Physiological Response And Transcriptome Analysis Of Rice Roots In Response To Cold Stress At Seedling Stage

In recent years,the harm of low temperature at seedling stage in southern rice region has become the main factor affecting the safe production of rice in the Yangtze River Basin in China.Mining and using rice low temperature tolerance genes has important theoretical and practical significance for the genetic improvement of rice cold tolerance.In this paper,the effects of low temperature stress on active oxygen metabolism and related gene expression in rice seedling roots were studied by artificially simulating low temperature;Secondly,transcriptome sequencing was used to analyze the differential gene expression of seedling roots under low temperature stress at seedling stage,and the function of target gene lts1 was preliminarily analyzed.The main results are as follows:1)The total root length,root surface area,root diameter,root volume and root activity of seedlings decreased under low temperature stress compared with that under moderate temperature stress,and the decrease amplitude increased with the extension of treatment time,while the decrease amplitude of seedlings of control variety Xiangzaoxian45 was greater than that of cold-tolerant cultivar Sanqishiluo.Under low temperature stress,the activities of CAT and POD in roots increased at first and then decreased,and the relative conductivity,MDA content,superoxide anion content and H₂O₂content in roots increased,and the variation amplitude of above physiological indexes in roots of Sanqishiluo was smaller than that of Xiangzaoxian45.Under low temperature stress,root proline content increased,soluble protein content increased first and then decreased,and the variation range of Xiangzaoxian45 was larger than that of Sanqishiluo.The expression levels of POD,CAT,APX and MDAR genes in roots showed a decreasing trend,while the expression levels of POD,CAT,APX and MDAR genes in roots of Rhizogenesis were higher than those of Xiangzaoxian45.The above physiological results indicated that the cold tolerance of Sanqishiluo was stronger than that of Xiangzaoxian45.2)The results showed that 1409 differencing genes were identified in Xiangzaoxian45seedlings under low temperature stress on the first day,including 1080 up-regulated genes and 329 down-regulated genes.There were 1218 genes,including 740 up-regulated genes and 478 down-regulated genes,among Sanqishiluo.Among them,there were 577up-regulated genes and 118 down-regulated genes.On day 3 of low temperature treatment,there were 3372 differentially expressed genes in Xiangzaoxian45,including 1324up-regulated genes and 2048 down-regulated genes.There were 1782 differentially expressed genes,including 540 up-regulated genes and 1242 down-regulated genes.1336common differential genes were screened from the two cultivars,including 434 up-regulated genes and 902 down-regulated genes.On day 5 of low temperature treatment,there were3548 differentially expressed genes in Xiangzaoxian45,including 1507 up-regulated genes and 2041 down-regulated genes.There were 1,810 differentially expressed genes,including485 up-regulated genes and 1,325 down-regulated genes.A total of 1192 common differential genes were screened,including 372 up-regulated genes and 820 down-regulated genes.On the first,third and fifth days of low temperature treatment,228 common differentially expressed genes were screened,among which 146 genes were up-regulated and82 genes were down-regulated.GO and KEGG enrichment analysis showed that these differential genes mainly performed catalytic,binding and transport functions in cell membrane and cell site,participated in metabolism,cellular processes and response to stimuli,and mainly affected cell membrane and transcriptional regulatory factors under low temperature stress.These genes were mainly enriched in the biosynthesis of secondary metabolites,MAPK plant signal,plant hormone signal transduction and phenylpropane biosynthesis,suggesting that these pathways play an active role in cold stress response of rice seedlings.3)Cloning primers were designed in the CDS region of LTS1 gene,and the LTS1 gene was amplified using c DNA of Zhonghua11 genome as template.Analysis of LTS1 gene expression characteristics showed that LTS1 gene could be induced under low temperature stress.The expression level of LTS1 gene was highest in stem on the first and third days of low temperature stress,followed by leaves and roots.The expression level of LTS1 gene was highest in leaf on the fifth day of low temperature stress,followed by stem and roots.The expression level of all organs under low temperature was higher than that under moderate temperature.The p LTS1-PCHCRISPR/Cas9 gene knockout vector was constructed and transformed into Zhonghua11,and 30 LTS1 gene knockout homozygous lines were obtained.Two types of mutation were found in homozygous lines by knockout site analysis.lts1-1,lts1-2 and wild-type seedlings were treated at 8℃for 5 days and recovered for 10 days.The survival rate of lts1-1 and lts1-2 seedlings was 98.2%and 96.6%,respectively,while that of wild-type seedlings was 20.3%.The relative conductivity of lts1-1 and lts1-2 seedlings was21.35%and 19.23%,respectively,while that of wild-type seedlings was 56.43%.The MDA content of lts1-1 and lts1-2 was 7.83μmol·g^-1and 7.03μmol·g^-1,respectively,and13.89μmol·g^-1in wild type.The root activities of lts1-1 and lts1-2 were 300.21μg·g^-1·h^-1and 294.74μg·g^-1·h^-1,respectively,and 250.56μg·g^-1·h^-1in wild type.The relative conductivity,MDA content and root activity of mutant lines were significantly higher than those of wild type,indicating that LTS1 gene knockout can improve rice seedling cold tolerance.LTS1 gene negatively regulates rice seedling cold tolerance,but its molecular mechanism needs further study.