Cloning and Expression Pattern Analysis of the ApNAP Gene in Asarina procumbens
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摘要: 金鱼藤（Asarina procumbens）是玄参科金鱼藤属多年生藤本植物，在园林中常用于垂直绿化，是良好的观赏植物，但金鱼藤叶片衰老后很快干枯并脱落，仅留光秃的茎蔓，使其观赏价值大大降低。AtNAP是拟南芥NAC基因家族中与衰老相关的转录因子，在调控叶片衰老过程中有重要的作用。本研究以金鱼藤衰老叶片为材料，运用RT-PCR和RACE技术克隆金鱼藤中ApNAP基因cDNA序列。结果显示，该基因位于细胞核中，cDNA全长为1341 bp，开放阅读框为936 bp，编码311个氨基酸，相对分子量为35.21 kD，等电点为9.13；ApNAP基因组全长1526 bp，含有2个内含子和3个外显子。多重序列比对表明，ApNAP属于NAC转录因子家族。系统进化树分析显示，ApNAP属于NAP亚组，与AtNAM的同源关系最近。实时荧光定量PCR结果表明，受自然状态下衰老和黑暗处理的诱导，ApNAP基因表达量增加，这进一步说明ApNAP基因参与调控金鱼藤叶片衰老的过程。该基因的分离对利用转基因技术延缓金鱼藤叶片衰老，提高金鱼藤在园林绿化中的观赏价值具有实际意义。Abstract: Asarina procumbens, Scrophulariaceae, is a perennial vine and good ornamental plant for vertical greening in the garden. However, when the leaves of A.procumbens become dry and abscised after senescing, the bare tendrils greatly reduce its ornamental value. AtNAP is a NAC family transcription factor gene that plays a key role in regulating leaf senescence in Arabidopsis. In this study, ApNAP, a homologous gene of AtNAP, was cloned from A.procumbens senescent leaves by RT-PCR and RACE. Results showed that the ApNAP gene was located in the nucleus and had a total length of 1341 bp with an open reading frame of 936 bp, encoding 311 amino acids, of which the relative molecular weight was 35.21 kD and the isoelectric point was 9.13. The length of the genome was 1526 bp with two introns and three exons. Multiple sequence alignment indicated that the ApNAP gene was a NAC transcription factor. Phylogenetic analysis indicated that the ApNAP gene belonged to the NAP subfamily and shared highest similarity with AtNAM in Arabidopsis. The results of real-time qPCR showed that the expression level of ApNAP was increased under treatments induced by natural and dark-induced senescence, indicating that it was involved in the regulation of the leaf senescent process of A.procumbens. The isolation and cloning of ApNAP had an important value in delaying leaf senescence and improving the greening value of A.procumbens.
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Keywords:
- Asarina procumbens /
- Gene cloning /
- ApNAP /
- Real-time qPCR
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