| Rice feeds around half of the population in the world and abiotic stresses such as high temperature,cold,drought,salinity,can affect the growth of rice,which in turn affects rice production.Therefore,it is of great significance for stable grain production by breeding rice varieties with enhanced stress tolerance.The purpose of this study is to understand the function of A20/AN1 zinc finger proteins ZFP173 and ZFP157 and the mechanisms of stress responses they may be involved in.The main findings obtained are as follows:Structure and expression of ZFP173:Unlike the conventional A20/AN1 type zinc finger protein,ZFP173 has only one AN 1 zinc finger structure and does not contain A20 zinc finger domain.Real-time quantitative PCR analysis showed that ZFP173 was expressed in different tissues during rice growth and development,and has higher expression in stems,node and flag leaves while less in roots and leaf.Expression of ZFP173 was induced by mutiple abiotic stresses,such as high salt,drought stress and exogenous ABA treatment.The subcellular localization assays with rice protoplasts and tobacco cells showed that ZFP 173was located in the nucleus.The results of transcriptional activity experiments indicate that ZFP 173 does not have transcriptional activation activity in yeast cells.Yeast two-hybrid assay showed that ZFP 173 could interact with ubiquitin protein,indicating that ZFP 173 may be involved in the 26S ubiquitin proteasome pathway.Access and analysis of genetically modified materials:ZFP 173 overexpressing transgenic plants,RNA interference-inhibiting expression transgenic plants and CRISPR/Cas9 gene edited transgenic plants were obtained by agrobacterium-mediated genetic transformation,and homozygous lines were obtained.At seedling stage and adult stage,the the plant height of ZFP 173 overexpressing transgenic plants was significantly lower than that of wild type,while the plant height of RNAi plants was slightly higher than that of wild type.Meanwhile,when ZFP173 was overexpressed,the grain length were shorter.The haplotype analysis was carried out using rice 3K genome resequencing data,and it was found that there were different haplotypes with significant differences in 1000-grain weight in the ZFP 1 73 coding region.The above results indicate that ZFP 173 may be involved in the regulation of rice growth and development.ZFP173 is involved in abiotic stress:ZFP173 can improve the drought tolerance of transgenic rice.Under drought treatment,the survival rate of ZFP173 overexpressing transgenic plants was significantly higher than that of wild type,while the survival rate of ZFP173 RNAi plants was lower than that of wild type.At the same time,the leaf conductivity of ZFP173 overexpressing plants was also significantly lower than that of wild type.It indicates that over-expression of ZFP173 can alleviate the damage of cells caused by drought stress and improve drought resistance.At the bud stage,ZFP173 over-expressing transgenic plants were more sensitive to ABA,and the relative germination rates were more significantly inhibited after ABA treatment.After ABA treatment at the seedling stage,the seedling height and root length of ZFP173 transgenic plants were not significantly different from wild type.Under low temperature,the survival rates of ZFP173 RNAi plants iwere significantly higher than that of wild-type plants,while the survival rates of over-expressing plants were not significantly different from that of wild-type plants,indicating that inhibition of ZFP173 expression could increase the tolerance of plants to low temperature stress.Under salt treatment conditions,the survival rates of ZFP173 overexpressing plants was much higher than that of wild type,while the survival rates of RNAi plants were lower than that of wild type,indicating that ZFP173 was positively involved in plant salt tolerance regulation.RNA-seq analysis:The RNA-seq results showed that ZFP173 overexpressing plants had 656 differentially expressed genes with wild type.Among them,the expression of 358 genes was up-regulated and the expression of 298 genes was down-regulated.GO enrichment of differentially expressed genes revealed that ZFP173 may be involved in oxygen binding,transferase activity,catalytic activity,secondary metabolic processes,biological regulation,tropism,metabolic processes,lipid metabolism processes,response to biotic stress,and abiotic stress.This indicates that ZFP173 may respond to abiotic stress by affecting the expression of stress-related genes in rice.Structure and expression of ZFP157:The biological function of another A20/AN1 zinc finger protein ZFP157 was studied.ZFP157 is expressed in different tissues of rice growth and development,and is located in the cytoplasm.ZFP157 interacts with zinc finger protein ZFP157YH1 and an ubiquitin protein RAD23.Four ZFP157 overexpression lines and three CRISPR/Cas9 edited lines have been obtained in the laboratory.ZFP157 is involved in abiotic stress:Under high salt stress,ZFP157 transgenic plants showed sensitivity to salt stress,and the survival rates were decreased,while the overexpressing plants were more sensitive than the gene edited plants.Increasing the expression of ZFP157 can increase the drought resistance of plants.Moreover,overexpression of ZFP157 reduced the plant’s low temperature tolerance.RNA-seq analysis:The RNA-seq results showed that ZFP157 overexpressing plants had 1461 differentially expressed genes with wild type.Among them,the expression of 822 genes was up-regulated and the expression of 639 genes was down-regulated.Differentially expressed genes are mainly involved in cell wall structure building,oxygen binding,transferase activity,metabolic processes,stress processes,catalytic activities,biological stimulation,non-biological stimulation,endogenous stimulation and so on.This indicates that ZFP157 may respond to abiotic stress by affecting the expression of rice stress-related genes. |
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