[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.
Cloning,expression and functional analysis of longan DlSWEET1 gene
[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-tive PCR(qRT-PCR)to detect the expression level of DlSWEET1 in different tissues and organs of lon-gan,as well as its expression level under hormone,cold,heat,and drought stress.All the experiments were repeated three times in terms of biology and technology,and the relative expression levels of the genes were calculated using the 2-∆∆CTmethod,and then statistically analyzed by t test.p<0.05 indicat-ed significant difference,and the error line represented the standard deviation of three biological re-peats.the CDS sequence of the DlSWEET1 was cloned and connected to the pMD18-T vector.Using this plasmid as a template,PCR amplification of the DlSWEET1 was performed using primers.The pH7LIC5.0-ccdBrc-N-eGFP vector enzyme was cleaved using Stu Ⅰ enzyme cleavage,the pSAK277 vector enzyme was cleaved +using EcoR Ⅰ and Hind Ⅲ enzyme cleavage,and the pDR196 vector en-zyme was cleaved using Pst Ⅰ and Spe Ⅰ enzyme cleavage.The amplification product was inserted in-to the multi clone sites of each vector.The first two recombinant vectors were transformed into GV3101 strain,and the last recombinant vector was transformed into EBYVW4000 yeast strain.The lower epi-dermis of tobacco leaves was injected with Agrobacterium and cultured in the dark room(25℃)for 2 days.The distribution of green fluorescence was observed by confocal laser microscope.The strawber-ries injected with Agrobacterium were cultured under 16 h light/8 h darkness in a greenhouse at 25℃ for 9 days.The relative expression of the DlSWEET1 in strawberries and the determination of soluble sugar content were analyzed by qRT-PCR.The bacterial solutions with OD600 of 0.1,0.01 and 0.001 were taken 5µL into various sugar substrate media,and the media were cultured in a constant tempera-ture incubator at 28℃ for 2-3 days,and the growth was observed and recorded.The function of the DlSWEET1 gene was investigated by subcellular localization,sugar transport activity analysis and tran-sient transformation of strawberry.[Results]The DlSWEET1 contained 750 bp of ORF(open reading frame)and encoded 249 amino acids,which contained a PQ-loop conserved domain and a protein typi-cal conserved domain MtN3_slv.Further analysis indicated that DlSWEET1 protein contained seven transmembrane domains.The qRT-PCR analysis results showed that the DlSWEET1 was expressed in different tissues such as the roots,stems,leaves,and pulp,with higher expression levels in the leaves,followed by in the pulp,and lower expression levels in the stems and roots.After treating the leaves with different concentrations of sucrose,glucose,and fructose,the expression level of the DlSWEET1 showed varying degrees of increase.The expression level of the DlSWEET1 in the leaves treated with sucrose was significantly higher than that in the control group,but there was no significant difference among different concentrations.The expression of glucose increased significantly with the increase of glucose concentration.In the treatment of fructose,lower concentration(0.5 g·L-1)and higher concen-tration(5 g·L-1)could significantly increase the expression of the DlSWEET1.The expression of the DlSWEET1 was significantly increased under low temperature,drought and MeJA treatments,and sig-nificantly decreased under ABA treatment.However,there was no significant change in its expression after high temperature,6-BA and GA3 treatments.The fluorescence signals of GFP were mainly concen-trated and overlapped in the cell membrane and nucleus.The sugar transport activity analysis showed that the DlSWEET1 protein could transport glucose,fructose,sucrose and mannose.After transient transformation,the contents of sucrose,glucose and fructose in the strawberry were significantly higher than those in the control treatment,and the expression level of the DlSWEET1 was significantly in-creased.[Conclusion]The DlSWEET1 gene was cloned from longan fruit,and its expression level could be induced by different sugar components(sucrose,glucose,and fructose),stress(low tempera-ture and drought),and hormones(MeJA and ABA).The subcellular localization revealed that the gene is localized on the cell membrane and nucleus.The analysis of sugar transport activity showed that it 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 in fruits of longan.The article would provide a theoretical reference for improving fruit quality of longan.
国家科技期刊平台
NSTL
万方数据
维普
