Function Analysis Of Genes Up-regulated Expression During Somatic Embryogenesis Using RNA Interference

Advanced by Fire in 1998, RNA interference which was characteristic of simple, high-efficient, high-quantity has been paid more and more attention and its characteristics of identify gene function became a hot spots of bio-technology. New research emerged in endlessly in pace with the expansion of its application. This technology has been applied in the research of cotton gene function successfully and lots of pleasantly surprised achievements has obtained in disease-resistant of cotton, resistant of insect, resilience, improvement of fiber quality and so on. Our research was to study the effect of RNAi in the course of cultivating cotton tissue by RNAi vector which was constructed with pCRI1210 and the valuable ESTs chose from SSH (Suppression Subtractive Hybridization) library of our lab. The results are as follows:1. To identify the function of ORF sequence that was cloned into pCRI1210 vector, the pCRI1210-GUS vector was transformed into tobacco (NC89) via leaf disc:Replaced theβ-tubulin intron cloned from cotton with GUS, after transforming into tobacco, induced bud, leaves and roots,then colored them with GUS stains, after bleaching with 70% alcohol, positive tissue showed significant dark blue, while wild-type tissue white.The results, proved pCRI1210 was a new vector with wide range of MCS, high-efficient promotion and easy to test, suggested that CaMV 35S promotor can promote vitro gene in a high-efficient and stable way and the reporter gene can tested.2. Four ESTs interference vectors were constructed which came from the SSH library, using interference expression vector pCRI1210.Transformed them into the asepsis seedling hypocotyls of CCRI24 mediated by Agrobacterium transformation, Semi-quantitative RT-PCR results showed that after the case of interfering, RNA levels of the callus decreased in varying degrees, dedifferentiation rate changed compared with the control. Among them, the callus rate of the interference vector pCRI210-418 corresponding callus has increased, while the pCRI210-447, pCRI210-496 and pCRI210-653 corresponding callus decreased. Meanwhile, the vectors were transformed into embryogenic callus with particle bombardment, the number of embryoidsis was different between different vectors unit mass of tissue.Therefore, these ESTs determine the cotton callus and embryogenic callus to some extent.