| Low temperature stress has seriously inhibited the growth and metabolism of rice.The phenomenon of major losses in rice productivity is an urgent problem in China.Therefore,researching the molecular mechanism of response-to-low temperature stress provides the theoretical basis for the breeding of cold resistance of rice and plays a critical role in improving sustainability of rice yield.Transcription factors which activate or inhibit the gene transcription and expression play important roles in the plant response to abiotic stress.This study was based on previous field experiment of psychrotolerant agronomic traits in different varieties.We found that there were phenotypic differences among the transgenic rice NV-ZFP151(plant expression vector of enhanced transcription),NE-ZFP151-1(plant expression vector of inhibited transcription)and NE-ZFP151-2(plant expression vector of inhibited transcription),as well as wild type Kitaake.We studied the bioinformatics,expression patterns and biological functions,at the same time,we analyzed the molecular mechanisms of the C2H2-type transcription factor Os ZFP151(for Oryza sativa Zinc Finger Protein 151)to response low temperature stress in order to provide theoretical basis of transcription factors in the application of low temperature stress.1.The bioinformatic analysis of Os FP151 geneAccording to the analysis of the multiple sequence alignment and phylogenetic tree,Os FP151 and five homologous genes were C2H2-type protein included two zinc finger structures.The N-terminal region of all proteins contained a nuclear localization signal(NLS)of sequences KKKKR.2.Subcellular localization of Os FP151 proteinThe vector that Os FP151 fused with GFP was constructed,and it was transfered into Arabidopsis protoplasts.We observed that Os ZFP151 protein was located to the nucleus using a Confocal Scanning Microscope.3.Functional analysis of Os ZFP151 in rice under low temperature stressThree positive transgenic plants including NV-ZFP151,NE-ZFP151-1 andNE-ZFP151-2 were identified with molecular detection,which provided the premise and guarantee of function research.Os ZFP151 had no tissue-specific expression characteristic.There was a low expression of Os ZFP151 gene in coleoptile and young roots,on the contrary,the mature leaves and spikes had a higher expression level.The phenomenon of low temperature resistance revealed the cold resistance of transgenic rice NV-ZFP151 was significantly higher than NE-ZFP151-1,NE-ZFP151-2,and wild type Kitaake.At the same time,the expression pattern of transgenic rice Os ZFP151 gene was analyzed,expression of Os ZFP151 was strongly induced by low temperature stress,and the expression level of Os ZFP151 gene in NV-ZFP151 significantly higher compared with other lines.Under low temperature stress,the expression levels of four Marker genes which including CBF1,CBF2,CBF3 and CDPK7 related to low temperature stress were significantly up-regulated in overexpression transgenic line NV-ZFP151.The Chlorophyll content was distinct from other lines after low temperature treatment,and may be that Os ZFP151 had lesser degradation effect on Chlorophyll under low temperature.However,the contents of soluble sugar and proline in NV-ZFP151 were significantly higher than other plants.The content of MDA in NV-ZFP151 was significantly lower than other plants.To sum up,Os ZFP151 gene directly or indirectly involved in regulating the expression of cold-related genes and contents change of physiological and biochemical indexes,which weaken the extent of the damage in overexpression transgenic line NV-ZFP151.Consequently,cold resistance of transgenic line NV-ZFP151 was improved.4.Screening and verification of proteins interacting with Os ZFP151We found a DNA directed RNA polymerase(Os RPB6)that can interact with Os ZFP151 through yeast two hybrid screening.The interaction between the two proteins was confirmed using yeast two-hybrid assay,CPRG liquid color test and Bi FC assay respectively. |
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