Analysis Of The Expressions And Function Of Cold Resistance Related MiR319 And Its Target(TC444396) In Winter Wheat

Micro RNAs(mi RNAs) are a class of endogenous, single-strand, non-coding RNAs with about20-24 nt in length. Mi RNAs are widespread regulators of gene expression in animals, plants and microorganisms. Plant mi RNAs play an important regulatory role in plant growth, development,hormone regulation and immune response, and response to a variety of biotic and abiotic stresses(such as nutrient stress, drought stress, salt stress, cold stress, etc.).Winter Wheat is an important crop, the most important reason is to limit cold Heilongjiang area planted winter wheat, Dongnongdongmai the 1st due to its strong cold hardiness of winter wheat cultivars become Heilongjiang region’s first safe wintering. Therefore important to study mi RNA Dongnongdongmai cold resistance of the 1st pair. Currently the regulation of mi RNA research adversity more concentrated in genomes sequenced, and stress conditions in a single simulation. East of the present study Dongnongdongmai 1 materials to 6 expression abundance difference in our laboratory was significant pre-mi RNA as an object, first explored cold mi RNA and its target genes in the field of natural cooling to 5 ℃,-10 ℃,-25 ℃ low temperature when the expression pattern of response is the key to get-10 ℃ temperature point Dongnongdongmai the 1st cold hardiness, and then cloned into Arabidopsis miR319 precursor, at 5 ℃ for indoor simulation By measuring the amount of plants overexpressing mi R319 expression of its target genes. The results are as follows:(1) natural cooling under field conditions, real-time fluorescence quantitative analysis that:As the temperature decreases, the sample mi R319,1118,1120,1128,1133 and 1139, the trend was down after the first rise, the target gene expression to the contrary trends and then decreased, at very low temperatures such changes may be associated with different functional mi RNA target gene, also found that the temperature of-10 ℃ is an important point Dongnongdongmai the 1st cold mechanism.(2) Online psRNATarget software to predict response to cold stress after significant differential expression of said six mi RNAs target gene, and contains a total of 89 target genes.(Rapid amplification of c DNA ends) method to verify their mi R319 target gene TC444396(CBF transcription factors) mi R319,5’RACE cleavage sites selected from a strong conservative six mi RNAs is 5’-end of the first 10 mi R319 one, among the first 11 nucleotides, this action site islocated TC444396 coding region.(3) Cloning Dongnongdongmai 1 Pre-mi R319 and insert user vector was successfully constructed after the fusion Pre-mi R319 overexpression vector pCMBIA230035 Su with transfected Arabidopsis pollen tube channel to obtain overexpression plants. After transformation of Arabidopsis plants 0h after 4 ℃ stress 0h, 24 h, 48 h, 72 h after detection and instantaneous leaves mi R319 target gene expression results showed that: within 0~72h treated 4 ℃, miR319 expression was first increased and then decreased, 4 ℃ for 48 h reached a peak when the mi R319 expression;mi R319 target gene expression after the amount was down then up trend within 0~72h treated 4 ℃,4 ℃ for 24 h at the lowest mi R319 expression. Arabidopsis thaliana, mi R319 response to temperature stress, then decreased expression, and the expression of negative regulation of its target genes. Presumably in response to cold winter wheat mi R319, and through regulation of target genes to enhance the cold hardiness of winter wheat.