尾叶桉苯丙氨酸解氨酶基因的克隆、表达与单核苷酸多态性分析

摘要

[目的]通过获得尾叶桉Eucalyptus urophylla苯丙氨酸解氨酶(PAL)基因序列,分析其在不同水肥处理下的表达模式及在尾叶桉群体中的单核苷酸多态性(SNP),为研究该基因与尾叶桉抗逆性的相关性提供基础.[方法]根据巨桉E.grandis的PAL基因序列设计引物,通过基因克隆、测序获得尾叶桉PAL基因序列;采用逆转录定量聚合酶链式反应(RT-qPCR)分析该基因在不同水肥处理下的表达模式.[结果]PAL基因组全长4507 bp,包括166 bp的5′非编码区、411 bp的3′非编码区、2172 bp的氨基酸编码序列(由2个外显子组成,分别为422和1750 bp)和1759 bp的内含子区域.PAL基因在AT4处理中表达量最高,在AT5处理中表达量最低.尾叶桉PAL基因与巨桉具有高度相似性.尾叶桉PAL基因的SNP位点主要集中在内含子区域,可见尾叶桉PAL基因在长期进化过程中保持着稳定的遗传.[结论]获得尾叶桉PAL基因全长4507 bp,该基因具有遗传稳定性,且可能在尾叶桉抗逆性中起重要作用.%[Objective]Through obtaining phenylalanine ammonia-lyase (PAL) gene sequence of Eucalyptus urophylla, to analyze the expression patterns of PAL gene in nine different water-fertilizer treatments and its single nucleotide polymorphism (SNP) in E. urophylla populations, and provide a basis for studying the correlation between PAL gene and stress resistance of E. urophylla.[Method]Primers were designed according toPAL gene sequence of E. grandis, and PAL gene sequence of E. urophylla was obtained by gene cloning and sequencing. The expression patterns of PAL gene in different treatments were obtained by reverse transcription quantitative polymerase chain reaction (RT-qPCR).[Result]The total length of PAL gene sequence was 4507 bp, including 166 bp 5′-untranslated region, 411 bp 3′-untranslated region, 1759 bp intron region and 2172 bp protein coding region (with two exons, 422 and 1750 bp respectively). The expression ofPAL gene was the most abundant in AT4 treatment, and lowest in AT5 treatment. The PAL gene of E. urophylla washighly similar to that of E. grandis. The SNP sites of PAL gene in E. urophylla were mainly concentrated in intron region, indicating that PAL gene was stably inherited in long-term evolution history.[Conclusion]The complete length of PAL gene sequence inE. urophylla is 4507 bp.PAL gene is stably inherited and plays an important role in stress resistance of E. urophylla.