Expression Vector Construction and Functional Analysis of Solanum tuberosum StCHS4 and StCHS5 Genes

doi:10.7668/hbnxb.20195410

Abstract

Abstract:

Chalcone synthase(CHS)is an important structural gene that regulates the early biosynthesis of flavonoid pathways in plants,and plays a role in plant growth and development and stress response.Previously,the key genes StCHS4 and StCHS5 for anthocyanin biosynthesis were identified in the potato CHS family by expression analysis.To further explore the function of potato StCHS4 and StCHS5 in the biosynthesis of flavonoids and anthocyanins,the characterization of StCHS4 and StCHS5 proteins was analyzed by online website.The 35S∷StCHS4-GFP and 35S∷StCHS5-GFP recombinant vectors were constructed by homologous recombination method based on the pRI101 binary vector,and then were transformed into Agrobacterium GV3101 strain.The subcellular localization of StCHS4 and StCHS5 proteins was determined by transient transformation of Nicotiana benthamiana.N.tabacum was used as the experimental material for transient overexpression and stable genetic transformation to analyze the content of total flavonoids and anthocyanins after overexpression of StCHS4 and StCHS5 genes.The results showed that the secondary structures of StCHS4 and StCHS5 proteins were mainly α-helix and random coil.StCHS4 was an unstable hydrophilic protein,and StCHS5 was a stable hydrophilic protein.The sequence alignment revealed that StCHS4 and StCHS5 had a close relationship with the CHSs of Capsicum annuum and Solanum lycopersicum,respectively.The results of subcellular localization revealed that StCHS4 and StCHS5 proteins were localized in the cytoplasm and cell membrane.In transient overexpression of tobacco,StCHS4 and StCHS5 genes significantly enhanced anthocyanin accumulation at 3—5 days after injection.Three transgenic N.tabacum lines of StCHS4 and StCHS5 gene were obtained,respectively.Compared with the wild type,the expression of StCHS4 and StCHS5 in transgenic plants was significantly higher,and the contents of total flavonoids and total anthocyanins were higher than those in the wild type.The total flavonoid content in StCHS4-OE3 and StCHS5-OE1 transgenic plants was significantly increased.The anthocyanin content in StCHS5-OE1 and StCHS5-OE2 plants increased by 89%,131%,respectively.The above results demonstrated that StCHS4 and StCHS5 were the key CHS genes in the flavonoid pathway of Solanum tuberosum,and the overexpression of StCHS4 and StCHS5 contributed to the biosynthesis of anthocyanins and flavonoids.

Key words: Solanum tuberosum, StCHS4, StCHS5, Flavonoid, Anthocyanin, Genetic transformation, Subcellular localization, Functional analysis

WANG Tongtong, WANG Wenjing, DONG Xinyu, SONG Jiafeng, SHENG Suao, CHENG Jielan, ZHENG Tingting, LYU Zhaoyan, ZHU Xiaobiao, HOU Hualan. Expression Vector Construction and Functional Analysis of Solanum tuberosum StCHS4 and StCHS5 Genes[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 74-83. doi: 10.7668/hbnxb.20195410.

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Figures/Tables 10

Tab.1 Primer sequences used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')	用途 Purpose
pRI101-StCHS4-F	GTTGATACATATGCCCGTCGACATGGTCACCGTAGAGGAGGTTCG	过表达载体构建
pRI101-StCHS4-R	CGCCCTTGCTCACCATGGATCCAGTAGACACACTATGGAGCACAAC
pRI101-StCHS5-F	GTTGATACATATGCCCGTCGACATGGTCACCGTGGAGGAGTATC
pRI101-StCHS5-R	CGCCCTTGCTCACCATGGATCCAGTAGCAACACTGTGAAGAACAAC
qStCHS4-F	TGGTCACCGTAGAGGAGGTT	实时荧光定量PCR
qStCHS4-R	TCATTGATTTATCACACATGCGCT
qStCHS5-F	TCACCGTGGAGGAGTATCGT
qStCHS5-R	TCATTGATTTATCACACATGCGCT
NtActin-F	CGTTTGGACCTTGCTGG	烟草内参基因
NtActin-R	TCTGGGCAACGGAACCT

Fig.1 Secondary and tertiary structure of StCHS4 and StCHS5 protein A.Secondary structure of StCHS4 protein;B.Secondary structure of StCHS5 protein; C.Tertiary structure of StCHS4 protein;D.Tertiary structure of StCHS5 protein.

Fig.2 Multiple sequence alignment of StCHS4 and StCHS5 proteins OsCHS.Oryza sativa,CAA61955.1;AtCHS.Arabidopsis thaliana,AAA32771;VvCHS.Vitis vinifera, NP_001267879.1;NtCHS2.Nicotiana tabacum, ANA78328.1;PhCHS.Petunia hybrida, AAF60297.1;SlCHS1.Solanum lycopersicum, NP_001234033.2;CaCHS.Capsicum annuum, ACN60400.1;StCHS4 and StCHS5.Solanum tuberosum.The highly conserved CHS sequence are underlined,the malonyl-CoA binding site are boxed with red line.

Fig.3 Phylogenetic relationship of StCHS4 and StCHS5 proteins

Fig.4 Construction of overexpression vector of StCHS4 and StCHS5 genes A.PCR amplification product of StCHS4 gene;B.PCR amplification product of StCHS5 gene; C.Dual-enzyme digestion of pRI101 vector;M.DL2000 DNA Marker.

Fig.5 Subcellular localization of the StCHS4 and StCHS5 proteins

Fig.6 Flavonoid and anthocyanin content of StCHS4 and StCHS5 transient overexpression tobacco Error bars indicate standard deviation of three biological replicates,different letters represent statistical difference(P<0.05,ANOVA).The same as Fig.8—9.

Fig.7 Genetic transformation of StCHS4 and StCHS5 in tobacco A.Tobacco genetic transformation of StCHS4;B.Tobacco genetic transformation of StCHS5.a.Tobacco leaf co-cultures;b.Callus tissue induction;c.Differentiation process;d.Seedlings of transgenic plants.

Fig.8 Identification of StCHS4 and StCHS5 transgenic lines A.PCR identification of transgenic tobacco:M.DL2000,1,7.Positive plasmid,2,8.Wild-type tobacco,3,9.H₂O,4—6.Transgenic tobacco of StCHS4,10—12.Transgenic tobacco of StCHS5;B.Semi-quantitative analysis of transgenic lines:WT.Wild-type tobacco,StCHS4-OE1/2/3 and StCHS5-OE1/2/3 indicates different lines of transgenic tobacco;C.Relative expression of StCHS4 in transgenic tobacco;D.Relative expression of StCHS5 in transgenic tobacco.

Fig.9 Flavonoid and anthocyanin content of transgenic tobacco leaves

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