林业科学2017，Vol.53Issue(5)：23-32,10.DOI:10.11707/j.1001-7488.20170504
青杄MYB转录因子基因PwMYB20的克隆及表达分析
Cloning and Expression Analysis of MYB Homologous Gene PwMYB20 from Picea wilsonii
摘要
Abstract
[Objective] MYB is the largest family of transcription factor in plants,which is widely involved in the regulation of plant life and play an important role in both plant development and growth,and in the regulation of stress resistance.Cloning and analysis of MYB homologous gene PwMYB20 in Picea wilsonii is propitious to explore the function of PwMYB20 in plant growth and development,for the purpose of efficient use of high-qualified genes in Picea wilsonii.[Method] The PwMYB20 was cloned and verified based on the cDNA library of the EST sequence of PwMYB20 with RACE-PCR method.ProtParam,ProtScale,FoldIndex and other bioinformatics software were used to analyze and predict the physical and chemical properties of PwMYB20.The homologous proteins were obtained by BLAST online tools,and their comparative analysis and phylogenetic tree analysis were carried out.The tissue specific expression of PwMYB20 in different tissues,as well as the changes of PwMYB20 expression with drought,cold,NaCl and ABA treatments were analyzed using real-time quantitative PCR.Furthermore,subcellular localization and transcriptional activation assay were carried out to reveal its biological properties.[Result] The full length of PwMYB20 cDNA was 966 bp with an open reading frame (ORF) of 675 bp encoding 225 amino acids.ProtParam analysis showed that the protein molecular formula is C1104H1740N340O330S8,molecular weight is 25.3 kDa and isoelectric point is 9.11.Hydrophobicity analysis with Protscale showed that the hydrophobic sites of PwMYB20 were uniformly distributed,suggesting that the protein is hydrophilic.No protein peptide domain was found with SignalP.Furthermore,Protein inherent disorder analysis showed the protein contains many inherently disordered sequences.In addition,TMHMM tools predicted that the protein has no transmembrane domain.BLAST online tools analysis showed that PwMYB20 belongs to the MYB family gene,which encodes a R2R3-MYB protein.The results of phylogenetic tree analysis showed that PwMYB20 and PgMYB20 were clustered into one cluster.The real-time quantitative PCR analysis indicated that PwMYB20 expressed constitutively at a high level in seed,followed by needle,and the least was in pollen.The expression of PwMYB20 displayed responses to drought,cold and ABA treatments,but slightly to NaCl treatment.With drought treatment,the expression of PwMYB20 was up-regulated at the early stage,and then decreased after 6 h.Additionally,the expression of PwMYB20 was induced when it was 4 ℃ treated for 3 h,12 h and with a fluctuation in 6 h,the expression showed an up-down-up trend.Moreover,the expression of PwMYB20 was induced by ABA continuously.Subcellular localization analysis showed that PwMYB20 was mainly localized in the nucleus.Transcriptional activation analysis revealed that C terminal of PwMYB20 had a transcriptional activation activity,whereas the full-length PwMYB20 and its N terminal had no transcriptional activation activity.[Conclusion] The results indicated that the expression of PwMYB20 was constitutive in different tissues,and induced by drought,cold and ABA.In addition,PwMYB20 was located in the nucleus.Its C terminal had a transcriptional activation activity,although its full length is not activated.It is widely involved in responding to different stresses.关键词
青杄/MYB转录因子/基因克隆/胁迫响应/基因表达Key words
Picea wilsonii/MYB transcription factor/gene cloning/stress response/gene expression分类
农业科技引用本文复制引用
游韩莉,袁义杭,李长江,张凌云..青杄MYB转录因子基因PwMYB20的克隆及表达分析[J].林业科学,2017,53(5):23-32,10.基金项目
转基因生物新品种培育重大专项(2016ZX08009003-002). （2016ZX08009003-002）
