青杄MYB转录因子基因PwMYB20的克隆及表达分析

doi:10.11707/j.1001-7488.20170504

摘要/Abstract

摘要： [目的] MYB转录因子家族是植物中最大的一类转录因子，在植物生长发育及抗逆调控网络中发挥重要作用。对青杄中MYB同源基因PwMYB20的克隆与分析，有利于进一步探究PwMYB20在植物生长发育及逆境响应中的功能，挖掘与利用青杄中的优质基因。[方法] 采用RACE-PCR技术，从青杄cDNA文库中克隆得到PwMYB20基因，并通过PCR技术克隆验证。利用ProtParam、ProtScale、FoldIndex等生物信息学软件对PwMYB20理化性质进行分析预测。通过BLAST在线工具得到植物同源蛋白，并对其进行比对分析和进化树分析。采用实时荧光定量PCR技术分析PwMYB20基因在不同组织中的表达性，以及干旱、低温、盐、ABA等非生物逆境胁迫处理后的表达变化。通过亚细胞定位及转录激活活性验证试验，揭示其生物学特性。[结果] 通过RACE-PCR克隆得到PwMYB20cDNA全长966 bp，含675 bp的完整开放阅读框，编码225个氨基酸。ProtParam工具计算蛋白分子式为C₁₁₀₄H₁₇₄₀N₃₄₀O₃₃₀S₈，分子质量为25.3 kDa，等电点为9.11；Protscale工具疏水性分析发现，PwMYB20的疏水位点与亲水位点均匀分布，推测该蛋白为亲水蛋白；SignalP工具预测发现该蛋白没有信号肽结构域；利用FoldIndex工具对蛋白质固有无序化进行分析，结果表明该蛋白固有无序化序列较多，推测在生理环境下蛋白的动态活性较大；TMHMM工具预测发现该蛋白没有跨膜结构域。通过对比分析发现PwMYB20属于MYB家族基因，编码1个R2R3-MYB蛋白。进化树分析结果显示，青杄PwMYB20与白云杉PgMYB20聚为一簇。实时荧光定量PCR结果表明，PwMYB20在种子中的表达量最高，其次是在针叶中，在花粉中的表达相对较少。PwMYB20对干旱、4℃和ABA处理均有响应，而对NaCl处理响应相对较弱。在干旱处理下，PwMYB20表达量先上升后下降；4 ℃低温处理3 h和12 h时PwMYB20的表达量上升，在4 ℃处理6 h时存在波动，呈现上升—下降—上升的趋势；PwMYB20的表达受ABA处理持续诱导。亚细胞定位分析表明，PwMYB20是一个主要定位于细胞核中的蛋白质。转录激活活性分析结果显示，PwMYB20的C端存在转录激活活性，而PwMYB20全长及其N端没有转录激活活性。[结论] 青杄PwMYB20，作为一个转录因子发挥作用，其转录激活活性位于C端；受干旱、低温和ABA诱导，普遍参与了植物应对逆境胁迫的响应过程。

关键词: 青杄, MYB转录因子, 基因克隆, 胁迫响应, 基因表达

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.

Key words: Picea wilsonii, MYB transcription factor, gene cloning, stress response, gene expression

中图分类号:

S718.46

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.

You Hanli, Yuan Yihang, Li Changjiang, Zhang Lingyun. Cloning and Expression Analysis of MYB Homologous Gene PwMYB20 from Picea wilsonii[J]. Scientia Silvae Sinicae, 2017, 53(5): 23-32.

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