| Cassava(Manihot esculent Crantz)is a tropical and subtropical economic crop,which has the characteristics of drought tolerance and barren resistence,but it is sensitive to low temperature.Chilling injury is one of the key factors affecting the yield of cassava.Thus far,there is limited information about gene regulation and signaling transduction pathways related to the cold stress response in cassava.Therefore,studying the molecular mechanism responsed to low temperature would provide a theoretical basis for molecular breeding of cassava,which is of great significance to promote the northward movement of cassava planting area.Zinc finger protein(ZFP)has specific structure,and is one of the important transcription factors in eukaryotic organisms.C2H2 zinc finger protein,also known as TFIIIA zinc finger protein,regulates various physiological reactions through interaction with other factors in the nucleus,and participates in plant growth and development and biological and abiotic stress.However,there are few studies on C2H2 zinc finger protein responsed cold stress in cassava.The results are as follows:(1)Me ZAT11,a C2H2 zinc finger transcription factor,was selected from the cold stress expression profile of An Dong(2012).Bioinformatics analysis showed that Me ZAT11 was a typical C2H2 zinc finger protein with two zinc finger domains and one ERF-like domain.Phylogenetic tree analysis showed that Me ZAT11 was closely related to Hevea brasiliensis and Ricinus communis of Euphorbiaceae.Protein subcellular localization showed that Me ZAT11 protein was located in the nucleus.(2)Tissue expression pattern analysis showed that Me ZAT11 had the highest expression in mature leaves,followed by old leaves and roots.When cassava was treated with low temperature,Me ZAT11 was induced to express,with the highest expression level at 24 h.However,the expression of Me ZAT11 was inhibited after ABA and Me JA treatment.(3)Me ZAT11-RNAi transgenic lines were obtained by transgenic technology,and three RNAi lines were screened by Real-time fluorescence quantitative PCR(q RT-PCR)and Southern Blot.Compared with the wild type,Me ZAT11-RNAi showed a cold resistant phenotype.In order to further study the molecular mechanism,the MDA content in the leaves of potted seedlings under cold stress was determined.It was found that the MDA content of transgenic lines was significantly lower than wild type.The cold resistance phenotype of transgenic lines was caused by transient low temperature under artificial culture conditions.In the process of natural cooling,the leaves of transgenic lines appeared yellow spots in advance,and the percentage of fallen leaves was higher than wild type,which indicated that transgenic lines perceived cold stress and avoided cold injury by falling leaves in advance.(4)q RT-PCR detection of MDA pathway related genes showed that there was significant difference in the expression of Me LOX3.In vitro experiments verified that Me ZAT11 indeed regulated the expression of Me LOX3.It is further proved that Me ZAT11 regulates Me LOX3 and affects membrane fluidity by regulating unsaturated fatty acid oxidation.In addition,Me LOX3 is involved in JA synthesis,and Me JA treatment inhibits the expression of Me ZAT11,forming a feedback regulation to maintain cell homeostasis.In addition,Me ZAT11 can regulate the expression of chlorophyll and senescence related genes to affect the senescence process and participate in the cold acclimation process.In conclusion,we cloned a C2H2 type zinc finger protein transcription factor from cassava and verified its function.We found that Me ZAT11 responded to low temperature and played an important role in low temperature stress by affecting lipid membrane fluidity.It provides a theoretical basis for the study of cold acclimation mechanism of cassava. |
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