龙眼NF-YB转录因子DlNF-YB1、DlNF-YB10的克隆和表达分析

Cloning and expression analysis of NF-YB transcription factors DlNF-YB1 and DlNF-YB10 in longan

【目的】克隆龙眼2个NF-YB转录因子DlNF-YB1和DlNF-YB10 cDNA编码区全长,了解DlNF-YB1和DlNFYB10在龙眼中的功能。【方法】RT-PCR克隆DlNF-YB1和DlNF-YB10的CDS序列,进行DlNF-YB1和DlNF-YB10蛋白序列理化性质以及转录起始位点前1500 bp的启动子序列分析;qRT-PCR分析DlNF-YB1和DlNF-YB10在龙眼不同组织的表达,DlNF-YB10在龙眼花芽分化期的表达,DlNF-YB1在水分胁迫条件下的表达。【结果】以‘红核子’cDNA为模板克隆得到DlNF-YB1(GenBank登录号:MK372373)和DlNF-YB10(GenBank登录号:MK359142)的CDS序列,分别编码175和176个氨基酸;蛋白理化性质分析结果显示DlNF-YB1与DlNF-YB10的理化性质基本相近,三维结构存在一定的区别;DlNF-YB1启动子序列中含有与干旱响应相关的MBS顺式作用元件,DlNF-YB10启动子序列中含有生长素响应以及ABA响应相关的顺式作用元件;表达分析结果,DlNF-YB10在龙眼成年叶芽中表达量较高,且表达量从10月到翌年1月呈现先升后降的趋势,在12月达到峰值;DlNF-YB1在根中表达量较高,根系水分胁迫试验表明DlNF-YB1在干旱胁迫下表达量显著升高。【结论】DlNF-YB10的功能可能与龙眼花芽分化早期的生理分化有关;DlNF-YB1的功能可能与龙眼的抗旱有关。

【Objective】Longan(Dimocarpus longan L.)is a subtropical fruit species of the Sapindaceae.Longan,a typical drought-tolerant plant,is widely cultivated in tropical and subtropical region and has great economic values.Transcription factor NF-YBs are important regulators during plant growth and development,participating in regulating embryonic development,plant drought resistance,floral meristem growth and flowering time under photoperiod pathway.Two NF-YB transcription factors DlNF-YB1 and DlNF-YB10 in longan were identified and their putative functions were investigated in this study.【Methods】Two NF-YB homolog genes,UN15136 and CL2518 were identified in longan transcriptome data and named DlNF-YB1 and DlNF-YB10.Primers were designed by DNAMAN8.0.Using cDNA of‘Honghezi’longan as the template,CDS sequences of DlNF-YB1 and DlNF-YB10 were amplified by RT-PCR.The bioinformatics analysis of two DlNF-YB sequences was carried out for further understanding of their putative functions.ORF regions of two DlNF-YB genes were founded by ORF Finder program in NCBI.Phylogenetic tree analysis of two DlNF-YBs and the Arabidopsis thaliana NFYB family were obtained by MEGA6.0 software.Multiply amino acid alignment of DlNF-YB genes with other NF-YB genes of 5 species was obtained by DNAMAN8.0.Information about NF-YB protein sequence in different plants was analyzed by PredictProtein online software.The basic physicochemical properties of the proteins were analyzed by ExPASyProtParam.The secondary structure analysis about the content ofα-helix,β-sheet,β-turn and random in DlNF-YB proteins were obtained by SOPMA online software.The distribution of curl and the homology modeling of the tertiary structure of DlNF-YB1 and DlNF-YB10 were performed using Swiss-Model online software.In addition,promoter sequences of DlNF-YB1 and DlNF-YB10 were analyzed by PLANTCARE online software.Further,expression patterns of DlNF-YB1 and DlNF-YB10 in different organs and different developmental period of longan were analyzed by qRT-PCR.The expression levels of DlNF-YB10 during longan flower bud differentiation and DlNF-YB1 under water stress were also analyzed by qRT-PCR.When water stress experiment was started,seeds of‘Honghezi’longan were sown in trays containing nutrient soil,vermiculite and perlite at the proportion of 2:1:1.Seedlings were cultivated under the growth chambers at a temperature of(25±2)℃,and the seedling growth was kept with 20.0%-30.0%soil moisture and 50%-55%air humidity.After 6 months,the seedlings were transplanted to 20 cm-diameter plastic pots for root water stress trial.Treatments of three water contents were set.The soil water content of 10%was set as drought treatment;the water level line was kept 5 cm above the bottom(at this time,the soil water content is more than 70%)was set as water immersion treatment;the water content between 20%and 30%was set as the control.In this study,the test was performed in a completely randomized design using Excel 2016,and the LSD test was performed using the Duncan test in SPSS 22.0(p<0.05).【Results】The CDS sequences of DlNF-YB1(GeneBank accession number:MK372373)and DlNF-YB10(GeneBank accession number:MK359142)were cloned.Multiply protein sequence analysis of DlNF-YB1 and DlNF-YB10 with NF-YB family of Arabidopsis thaliana showed that DlNF-YB1 and DlNF-YB10 had the highest similarity with AtNF-YB1(80%)and AtNF-YB10(69%),respectively.DlNF-YB1 contained528 bp and DlNF-YB10 contained 531 bp open reading frames(ORF),encoding 175 amino acids and176 amino acids,respectively.Both DlNF-YB amino acid sequences included a typical NF-YB/HAP3 domain and five potential functional sites:N-myristoylation site,N-glycosylation site,Casein kinaseⅡphosphorylation site,protein kinase C phosphorylation site,and cAMP and cGMP-dependent protein kinase phosphorylation sites.Multiple protein sequence alignment revealed a high similarity among DlNF-YB1,DlNF-YB10,CpNF-YB1,TcNF-YB1,AtNF-YB1,GmNF-YB10 and AdNF-YB10.Protein physicochemical properties analysis showed that DlNF-YB1 and DlNF-YB10 had similar physical and chemical properties,and both were unstable and hydrophilic proteins.The secondary structure of DlNF-YB1 and DlNF-YB10 included the form ofα-helix and random coil mainly.However,the three-dimensional structure of DlNF-YB1 and DlNF-YB10 proteins differed greatly.Promoter sequence analysis showed that both of DlNF-YB1 and DlNF-YB10 promoters contained ABRE abscisic acid response elements,and specially,DlNF-YB1 promoter contained the drought response MBS cis-acting element,while the DlNF-YB10 promoter sequence contained the auxin response-related AuxRR-core cis-acting element.The expression level of both genes in‘Honghezi’seedling buds were similar with the expression level in the new shoots of‘Sijimi’longan.But the relative expression of DlNF-YB10 and DlNF-YB1 varied in different organs and months,DlNF-YB10 was highly expressed in the vegetative buds of adult longan tree and the expression level of DlNF-YB10 during physiological differentiation of flower bud gradually increased from October to December,with a peak in December,and began to decline in January of the following year.Differently,DlNF-YB1 was highly expressed in roots.Relative expression of DlNF-YB1 varied under different water stresses and it significantly increased under drought stress.【Conclusion】DlNF-YB1 and DlNF-YB10 have different functions in longan.The function of DlNF-YB10 may be related to the physiological differentiation of flower bud,while DlNF-YB1 may play an important role in the drought resistance of longan.