Molecular Cloning And Analysis Of Several Important Genes In Nelumbo Nucifera(Lotus)

Lotus(Nelumbo nucifera) is a very old water flower plant originated from China. As a very important economic water plant with beautiful flowers,it can be also eaten as a delicious vegetables,useful medical herbs.Lotus,which has a long planting history in the most river area of China,has always been paid a great attention.Traditional biological researches of lotus mostly concerned morphology,plant taxology and genetic breeding. So it has a great sense to do the lotus molecular biological researches.In the first step of lotus molecular biological researches,a highly efficient RNA extraction method for lotus tissues was modified according to the prior researches,then by using RT—PCR method,partial sequences of three different genes(chs,expansin,ppo) were amplified from total RNA of Lotus (Nelumbo nucifera)' s young folded leaves.1. RNA extractionThe lotus tissues contain a lot of secondary metabolites such as polysacharides,phenolic compounds, lipids etc.Therefore it is difficult to isolate high-quality RNA from the metabolites with routine methods.According to the prior researches,a highly efficient RNA extraction method for lotus tissues was established by a modification.RNA,which is high concentration, good purity and unconspicuous degradation,could be attained by using a novel and simple CTAB-LiCl-based extraction method.This method is also suitable for further RT-PCR and other molecular biology experiments.Obviously, it is also useful for the RNA extraction of other plant materials which have plenty of polysaccharide,phenolic compounds.2. Molecular cloning of chs genesChalcone synthase (CHS) is a key enzyme in the biosythesis of all classes of flovonoids,and variation of its expression might affect the color of flowers.By using PCR method,partial sequences of chs gene exon2 were amplified from cDNA of Nelumbo /Jwci/era(lotus).After cloning and sequencing,? sequences ranging from 844bp to 936bp in size were obtained,and they encode 282 to 312 amino acids.Compared these sequences with other chs genes from GenBank,it reveals a similarity higher than 80% at the nucleotide level,and 85% at the amino acid level,so they should be the members of chs gene family.The success of cloning chs gene from lotus has laid the foundations ofstuding its functions,especially in flower colors.to3. Molecular cloning of expansin genesExpansin is a group of cell-wall-localized proteins which inducing extension of cell wall.So it is believed to play a great important role in plant development and growth of vegetative organs, the reactions to the environment,fruit development and ripening. By using PCR method,partial sequences of expansin genes were amplified from cDNA of Nelumbo nucifera(\otas).After cloning and sequencing^ sequences ranging from 531bp to 532bp were obtained,and all of they encode 177 amino acids.Compared these sequences with other expansin genes from GenBank,it reveals a similarity higher than 83% at the nucleotide level,and 84% at the amino acid level.so they should be the members of a -expansin gene family.4. Molecular cloning of ppo genesPolyphenol oxidase (PPO) is a ubiquitous copper metalloprotein in the plastids of many plant species,fungi and insects.lt is believed to be the primary response to the deleterious browning of many fruit and vegetable crops and is thought to be involved in plant photosynthesis,plant-pest interactions,grown and development,flower coloration. By using PCR method,partial sequences of ppo genes were amplified from cDNA of Nelumbo ?wc//era(lotus).After cloning and sequencing, 7 sequences ranging from 978bp to 1057bp were obtained,and they encode 326 to 352 amino acids. Compared these sequences with other ppo genes from GenBank,it reveals a similarity higher than 79% atthe nucleotide level,and 61% at the amino acid level, so they should be the members of ppo gene family.