南美蟛蜞菊内参基因Actin的克隆及其分析  

Cloning and Analysis of The Reference Gene Actin from Wedellia trilobata

作　　者：何伟杰 何莉莉 沈芳圆 郑乾璐 黄萍 He Weijie;He Lili;Shen Fangyuan;Zheng Qianlu;Huang Ping(Institute of Environment and Ecology,School of the Environment and Safety Engineering,Jiangsu University,Zhenjiang,212013)

机构地区：[1]江苏大学环境与安全工程学院,环境生态研究所,镇江212013

出　　处：《基因组学与应用生物学》2021年第1期308-314,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31200316,31770446);江苏大学高级人才基金项目(11JDG150)共同资助。

摘　　要：看家基因Actin常被用作定量、半定量PCR试验的内参基因。为研究其他基因在南美蟛蜞菊响应环境变化的表达调控机制,根据GenBank上已登录的肌动蛋白基因(Actin)的同源核苷酸保守序列,设计特异性引物,利用RT-PCR的方法克隆获得了南美蟛蜞菊Actin基因的全长序列,并将该序列命名为WtAct。序列分析结果表明WtAct基因全长为1 134 bp,编码377个氨基酸,且与已发表的蓖麻、麻风树、青葙、向日葵等植物的核苷酸序列同源性分别在82.9%~93.6%,与这些植物的氨基酸序列相似度在94.0%~99.7%,其中南美蟛蜞菊WtAct与向日葵Actin基因的氨基酸序列相似度最高,达到了99.7%。进化树分析也表明,两者的同源性最高。采用RT-qPCR方法检测以WtAct为内参基因时南美蟛蜞菊热休克蛋白基因HSP70在不同处理条件下的表达情况,表明HSP70的确受到不同环境变化的诱导表达,说明WtAct可以作为有效的内参基因。这些结果为深入研究南美蟛蜞菊相关功能基因的表达提供了理论基础,也为其他非模式植物Actin基因的克隆提供了借鉴。Actin gene is often used as the reference gene in qPCR and PCR. In order to study the expression and regulation mechanism of other genes in the adaptation of environmental changes in Wedellia trilobata, a pair of specific primers were designed based on the conserved sequence of the homologous nucleotides of Actin genes registered in GenBank and the full-length of Actin gene was cloned by reverse transcription polymerase chain reaction(RT-PCR). The full-length sequence of the Actin gene of Wedellia trilobata was named WtAct. The sequence analysis results revealed that the full-length of WtAct gene is 1 134 bp, which encodes 377 amino acids. The homology of Actin gene nucleotide sequence between Wedellia trilobata and those plants(Ricinus communis, Jatropha curcas, Celosia argentea, Helianthus annuus, etc) shared from 82.9% to 93.6%, when the similarity of amino acid sequence is ranged from 94.0% to 99.7%, among which the highest similarity was found between Wedellia trilobata and Helianthus annuus and was 99.7%. The phylogenetic tree analysis showed that the genetic relationship between Wedellia trilobata and Helianthus annuus is the closest. The RT-qPCR method was used to detect the expression of HSP70, a heat shock protein gene of Wedellia trilobata, under different treatments. The results of RT-q PCR suggested that HSP70 was indeed induced by different environmental changes, indicating that WtAct could be used as an effective reference gene. These results provided a theoretical basis for the in-depth study of the expression of related functional genes in Wedellia trilobata, and a reference for the cloning of Actin genes in other non-model plants.

关 键 词：南美蟛蜞菊(Wedellia trilobata) ACTIN基因 克隆 表达分析 

分 类 号：Q943.2[生物学—植物学]