黄花苜蓿铁蛋白cDNA的克隆与序列分析

Cloning and sequence analyzing of ferritincDNA from Medicago falcata L.

为克隆黄花苜蓿铁蛋白基因,根据已报道的植物铁蛋白基因序列设计引物,利用RT-PCR法扩增得到大小约为750 bp的特异性片段,并将其克隆到pBlueskript II SK+上.序列分析结果表明,黄花苜蓿铁蛋白基因cDNA全长为756 bp,编码252个氨基酸,与紫花苜蓿铁蛋白基因的核苷酸、氨基酸的同源性均为97%.该基因编码的蛋白质是一个定位在叶绿体上的球形蛋白,理论相对分子质量为28 ku,等电点为5.47,二级结构为α/β混合型蛋白,某些氨基酸在密码子的选择上存在一定的偏向性.该蛋白具有1个FER-RITIN-LIKE功能区、2个铁蛋白基因家族铁结合区域的信号序列、3个潜在的N-糖基化位点、1个蛋白激酶C磷酸化位点、1个依赖于cAMP和cGMP的蛋白激酶磷酸化位点以及3个酪蛋白激酶Ⅱ磷酸化位点等重要的功能基序.黄花苜蓿铁蛋白基因的克隆,为今后利用该基因进行改良植物铁营养成分及提高植物抗氧化胁迫能力的基因工程奠定了基础.

In order to clone theferritin gene from Medicago falcata L. , a pair of primers was designed based on theferritin gene sequence ofMedicago sativa L. , and a 750 bp specific fragment was amplified by RT- PCR technique. Then it was cloned into pBlueskript II SK ＋. The results of sequence analysis show that the full length of ferritin gene from Medicagofalcata L. is 756 bp; it encodes 252 amino acids and shares homology of 97% both in nucleotide acid and amino acid sequence compared with that of Medicago sativa L. The ferritin from Medicago falcata L. is a domain globular protein located in chloroplast, the molecular mass and isoelectric points of the putatire protein calculated theoretically are 28 ku and 5. 47 respectively, and amino acids exist codon bias. Its second structure is α/β mixed protein. Sequence analysis reveals that it has one FERRITIN_LIKE domain, two signature sequences of ferritin iron-binding regions, three potential N-glycosylation sites, one protein kinase C phosphorylation site, one cAMP-and cGMP-dependent protein kinase phosphorylation site, three casein kinase II phosphorylation sites and so on. The cloning offerritin gene from Medicagofalcata L. provides a fundamental basis for further improvement of iron content and anti-oxidation stress of plants through genetic engineering.