Differential Co-Expression Analysis Reveals Additional Regulatory Mechanisms Of CSA On Rice Anther Development

Pollen fertility of rice photo-thermo sensitive genic male sterile(P/TGMS)line is controlled by light or temperature,which is sterile in long day and high temperature and fertile in short day and low temperature.Thus,the P/TGMS line can be employed to two-line hybrid breeding system,making up the drawback of three-line system.Though there are about 70 rice P/TGMS line was reporter,systematical knowledge on P/TGMS mechanism and regulatory network is still unknown.Previous study in out lab shows that rice R2R2 MYB transcriptional factor csa(carbon starved anther)can create a novel PGMS line and be use in rice two-line system,however,the underlying mechanism of csa still need further study.Herein,we further studied the CSA with the co-expression network by combining the public and csa specific microarrays to systematically analyze the possibly function of CSA in regulating the pollen development,which could help further understand the mechanism of PGMS.The main findings of this study are as follows:(1)We came up with the method of expression coherence(EC)to analyze the rice reproductive and development transcriptional profiling to detect if there is any correlation between expression profiling and biological function.Moreover,after choosing the microarrays in GEO database,we constructed a co-expression platform to study specific biological events in rice reproductive and development process,which contained 27,673 genes and 243,671 gene pairs and could be used to search for genes with similar expression profile.(2)By analyzing the csa microarrays,we detected 2,085 differential expression genes consisting of 1,210 up-regulated genes and 875 downregulated genes with a threshold of 2 fold change and significant level of 0.005.Functional enrichment analysis and hub genes analysis of the network indicated that CSA could impact on the lipid(GO:0006633,fatty acid biosynthetic process;GO:0080019,fatty-acyl-CoA reductase activity)and sugar(GO:0008643,carbohydrate transport;GO:0019252,starch biosynthetic process)metabolism processes,resulting in abnormal anther development and pollen sterility in csa.(3)By combining the expression profile in rice reproductive and development process with the DEGs in csa microarrays,we constructed a high-resolution differential co-expression network(DCN)to systematically study the events during rice reproductive and development.By calculating the DEGs enrichment ratio for all genes in the network,we found 718 qualified genes and named them as hub genes and extracted the network for further study.DCN analysis showed that CSA had highly correlation with tapetum-associated genes(TDR,OsC6 and GAMYB)and sugar-associated genes(MST8 and RTS),indicating the important role of CSA in these biological processes.(4)From the result of network analysis,we also found some candidate genes,which could interact with CSA.We choosed one of the candidate genes MYB5,also a R2R3 MYB transcriptional factor,to perform the CRIPSPR-Cas9,and primary result showed that the myb5 was fertile in long day condition,while partially sterile in short day condition,which was similar to the phenotype of csa,indicating MYB5 could execute similar function with CSA.In our study,we constructed the DCN to systematically study the biological events during rice reproductive and development process and tried to find out candidate genes influencing the phenotype of CSA,which could help to illustrate the phenotype transition in bioinformatics level.In addition,in our study,we innovatively employed the DEGs enrichment ratio analysis to the network,which was proved to be an effective method in detecting functional modules.Our method in studying the transcriptome data could provide novel insight for biologist in the future.