甜菊SrUGT76G1上游负调控因子SrMYB1的克隆与功能研究

doi:10.1016/j.jia.2023.03.001

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (4): 1058-1067.DOI: 10.1016/j.jia.2023.03.001

甜菊SrUGT76G1上游负调控因子SrMYB1的克隆与功能研究

收稿日期:2022-07-22 接受日期:2023-01-13 出版日期:2023-04-20 发布日期:2023-01-13

Isolation and functional analysis of SrMYB1, a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

ZHANG Ting^{1, 2}, ZHANG Yong-xia^{1, 2}, SUN Yu-ming^{1, 2}, XU Xiao-yang^{1, 2}, WANG Yin-jie^{1, 2}, CHONG Xin-ran^{1, 2}, YANG Yong-heng^{1, 2#}, YUAN Hai-yan^{1, 2#}

¹Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-sen), Nanjing 210014, P.R.China
²Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Nanjing 210014, P.R.China

Received:2022-07-22 Accepted:2023-01-13 Online:2023-04-20 Published:2023-01-13
About author:ZHANG Ting, E-mail: zhangting901014@163.com; #Correspondence YANG Yong-heng, Tel: +86-25-84347086, E-mail: yyh8576@126.com; YUAN Hai-yan, Tel: +86-25-84347086, E-mail: yuanhaiyan@cnbg.net
Supported by:
This work was supported by the National Natural Science Foundation of China (31901597) and the Natural Science Foundation of Jiangsu Province, China (BK20201243).

摘要/Abstract

摘要：

SrUGT76G1对于合成优质甜菊糖苷至关重要，也是目前甜菊中研究最为深入的糖基转移酶基因，但是关于它的转录调控机制目前还不甚了解。本研究通过酵母单杂交手段鉴定得到了一个SrUGT76G1的上游调控因子SrMYB1。SrMYB1属于典型的R2R3类型的MYB类转录因子，其定位在细胞核并且具有转录激活活性。SrMYB1在花中的表达量较高而在叶片中较低。酵母单杂（Y1H）和凝胶阻滞（EMSA）实验证实SrMYB1可以结合在SrUGT76G1启动子的+50～-141区域即F4-3区段。进一步研究发现在烟草表皮细胞和甜菊愈伤组织中SrMYB1均可显著抑制SrUGT76G1的表达。综上所述，本研究不但发现了一个SrUGT76G1的潜在上游调控因子并且丰富了甜菊中糖苷代谢途径的调控网络。

Abstract:

SrUGT76G1, the most well-studied diterpene glycosyltransferase in Stevia rebaudiana, is key to the biosynthesis of economically important steviol glycosides (SGs). However, the molecular regulatory mechanism of SrUGT76G1 has rarely been explored. In this study, we identified a MYB transcription factor, SrMYB1, using a yeast one-hybrid screening assay. SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity. The transcript of SrMYB1 is predominantly accumulated in flowers, but is also present at a lower level in leaves. Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment (+50–(–141)) of the SrUGT76G1 promoter. Furthermore, we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus. Taken together, our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S. rebaudiana.

Key words: Stevia rebaudiana , SrUGT76G1 , MYB transcription factor , transcriptional regulation , steviol glycosides

. 甜菊SrUGT76G1上游负调控因子SrMYB1的克隆与功能研究[J]. Journal of Integrative Agriculture, 2023, 22(4): 1058-1067.

ZHANG Ting, ZHANG Yong-xia, SUN Yu-ming, XU Xiao-yang, WANG Yin-jie, CHONG Xin-ran, YANG Yong-heng and YUAN Hai-yan.

Isolation and functional analysis of SrMYB1, a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana [J]. Journal of Integrative Agriculture, 2023, 22(4): 1058-1067.

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甜菊SrUGT76G1上游负调控因子SrMYB1的克隆与功能研究

Isolation and functional analysis of SrMYB1, a direct transcriptional repressor of SrUGT76G1 in Stevia rebaudiana

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