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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (10): 1468-1476.doi: 10.3724/SP.J.1006.2018.01468

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

Construction of Hairy Root Induction System and Functional Analysis of TTG1 Gene in Brassica juncea

Long LI1,2,Cheng CHENG1,2,Xiao-Fang WU1,2,Da-Wei ZHANG1,3,Li-Li LIU1,3,Jing ZHOU1,Mei-Liang ZHOU2,Kai-Xuan ZHANG2,*(),Ming-Li YAN1,3,*()   

  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 Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-Polluted Soils, Hunan University of Science and Technology, Xiangtan 411201, Hunan, China
  • Received:2018-02-14 Accepted:2018-06-12 Online:2018-10-10 Published:2018-07-02
  • Contact: Kai-Xuan ZHANG,Ming-Li YAN E-mail:zhangkaixuan@caas.cn;ymljack@126.com
  • Supported by:
    This study was supported by the National Key Research and Development Program(2016YFD0100202);the Natural Science Foundation of Hunan Province(2016JJ1010);the National Natural Science Foundation of China(31572457);the Foundation of Hunan Education Department(17K035)

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Abstract:

Yellow seed rape has the advantages of thin seed coat and high oil yield. It is of great significance to study the formation of yellow seed in rapeseed. Previous studies showed that TTG1 (TRANSPARENT TESTA GLABRA 1) gene was involved in the formation of seed coat color. In this study, Agrobacterium rhizogenes A4 strain was used to induce hairy roots of Sichuan yellow, and investigate the effects of bacterial concentration and different sources of explants on hairy root induction. The highest average rooting rate was 71.5% when the OD value of A. rhizogenes A4 reached to 0.8. The average rooting rate of hypocotyls was significantly higher than that of cotyledons and up to 87.3% after the infection. Overexpression of TTG1 gene in Sichuan yellow hairy roots inhibited the expression of DFR (Dihydroflavonol4-reductase), ANS (Anthocyanidin synthase), and BAN (Anthocyanidin reductase) genes, encoding key enzymes of the proanthocyanidins biosynthesis. In this study, we optimized the induction system of hairy roots in B. juncea, and analyzed the function of TTG1 in the regulation of proanthocyanidins synthesis pathway, which provide a new idea and method for the functional analysis of genes in the hairy root system.

Key words: Brassica juncea, hairy root, inducing condition, TTG1 gene, proanthocyanidins

Table 1

List of qRT-PCR primers"

基因
Gene		 名称
Name		 引物
Primer (5°-3°)		 PCR长度
PCR length (bp)
BAN Anthocyanidin reductase F: ATCTTCCATGTCGCAACTCC
R: CGAGAGAGATAAACTGCTAG		 200
DFR Dihydroflavonol4-reductase F: AGGATTCATTGGTTCATGGCTCG
R: TCCGTTTATGGCGTCATCGTAGC		 189
ANS Anthocyanidin synthase F: GGACAACTTGAGTGGGAAGATTA
R: GCAAACGAAGACACTTAGCGTAC		 132
TTG1 protein (“WD40” or “WDR”) F: GGCTGTAAGCATATCTGTTCCG
R: GAGAAAGCAATCCCAATCCAATG		 173
内参
Internal reference		 Actin7 F: GCTGACCGTATGAGCAAAG
R: AAGATGGATGGACCCGAC		 182

Table 2

Effects of different explants on the induction rate of hairy roots"

外植体
Explant		 感染外植体数量
Number of infected explants		 外植体发根数量
Number of explants rooted		 诱导率
Induction rate (%)
下胚轴 Hypocotyl 30 26.2±0.3 a 87.3±0.01 a
子叶 Cotyledon 30 12.0±0.5 b 40.0±0.02 b

Fig. 1

Process of the hairy root induction A: 14 d SY rape sterile seedling; B: 7 d explants with hairy roots; C: 14 d hairy roots; D: 21 d hairy roots on the screening medium; E: 28 d hairy roots in the liquid medium; F: 21 d hairy roots in the liquid medium."

Fig. 2

Induction efficiency of different concentrations of A. rhizogenes A4 on hairy roots"

Fig. 3

Vector construction A: pCAMBIA3301 vector; B: pCAMBIA3301 vector containing TTG1 gene."

Fig. 4

Identification of the transformed A. rhizogenes A4 A: identification of TTG1 gene in A. rhizogenes A4. +: pCAMBIA3301-TTG1, -: H2O; B: identification of GUS gene in A. rhizogenes A4. +: pCAMBIA3301 plasmid, -: H2O."

Fig. 5

Identification of transgenic hairy roots A: three lines of hairy roots overexpressing GUS gene tested by PCR. +: pCAMBIA3301 plasmid; -: wild type hairy roots; B: three lines of hairy roots overexpressing TTG1 gene tested by PCR. +: pCAMBIA3301-TTG1; -: wild-type hairy roots."

Fig. 6

Staining of wild type and transgenic hairy roots L1, L2, L3: three lines of wild-type hairy roots; Z1, Z2, Z3: three lines of hairy roots overexpressing GUS genes."

Fig. 7

Growth comparison of wild type and transgenic hairy roots A1: wild-type hairy root tip; B1: GUS-overexpression hairy root tip; C1: TTG1-overexpression hairy root tip; A2: 14 d old wild-type hairy roots; B2: 14 d old GUS-overexpression hairy roots; C2: 14 d old TTG1-overexpression hairy roots."

Fig. 8

Related genes expression tested by qRT-PCR A: TTG1 gene; B: DFR gene; C: ANS gene; D: BAN gene."

Fig. 9

MBW complex formed by TT2, TT8, and TTG1 regulates the expression of DFR, ANS, and BAN genes in the proanthocyanidin metabolism pathway of Arabidopsis thaliana The enzyme genes of the PA metabolic pathway are chalconesynthase (CHS), chalconeisomerase (CHI), flavanone3-hydroxylase (F3H), flavonoid3'-hydroxylase (F3’H), dihydroflavonol4-reductase (DFR), anthocyaninsynthase (ANS), anthocyanidinreductase (BAN), MATE secondary transporter (TT12), and glutathione S-transferase (TT19)."

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