Mengliang Cao

Comparative transcriptome analysis of RNA-seq data for cold-tolerant and cold-sensitive rice genotypes under cold stress

Cold stress greatly affects plant growth and crop yield. To identify novel genes and possible mechanisms involved in chilling tolerance responses in rice seedlings, RNA sequencing (RNA-seq) technology was used for genome-wide gene expression profiling analysis to compare three cold-tolerant genotypes and one cold-sensitive genotype under both normal temperature and cold stress treatments. The results showed that in response to cold, a total of 2242 common regulated differentially expressed genes (DEGs) were detected during cold stress in all of the genotypes, and a total of 318 DEGs were detected as common DEGs related to cold tolerance in the three cold-tolerant genotypes. Gene ontology (GO) enrichment analyses were performed for these DEGs. By combining protein function clustering analysis and significantly enriched GO terms analysis, we suggest that calcium signal transduction may play a dominating role in the cold stress response of rice. Further, we suggest that RNA helicases may be directly involved in temperature sensing in rice under cold stress, and that compensation of ATPase activity, alteration of epigenetic processes, and further reduction of functional photosynthesis proteins may serve as mechanisms for cold tolerance regulation in the cold-tolerant rice genotypes. These results expand our understanding of the complex mechanisms involved in chilling tolerance in rice, and provide a foundation for future studies on chilling tolerance in rice and other cereal crops.

Knock out of the annexin gene OsAnn3 via CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice

Plant annexins are Ca2+-dependent phospholipid-binding proteins and exist as multigene families in plants. They are implicated in the regulation of plant development as well as protection from environmental stresses. In this study, the rice annexin gene OsAnn3 knockout was performed via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing. Thus, mutant plantlets were successfully obtained. We identified cold tolerance phenotype of T1 mutant lines from T0 biallelic mutants using the 4∼6°C for 3 days cold treatment. The results showed that REC (the relative electrical conductivity) of T1 mutant lines was increased, and the survival ratio of T1 mutant lines was decreased dramatically compared with the wild type after the exposure to cold treatment. It was suggested that OsAnn3 was involved in cold tolerance of rice.

Isolation of candidate disease resistance genes from enrichment library of Oryza minuta based on conserved domains

A strategy was described for the isolation of disease resistance genes from Oryza minuta by integrating the techniques of transformation-competent genomic library, RecA-mediated magnetic bead enrichment library and candidate disease resistance gene cloning. The principal advantages of this method were: simple, rapid and suitable. In this research, a transformation-competent genomic library with a volume of 2.68 × 10 5 clones was constructed for O. minuta ; an enrichment library of disease resistance genes was further constructed with a volume of 4992 clones, from which 26 positive clones were screened by colony in situ hybridization. The end-clone sequencing of 13 representative positive clones showed that 6 clones were well matched with cloned disease resistance genes or located near the existing disease resistance genes. Full sequencing of a clone revealed a gene similar to a putative brassinosteroid LRR receptor kinase in japonica rice; the protein structure analysis suggested that it may be a disease resistance gene or functionally involved in a signal transduction pathway. These results indicate these clones may include new R genes and the strategy is feasible to clone new R genes from wild rice species. Key words : Oryza minuta, disease resistance gene, transformation-competent genomic library, magnetic bead enrichment library, colony in situ hybridization, sequence analysis.