Functional Analysis Of Heat Shock Transcription Factor OsHsfc2a In Rice(Oryza Sativa L.)

Temperature plays a key role in plant growth and survival.In the face of more and more frequent extreme weather caused by global climate change,plant growth is facing more and more challenges.Chilling injury and low temperature stress has become one of the main reasons restricting cash crops.Rice,as the main food crop in China,is a kind of coldsensitive economic crop.Improving the low temperature tolerance can ensure the normal growth and development of rice at low temperature.Therefore,to study the response of rice to low temperature and improve rice cold tolerance is one of the important goals of rice breeding.At present,it has been reported that Hsf Cs in rice can be induced by low temperature,however,there are few reports on their specific functions response to low temperature stress.In order to clarify the function of Os Hsf C2 a,the CRISPR/Cas9 vector of Os Hsf C2 a was constructed and the transgenic plants with down-regulated Os Hsf C2 a expression were obtained by genetic transformation.Through bioinformatics analysis and low temperature stress analysis of Os Hsf C2 a,we revealed the role of Os Hsf C2 a in regulating low temperature tolerance of rice.The key results are as follows,1.Sequence analysis of Os Hsf C2 a in japonica rice ZH11 and indica rice NGZ indicated that there are differences in nucleic acid sequence and amino acid sequence.Homologous analysis of the low temperature stress associated HSF in Arabidopsis and rice with Os Hsf C2 a suggested that Four HSFs of class C have the highest homology.2.The expression profile of Os Hsf C2 a was analyzed by q RT-PCR and RNA in situ hybridization.The results showed that Os Hsf C2 a exhibits difference in ZH11 and NGZ.Os Hsf C2 a is highly expressed in the leaves at flowering stage in ZH11 and leaves at filling stage in NGZ.3.By analyzing the expression of Os Hsf C2 a at different temperatures,we found that Os Hsf C2 a showed different responses to different temperatures.High temperature stress(45℃)inhibited the expression of Os Hsf C2 a,while low temperature stress(8℃)could induce the expression of Os Hsf C2 a.4.The seed germination of the indica rice NGZ and its CRISPR/Cas 9 knockout mutant lines of Os Hsf C2 a was observed and analyzed.The results showed that the downregulated expression of Os Hsf C2 a could accelerate the germination rate of seeds under normal and low temperature initiation,and improve the seed vigor.5.The seedlings of the indica rice NGZ and its CRISPR/Cas 9 knockout mutant lines of Os Hsf C2 a were planted outdoor in Guangzhou winter season and treated by low temperature stress(8℃)respectively,and their phenotype and physiological and biochemical characteristics were observed and analyzed.The results showed,compared with the NGZ wild type plants,the CRISPR/Cas9 lines showed milder symptoms,lower R1/R2,higher Fv/Fm and less reactive oxygen species accumulation under low temperature stress,while up-regulating the expression of cold response genes such as COLD1.6.The CRISPR/Cas9 mutant of Os Hsf C2 a were phenotypically observed in japonica rice ZH11 and indica rice NGZ.It was found that the agronomic traits such as plant height,tiller number and panicle length of ZH11 were not affected by knockout of Os Hsf C2 a,however,the plant height and panicle length of NGZ significantly decreased,and the the harvest index was increased by knockout of Os Hsf C2 a.In summary,Os Hsf C2 a participates in the response to low temperature for indica rice NGZ and may play a negative regulatory role.Os Hsf C2 a has an important application value in the production of indica rice.