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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (5): 1169-1180.doi: 10.3724/SP.J.1006.2022.14058

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

Regulation of flavonoid synthesis by BjA09.TT8 and BjB08.TT8 genes in Brassica juncea

HUANG Wei1,2(), GAO Guo-Ying1(), WU Jin-Feng1,5, LIU Li-Li1,5, ZHANG Da-Wei1,5, ZHOU Ding-Gang1,5, CHENG Hong-Tao2, ZHANG Kai-Xuan3, ZHOU Mei-Liang3, LI Mei4, YAN Ming-Li1,4,5,*()   

  1. 1School of Life Science, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
    2Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
    3Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    4Crop Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China
    5Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Xiangtan 411201, Hunan, China
  • Received:2021-04-11 Accepted:2021-07-12 Online:2022-05-12 Published:2021-08-05
  • Contact: YAN Ming-Li E-mail:2646213662@qq.com;1019982091@qq.com;ymljack@126.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Key Research and Development Program of China(2016YFD0100202);National Natural Science Foundation of China(31971980);Key Research and Development Program of Hunan(2020NK2045)

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Abstract:

bHLH transcription factors TT8 can effectively regulate the biosynthesis of plant flavonoids. In this study, two copies of TT8 were obtained in Brassica juncea by homologous cloning method, and named BjA09.TT8 and BjB08.TT8, which encoded 521 and 517 amino acids, respectively. Interestedly, qRT-PCR showed that the relative expression levels of the two copies in leaves were significantly higher than those in stems and roots, both responded to jasmonic acid (JA) signal, and the relative expression levels of Bju.TT8 reached maximum after 50 µmol L-1methyl jasmonate treatment for 0.5 hour. Furthermore, we found that both Bj.TT8 can increase the contents of total flavonoids in hairy roots of purple leaf mustard and green leaf mustard cultiver Sichuan Yellow Seed by hairy root system overexpression assay. It promoted the expression of flavonoid synthesis Bj.CHS, indicating they acted as redundant function. Over expression of BjA09.TT8 and BjB08.TT8 of wild-type Arabidopsis and Arabidopsis tt8 mutant revealed that the leaf colour of overexpressed Arabidopsis plant turned purple. Moreover, the contents of total flavonoid and proanthocyanidins increased significantly. In summary, BjA09.TT8 and BjB08.TT8 genes could promote the synthesis of flavonoids in Brassica juncea, which provided a reference for further analysis of the regulatory mechanism of proanthocyanidin synthesis in Brassica.

Key words: Brassica juncea, TT8, regulate, flavonoid, proanthocyanidin

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Primer purpose
TT8-A-F ATGGATGAATTAAGTATTATACCG 基因克隆 Gene cloning
TT8-A-R CTAGAGTTTATTATTATATATGA
TT8-B-F ATGGATGAATCAAGTATTATACAG
TT8-B-R CTAGAGTTTATTTTGAGATATG
3301-TT8-A-NcoIF GGGGACTCTTGACCATGGTAATGGATGAATTAAGTATTATACCG TT8载体构建引物
3301-TT8-A-Eco91IR GAAATTCGAGCTGGTCACCCTAGAGTTTATTATTATATATGA TT8 vector primer
3301-TT8-B-NcoIF GGGGACTCTTGACCATGGTAATGGATGAATCAAGTATTATACAG
3301-TT8-B-Eco91IR GAAATTCGAGCTGGTCACCCTAGAGTTTATTTTGAGATATG
qRT-PCR-Actin7-F GCTGACCGTATGAGCAAAG qRT-PCR检测引物
qRT-PCR-Actin7-R AAGATGGATGGACCCGAC qRT-PCR detection primers
qRT-PCR-TT8-A-F ACCAAAAGCGGATGCGTAT
qRT-PCR-TT8-A-R TCTATACCCAGCTCCTTAAGTACCT
qRT-PCR-TT8-B-F GAAGCAACCAATAGTTTAGTAGATACACA
qRT-PCR-TT8-B-R CGATGATTGAACGTAAGAAGATGTGGA
qRT-PCR-PAL-F AGAGCTTTTGACCGGAGAGA
qRT-PCR-PAL-R TTAATCACTCTTAACATATAGGAATGGGAG
qRT-PCR-CHS-F TCTTCATATTGGACGAGATGAGGA
qRT-PCR-CHS-R GCGTTTCTGTTCAAACAGGAA
qRT-PCR-CHI-F CTTTGGAGCGACCATTAGAG
qRT-PCR-CHI-R AGACAAAGCTTAACAAGAGAGGT
qRT-PCR-F3’H-F TGATTGGGAATTAGCTGGAGGA
qRT-PCR-F3’H-R AGTTAAATTTTAACCCGACCCGA
qRT-PCR-F3H-F ATCTTGGAGGAGCCAATTACGT
qRT-PCR-F3H-R ACACAAGGAGTCTAAGCGATGA
qRT-PCR-FLS-F ACTAGGAATGTGATCGCACCA
qRT-PCR-FLS-R TCAGAGGGATTAGGTTTACGG
qRT-PCR-DFR-F TCTTTGGAACAGGTTTGAAGGA
qRT-PCR-DFR-R TAAAGTGACAGGGAGAAAACCCT
qRT-PCR-ANS-F AAGCCGTTGCCTGAGA
qRT-PCR-ANS-R AGAGTTTCAGACTCAGACTTCA
qRT-PCR-BAN-F GGTTTTTGTTGTTAGGGAAAGA
qRT-PCR-BAN-R ATATGCTTACTCTGACAAAACAT

Fig. 1

Secondary structure and three-dimensional structure prediction of BjA09.TT8 and BjB08.TT8 in purple leaf mustard A: secondary structure of BjA09.TT8 and BjB08.TT8 in purple leaf mustard; B: three-dimensional structure prediction of BjA09.TT8 and BjB08.TT8 in purple leaf mustard."

Fig. 2

Relative expression patterns of BjA09.TT8 and BjB08.TT8 in PM and SY A: expression analysis of BjA09.TT8 gene in PM; B: expression analysis of BjA09.TT8 gene in SY; C: expression analysis of BjB08.TT8 gene in PM; D: expression analysis of BjB08.TT8 gene in SY. PM: purple-leaf mustard rape; SY: Sichuan yellow seed. *, ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 3

Relative expression patterns of BjA09.TT8 and BjB08.TT8 gene induced by MeJA in purple-leaf mustard rape * indicates significant difference at the 0.05 probability level."

Fig. 4

Relative expression patterns of BjA09.TT8 and BjB08.TT8 gene in hairy roots of Brassica juncea A: relative expression of BjA09.TT8 gene in hairy roots of PM; B: relative expression of BjA09.TT8 gene in hairy roots of SY; C: relative expression of BjB08.TT8 gene in hairy roots of PM; D: relative expression of BjB08.TT8 gene in hairy roots of SY. A4: wild-type hairy roots; 3301: hairy roots transfected with pCAMBIA3301 empty vector; PTT8-A1-A3: transgenic PM hairy roots; PTT8-B1-B3: transgenic PM hairy roots; STT8-A1-A3: transgenic SY hairy rootsr; STT8-B1-B3: transgenic SY hairy roots; PM: purple-leaf mustard rape; SY: Sichuan yellow seed. Each set of data represents the mean ± SD of three biological replicates. * and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 5

Relative expression patterns of the key of enzyme genes in flavonoids metabolism pathway in transgenic with BjA09.TT8 and BjB08.TT8 gene hairy roots A: relative expression of the key of enzymes in flavonoids metabolism pathway in PM hairy roots from A4, pCAMBIA3301 and BjA09.TT8 roots were detected; B: relative expression of the key of enzyme genes of flavonoid metabolism pathway in SY hairy roots from A4, pCAMBIA3301 and BjA09.TT8 roots were detected; C: relative expression of the key of enzymes in flavonoids metabolism pathway in PM hairy roots from A4, pCAMBIA3301 and BjB08.TT8 roots were detected; D: relative expression of the key of enzyme genes of flavonoid metabolism pathway in SY hairy roots from A4, pCAMBIA3301 and BjB08.TT8 roots were detected. ACTIN7 was used as internal control. PM: purple-leaf mustard rape; SY: Sichuan Yellow seed. 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. Error bars represent the standard deviation from three replicates. * and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 6

Detection of the total amount of flavonoids content in transgenic with BjA09.TT8 and BjB08.TT8 hairy roots A: measurement of the total amount of flavonoids content in PM hairy roots from A4, pCAMBIA3301 and BjA09.TT8; B: measurement of the total flavonoids content in SY hairy roots from A4, pCAMBIA3301 and BjA09.TT8; C: measurement of the total flavonoids content in PM hairy roots from A4, pCAMBIA3301 and BjB08.TT8; D: measurement of the total flavonoids content in SY hairy roots from A4, pCAMBIA3301 and BjB08.TT8; PM: purple-leaf mustard rape; SY: Sichuan yellow seed. Each set of data represents the mean ± SD of three biological replicates. * and ** indicate significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 7

Relative expression patterns of BjA09.TT8 and BjB08.TT8 in transgenic Arabidopsis leaves A: relative expression of transgenic with BjA09.TT8 Arabidopsis; B: relative expression of transgenic with BjB08.TT8 Arabidopsis. WT: wild-type Arabidopsis; 3301: transfer pCAMBIA3301 vector to Arabidopsis; TT8-A1-A3, TT8-B1-B3: transgenic positive lines. ** indicate significant difference at the 0.01 probability level."

Fig. 8

Detection of the total amount of flavonoids and proathocyanidins content in transgenic Arabidopsis A: measurement of the total amount of flavonoids content in Arabidopsis of WT, pCAMBIA3301 and BjA09TT8; B: measurement of the proathocyanidins content in Arabidopsis of WT, pCAMBIA3301 and BjB08TT8. TT8-A9, TT8-B8: transgenic positive lines. Each set of data represents the mean ± SD of three biological replicates. ** indicates significant difference at the 0.01 probability level."

Fig. 9

Phenotypic identification of transgenic Arabidopsis a: wild-type Arabidopsis plants; b: BjA09.TT8 gene Arabidopsis; c: BjB08.TT8 gene Arabidopsis."

Fig. 10

Detection of gene expression after overexpression of BjA09.TT8 and BjB08.TT8 in Arabidopsis tt8 WT: wild-type Arabidopsis; 3301: transfer pCAMBIA3301 vector to Arabidopsis; tt8-TT8-A1-A3: positive lines of BjA09.TT8 gene was replenished; tt8-TT8-B1-B3: positive lines of BjB08.TT8 gene was replenished. ** indicates significant difference at the 0.01 probability level."

Fig. 11

Detection of the total amount of flavonoids content and proathocyanidins content after overexpression of BjA09.TT8 and BjB08.TT8 in Arabidopsis tt8 A: measurement of the total amount of flavonoids content in replenish with BjA09.TT8 and BjB08.TT8 Arabidopsis tt8 mutant; B: measurement of the proathocyanidins content in replenish with BjA09.TT8 and BjB08.TT8 Arabidopsis tt8 mutant. TT8-A1-A3: positive lines of BjA09.TT8 gene was replenished; TT8-B1-B3: positive lines of BjB08.TT8 gene was replenished. Each set of data represents the mean ± SD of three biological replicates. ** indicates significant difference at the 0.01 probability level."

Fig. 12

Phenotypes of BjA09.TT8 and BjB08.TT8 overexpressed in Arabidopsis tt8 a: tt8 mutant Arabidopsis plants; b: Arabidopsis tt8 mutant of BjA09.TT8 gene was replenished; c: Arabidopsis tt8 mutant of BjB08.TT8 gene was replenished."

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