The Analysis Of Physiological Response And Transcriptome Of Rice Anthers Under Freezing Stress At Booting Stage

With the obvious increase frequency of extreme weather,abiotic stress such as chilling,drought and others have lead serious damage to agricultural production.Rice is one of the most important food crops.It is a typical warm-light crop,sensitive to temperature conditions and vulnerable to suffer chilling.The chilling impact has caused frequent cold damage especially in Heilongjiang in recent years,which seriously restricts the production of rice in Heilongjiang Province.Low temperature chilling mainly causes rice anther abortion to form empty mites,resulting in reduced yield.Therefore,it is of great significance to carry out research on cold tolerance of anthers at the booting stage of rice,screen cold-tolerant germplasm resources,explore the mechanism of cold tolerance physiological regulation at the booting stage of rice,and explore the cold tolerance genes of rice at booting stage.In this study,190 inbred lines of Dongnong422×Kongyu 131 RIL population were used to evaluate the cold tolerance of rice at the booting stage of rice,and 19 cold-tolerant inbred lines and 19 cold-sensitive inbred lines were screened by seed setting rate.38 lines were used to identify the genetic consistency by 88 pairs of SSR primers.And the cold-tolerant line E176 and the cold-sensitive line E186 with the highest genetic consistency and different cold tolerance were selected.The transcriptome sequencing technique was used to analyze the anthers RNA-seq data,and the cold stress response genes and related metabolic pathways of the cold tolerance rice inbred lines at booting stage were compared and analyzed.The results are as the following;?1?In this study,the chilling treatment was carried out for 10 days in cold water at 17 °C,and then the normal irrigation conditions were restored.The seedling rate of the main stem was evaluated as the cold tolerance index,and the seedling rate of the main stem was counted after maturity.The results showed that the seed setting rate of cold stress treatment ranged from 41% to88%,which could distinguish the cold tolerance of different lines.Therefore,17 °C was used as the effective temperature for identification of cold tolerance at the booting stage of rice,and 88 SSR molecular markers were used to screen out two lines.The two lines with extremely high genetic similarity were used for follow-up studies,with line E176 being cold tolerant genotype and line E186 being cold sensitive genotype.?2?The soluble sugar content in the stems of the two materials was significantly higher than that of other organs under cold stress,and the soluble sugar content in the anthers was the lowest,indicating that a large amount of soluble sugar was transferred from the vegetative organs into the reproductive organs during meiosis from the soluble sugar measurement results.Compared with the control,low temperature treatment reduced the soluble sugar content of various organs in rice.although the total sugar content in the anthers of the cold-tolerant and cold-sensitive lines reached a significant level between the treatment and the control,but the total sugar increase in anthers of cold-tolerant lines was equivalent to 29.35% of the control,while the total sugar increase in the anthers of the cold-sensitive lines was only 9.7% of the control,indicating that the transport efficiency of the soluble sugar of the cold-tolerant lines was higher than that of the cold sensitive ones.From the multiple comparison results of proline content,the proline content in anthers was significantly higher than other organs.This suggests that during the meiosis phase,the synthetic center of proline is in the reproductive organs.Except for the proline content of the cold-tolerant lines and the proline content of the cold-sensitive strains,which were significantly higher than the control level,the proline content of the other organs did not change significantly.The anthocyanin content of the anthers in the cold-tolerant lines increased significantly,but the anthers in the cold-sensitive linesdid not change significantly,indicating that the cold-tolerant lines had a higher proline-infiltration regulation mechanism than the cold-sensitive lines.?3?A total of 97.42 Gb data were obtained from all anther samples,and 95.37% of the sequences were aligned to the rice reference genome.RNA-seq results showed that 23756 genes were detected in all samples,of which 375 specifically expressed in cold-tolerant line E176,and341 genes were specifically expressed in cold-sensitive line E186.Analysis of differentially expressed genes?DEGs?between different materials and treatments showed that there were 54 genes before and after cold stress in E176.There were 2455 DEGs in cold-sensitive lines before and after cold stress.GO enrichment analysis performed on DEGs showed that there were differences in the number of enriched GO entries and the number of genes they contained,and two materials responded cold.There are 15 common DEGs of biological processes in the two materials before and after cold stress.We found two rice anther cold tolerance candidate genes?Os03g0308800 and new gene Os new Gene₁0238?;15 DEGs were screened for real-time quantitative PCR?q RT-PCR?verification,and 14 genes were same between q RT-PCR and RNA-seq.It showed that the transcription data was true and accurate.