Crops ›› 2021, Vol. 37 ›› Issue (4): 1-9.doi: 10.16035/j.issn.1001-7283.2021.04.001

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The Overexpression of FtbZIP5 Improves Accumulation of Flavonoid in the Hairy Roots of Tartary Buckwheat and Its Salt Tolerance

Weng Wenfeng1,2(), Wu Xiaofang2, Zhang Kaixuan2, Tang Yu3, Jiang Yan1, Ruan Jingjun1(), Zhou Meiliang2()   

  1. 1College of Agronomy, Guizhou University, Guiyang 550025, Guizhou, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3College of Food Science and Technology, Sichuan Tourism University, Chengdu 610100, Sichuan, China
  • Received:2021-01-12 Revised:2021-05-08 Online:2021-08-15 Published:2021-08-13
  • Contact: Ruan Jingjun,Zhou Meiliang E-mail:1332721469@qq.com;jjruan@gzu.edu.cn;zhoumeiliang@caas.cn

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Abstract:

The bZIP transcription factors, with an important role in salt stress network, can regulate flavonoid accumulation in plant. In this paper, the bZIP family gene FtbZIP5, with transcriptional activation activity, was cloned from the ‘Chuanqiao 1’. Expression of FtbZIP5 gene in stems and leaves was higher than that in root when treated by NaCl and abscisic acid. Flavonoid detection was carried out on hairy roots of overexpressing the FtbZIP5 gene strain, and results showed that flavonoid accumulation in overexpressing strains was significantly higher than that in wild-type plants. And the expression of key enzyme gene flavanone-3-hydroxylase (F3H) in flavonoid synthesis pathway was high, which can speculate that accumulation of total flavonoids in overexpressed hair root strains was associated with F3H expression. Under the stress of 100mmol/L NaCl, the accumulation of total flavonoids in all lines was suppressed, and the content of total flavonoids in overexpression lines decreased to 0.63mg/g, and the expression of F3H was still higher than that of the control. Catalase activity of the control was significantly lower than that of the overexpressing strains after stressing. After stressing by NaCl, the content of malondialdehyde in wild-type plants increased and that in overexpressing strains was stable. The above results indicated that overexpressing the FtbZIP5 gene in the hairy roots, the increase in total flavonoid content may be regulated by the up-regulation of the key enzyme gene F3H; and FtbZIP5 also improved the salt tolerance of tartary buckwheat roots. This paper analyzed the role of FtbZIP5 in the accumulation of total flavonoids and salt tolerance in the hairy roots of tartary buckwheat, and laid a foundation of studying the salt tolerance of buckwheat and analyzing the salt tolerance mechanism of buckwheat.

Key words: Tartary buckwheat, bZIP, Flavonoids, Salt stress

Table 1

Summary of primer sequence"

引物名称Primer 引物序列Primer sequence (5′-3′) 用途Function
1307-FtbZIP5-F CTAGAACTAGTGGATCCATGGGAAGTAACCTG 构建过表达载体
1307-FtbZIP5-R GCGGAGTACCCGGGTACCTCACCACACACCAG
pAS-FtbZIP5-F TACCCAGCTTTGACTCATATGATGGGAAGTAACCTG 转录活性分析
pAS-FtbZIP5-R TTAGCTTGGCTGCAGGTCGACTCACCACACACCAG
FtbZIP5-F ATGGGAAGTAACCTG 基因克隆
FtbZIP5-R TCACCACACACCAG
TLF CTCAAGCAA TCAAGCA TTCTAC 转基因毛状根阳性检测
RT1-QF TCAAA TAAGCTCGCCTCCCA
RT1-QR GCTGCA TTTTGTCAAGAGCG
FtbZIP5-QF CAAGTTCCTTTGCAGTCGCC
FtbZIP5-QR TAATCCCTGTGCAACCCACC
FtCHS-QF AGACGCATGTGCGACAAATC
FtCHS-QR CCACTCCTTGATGGCCTTCT
FtCHI-QF GGCACAGTACTCTGAAAAGGT
FtCHI-QR CTTCCCGAATGCAATCCTTAATG
FtF3H-QF AACAGCAGCCGTTTGTCAAT
FtF3H-QR TGCTCCTTAGCCAGCTTCTT
FtPAL-QF ACAAGGCGTTACATGGAGGA
FtPAL-QR CCAAGCTAGGGTTTCTCCCA

Table 2

Summary of online platforms"

在线平台Online platform 网址Website 功能Function
Expasy https://web.expasy.org/compute_pi 蛋白理化性质
SOMPA https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html 蛋白二级结构
Swissmodel https://swissmodel.expasy.org/interactive 蛋白三级结构
Cell-ploc 2.0 http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ 亚细胞定位

Fig.1

CDS sequence amplification and bioinformatics analysis of FtbZIP5 in tartary buckwheat a: the secondary structure prediction of FtbZIP5 protein; alpha helix (blue); extended strand (red); beta turn (green); random coil (purple). b: the tertiary structure of FtbZIP5 protein. c: phylogenetic tree analysis of FtbZIP5 protein; d: amplification of FtbZIP5 CDS sequence"

Fig.2

Transcription activation assay of FtbZIP5 1×: stock solution; 10×: diluting 10 times"

Fig.3

Expression specificity of FtbZIP5 gene under NaCl and ABA induction The significant difference is represented by“**”, indicating P < 0.01, the same below"

Fig.4

Induction of tartary buckwheat hairy roots (a) and expression detection of FtbZIP5 (b) ① Chuanqiao 1 aseptic seedlings for 10 days; ② co-culture of explants; ③ explants cultured for 10 days; ④ positive hairy root"

Fig.5

Determination of total flavonoids and gene expression of key enzymes in the flavonoid synthesis pathway PAL: phenylalanine ammonia lyase gene; CHS: chalcone synthase gene; CHI: chalcone isomerase gene; F3H: flavone 3-hydroxylase gene; RT1: rhamnosyl transferase gene. “*”indicates P < 0.05, the same below"

Fig.6

Effects of salt stress on secondary metabolites of hairy roots"

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