| The lotus(Nelumbo nucifera)is a perennial aquatic herb and is also an important economic plant.It is widely planted in North and South China,with many varieties in Wuhan and Hangzhou.According to the use of various parts of the lotus,the lotus can be divided into rhizome lotus,seed lotus and flower lotus.The rhizome is the storage organ of lotus and its main economic organ.Understanding its development mechanism will provide theoretical basis for lotus cultivation and breeding.The development of lotus rhizome is a complicated process,which can be divided into four stages: the stolon stage(elongation in a single direction),the initial swelling stage(longitudinal growth stops and begins to increase the perimeter),the intermediate swelling stage(the rhizome continues to swell,and the starch gradually accumulation),and the later swelling stage(the rhizome stops growing and starch accumulates quickly).Although increasing people are concerning about the mechanism of storage organ enlargement,the molecular mechanism of lotus rhizome formation is still largely unknown.At present,the most commonly-used method for swelling gene mining is finding out the differentially expressed genes according to the change of gene expression.However,this method usually ignores genes that are essential for the expansion of lotus rhizome but with small changes in expression.In this study,we used transcriptome sequencing data from different lotus tissues and applied sample-specific network analysis method to mine key genes of lotus rhizome enlargement.First,we downloaded the sequencing data of 11 different organizations of lotus and three development stages including stolon stage,middle swelling stage,and later swelling stage of lotus rhizome from the NCBI database.Secondly,HISAT2 was used to align reads to the reference genome,StringTie was used to assemble the alignments into transcripts,quantify gene expression levels and obtain gene expression matrix.Then,a reference network was constructed using gene expression matrix of a total of 47 samples from 11 different tissues of lotus,and a perturbation network was constructed from gene expression matrix of a total of 6 samples at three developmental stages of lotus rhizome,that is,lotus root specific networks at different development stages.Finally,The correlation between genes was calculated based on Pearson’s correlation coefficient,and the difference network of three different development stages of lotus rhizome was constructed.It was found that the difference network between stolon stage and middle swelling stage of lotus rhizome contained 427 genes,the difference network between the later swelling stage and middle swelling stage contained 1,222 genes,and the difference network between the later swelling stage and stolon stage contained 1,284 genes.What’s more,Enrichment analysis of genes in the difference network between the later swelling stage and middle swelling stage found that these genes were mainly enriched in organic substance metabolic process,primary metabolic process,purine nucleoside monophosphate metabolic process in terms of biological processes;in terms of cellular component,these genes were significantly enriched in macromolecular complex,intracellular part,cytoplasm;in terms of molecular function,these genes were mainly enriched in threonine-type endopeptidase activity,RNA polymerase activity.Among these genes,11 genes related to starch,1 gene related to sugar metabolism,11 genes related to cell division,and 9 genes related to cell shape were found.In addition,three tissue-specific regulatory networks for lotus leaf,petal,and lotus rhizom were constructed using sample-specific networks.Among the three tissuespecific networks,the top ten genes with higher degree were selected as tissue-specific candidate genes for lotus leaf,petal,and lotus rhizome respectively.We found that most of the genes with high degree values in lotus leaf are related to chloroplastic-like;genes with high degrees found in petal are related to oxygenase and acetyl-CoA;genes with high degrees found in lotus rhizome are similar to vacuolar and neutral ceramideaselike.The sample-specific network used gene interaction analysis to analyze the key genes of lotus rhizome enlargement,which provides new ideas for botanical research.It can be applied to the construction of a plant network with a small sample size and can be used to find key genes for stress resistance and control different important genes of flower color,tissue-specific genes of different developmental stages,etc,which will facilitate subsequent research efforts in botany. |
