甘蔗烟酰胺转氨酶基因(SoNAAT1)的克隆及其在缺Fe胁迫下表达量变化  

Cloning of Nicotianamine Aminotransferase Gene(SoNAAT1)from Sugarcane and Its Expression Analysis under Fe Deficiency

作　　者：农倩[1] 李长宁 Mukesh Kumar Malviya 苏琴[1] 张艳[1] 陈艳露[1] 覃丽萍[1] 谢玲[1] NONG Qian;LI Chang-ning;Mukesh Kumar Malviya;SU Qin;ZHANG Yan;CHEN Yan-lu;QIN Li-ping;XIE Ling(Microbiology Research Institute, Guangxi Academy of Agricultural Sciences, Guangxi Nanning 530007, China;Key Laboratory of Sugarcane Biotechnology and Genetic Improvement （Guangxi） , Ministry of Agriculture / Guangxi Key Laboratory of Sugarcane Genetic Im-provement ,Guangxi Nanning 530007 , China;Guangxi Crop Genetic Improvement and Biotechnology Lab, Guangxi Nanning 530007,China)

机构地区：[1]广西农业科学院微生物研究所,广西南宁530007 [2]农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室,广西南宁530007 [3]广西作物遗传改良生物技术重点开放实验室,广西南宁530007

出　　处：《西南农业学报》2018年第4期653-658,共6页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31701489);广西自然科学基金项目(2016GXNSFAA380126;2017GXNSFBA198123)

摘　　要：【目的】克隆甘蔗烟酰胺转氨酶(Nicotianamine Aminotransferase,NAAT)基因,分析其基本生物学信息及在缺Fe胁迫下的表达量变化,为进一步探讨SoNAAT1基因在甘蔗麦根酸类Fe载体生物合成途径中的作用提供理论依据。【方法】根据前期获得的EST序列设计引物,利用RT-PCR和RACE-PCR获得甘蔗SoNAAT1基因c DNA全长,应用在线软件程序对氨基酸序列进行分析,通过实时荧光定量PCR(qRT-PCR)分析基因在不同缺Fe胁迫时长下的表达情况。【结果】甘蔗SoNAAT1基因全长1593 bp,编码区长度1206 bp,编码401个氨基酸,理论分子量为44.2 kD,等电点为5.64,主要在细胞质中表达,蛋白结构以α-螺旋为主。SoNAAT1与玉米、水稻、高粱的NAAT基因序列具有高度的同源性,系统发育进化树显示其与高粱Sb NAAT3优先聚在一起。qRT-PCR分析结果表明,SoNAAT1的表达随着缺Fe胁迫时间的延长而增强,缺Fe胁迫引起甘蔗根系内源ABA含量和相关抗氧化酶活性增加,并与SoNAAT1表达密切相关。【结论】甘蔗SoNAAT1基因编码序列具有较高的保守性,表达量随着缺Fe胁迫时间的延长而增加,推测该基因参与了甘蔗麦根酸类Fe载体生物合成途径的调控过程。【Objective】The nicotianamine aminotransferase gene（SoNAAT1） of sugarcane was cloned,and its biological information and expression under Fe deficiency stress was analyzed,in order to provide theoretical basis for further exploring the role of SoNAAT1 in sugarcane Fe-chelating compounds known as phytosiderophores biosynthesis pathway.【Method】Primers were designed according to the EST sequence obtained from the previous research.The sugarcane＇s gene SoNAAT1 was cloned by RT-PCR and RACE-PCR,and the bioinformatics analysis was performed by using various online software programs.Meanwhile,the real-time fluorescence quantitative PCR（qRT-PCR） method was adopted to discuss the expression of SoNAAT1 gene under Fe deficiency stress.【Result】The length of SoNAAT1 gene was 1593 bp,the length of the coding region was 1206 bp,and 401 amino acids were encoded.SoNAAT1 had a molecular weight of 44.2 k D and a theoretical isoelectric point of 5.64,which was mainly expressed in cytoplast,and its protein structure was mainly composed of α-helix.The sequence of the SoNAAT1 gene had high homology with the NAAT gene sequence of maize（Zea mays）,rice（Oryza sativa）,and sorghum（Sorghum bicolor）.A phylogenetic treeindicated the preferential clustering of SoNAAT1 with SbNAAT3 in a small branch.The qRT-PCR results showed that SoNAAT1 expression was significantly enhanced with the prolonged absence of Fe stress.,followed by increasing ABA content and antioxidant enzyme activities.【Conclusion 】The coding sequence of sugarcane SoNAAT1 gene was highly conserved,and its expression level in sugarcane root gradually increased under Fe deficiency stress.It was speculated that this gene is involved in the regulation process of the Fe phytosiderophores biosynthesis pathway of the sugarcane.

关 键 词：甘蔗 烟酰胺转氨酶 基因克隆 表达分析 

分 类 号：S566.1[农业科学—作物学]