农业生物技术学报2012，Vol.20Issue(9)：1028-1034,7.DOI:10.3969/j.issn.1674-7968.2012.09.006
苹果属山定子柠檬酸合成酶基因(MbCS1)的克隆及表达分析
Cloning and Expression Analysis of Citrate Synthase Gene (MbCS1) in Apple (Malus baccata Borkh)
摘要
Abstract
In order to study the iron absorption and utilization molecular mechanism of different Fe efficiency genotypes apple rootstocks, we used the Malus baccata Borkh as material, which is an iron-inefficient genotype apple rootstock. Through the full-length of citrate synthase gene MxCSl was obtained from M. Xiaojinensis, which is an iron-efficient the gene encoding citrate synthase in Golden Delicious (M. Domestica Borkh ) and M. Xiaojinensis, thus we designated it as MbCS1 (GenBank accession No. JQ898346). The bioinformatics analysis showed that citrate synthase gene from M. Baccata Borkh encoded 473 amino acids, whose relative molecular weight was 54.26 kD and theoretical isoelectric point was 8.95. Subcellular localization assay of the MbCSl protein, which used transient expression of MbCS1::GFP fusion protein in onion (Allium cepa) cells by particle bombardment, displayed that MbCSl encoding protein localized in cell membrane. RT-PCR and Real-time PCR analysis suggested that MbCSl gene was expressed in roots, shoots and leaves in M. Baccata Borkh under normal supply of iron; and the expression of MbCS1gene was enhanced obviously in roots, shoots and leaves under a low iron concentration (EDTA-NaFe, 4 μmol/L) and reached to the highest value at the ninth day, then decreased; the expression of MbCSl gene was different in roots, shoots and leaves, enriched in shoots than that in leaves and roots under ion deficiency induced. The expression pattern of MbCSl gene in M. Baccata Borkh was very different from MxCSl gene in M. Xiaojinensis. The result provides solid foundation for further study of the resistance mechanisms in higher plants and provides basis for improvement of iron-inefficient apple rootstocks.genotype apple rootstock and previous studies have shown that it is related to iron absorption and transport, and their specific primers were designed to clone the citrate synthase gene (CS) from cDNA of M. Baccata Borkh by using RT-PCR, which was composed of 1 422 bp and had high homology with关键词
山定子/柠檬酸合成酶基因/铁胁迫/Real-time PCRKey words
Malus baccata Borkh/ Citrate synthase gene/ Iron stress/ Real-time PCR引用本文复制引用
张柳霞,王忆,朱斌,王少甲,张新忠,许雪峰,韩振海..苹果属山定子柠檬酸合成酶基因(MbCS1)的克隆及表达分析[J].农业生物技术学报,2012,20(9):1028-1034,7.基金项目
国家高技术研究发展计划(863)项目(No.2011AA001204)、现代农业产业技术体系岗位专家(No.CARS-28)和国家转基因专项(No.2009ZX08009-122B) （863）
