Construction And EST Analysis Of Wheat Leaf Normalized Full-length CDNA Library

Drought stress is one of the premier limitations to global agricultural production due to the complexity of the water-limiting environment and changing climate.Plants have evolved a series of mechanisms to overcome water deficit or drought stress conditions.The researches that to detect plant drought resistance genes,uncovering the plant drought resistance mechanism and improving crop drought resistance are significant to maintain national stability and prosperity and will promote the development of agricultural production in arid and semiarid regions.The construction of a normalized full-length cDNA library of wheat leaf under drought stress and the expression analysis of wheat genes under drought would lay the foundation for subsequent wheat drought resistance genes mining and wheat drought resistance mechanism reveal and provide reference for molecular breeding,contributing to safeguard national food security.In order to discover wheat drought resitance genes and reveal the drought resitance mechanism of wheat,we constructed a normalized full-length cDNA library of wheat leaf under drought stress with SMART method and performed EST analysis of c DNA library: Firstly,we performed 20%PEG8000 treatment on wheat seedlings to simulate drought stress and harvested wheat leaves as material.Then,the normalized full-length cDNA library was constructed via RNA isolation,first-strand cDNA synthesis,Long Distance PCR,Protease K digestion,cDNA normalization,ds cDNA purification and in vivo recombination,we transformed the cDNA library plasmids into yeast strain Y187 and the yeast cDNA library were diluted or concentrated into working density.Finally,the clones from the wheat normalized full-length cDNA library were selected randomly to perform sequencing.The obtained sequences were assembled into unigenes after removing the low quality sequences.Then,the unigenes were used in subsequent BLAST,GO annotation and KEGG pathway cluster analysis.The experiment results were displayed as follow:1.RNA concentration was 2.2860?g/?L,A260/280 was 2.17,and A260/230 was 2.41,reaching the study requirement.2.The capacities of cDNA library in E.coli and yeast were 5.3×105 and 1.36×106,respectively.The insert length of the cDNA library was averagely above 1000 bp.3.804 high quality ESTs were obtained from sequencing,these ESTs were assembled into 591 unigenes,64 of them were contigs,and 527 of them were singletons.The BLAST results revealed that 579 of unigenes had more homologous genes in Glycine max,Zea mays,Triticum aestivum,Oryza sativa,Hordeum vulgare,Brachypodium distachyo,Aegilops tauschii,Setaria italic,Sorghum bicolor and Triticum urartu,especially in Glycine max,Zea mays and Triticum aestivum.In addition,BLAST result revealed several stress response TFs,such as MYB and bHLH.GO annotation showed that 415 of 579 unigenes had GO terms and were involved in many biological functions,28.67% in cellular component,22.66% in molecular function and 48.67% in biological process.KEGG pathway analysis displayed that cDNA library contained several plant stress response pathways.Taken together,a high quality normalized full-length cDNA library of wheat leaves was constructed,804 high quality ESTs were obtained via sequencing,the functional annotation and classification analysis were performed,and several potential drought-related genes were discovered in wheat.