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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (9): 1322-1331.doi: 10.3724/SP.J.1006.2020.04008

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Regulation of flavonoid pathway by BjuB.KAN4 gene in Brassica juncea

GAO Guo-Ying1,2,3(), WU Xiao-Fang1,2,3, HUANG Wei1,3, ZHOU Ding-Gang1,3, ZHANG Da-Wei1,3, ZHOU Mei-Liang2, ZHANG Kai-Xuan2,*(), YAN Ming-Li1,3,*()   

  1. 1 College of Life Science, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
  • Received:2020-01-12 Accepted:2020-04-15 Online:2020-09-12 Published:2020-04-27
  • Contact: Kai-Xuan ZHANG,Ming-Li YAN E-mail:1019982091@qq.com;zhangkaixuan@caas.cn;ymljack@126.com
  • Supported by:
    National Key Research and Development Program of China(2016YFD0100202);National Natural Science Foundation of China(31971980);Foundation of Hunan Education Department(17K035)

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Abstract:

MYB transcription factors KAN4 can effectively regulate the biosynthesis of plant proanthocyanidins. In order to investigate the function of the MYB transcription factor KAN4 on the regulation of proanthocyanidin synthesis in Brassica juncea, we cloned BjuB.KAN4 gene from purple-leaf mustard rape (PM), which encoded 266 amino acids. The BjuB.KAN4 protein contained a highly conserved MYB-like DNA-binding domain belongs to the 1R-MYB transcription factor family. BjuB.KAN4 gene expression showed significantly higher level in root than in leaf and stem. GUS histochemical staining showed that this gene might be expressed in vascular tissues. Overexpression of BjuB.KAN4 in hairy roots of PM and Sichuan Yellow (SY) increased the expression level of some key enzyme genes in the flavonoids biosythesis pathway, such as Bju.CHS and Bju.DFR. The total flavonoids content was 2.798 mg g-1 in transgenic roots of PM, which was 1.3 times higher than that of the control, and 2.567 mg g-1 in transgenic roots of SY, which was 1.2 times higher than that of control. In transgenic Arabidopsis plants overexpressing BjuB.KAN4, total flavonoids was 0.237 mg g-1, which was 1.5 times higher than that of wild type, however, the proanthocyanidins content decreased. This study indicates that the BjuB.KAN4 gene is involved in the regulation of PM flavonoid synthesis, and provides a reference for the research of regulation mechanism of proanthocyanidins synthesis in Brassica.

Key words: Brassica juncea, BjuB.KAN4, flavonoid synthesis, hairy roots

Table 1

Primer sequences"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Purpose of primer
KAN4-B-F AGTGAGATGATCATGTTCGAGTC 基因克隆Gene cloning
KAN4-B-R CAATTAGCACTTGAGAAGGGTTA
KB-NcoIF GGGGACTCTTGACCATGGTAATGATCATGTTCGAG KAN4载体构建引物
KAN4 vector primers
KB-Eco91IR GAAATTCGAGCTGGTCACCCAATTAGCACTTGAGAAGGG
KB-pro-BamHIF GGATCCATTGTCGTTGGTGACAGAAAC KAN4启动子载体构建引物
KB-pro vector primer
KB-pro-NcoIR CCATGGTTGGAGTTTTCAGAACTTTGGC
Actin7-F GCTGACCGTATGAGCAAAG qRT-PCR检测引物
qRT-PCR detection primers
Actin7-R AAGATGGATGGACCCGAC
qKAN4-B-F AGGACCCAAAGATCTCCTTGGT
qKAN4-B-R TTACACATAGTCAATCCCCCAACT
qPAL-F AGAGCTTTTGACCGGAGAGA
qPAL-R TTAATCACTCTTAACATATAGGAATGGGAG
qCHS-F TCTTCATATTGGACGAGATGAGGA
qCHS-R GCGTTTCTGTTCAAACAGGAA
qCHI-F CTTTGGAGCGACCATTAGAG
qCHI-R AGACAAAGCTTAACAAGAGAGGT
qF3’H-F TGATTGGGAATTAGCTGGAGGA
qF3’H-R AGTTAAATTTTAACCCGACCCGA
qF3H-F ATCTTGGAGGAGCCAATTACGT
qF3H-R ACACAAGGAGTCTAAGCGATGA
qFLS-F ACTAGGAATGTGATCGCACCA
qFLS-R TCAGAGGGATTAGGTTTACGG
qDFR-F TCTTTGGAACAGGTTTGAAGGA
qDFR-R TAAAGTGACAGGGAGAAAACCCT
qANS-F AAGCCGTTGCCTGAGA
qANS-R AGAGTTTCAGACTCAGACTTCA
qBAN-F GGTTTTTGTTGTTAGGGAAAGA
qBAN-R ATATGCTTACTCTGACAAAACAT

Fig. 1

Phylogenetic tree of BjuB.KAN4 and its homologous proteins from other species AtKAN4: Arabidopsis thaliana NP199077; BjuB.KAN4: Brassica juncea; BolC.KAN4: Brassica oleracea XP013619450; BraA. KAN4: Brassica rapa XP009123685; CpKAN4: Carica papaya XP021896463; CrKAN4: Capsella rubella XP006280924; CsKAN4: Camelina sativa XP010442142; EsKAN4: Eutrema salsugineum XP006403368; GsKAN4: Glycine soja XP028198852; PeKAN4: Populus euphratica XP011030894; RsKAN4: Raphanus sativus XP018449685; ThKAN4: Tarenaya hassleriana XP010530376; VvKAN4: Vitis vinifera CBI19594; ZmKAN4: Zea mays NP001168849."

Fig. 2

Tissue-specific expression analysis of BjuB.KAN4 gene by qRT-PCR A: relative expression of BjuB.KAN4 in different tissues of PM; B: relative expression of BjuB.KAN4 in different tissues of SY. ** Significant at P < 0.01."

Fig. 3

Induction of hairy roots in Brassica juncea and identification of transgenic hairy roots A: induction process of hairy roots in Brassica juncea. B: DNA identification of PM and SY transgenic hairy roots; PKB1-PKB3: positive hairy roots of PM; SKB1-SKB3: positive hairy roots of SY; -: H2O (negative control); +: plasmid (positive control). C: relative expression of BjuB.KAN4 in hairy roots of PM. D: relative expression of BjuB.KAN4 in hairy roots of SY. A4: hairy roots infected by A4; 3301: hairy roots transfected with pCAMBIA3301 empty vector; KAN4-B1-B3: transgenic hairy roots. ** Significant at P < 0.01."

Fig. 4

Relative expression of the key enzyme genes involved in flavonoids biosynthesis pathway in transgenic hairy roots A: transgenic hairy roots of PM; B: transgenic hairy roots of SY. A4: hairy roots infected by A4; 3301: hairy roots transfected with pCAMBIA3301 empty vector; KAN4-B04: transgenic hairy roots. internal control: Actin7, error bars represent the standard deviation of triplicate runs for qRT-PCR. PAL: phenylalanine deaminase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavone 3-hydroxylase; F3’H: flavonoid 3’-hydroxylase; FLS: flavonol synthase; DFR: dihydroflavonol reductase; ANS: anthocyanin synthase; BAN: anthocyanin reductase. * Significant at P < 0.05; ** Significant at P < 0.01."

Fig. 5

Measurement of the total flavonoids content in transgenic hairy roots A: transgenic hairy roots of PM; B: transgenic hairy roots of SY. A4: hairy roots infected by A4; 3301: hairy roots transfected with pCAMBIA3301 empty vector; KAN4-B04: transgenic hairy roots. Each set of data represents the mean ± SD from three biological replicates. * Significant at P < 0.05."

Fig. 6

Identification of transgenic Arabidopsis plants overexpressing BjuB.KAN4 A: identification of transgenic Arabidopsis by PCR; B: relative expression of BjuB.KAN4 in transgenic Arabidopsis. WT: wild-type of Arabidopsis; KB-1-6: transgenic positive lines; 3301: Arabidopsis overexpressing empty vector; KAN4-B1-3: Arabidopsis lines overexpressing BjuB.KAN4 gene. ** Significant at P < 0.01."

Fig. 7

Measurement of the total flavonoids and the proathocyanidins contents in transgenic Arabidopsis A: measurement of the total flavonoids content in transgenic Arabidopsis; B: measurement of the proathocyanidins content in transgenic Arabidopsis. WT: wild-type Arabidopsis; 3301: Arabidopsis overexpressing empty vector; KAN4-B04-1-3: Arabidopsis lines overexpressing BjuB.KAN4 gene. Each set of data represents the mean ± SD of three biological replicates. * Significant at P < 0.05; ** Significant at P < 0.01."

Fig. 8

Histochemical staining of transgenic Arabidopsis plants A, B, C: independent Arabidopsis lines overexpressing 35Spro::GUS; D, E, F: independent Arabidopsis lines overexpressing BjuB.KAN4pro::GUS."

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