Biology 2022, 11, 1092. https://doi.org/10.3390/biology11081092 www.mdpi.com/journal/biology Article Isolation and Functional Characterization of a Green-Tissue Promoter in Japonica Rice (Oryza sativa subsp. Japonica) Mi Lin 1,† , Jingwan Yan 1,† , Muhammad Moaaz Ali 2 , Shaojuan Wang 2 , Shengnan Tian 2 , Faxing Chen 2, * and Zhimin Lin 1, * 1 Fujian Academy of Agricultural Sciences Biotechnology Institute, Fuzhou 350003, China; linmi1009@163.com (M.L.); yjw@fjage.org (J.Y.) 2 College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China; muhammadmoaazali@yahoo.com (M.M.A.); wsj975620wsj@163.com (S.W.); tsl0224@163.com (S.T.) * Correspondence: cfaxing@126.com (F.C.); linzhimin@faas.cn (Z.L.) † Equally contributed authors. Simple Summary: Transgenic applications have largely focused on constitutive promoters in plants. However, strong and continuous over-expression of certain genes may be redundant and even harmful to plant growth. Thus, tissue-specific promoters are the most suitable for regulating target gene expression. Although several tissue-specific promoters have been identified, the regula- tory mechanism of tissue-specific gene expression remains unclear. By a series of GUS staining of 5′ and 3′ deletions, we uncover tissue-specific cis-acting elements in GSX7R, including ten light-re- sponsive elements. The results reveal that GSX7R is a reverse green tissue-specific promoter, except in endosperm. In contrast, strong tissue-specific promoters that can be used for rice improvements are limited. In this study, we successfully showed that the GSX7R promoter can drive the Cry1Ab gene to resistant rice yellow stem borer. In addition, our study demonstrates an effective promoter to drive foreign genes for crop improvement. Abstract: Plant promoters play a vital role in the initiation and regulation of gene transcription. In this study, a rice protein/gene of unknown expression, named Os8GSX7, was gained from a rice T- DNA capture line. The semi-quantitative RT-PCR analysis showed that the gene was only expressed in root, glume, and flower, but not in stem, leaf, embryo, and endosperm of japonica rice. The GUS activity analysis of the GSX7R promoter showed that it was a reverse green tissue expression pro- moter, except in endosperm. The forward promoter of GSX7 cannot normally drive the expression of the foreign GUS gene, while the reverse promoter of GSX7 is a green tissue-specific expression promoter, which can drive the expression of the foreign GUS gene. The region from −2097 to −1543 bp was the key region for controlling the green tissue-specific expression. The regulatory sequences with different lengths from the 2097 bp reverse sequence from the upstream region of the Os8GSX7 were fused with the GUS reporter gene and stably expressed in rice. Furthermore, transgenic rice plants carrying Cry1Ab encoding Bacillus thuringiensis endotoxin, regulated by GSX7R, were re- sistant to yellow stem borer. The analysis suggested that 10 light responsive elements of tissue- specific expression were found, including ACE, Box4, CAT-box, G-Box, G-box, GATA motif, GC motif, I-box, Sp1, and chs-unit1 M1. In addition, the results of 5′ and 3′ deletions further speculated that ACE and I-box may be the key elements for determining the green tissue-specific expression of GSX7R promoter. Keywords: promoter; green-tissue specific expression; cloning; agrobacterium; RT-PCR Citation: Lin, M.; Yan, J.; Ali, M.M.; Wang, S.; Tian, S.; Chen, F.; Lin, Z. Isolation and Functional Characterization of a Green-Tissue Promoter in Japonica Rice (Oryza sativa subsp. Japonica). Biology 2022, 11, 1092. https:// doi.org/10.3390/biology11081092 Academic Editor: Cheng-Gui Han Received: 4 July 2022 Accepted: 21 July 2022 Published: 22 July 2022 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2022 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/).