The analysis of transcription factor CsHB1 effects on caffeine accumulation in tea callus through CRISPR/Cas9 mediated gene editing. (February 2021)

• Record Type: Journal Article
• Authors: Ma, Wenhui; Kang, Xin; Liu, Ping; Zhang, Yuanyuan; Lin, Xiaorong; Li, Bin; Chen, Zhongzheng
• Is Part Of: Process biochemistry. Volume 101(2021)
• Page Start: 304
• Page End: 311
• Publication Date: 2021-02
• Subjects: Tea -- Caffeine -- CsHB1 transcription factor -- Gene editing -- N-methyltransferase (NMT); Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2021.01.001
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: The expression of transcription factor CsHB1 was similar to that of caffeine synthase gene yhNMT1 in shoots of Yinghong 9. The transient overexpression of CsHB1 had no significant effect on yhNMT1 in tobacco leaves. The CsHB1 gene received effective mutation and its expression decreased by 65% in transgenic tea callus using CRISPR/Cas9 technology. The expression of yhNMT1 and the caffeine accumulation decreased by 93% and 97.5% respectively in transgenic callus. The decreased yhNMT1 expression and caffeine content in transgenic callus verified the regulation effect of CsHB1 on yhNMT1. Abstract: Caffeine is an important metabolite in tea. In previous study, transcription factor CsHB1 was cloned from tea plants via yeast one-hybrid using yhNMT1 as bait. In this study, the expression of CsHB1 and yhNMT1 in tea plants was analyzed by qRT-PCR, the effect of CsHB1 on the expression of yhNMT1 was determined by tobacco transient expression system, and the CsHB1 was assembled into pYLCRISPR/Cas9Pubi -H vector followed by transgene to tea callus. Our results showed that the expression of CsHB1 was similar to that of yhNMT1 . The overexpression of CsHB1 showed no significant effect on yhNMT1 in tobacco leaves. The constructed pYLCRISPR/Cas9Pubi -H-CsHB1 vector was successfully transformed into tea callus, and CsHB1 gene was mutated directionally. In transgenic callus, the expression of CsHB1 and yhNMT1 decreased by 65 % and 93 %, respectively, and the accumulation of caffeine decreased by 97.5 %, which indicated that yhNMT1 gene was regulated by CsHB1. Our results will help to understand the regulation mechanism of caffeine biosynthesis in tea.