Cloning And Identification Of Cold Stress Related MiRNAs In Citrus

China is a big country of citrus production where citrus originated, possessing abundant kinds of citrus and long history of planting. Citrus industry is key to regional economy development as well as our new country construction. Cold stress is an important environmental factors which limiting citrus plant growth, development, geographical distribution and gravely affect citrus production and quality.MicroRNA is an class of single-strand noncoding RNAs with about 21-22 nt length.By cleavaging target mRNA or inhibiting its translation, microRNA can coordinate expression of many genes invoving regulating plant hormone and signal conduction to adapte to adversity. There are some reports about plant adaptions to abiotic stress,but rare is about relationship between miRNA and cold stress. Exploring cold related miRNAs in citrus to find new miRNAs and their patterns of gene regulation is of great significance to development of citrus industry, while it can optimize citrus species, improving their adaption to cold. The main results are as follows:1 Focusing on 27 citrus miRNAs in previous study, we screen 5 cold stress related miRNAs by integrating microarray dates, predicting pre-miRNAs structure and analyzing cis-acting element in miRNAs promoter region.2 After total RNA extracted from young leaves of kumquat, reverse transcription reactions were performed using universal primer Olig(T), then we clone precursors of miRNA167a,miRNA167d and miRNAs171 by PCR amplification.3 Mature sequence of miRNA 156 and miRNA 166 are detected by two means. One is stem loop RT-PCR by designing special stem loop primer, the other is that adding poly(A) to small molecule miRNAs isolated from total RNA4 RNA are isolated from different samples kumquat; untreated, treated with cold stress 4°C for 0.5h,4h,8h and 14 days prior to extraction. We study expression difference of 5 miRNAs by semi-quantitative PCR and find that the expression patterns of miRNA167a and miRNA167d are similar, while miRNA156,miRNA166 and miRNA171 are different from each other.