摘要
以耐旱性较强的湘苎3号为材料,从苎麻转录组测序结果中获得了1个与P5CS基因高度相似的Unigene,该片段长1448bp,根据该序列设计5′RACE和3′RACE槽式PCR引物,利用RACE结合RT-PCR技术分别克隆得到590bp的5′端和293bp的3′端,拼接获得该基因全长cDNA序列。对该序列进行生物信息学分析表明,该基因全长为2318bp,其中开放读码框为2154bp,编码717个氨基酸,其编码蛋白质的等电点和分子量分别为6.57kD和77.56kD,与亚洲棉、麻风树、拟南芥和水稻的P5CS基因的核苷酸序列相似性分别为81%、81%、75%和72%,蛋白质序列的相似性分别为86%、85%、78%和77%,说明该基因与P5CS为同源,被命名为BnP5CS1。半定量RT-PCR分析表明,该基因在苎麻的茎尖中表达量最高,在根中其次,且受干旱胁迫的诱导。P5CS基因是植物体内合成脯氨酸的关键酶基因,BnP5CS1基因的克隆将为苎麻的抗逆分子育种和进一步的功能分析鉴定基础。
Proline is one of osmotic regulators in plant, which plays an important role in regulating ramie in response to drought stress. The synthesis of proline has been reported to be restricted by the enzyme of Δ1-pyrroline-5-carboxylate synthetase （P5CS）. In this study, taking Xiangzhu 3 with drought tolerance as an experimental material, we obtained a ramie Unigene highly identified with P5CS gene from the ramie transcription group sequencing, and the gene length was 1 448 bp. According to the known fragment, the 5’ and 3’ RACE PCR nest primers were designed, and a 590 bp 5′ terminal sequence and a 293 bp 3′ terminal sequence were ampli-fied by RACE and RT-PCR, separately. The results of bioinformatics analysis showed that the full length of the P5CS gene was 2 318 bp, the open reading frame was 2 154 bp, coding 717 amino acids, the isoelectric point and the molecular weight of coded protein were 6.57 and 77.56 kD, respectively. Nucleotide sequence Blast indicated that the Unigene of ramie shared an identity of 81%, 81%, 75% and 72% with P5CS genes of cotton, leprosy tree, Arabidopsis thaliana and rice, respectively, and the similarity in protein Blast was 86%, 85%, 78%, and 77%, respectively. The result showed it was a homologous gene of P5CS in ramie, and named BnP5CS1. Semi-quantitative RT-PCR analysis indicated that the expression of BnP5CS1 had a higher level in stem tip than in root, and the gene induced by the drought stress. P5CS is a key enzyme gene in the synthesis of proline in plants, so the cloning of BnP5CS1 gene might lay a foundation for the adversity resistance molecular breeding of ramie and further function analysis.
出处
《作物学报》
CAS
CSCD
北大核心
2012年第3期549-555,共7页
Acta Agronomica Sinica
基金
湖南省科技计划重点项目(2010TP4004-1)
湖南省教育厅重点项目(09A042)
湖南省教育厅优秀人才项目(10B050)
湖南农业大学人才引进科技资助项目(07YT03)
省部共建国家重点实验室培育基地科学基金开放项目(10KFXW09)资助
关键词
苎麻
P5CS
脯氨酸
干旱
表达分析
Boehmeria nivea； P5CS； Proline； Drought； Expression analysis
