Cloning and expression analysis of <i>OsSPL3</i> promoter in rice

doi:10.3785/j.issn.1008-9209.2022.05.091

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (3): 319-327 DOI: 10.3785/j.issn.1008-9209.2022.05.091

Crop sciences

Cloning and expression analysis of OsSPL3 promoter in rice

Huiling ZENG¹(

),Zuyi MO¹,Qiaoxian PU¹,Jiashu WANG¹,Kai FAN²,Zhaowei LI¹(

)

^1.College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
^2.College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

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Abstract

OsSPL transcription factor plays an important role in the development and stress response of rice (Oryza sativa) roots, leaves, floral organs, and ears. In this study, the OsSPL3 promoter was analyzed to explore the expression pattern of OsSPL3 transcription factor in rice and its response to drought stress. The Cis-acting elements in the OsSPL3 promoter region were analyzed by PLACE and Plant CARE online softwares, and the recombinant expression vector of OsSPL3 promoter and β-glucuronidase(GUS) gene was constructed, which was transformed into ZH11 rice callus, and positive transgenic plants were obtained by screening. The GUS expression activity of pOsSPL3-GUS transgenic plants and the expression patterns under drought stress and abscisic acid (ABA) treatments were detected. The results of promoter analysis showed that in addition to the necessary transcription initiation core elements and light-responsive elements, the OsSPL3 promoter region also included three MYB-involved drought-inducible elements, three gibberellin-responsive elements, two anaerobic induction essential elements, one low temperature response element, one endosperm expression regulatory element, one zein metabolism regulatory element and one meristem expression-related regulatory element. The results of GUS staining showed that the expression activity of GUS gene in young leaves, stem sheaths, coleoptiles and other young tissues was high, as well as in the vigorous growth parts of roots such as root cap, meristem zone, and elongation zone. In addition, the drought stress could significantly enhanced the GUS activity of transgenic rice leaves and roots. It shows that OsSPL3 transcription factor plays a regulatory role in the process of coleoptile growth, new leaf formation, root extension and stem sheath elongation after seed germination, and OsSPL3 transcription factor is also involved in the response process of rice drought stress.

Key words： rice (Oryza sativa) OsSPL3 transcription factor promoter β-glucuronidase (GUS) drought stress

Received: 09 May 2022 Published: 25 June 2023

CLC:

S511.22

Corresponding Authors: Zhaowei LI E-mail: 18349325508@163.com;lizw197@163.com

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	Huiling ZENG
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Cite this article:

Huiling ZENG,Zuyi MO,Qiaoxian PU,Jiashu WANG,Kai FAN,Zhaowei LI. Cloning and expression analysis of OsSPL3 promoter in rice. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(3): 319-327.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.05.091 OR https://www.zjujournals.com/agr/Y2023/V49/I3/319

水稻OsSPL3启动子克隆及表达分析

OsSPL转录因子在水稻（Oryza sativa）根系、叶片、花器官、穗等发育与逆境响应过程中起重要作用。本文通过对OsSPL3启动子的分析，探究了OsSPL3转录因子在水稻中的表达模式及其对干旱胁迫的响应方式。利用PLACE和Plant CARE在线软件分析OsSPL3启动子区的顺式作用元件，并构建OsSPL3启动子与β-葡糖醛酸糖苷酶（β-glucuronidase, GUS）基因的重组表达载体，转化‘中花11’（ZH11）水稻愈伤组织，筛选获得阳性转基因植株，且对pOsSPL3-GUS转基因植株的GUS表达活性以及在干旱胁迫与脱落酸（abscisic acid, ABA）处理下的表达方式进行检测。启动子分析结果表明，OsSPL3启动子区除包含必要的转录起始核心元件与光响应元件外，还包括3个MYB参与的干旱诱导元件、3个赤霉素响应元件、2个厌氧诱导必需作用元件、1个低温响应作用元件、1个胚乳表达调控元件、1个玉米醇溶蛋白代谢调控元件和1个分生组织表达相关调控元件。GUS染色结果显示，GUS基因在新生叶片、茎鞘、胚芽鞘等幼嫩组织及根冠、分生区、伸长区等根系旺盛生长部位中表达活性较高。此外，干旱胁迫能明显增强转基因水稻叶片与根系的GUS活性。本研究结果表明，OsSPL3转录因子在水稻种子萌发后的胚芽鞘生长、新叶发生、根系延伸等器官发育与茎鞘伸长等过程中发挥调控作用，同时，OsSPL3转录因子还参与水稻干旱胁迫响应过程。

关键词： 水稻, OsSPL3转录因子, 启动子, β-葡糖醛酸糖苷酶, 干旱胁迫

Fig. 1 Cloning of OsSPL3 promoter by PCR amplificationM: DL5000 DNA marker; 1: OsSPL3 promoter.

Table 1 Analysis of Cis-acting elements of OsSPL3 promoter

Fig. 2 Identification of positive transgenic plants by PCR amplificationM: DL2000 DNA marker; 1: Positive control; 2-22: Hygromycin genes; 23: Negative control.

Fig. 3 GUS staining for different tissues of transgenic rice at the seedling stage and detection of OsSPL3 expression levels in different tissues of wild-type ZH11 riceA. Tip of young leaf; B. Young leaf and fully-unfolded top 2nd leaf; C. Young leaf, top 2nd leaf, and top 3rd leaf; D. Top 2nd leaf, top 3rd leaf, and upper part of leaf sheath; E. Middle part of leaf sheath; F. Near base of leaf sheath; G. Radicle on the residual seed attached to roots; H-I. Primary roots, adventitious roots, branch roots, and lateral roots; J. Ear tissue located on the junction of leaf blade and leaf sheath; K. Coleoptile and young leaves; L. Adventitious roots after seed germination. The scale is 0.5 cm.

Fig. 4 GUS staining detection for the leaves and roots of transgenic rice under the PEG6000 stress and ABA treatmentA, D. Normal nutrient solution culture; B, E. 20% PEG6000 stress; C, F. 100 µmol/L ABA treatment. The scale is 0.5 cm.

Fig. 5 Expression levels of GUS gene in the leaves and roots of pOsSPL3-GUStransgenic rice under the PEG6000 stress and ABA treatmentA, C. Leaves; B, D. Roots. Different lowercase letters above bars indicate significant differences at the 0.05 probability level, and the same as below.

Fig. 6 Expression levels of OsSPL3 in the leaves of wild-type ZH11 rice under the PEG6000 stress and ABA treatment


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