Crops ›› 2020, Vol. 36 ›› Issue (5): 33-40.doi: 10.16035/j.issn.1001-7283.2020.05.005

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Study on the Cloning and Transformation of Rhamnose Transferase FtF3GT1 Gene in Tartary Buckwheat

Lu Xiaoling1,2(), He Ming2, Zhang Kaixuan2, Liao Zhiyong1(), Zhou Meiliang2()   

  1. 1College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, Zhejiang, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2020-01-19 Revised:2020-03-07 Online:2020-10-15 Published:2020-10-12
  • Contact: Liao Zhiyong,Zhou Meiliang E-mail:luxiaoling07@163.com;zyliao@wzu.edu.cn;zhoumeiliang@caas.cn

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Abstract:

Flavonoids in tartary buckwheat have high medicinal value and health function. Based on the tartary buckwheat Chuanqiao No.1, FtF3GT1 gene was cloned from tartary buckwheat and its CDS length was 1 401bp, encoding 467 amino acids. Phylogenetic analysis showed that FtF3GT1 was closely related to FeUF7GT protein in common tartary buckwheat. Specific expression analysis results showed that FtF3GT1 gene was expressed in all tissues, but the lowest in roots and the highest in stems. Treated with MeJA, expression of FtF3GT1 gene and GUS activity in pCAMBIA3301:FtF3GT1pro::GUS in transgenic hairy roots of tartary buckwheat were significantly induced by MeJA, it is speculated that FtF3GT1 gene was induced by MeJA and promoted the biosynthesis of flavonoids synthesis.

Key words: Cloning, Tartary buckwheat, Flavonoids synthesis

Table 1

Primer sequence"

引物名称Primer name 引物序列(5'-3') Primer sequence (5'-3')
FtF3GT1-F(CDS) 5'-ATGGGAACCCAATCAAGC-3'
FtF3GT1-R(CDS) 5'-CTACTGCTTACCAACCAAAC-3'
pCAMBIA1307-FtF3GT1-F 5'-GGGGGCGGCCGCTCTAGAATGGGAACCCAATCAAGC-3'
pCAMBIA1307-FtF3GT1-R 5'-AAGCTTGATATCGAATTCCTACTGCTTACCAACCAAAC-3'
pCAMBIA3301-FtF3GT1-F 5'-GAATCCTTAAATGGTTCATATTACTATGCAATTAG-3'
pCAMBIA3301-FtF3GT1-R 5'-TCTAGATTTGTAAAATGCTTGATTTGTTTCTTG-3'
FtH3-QF 5'-GAAATTCGCAAGTACCAGAAGAG-3'
FtH3-QR 5'-CCAACAAGGTATGCCTCAGC-3'
FtF3GT1-QF 5'-TCAAATAAGCTCGCCTC-3'
FtF3GT1-QR 5'-GCTGCATTTTGTCAAGAGCG-3'
FtActin-QF 5'-GAAATTCGCAAGTACCAGAAGAG-3'
FtActin-QR 5'-CCAACAAGGTATGCCTCAGC-3'

Fig.1

Cloning of CDS and promoter of FtF3GT1 gene A: M: DL 2 000 DNA marker; 1: PCR product of FtF3GT1 gene CDS; B: M:DL 2 000 DNA marker; 1: PCR product of FtF3GT1 gene promoter"

Fig.2

Multi-alignment of FtF3GT1 with GTs type proteins from other plants PhUF5GT (Petunia hybrida BAA89009); AtUF7GT (Arabidopsis thaliana NP567955); FtUF7GT (Fagopyrum esculentum BBF24939); FtUF7GT (F.esculentum BBF24939); FtUFGT2 (F.tataricum KX216513); FtUFGT3 (F. tataricum KX216514); GeUF7GT (Glycyrrhiza echinata BAC784380); GhUF5GT (Glandularia hybrida AB013598); GtUF3GT (Gentiana triflora BAA12737); HvUF3GT (Hordeum vulgare X15694); IhUF5GT (Iris hollandica AB113664); LbUF7GT (Lycium barbarum BAG80536); NtUF7GT (Nicotiana tabacum AAB36653); PyUF3GT (Petunia hybrida BAA89008); SbUF7GT (Scutellaria baicalensis BAA83484); ThUF5GT (Torenia hybrid AB076698); VmUF3GT (Vigna mungo BAA36972); VvUF3GT (Vitis vinifera AAB81683) "

Fig.3

Phylogenetic analysis of amino acid sequences of FtF3GT1 and GTs from other plants"

Table 2

Cis-elements in FtF3GT1 gene promoter sequence"

位点名称Site name 序列Sequence 位点功能Function of site
TGACG-motif TGACG 参与MeJA反应的顺式作用调控元件cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif CGTCA 参与MeJA反应的顺式作用调控元件cis-acting regulatory element involved in the MeJA-responsiveness
ABRE ACGTG 参与ABA反应的顺式作用调控元件cis-acting element involved in the abscisic acid responsiveness
GARE-motif TCTGTTG 响应GA元素gibberellin-responsiveness
P-box CCTTTTG 响应GA元素gibberellin-responsiveness
TCA-element CCATCTTTTT 参与SA反应的顺式作用调控元件cis-acting element involved in salicylic acid responsiveness
G-box CACGTC 光调控元件cis-acting regulatory element involved in light responsiveness
ARE AAACCA 厌氧诱导所必需的顺式调节元件cis-acting regulatory element essential for the anaerobic induction
TATA-box TATATA 转录起始-3-O核心启动子元件core promoter element of 3-O transcription start

Fig.4

Relative expression levels of FtF3GT1 gene in different tissues FtH3 was used as an internal control. Error bars represent the standard deviation of triplicate runs for qRT-PCR. * indicate significant differences using t-test (P < 0.05), the same below"

Fig.5

Expression of FtF3GT1 gene induced by MeJA"

Fig.6

Identification of transgenic roots and GUS staining A: Molecular identification of transgenic FtF3GT1pro::GUS hairy root lines. M: DL 2 000 DNA marker; 1: pCAMBIA3301:FtF3GT1pro::GUS plasmid; 2-4: transgenic lines DNA of A4 rhizogenes; 5-7: transgenic lines DNA of pCAMBIA3301-35S::GUS; 8-11: transgenic lines DNA of FtF3GT1pro::GUS; B: GUS staining of FtF3GT1pro::GUS hairy root treated with MeJA"

Fig.7

Induction process of hairy roots of tartary buckwheat and identification of transgenic root lines A: Induction process of hairy roots of tartary buckwheat; B: Identification of transgenic root lines; M: DL 2 000 DNA Marker; 1: pCAMBIA1307-FtRT1 plasmid; 2: natural root of tartary buckwheat (WT); 3: pCAMBIA1307 transgenic root lines; 4-8: DNA detection of pCAMBIA1307-FtRT1 transgenic root lines"

Fig.8

Determination of total flavonoids in A4, pCAMBIA1307 empty vector and pCAMBIA1307-FtF3GT1 in overexpression of root system"

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