甘蔗割手密种糖转运蛋白基因SsSWEET11的克隆与功能分析

doi:10.3724/SP.J.1006.2023.24232

作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2385-2397.doi: 10.3724/SP.J.1006.2023.24232

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蔗割手密种糖转运蛋白基因SsSWEET11的克隆与功能分析

杜翠翠¹(), 吴明星¹(), 张雅婷¹, 谢婉婕¹, 张积森²^,^*(), 王恒波¹^,^*()

¹农业农村部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗工程技术研究中心 / 福建农林大学农学院, 福建福州 350002
²广西大学亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530004

收稿日期:2022-10-16 接受日期:2023-02-10 出版日期:2023-09-12 网络出版日期:2023-02-21
通讯作者: *张积森, E-mail: zjisen@126.com; 王恒波, E-mail: wanghengbo_0354@126.com
作者简介:杜翠翠, E-mail: 1875327155@qq.com
吴明星, E-mail: 1622076451@qq.com第一联系人：^**同等贡献
基金资助:
国家重点研发计划项目(2021YFF1000104);大学生创新创业训练计划项目(X202210389051);财政部和农业农村部国家现代农业产业技术体系建设专项(糖料, CARS-17)

Cloning and functional analysis of sucrose transporter protein SsSWEET11 gene in sugarcane (Saccharum spontaneum L.)

DU Cui-Cui¹(), WU Ming-Xing¹(), ZHANG Ya-Ting¹, XIE Wan-Jie¹, ZHANG Ji-Sen²^,^*(), WANG Heng-Bo¹^,^*()

¹Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / National Sugarcane Engineering Technology Research / College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
²State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, China

Received:2022-10-16 Accepted:2023-02-10 Published:2023-09-12 Published online:2023-02-21
About author:First author contact:^**Contributed equally to this work
Supported by:
National Key Research and Development Program of China(2021YFF1000104);National Undergraduate Innovation and Entrepreneurship Training Program Project(X202210389051);China Agriculture Research System of MOF and MARA(Sugar, CARS-17)

摘要/Abstract

摘要：

SWEET蛋白通过调控植物体内糖分的运输、分配、转化和贮藏, 广泛参与植物生长发育及响应病原菌胁迫的生理生化过程。为揭示SWEET基因在甘蔗生长发育及其与赤条病菌互作中的生物学功能, 本研究基于甘蔗割手密种全长转录组文库和比较基因组学, 根据注释SsSWEET11基因序列设计特异引物, 利用RT-PCR技术从甘蔗割手密种cDNA文库中扩增该基因的全长序列, 运用多种生物信息工具分析其特征, 构建系统进化树; 采用不同组织和抗、感赤条病的甘蔗品种分析SsSWEET11的表达模式; 利用瞬时表达和亚细胞定位分析SsSWEET11的功能。结果表明, 从甘蔗割手密种克隆获得SsSWEET11基因(登录号为OP554214), 该基因全长927 bp, 编码308个氨基酸残基, 具有2个MtN3_saliva结构域和7次跨膜结构域。系统进化树分析显示, SsSWEET11属于SWEET蛋白家族第III亚家族成员, 与高粱SbSWEET11相似性高达97.41%。qRT-PCR分析表明, ShSWEET11基因在不同组织中组成型表达, 在蔗叶和根中表达量显著高于其他组织; 赤条病菌胁迫下, ScSWEET11基因在抗病品种ROC22和感病品种MT11-610中呈现完全不同的表达趋势, 与对照相比, 抗病品种中该基因的表达量显著下调, 而感病品种中, 在胁迫48 h和72 h后该基因的表达量显著上调, 分别为对照的5.90倍和5.43倍。亚细胞定位表明, SsSWEET11-GFP融合蛋白定位在质膜上。瞬时过表达SsSWEET11基因1 d后, 二氨基联苯胺(Diaminobenzidine, DAB)对本氏烟叶片进行染色, 叶片颜色没有变化, 再接种烟草青枯菌、茄病镰刀菌蓝色变种7 d后, 过表达植株叶片发病比对照组严重, 且过敏反应相关基因、茉莉酸和水杨酸代谢途径相关基因呈现上调表达, 而乙烯通路相关基因则没有响应, 表明SsSWEET11基因参与茉莉酸和水杨酸信号传导通路, 且病原菌侵染本氏烟草叶片能够诱发过敏反应。研究结果不仅为开发与甘蔗抗赤条病菌性状关联的分子标记提供积累, 也为深入解析赤条病侵染甘蔗的分子机制奠定一定的基础。

关键词: 割手密种, SWEET基因, 糖转运蛋白, 赤条病菌, 基因功能, 甘蔗

Abstract:

SWEET (Sugars Will Eventually be Exported Transporter) proteins are widely involved in the physiological and biochemical processes of plant growth and development and response to pathogen stress by regulating the transportation, distribution, transformation, and storage of sugar in plants. This study revealed the biological function of SWEET genes in the growth and development of sugarcane and its interaction with red stripe pathogen Acidovorax avenae subsp. avenae (Aaa). Firstly, based on the PacBio full-length transcriptome cDNA library of S. spontaneum SES208 and comparative genomics, the specific primers were designed according to the re-annotated SsSWEET11 gene sequence. The full-length sequence was mined from the cDNA library by quantitative RT-PCR technology. The characteristics of the SWEET proteins were analyzed using various biological information tools, and the SWEET proteins from some plants were constructed a phylogenetic tree. Secondly, RT-qPCR detected the relative expressions of the SsSWEET11 gene with different tissues and two cultivars, ROC22 (resistant to red stripe) and MT11-610 (susceptible to red stripe). Finally, transient overexpression and subcellular localization performed the function of the SsSWEET11 gene. The results showed that the full-length cDNA sequence of the SsSWEET11 gene (GenBank accession number: OP554214) was cloned from S. spontaneum SES208, with an open reading frame of 927 bp and encoding 308 amino acid residues, which contained two MtN3_saliva domains and seven transmembrane domains. Phylogenetic analysis revealed that the SWEET protein family could be divided into four subfamilies, and SsSWEET11 belonged to subfamily III. The amino acid sequence similarity between SsSWEET11 and SbSWEET11 protein from sorghum is 97.99%. qRT-PCR demonstrated that the SsSWEET11 gene was constitutively expressed in different tissues of S. spontaneum and the relative expression level in leaves and roots was significantly higher than that in other tissues. Under the stress of Aaa, the SsSWEET11 gene presented a different expression pattern between sugarcane cultivars (ROC22) and (MT11-610) and was significantly reduced in the resistant sugarcane cultivar compared with the blank control. However, the expression of ShSWEET11 was significantly up-regulated in the susceptible sugarcane cultivar after 48 hours post-inoculation (hpi) and 72 hpi, which were 5.90 times and 5.43 times higher than the control, respectively. Subcellular localization indicated that the SsSWEET11-GFP fusion protein was located in the plasma membrane. After transiently overexpression of the SsSWEET11 gene for one day, the color of Nicotiana benthamiana leaves remained unchanged by DAB staining, and seven days after inoculation with Pseudomonas solanacearum, and Fusarium solani var. coeruleum, the incidence of transient overexpression of ShSWEET11 gene in N. benthamiana leaves were more susceptible than that of the control. Allergic reaction-related genes, jasmonic acid, and salicylic acid metabolism pathway-related genes were up-regulated, but ethylene pathway-related genes did not respond, suggesting that the SsSWEET11 gene is involved in jasmonic acid and salicylic acid signal transduction pathways, and the infection of N. benthamiana leaves by pathogens can induce an allergic reaction. These results not only provided an accumulation for the development of molecular markers associated with sugarcane resistance to Aaa but also laid a foundation for in-depth analysis of the molecular mechanism in sugarcane in response to Aaa infection.

Key words: Saccharum spontaneum, SsSWEET11 gene, sucrose transporter protein, red stripe pathogen, gene function, sugarcane

杜翠翠, 吴明星, 张雅婷, 谢婉婕, 张积森, 王恒波. 甘蔗割手密种糖转运蛋白基因SsSWEET11的克隆与功能分析[J]. 作物学报, 2023, 49(9): 2385-2397.

DU Cui-Cui, WU Ming-Xing, ZHANG Ya-Ting, XIE Wan-Jie, ZHANG Ji-Sen, WANG Heng-Bo. Cloning and functional analysis of sucrose transporter protein SsSWEET11 gene in sugarcane (Saccharum spontaneum L.)[J]. Acta Agronomica Sinica, 2023, 49(9): 2385-2397.

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名称 Name	引物序列 Primer sequences (5°-3°)	备注 Note
CDS-SWEET11-F_27	AATCGAGCATCACCTTAGCAGTAGC	基因克隆 Gene cloning
CDS-SWEET11-R_1194	TCGTCCGAGTCGATCCGAGCC
CDS-SsSWEET11-F_72	GGAAGGAAGTCGCACTAGGAA
SsSWEET11-QF	AGGTGTACCGCAAGAAGTCG	定量PCR引物 qRT-PCR primers
SsSWEET11-QR	AGCGCGTAGAAGATCCACAG	定量PCR引物 qRT-PCR primers
GAPDH-F	CACGGCCACTGGAAGCA	内参基因 Reference gene
GAPDH-R	TCCTCAGGGTTCCTGATGCC	内参基因 Reference gene
pMDC202-SsSWEET11-F	CTCGACTCTAGAACTAGTATGGCAGGAGGCCTCTTCTCCAT	过表达载体构建 Construction of overexpression vector
pMDC202-SsSWEET11-R	ATTTTTTCTACCGGTACCCACCGCGGCGGCGGGGAC	过表达载体构建 Construction of overexpression vector
pSuper-1300-SsSWEET11-F	GGGGCCCGGGGTCGACATGGCAGGAGGCCTCTTCTCCAT	亚细胞定位载体构建 Construction of subcellular localization vector
pSuper-1300-SsSWEET11-R	CCCTTGCTCACCATGGTACCCACCGCGGCGGCGGGGAC	亚细胞定位载体构建 Construction of subcellular localization vector

甘蔗割手密种糖转运蛋白基因SsSWEET11的克隆与功能分析

Cloning and functional analysis of sucrose transporter protein SsSWEET11 gene in sugarcane (Saccharum spontaneum L.)

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