Study On Function Of MiR172, MiR319, MiR393, MiR402 And Transformation To Cassava

MicroRNA (miRNA) is a group of single-chain small RNA with stem-ring struct, which commonly includes in vivo of all biologies. It was highly conservative, timing series and organization specificity. MicroRNAs play important roles in regulating gene expression by specifically matching the target gene domain, which results in the target mRNA degradation or translational regulation. In contrast to animal miRNA, the research of the plant microRNA is comparably later. However, as the new technology develops, more and more plant microRNAs were found. Until September 2010, there were 3070 plant miRNAs to be publicly announced in miRbase database (Release 16.0), among them 1769 miRNAs were from dicotyledons and 887 miRNAs were from monocotyledons. Presently, the research of plant miRNA is focused on model plant of Arabidopsis. In the miRbase database, no miRNA was isolated from cassava. In this research, the function of the announced miRNA of miR319、miR393、miR402 and miR172 have been identified. Afterwards, some of the cold related miRNAs have been transformed to cassava. Meanwhile, the cold-related miRNAs have been isloated from cassava. The results are as the following: 1. The known miRNAs of miR319、miR393、miR402 and miR172 have been isolated fromArabidopsis; and their target genes have been analyzed by bioimformation. The resultsshowed that 20 target genes for miR172c,14 target genes for miR319c,8 target genes formiR393b, and 2 target genes for miR402.2. Construction of pVKH-35S-miR172c-pA、pVKH-35S-miR319+402+393-pA plant expression vectors, which were transformed to Arabidopsis. The result showed that the pVKH-35S-miR172 transgenic Arabidopsis grew normally in plant phenotype, but early flowering 20 d. The pVKH-35 S-miR319+402+393 transgenic Arabidopsis showed cold tolerance, but plants developed a suppressed terminal buds and few abnormally developed seeds.3. The precursors of miR172, miR319a, miR319c, miR393 and miR402 have been isolated from the cassava by PCR using the specific primers designed according the miRNA sequence in miRbase database, which the length of them is 124 bp、176 bp、179 bp、96 bp、121 bp, respectively. The mature sequences are 5’-agaaucuugaugaugcugcau-3’ for miR172; 5’-uuggacug aagggagcucccu-3’ for miR319a; 5’-uuggacugaagggagcuccuu-3’ for miR 319c; 5’-uccaaaggg aucgcau ugauc-3’ for miR393, and 5’-uucgaggccuauuaaacctgga-3’ for miR402. 4. Cassava is one of three major tuber crops with many excellent characteristics, such as being plantation during the whole year, simple cultivation, high drought and barren tolerance, high yield and biomass. Cassava is widely planted in tropical regions. But temperature seriously affects the growth of cassava. In this study, the pVKH-35S-miR319+ 402+ 393-pA has been transformrmed into cassava by the transgenic technology and 4 independent transgenic lines have been identified by PCR.