Study On The Function Of Rice(Oryza Sativa L.)ECT1 In Response To Low Temperature Stress

Rice is sensitive to temperature,and the problem of chilling injury is prominent in high latitude and high altitude areas.The cold tolerance of rice is determined by many factors,and its regulation mechanism is very complex,so it is difficult to solve the problem of low temperature stress of rice varieties by traditional breeding methods.At the same time,WRKY transcription factor family usually plays an important role in plant abiotic stress.Therefore,it is of great theoretical and practical significance to explore and identify rice genes related to cold tolerance,to study the mechanism of rice cold tolerance,and then to cultivate excellent rice varieties with strong cold tolerance by means of molecular breeding.In this paper,the function and molecular mechanism of cold tolerance of ECT1,a member of rice WRKY transcription factor family,were studied in order to provide a theoretical basis for the cultivation of coldtolerant rice varieties.The main conclusions are as follows:1、Through the analysis of its protein sequence and promoter sequence,it is found that there are many abiotic stress-related elements in the promoter region,such as MYB,MYC,ABRE,etc.,so it is speculated that the function of ECT1 may be related to abiotic stress response.Further analysis of the expression of ECT1 under low temperature,drought stress and plant hormone treatment showed that the expression of ECT1 was affected by low temperature,drought,JA and ABA treatment.2、The cold tolerance phenotypes of rice lines with stable inheritance of ECT1 overexpression in T3 generation were identified at bud stage,seedling stage and booting stage.At the budding stage,the germination rate of the overexpressed lines was significantly higher than that of the wild type at each time point;at the seedling stage,the growth of the overexpressed lines after low temperature treatment was significantly better than that of the wild type;at the booting stage,the fertile pollen rate and seed setting rate of the overexpressed lines after cold treatment were significantly higher than those of the wild type.The above results showed that the overexpressed lines had stronger cold tolerance than the wild type in these periods,which indicated that ECT1 was a positive regulator of cold tolerance in rice.In addition,we also found that rice lines with overexpression of ECT1 had a phenotype sensitive to ABA.3、In RNA-seq analysis,by drawing Venn diagram,it was found that the number of differentially expressed genes between the two ECT1 overexpression lines and wild type was 524,305 respectively under cold treatment and normal treatment.Then,by drawing the volcanic diagram of differentially expressed genes between two overexpressed lines and wild types under normal treatment,it was found that the expression levels of Os03g0299700,Os03g0168800 and Os03g0115800 in the two overexpressed lines were significantly higher than those in wild types.Finally,through GO enrichment analysis,it was found that the differentially expressed genes were mainly enriched in the assembly of some proteins,DNA and macromolecular complexes before cold treatment,while after cold treatment,the differential genes were mainly enriched in biological stress response,phosphotransferase and other signal transduction pathways,which was consistent with the cold tolerance phenotype of overexpressed lines.4、In the experiment of screening downstream genes by q RT-PCR,we found that the expression of 6 cold tolerance genes increased significantly in overexpressed lines,while the expression of 12 cold sensitive related genes decreased significantly in overexpressed lines,indicating that these cold related genes may be used as direct or indirect downstream of ECT1 transcription factors to help ECT1 achieve the function of improving cold tolerance of transgenic lines.To sum up,ECT1 overexpression lines have strong cold tolerance phenotype,and ECT1 may be involved in rice cold tolerance regulation mechanism dependent on ABA pathway to improve rice cold tolerance.The above results lay a foundation for screening differential genes and verifying downstream genes.