Study On Clone, Expression And Promoter Of MiRNA-novel4 From Cassava Responding To Cold And Drought Stress

MicroRNA (miRNAs) is a type of non-coding RNAs (-22nt) which widely exist in plant genome and play an important role in the gene regulation gene in eukaryote. It is involved in almost all crucial biological process. The mechanism of miRNAs regulate endogenic gene expression is to repress target protein or degrade mRNA by basepair with target gene at post-transcriptional level. so function analysis of target gene become an effective way to understand the mechanism and function of miRNAs. Cassava is a typical energy crop in tropical area. The mechanism studies of drought and cold resistance have been the important and basic works for cassava genetic improvement on cold and drought resistance. Thus, isolation of miRNAs and target genes correlated to drought resistance could also provide another way for cassava genetic improvement.This study took novel4 as target miRNA which selected by miRNA transcription analysis as study object. We analysis the expression,negative regulation relationship and the cleavage site of novel4 and target mRNA by real time PCR and 5’RACE technology. In addition, the promoter was cloned and analysed to annotation the function of novel4. The conclusions we found are listed as follows:1. The correlation between novel 4 and target mRNA was proved by fluorescence real time PCR technology. In addition,5’RACE technology was further taken to demonstrate the negative regulation relationship between novel 4 and target mRNA.2. The expression certified novel 4 could respond to and regulate drought and cold stress, but shown different regulating pattern in drought and cold stress adaption, being down-regulation in drought stress while up-regulation in cold stress, which agreed with expression profile.3. On the other hand, analysis of novel 4 5’regulating domain found the cis-element responding to cold, defense and stress,which was consistent with the results from expression profile analysis and expression test. All those conclusions verified novel 4 regulated the drought and cold stress.4. The results show that drought treatments have significant differences on K+ and ABA content in the two varieties, while less significant differences on Ca2+; In detail, K+ content of SC124 has significant difference between DH and DNH treatments, ABA content of the two varieties shows significant differences with different trends between DH and DNH; Moreover, the correlation co-efficient becoming significant among K+, Ca2+ and ABA when cassava suffered drought stress.The result showed that novel 4 performed different regulated model in drought and cold stress, with down-expression in drought stress but up-expression in cold stress. Moreover, according to analysis of novel 4 5’regulation region, novel 4 was deduced to hold better function in cold stress than drought stress. However,there were more works needed us to prove the speculation. All conclusion verified novel 4 regulated the drought and cold stress, and it is very possible that the mechanism of novel4 is the overlapping pathway of response drought and cold stress. The study of promoter demonstrate that the up-stream of novel4 was regulated by stress signals, which provided proof for further study of novel4 function and regulation mechanism.