白屈菜CmTLP基因克隆及植物转化载体构建

Cloning of CmTLP gene from Chelidonium majius L.and construction of plant expression vector

【目的】克隆白屈菜CmTLP基因并进行植物表达载体构建,为进一步研究CmTLP基因的功能及探索耐寒抗寒药用植物分子育种奠定基础。【方法】基于白屈菜转录组数据,分析得到白屈菜TLP基因开放阅读框,以18S RNA为内参基因,采用qRT-PCR法检测CmTLP基因在不同温度(20(CK),10,0,-7,-20℃)下白屈菜植株中的相对表达量。用RT-PCR法克隆该基因并构建植物表达载体,电击法转化根癌农杆菌。【结果】qRT-PCR结果表明,CmTLP基因在-7℃、12 h时,相对表达量最高;-7℃、24 h时,相对表达量最低。克隆白屈菜TLP基因,命名为CmTLP。该基因开放阅读框长度为696 bp,编码由231个氨基酸组成的肽链;分子质量为24.66 ku,为酸性蛋白。系统发育树表明,CmTLP基因与博落回、月季、胡桃的氨基酸序列有较高的同源性。构建了植物表达载体pRI201-AN-CmTLP,并成功转化根癌农杆菌。【结论】克隆了白屈菜CmTLP基因,构建植物表达载体pRI201-AN-CmTLP,并成功转化根癌农杆菌。

【Objective】Cloning of the CmTLP gene of Chelidonium majius L.and construction of plant expression vector were conducted to provide basis for further study of the function of CmTLP gene.【Method】Based on the analysis of celandine transcriptome data,the open reading frame of Chelidonium majius L.TLP gene was obtained.Using 18S RNA as the internal reference gene,the relative expression of CmTLP gene in Chelidonium majius L.plants at different treatment temperatures(20(CK),10,0,-7,-20℃)was detected by qRT-PCR.The gene was cloned by RT-PCR and a plant expression vector was constructed.Agrobacterium tumefaciens was transformed by electric shock.【Result】The results of qRT-PCR showed that the highest expression of CmTLP gene was at-7℃and 12 h and the lowest was at-7℃and 24 h.The Chelidonium majius L.TLP gene was cloned and named CmTLP.The gene had an open reading frame length of 696 bp encoding a peptide chain of 231 amino acids.The molecular weight was 24.66 ku and it was an acidic protein.Phylogenetic analysis showed that the CmTLP gene had high homology with the amino acid sequences of Macleaya cordata,Rosa chinensis Jacq.and Juglans regia L..The plant expression vector pRI201-AN-CmTLP was constructed and successfully transformed into Agrobacterium tumefaciens.【Conclusion】The CmTLP gene of Chelidonium majius L.was cloned,its plant expression vector pRI201-AN-CmTLP was constructed,and Agrobacterium tumefaciens was successfully transformed.