

CiMYB15 from Caragana Intermedia Positively Regulates Flavonoids Metabolism of Arabidopsis

Wen-juan CHAI¹,Qi YANG^1,^2,³,Guo-jing LI^1,^2,³,Rui-gang WANG^1,^2,^3,^**(

)

1 College of Life Sciences,Inner Mongolia Agricultural University,Hohhot 010018,China
2 Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology,Hohhot 010018,China
3 Inner Mongolia Scientific Innovation Team of Genetic Resource Utilization and Molecular improvement of Stress Resistant Plants, Hohhot 010018, China

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Abstract

Objective: R2R3-MYB transcription factors regulate primary and secondary metabolism in plants.Methods: A R2R3-MYB encoding sequence, characterized and cloned from drought treated transcriptome of Caragana intermedia, was named as CiMYB15. The gene was transferred into Arabidopsis thaliana, and the total flavonoids contents of transgenic lines and wild type were measured by spectrophotometric method, and the expression of AtCHS in transgenic plants was analyzed by qRT-PCR. A 1 580bp fragment of CiMYB15 promoter was isolated by genome walking. The results revealed that: (1) the length of CiMYB15 gDNA was 1 960bp, it’s consisted of three exons (134, 131 and 521 bp) and two introns (281 and 893 bp). The open reading frame (ORF) encodes a polypeptide of 262 amino acids. (2)The main cis-elements of CiMYB15 promoter include abiotic stress responded elements (G-box, P-box, GT1-motif and MBS), biotic stress responded elements (BOX-W1 and EIER), and MYB binding sites of flavonoids synthase regulatory genes. (3) The expression of CiMYB15 was induced by UV-B. (4) Total flavonoids contents of CiMYB15 overexpression plants were higher than that of wild type Arabidopsis. (5) Furthermore, the expression level of AtCHS was increased in transgenic Arabidopsis. In brief, CiMYB15 positively regulated the flavonoids metabolism.

Key words： Caragana intermedia Total flavonoids CiMYB15 Promoter Chalcone synthase (CHS)

Received: 07 July 2018 Published: 09 November 2018

ZTFLH:

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Corresponding Authors: Rui-gang WANG E-mail: ruigangwang@126.com

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Cite this article:

Wen-juan CHAI,Qi YANG,Guo-jing LI,Rui-gang WANG. CiMYB15 from Caragana Intermedia Positively Regulates Flavonoids Metabolism of Arabidopsis. China Biotechnology, 2018, 38(10): 8-19.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181002 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I10/8

Table 1 Primers used in gene cloning and functional analysis

Fig.1 Gel electrophoresis of PCR products of CiMYB15 1, 2: The amplified cDNA; 3, 4: The amplified gDNA; M: 1kb DNA ladder

Fig.2 AAAcDNA, gDNA and deduced amino acid sequence of CiMYB15 The underlined parts indicate intron regions

Fig.3 The alignment of amino acid sequence of CiMYB15 and other plant MYBs Sequences in this map are obtained from C. intermedia, A. thaliana, G. max, and M. truncatula from top to bottom; The black full line and dotted line indicates R2 domain and R3 domain respectively, black points indicate IDxSFW-MxFWFD

Fig.4 Phylogenetic analyses of CiMYB15 and other plant MYBs Phylogenetic tree was constructed by Neighbor-Joining method; the value of bootstrap replication was set as 1000.

Fig.5 PCR products of CiMYB15 promoter 1: Product of the first extension; 2: Product of the second extension; 3, 4: Products of the third extension; 5, 6: Products of specific primers; M1: 1kb DNA ladder; M2: DL5000

Table 2 Prediction analysis of the CiMYB15 promoter

Fig. 6 CiMYB15 gene expression analysis under UV-B treatment by qRT-PCR CiEF1α was selected as reference gene. The result was calculated by 2^-ΔΔCT

Fig.7 The construction of recombinant vector pCanG-CiMYB15 and expression analysis of CiMYB15 in transgenic lines (a) Identification of the recombinant vector pCanG-CiMYB15: 1, Vector control; 2, The recombinant vector digested by Spe I and Sal I (b) RT-PCR assay of CiMYB15 transgenic lines: C-, wild type Arabidopsis cDNA as negative control; C⁺, Caragana intermedia cDNA as positive control; Other numbers are overexpression lines (c) Expression level of CiMYB15 in transgenic lines was analyzed by qRT-PCR (AtEF1α was selected as reference gene. The result was calculated by 2^-ΔCT. Y-axis was Logarithmic). M: 1kb DNA ladder

Fig.8 Total flavonoids content of wild type and transgenic Arabidopsis lines

Fig.9 The detection of flavonoids metabolism related genes from wild type and CiMYB15 transgenic lines AtEF1α was selected as reference gene. The result was calculated by 2^-ΔΔCT


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