Cloning Andanalysis Of Lfy And Ap1 Gene From Lotus

By using RT-PCR method, partial sequences of LFY and AP1 Swere cloned from Nelumbo nucifera'Hongxiamantian',which provided necessary foundation for obtaining the whole cDNA and understanding the molecular mechanism of flowering and flower regulation in lotus.(1)An improved method for RNA extraction from lotus samples was developed. In order to extract high quality RNA from lotus materials, three methods were compared, which included modified Trizol, CTAB-LiCl and modified CTAB-LiCl, and the modified CTAB-LiCl was the best one. Based on the method of modified CTAB-LiCl, we obtained the pure and clean total RNA that could be used for further studies in the field of molecular biology such as PCR.(2) By using RT-PCR method, partial cDNA sequences of LFY were cloned from lotus.The sequences were 431bp long and encoded a protein of 143 amino acids. Sepquence analysis showed that the deduced amino acid sequence shared 95﹪, 95﹪, 94﹪, 94﹪, 94﹪and 93﹪identity with that of LFY of Chenopodium rubrum, Malus×domestica, Vitis vinifera, Nicotiana tabacum, Petunia×hybrida, Chrysanthemum lavandulifolium.Phylogenetic tree analysis also indicated that lotus had the closest genetic relationship with Eriobotrya japonica and Malus×domestica.(3) By using RT-PCR method, two partial cDNA sequences of homologous gene AP1 were cloned from lotus, which named as NeAP1-1 and NeAP1-2.The NeAP1-1 and NeAP1-2 were both 216bp long and encoded a protein of 72 amino acids.With the DNAMAN software,the nucleotides and amino acids sequences of NeAP1-1 and NeAP1-2 were analyzed.The homologies aimed to 89.35﹪and 88.89﹪respectively. It was also found that the homology reached 62﹪to most of the other AP1 homologous gene. Phylogenetic tree analysis also indicated that lotus had the closest genetic relationship with Chelidonium majus.(4)Undertaking conservative domain-search in the amino acid sequences of NeAP1-1 and NeAP1-2,the results indicated that NeAP1-1 had MADS-box region between the 1st and 21th amino acid and K-box region between the 53th and 72th amino acid, NeAP1-2 had MADS-box region between the 1st and 22th amino acid and K-box region between the 45th and 72th amino acid.