蔗糖转运蛋白OsSUT5在水稻花粉发育及结实中的作用

doi:10.3864/j.issn.0578-1752.2021.16.001

摘要/Abstract

摘要：

【目的】蔗糖是植物体内光合产物运输的主要形式。蔗糖-质子同向运输蛋白（sucrose transporter/sucrose carrier,SUT/SUC）在植物细胞之间的蔗糖跨膜运输以及植物组织和器官之间的蔗糖分配中具有重要作用。水稻SUT家族共有5个成员。已有研究表明,敲除OsSUT1、OsSUT2、OsSUT3、OsSUT4对水稻的生长发育产生显著影响,说明这些基因不可替代。然而,OsSUT5的功能还未见报道。阐明OsSUT5在水稻生长发育中的作用,将为全面了解SUT蛋白在模式植物水稻中的生理功能提供新的依据。【方法】通过对水稻OsSUT5的时空表达特性进行实时荧光定量PCR分析,同时利用OsSUT5的推测启动子驱动GUS在水稻体内表达,获得其基因编码蛋白的组织定位信息,以及在烟草叶片表皮细胞内进行OsSUT5-GFP融合蛋白瞬时表达,对蛋白进行亚细胞定位,比较基因编辑技术（CRISPR-Cas9）创制的OsSUT5不同纯合突变株系与野生型对照水稻的形态和生理特征,获得OsSUT5的功能信息。【结果】转录水平上,OsSUT5在水稻茎、叶、花序和颖果中均有表达,并且该基因编码蛋白在营养器官中的表达集中于维管组织。在生殖器官中,OsSUT5的表达主要位于花药及发育的颖果内。该基因编码蛋白在水稻发育的颖果特别是盾片和胚根鞘中高表达。烟草叶片表皮细胞内的瞬时表达显示OsSUT5-GFP融合蛋白定位于细胞质膜。与野生型水稻相比,3个OsSUT5纯合突变株系均表现为花粉活性以及离体萌发率明显降低。与此相吻合的是,OsSUT5突变株系未授粉的小穗比例相对于野生型显著增加,同时突变株系结实率显著下降。通过对OsSUT5突变株系颖果的观察和比较显示,OsSUT5突变体水稻颖果的垩白相对于野生型明显增多,其颖果长度相对于野生型对照也有一定程度的增加,但统计结果表明OsSUT5突变体水稻的千粒重与野生型无显著区别。【结论】OsSUT5在水稻花粉发育甚至受精过程中可能发挥重要作用;敲除OsSUT5对水稻的结实率、籽粒的形态和品质有明显影响。推断OsSUT5在水稻开花结实中的作用（包括对花粉活性和颖果内胚乳发育的影响）与其蔗糖运输功能密切相关。

关键词: 水稻, OsSUT5, CRISPR-Cas9, 花粉萌发, 结实率, 垩白

Abstract:

【Objective】 Sucrose is the main form of photosynthates transported in the plant, and sucrose transporters (or sucrose carrier, SUT/SUC) play important roles in the transport of sucrose across the plasma membrane between cells and allocation of sucrose among different tissues and organs. The rice SUT family possesses 5 members. Knockout of the genes encoding OsSUT1, OsSUT2, OsSUT3 or OsSUT4 confers significant effects on rice indicating that the functions of these genes are indispensable. However, the physiological role of OsSUT5 has not been systematically characterized. This study aims at elucidating the function of OsSUT5 in rice growth and development and provides new evidence for a comprehensive understanding of SUT’s role in model plant rice. 【Method】In this study, the temporal and spatial expression pattern of OsSUT5 was analyzed via quantitative real-time PCR. The tissue localization of OsSUT5 in rice was tested via GUS represented expressions driven by the putative promoter of OsSUT5 and subcellular localization of transiently expressed OsSUT5-GFP fusion protein was observed in leaf cells of tobacco. In addition, CRISPR-Cas9 mediated gene editing was employed to create mutant lines of the gene for the characterization of OsSUT5. 【Result】Our results show that OsSUT5 was expressed in culm, leaf, inflorescence, and caryopsis of rice but it was predominantly expressed in inflorescence and developing caryopsis at the transcriptional level. At the protein level, it was prominently expressed in the vascular bundles of rice vegetative organs. In reproductive organs, the protein was mainly expressed in the anther and developing caryopsis, particularly in the scutellum and coleorhiza. Transient expression of OsSUT5-GFP fusion protein in epidermis cells of tobacco leaf indicated that it was localized on the plasma membrane. Compared with the wild-type control, three homozygous mutant lines of OsSUT5 created via CRISPR-Cas9 gene-editing system consistently showed reduced pollen viability, a lower percentage of germination rates. Accordingly, the percentage of unpollinated florets and seed-setting rate decreased significantly. Comparison of OsSUT5 mutant lines and the wild-type control showed that more chalk was observed in the mutant caryopses than that of the wild type. In the mutant lines, caryopsis length increased but 1000 grain weight didn’t show a significant difference between the mutants and the wild-type control based on statistics. 【Conclusion】These results indicate that OsSUT5 played an important role in pollen development and probably also in the fertilization process. Knockout of the gene affected the morphology and quality of rice caryopsis. Given the sucrose transport capacity of OsSUT5 and its plasma membrane localization, it can be deduced that function of OsSUT5 including its influence on rice pollen viability and endosperm development is related to its sucrose transport activity at the cellular level.

Key words: rice, OsSUT5, CRISPR-Cas9, pollen germination, seed setting rate, chalk

张雅文, 包淑慧, 唐振家, 王小文, 杨芳, 张德春, 胡一兵. 蔗糖转运蛋白OsSUT5在水稻花粉发育及结实中的作用[J]. 中国农业科学, 2021, 54(16): 3369-3380.

ZHANG YaWen, BAO ShuHui, TANG ZhenJia, WANG XiaoWen, YANG Fang, ZHANG DeChun, HU YiBing. Function of Sucrose Transporter OsSUT5 in Rice Pollen Development and Seed Setting[J]. Scientia Agricultura Sinica, 2021, 54(16): 3369-3380.

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