新麦草IPT基因亚细胞定位、过表达载体构建及鉴定

摘要：为了验证IPT基因在新麦草中的功能，研究以DT型和ST型的新麦草分蘖节为材料，通过RNA-seq分析和qRT-PCR试验验证IPT的相对表达量并进行GO富集分析，采用PCR法克隆IPT基因，构建1300-cYFP-IPT过表达载体，并进行生物信息学分析；通过烟草瞬时转染法和蛋白质印迹法鉴定IPT蛋白的亚细胞定位及表达情况。结果显示：IPT与tRNA二甲基烯丙基转移酶活性相关，在新麦草分蘖中起上调作用；克隆得到新麦草IPT基因全长，并构建了1300-cYFP-IPT过表达载体，其开放阅读框（ORF）为1362bp；多序列比对与保守结构域分析表明，新麦草IPT蛋白存在PLN02840蛋白保守结构域，与二粒小麦、小麦及硬粒小麦IPT蛋白的亲缘关系较近；蛋白质二级结构预测显示新麦草IPT蛋白二级结构由α-螺旋、延伸链、β-折叠和不规则卷曲组成；分析显示丝氨酸的磷酸化位点最有可能是蛋白发挥功能的潜在磷酸化位点；烟草瞬时转染显示IPT蛋白正常表达，定位于叶绿体中；Western blot结果显示连接载体的目的蛋白IPT正常表达，大小为76.8kDa。研究表明IPT基因在新麦草分蘖过程中上调分蘖数，1300-cYFP-IPT载体可在植株叶绿体中正常表达；该研究为后续新麦草IPT基因进一步功能验证提供试验材料和理论依据。

Subcellular Localization, Overexpression Vector Construction and Identification of IPT Gene in Psathyrostachys juncea

Abstract :In order to verify the function of IPT gene in Psathyrostachys juncea, the tillering nodes of DT type and ST type were used as materials. The relative expression of IPT was verified by RNA-seq analysis and qRT-PCR test, and GO enrichment analysis was carried out. The IPT gene was cloned by PCR method, and the 1300-cYFP-IPT overexpression vector was constructed and analyzed by bioinformatics. The subcellular localization and expression of IPT protein were identified by tobacco transient transfection and Western blot. The results showed that IPT was related to the activity of tRNA dimethylallyl transferase and up-regulated in the tillering of Psathyrostachys juncea. The full-length IPT gene of Psathyrostachys juncea was cloned, and the 1300-cYFP-IPT overexpression vector was constructed with an open reading frame (ORF) of 1362 bp. Multiple sequence alignment and conserved domain analysis showed that the IPT protein of Psathyrostachys juncea had a conserved domain of PLN02840 protein, which was closely related to the IPT protein of Triticum dicoccoides, Triticum aestivum and Triticum turgidum subsp. Durum. Protein secondary structure prediction showed that the secondary structure of IPT protein was composed of alpha helix, extended strand, beta turn and random coil. Analysis showed that serine phosphorylation sites were most likely to be potential phosphorylation sites for protein function. Transient transfection of tobacco showed that IPT protein was normally expressed and localized in chloroplasts. Western blot results showed that the target protein IPT of the connected vector was normally expressed, with a size of 76.8 kDa. The results showed that IPT gene up-regulated the number of tillers during the tillering process of Psathyrostachys juncea, and the 1300-cYFP-IPT vector could be normally expressed in the chloroplast of plants. This study provides experimental materials and theoretical basis for further functional verification of IPT gene in Psathyrostachys juncea.