龙眼DlSWEET1基因的克隆、表达及功能分析  

Cloning,expression and functional analysis of longan DlSWEET1 gene

作　　者：包雨莹 李韵 江文洁 谢涛 方庭 BAO Yuying;LI Yun;JIANG Wenjie;XIE Tao;FANG Ting(College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China;Institute of Horticultural Plant Genetics and Breeding,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建农林大学园艺学院,福州350002 [2]福建农林大学园艺植物遗传育种研究所,福州350002

出　　处：《果树学报》2024年第4期679-689,共11页Journal of Fruit Science

基　　金：国家自然科学基金项目(32102343);福建省高原学科建设经费(102/71201801101)。

摘　　要：【目的】SWEET(sugars will eventually be exported transporters)是一类参与植物生长发育多个过程的糖转运蛋白,分析DlSWEET1基因在龙眼不同组织和处理下的表达,探究其在果实糖积累中的功能。【方法】以龙眼松风本果实为材料,克隆DlSWEET1基因,采用实时荧光定量PCR分析DlSWEET1在龙眼不同组织器官的表达以及在激素、冷、热、干旱胁迫下的表达模式。通过亚细胞定位、糖转运活性分析及草莓瞬时转化研究DlSWEET1基因的功能。【结果】DlSWEET1基因开放阅读框(ORF)全长为750 bp,编码249个氨基酸,包含一个PQ-loop保守结构域和蛋白典型保守结构域MtN3_slv。DlSWEET1在龙眼根、茎、叶、果肉等组织中均有不同程度的表达,在叶中的表达量较高,在果肉中的表达量次之,而在茎和根中表达量较低;不同浓度的蔗糖、葡萄糖和果糖处理龙眼叶片后,DlSWEET1在叶片中的表达量均有显著升高;低温、干旱及MeJA(茉莉酸甲酯)处理可显著提高DlSWEET1的表达。农杆菌侵染本氏烟草发现DlSWEET1蛋白定位在细胞膜和细胞核。糖转运活性分析证明DlSWEET1蛋白可以转运葡萄糖、果糖、蔗糖和甘露糖。草莓中瞬时转化DlSWEET1可以显著提升果实中的可溶性糖含量。【结论】瞬时过表达DlSWEET1导致转基因草莓果实的可溶性糖含量增加,为进一步解析DlSWEET1在龙眼果实糖积累中的作用提供理论依据。【Objective】Longan(Dimocarpus longan L.)is one of the important economic fruit crops in southern China.Longan has the function of nourishing the heart,spleen and blood,and calming the mind of peaple.It has been regarded as a precious supplement since ancient times.The sugar content in fruits is a key factor affecting fruit quality,and improvement of fruit sugar content is of great signifi-cance for promoting the high-quality and efficient development of China’s longan industry.The SWEET sugar transporters protein(SWEETs)not only plays an important role in plant stress and hor-mone response,but also plays a crucial role in the normal growth and development of plants,especially in promoting sugar accumulation.However,there has been limited research on the DlSWEETs in lon-gan,especially on sugar accumulation.The purpose of this study is to screen and validate the functions of the candidate DlSWEETs that may be involved in sugar accumulation processes.【Methods】The CDS sequence of the DlSWEET1 was cloned using the cDNA of the preserved Songfengben fruit in the laboratory as a template.The DNAMAN software was used to translate the correctly sequenced DlS-WEET1 gene nucleotide sequence into amino acid sequence,and its conserved domain was predicted by NCBI.The protein transmembrane domains were analyzed using the TMHMM2.0.We extracted the to-tal RNA from different tissues(roots,stems,leaves,and fruits)of longan and leaf samples after differ-ent treatments,reversed the transcribe to obtain cDNA,and then used real-time fluorescence quantita-could transport various sugar components,such as sucrose,glucose,fructose and mannose,but could not transport the toxic substrate deoxyglucose.The transient overexpression of the DlSWEET1 resulted in increased soluble sugar content in the transgenic strawberry fruits.The transient conversion of this gene in the strawberry significantly increased the relative expression of the DlSWEET1.These results in-dicated that the DlSWEET1 has the function of promoting sugar accumulation i

关 键 词：龙眼 DlSWEET1 表达分析 亚细胞定位 糖积累 

分 类 号：S667.2[农业科学—果树学]