Cloning of CsANS Gene from Tea Plant (Camellia sinensis) and Its Functional Analysis in Transgenic Tobacco (Nicotiana tabacum)
QI Yong1, 2, ZHAO De-Gang1, 2, LV Li-Tang1, 3, *
1 The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education)/Guizhou Key Lab of Agro-Bioengineering, Institute of Agro-Bioengineering and College of Life Sciences, Guizhou University, Guiyang 550025, China; 2 The 2011 Collaboration Innovation Center for Mountain Ecology and Agro-Bioengineering in Guizhou Province, Guizhou University, Guiyang 550025, China; 3 Tea Academy, Guizhou University, Guiyang 550025, China
Abstract：Anthocyanin synthase (ANS) is a key enzyme at the end of plant anthocyanin biosynthetic pathway,which catalyzes leucoanthocyanins into anthocyanins.In this study, the ANS gene of Camellia sinensis was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on tea full-length transcriptome sequencing data (GenBank No. AY830416).The full-length cDNA coding region was 1 068 bp and encoded 355 amino acids. The plant expression vector pSH-CsANS was constructed, and Agrobacterium tumefaciens was used to infect tobacco (Nicotiana tabacun) leaves for genetic transformation. Thirty-five transgenic plants were obtained by tissue culture. And after resistance selection and PCR identification, three PCR-positive tobacco lines TP-1, TP-2 and TP-4 were selected for gene expression and anthocyanin and proanthocyanidin content analysis. Quantitative real-time PCR (qRT-PCR) analysis showed that over-expression CsANS gene resulted in up-regulation of endogenous flavonoid biosynthesis pathway genes chalcone isomerase(CHI), (2S)-flavanone 3-hydroaylase (F3H) and dihydroflavonol4-reductase (DFR) and down-regulation of flavonol synthase (FLS) gene in tobacco. The results showed that over-expression of CsANS gene promoted the synthesis of tobacco anthocyanins, and the anthocyanin content was increased by about 45% compared with wild type. Moreover, over-expression of CsANS gene could increase the content of flavan-3-ol, a prerequisite material for the synthesis of proanthocyanidins, and increase the content of proanthocyanidins in transgenic plants by about 34% compared with wild type. This research has provided a foundation for future study on gene expression and functional analysis.
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