Effect Of Silenting Expansin Gene On Suspension Culture Cells Of Licorice (Glycyrrhiza Uralensis Filch)

In order to obtain high-density cell culture, RNA interference was used to inhibit the expansion gene expression of Glycyrrhiza uralensis filch whose expansin was decreased and cell volume was reduced.Expansion of Glycine max Exp2(Genbank AF516880) nucleotide sequence being the object, the positive fragments(I and II)was selected into two conserve regions of about50base pairs of nucleotide sequences and the expansin genes were got from different plant. Then the reverse sequences(Ⅰ’ and Ⅱ’) were designed based on the principle of positive fragments. According to two pairs of positive and negative sequences, it was designd an appropriate primer that restriction endonuclease sites(EcoR I and Hind III)and the corresponding GU-AG intron marginal sequences were inserted in the positive and negative sequence in the PCR process, respectively. After Ⅰ and Ⅱ’ or I and Ⅱ’ two fragments ligated and PCR screened, we have got two kinds of ligation products Ⅰ-Ⅱ’ and Ⅱ-Ⅰ’. By digestion, re-ligating, transformating, cloning and identification, we may get a four fragments of ligation product-Ⅰ-Ⅱ’-Ⅱ-Ⅰ’. The product is inserted into plant expression vector pCAMBIA2300and the recombinant vector was used to be imported by A.tumefaciens strains(EHA105). The plant engineering bacteria was screened out which can be transformed.The epicotyl of liquorice was precultured for2d, co-cultivated liquorice callus with A.tumefaciens at25℃for2d in MS solid medium and selective medium(50mg/L hygromycin) for15d. Then the resistant callus were selected and subculture so that the the loose one of resistant callus could be suspension culture in the MS liquid medium,25℃,120r/min. It is compared that suspension cells and non-transformed suspension cells’size, shape and growth curves.The results show that:1) In accordance with design requirements and sequencing proved, that we have obtained recombinant DNA fragments of interference (Ⅰ-intron Ⅱ’-Ⅱ-intron-Ⅰ’).The mRNA of transcribed can complementary condition form a hairpin. This is meet the requirements of construction of RNAi fragment;2) when the suspension culture of A.tumefaciens concentration OD600of0.4, co-cultivated liquorice callus with A.tumefaciens at25℃for2d.After dried treatment can greatly increase the rate of resistant callus;3)The resistant callus grow slowly;4)Transformed suspension cells volume are smaller than non-transformed suspension cells Under the optical microscope observation;5)Compared with the control material, transformed suspension cells have a longer growth cycle;6)Compared with the control material, the expansins of transformed suspension cells was decreased;7) DNA of suspension cells was extracted for PCR analysis showed that interference fragments have been inserted into the genome of liquorice cells.This paper shows that, the sequence is transcribed in the cell of mRNA, which will form a double-stranded RNA fragments. The transcription products of mRNA produced interference effect to expansion gene so that the synthesis of expansin was inhibited, so it made a result that the non-covalent fracture of cellulose and hemicellulose of the cell wall were different. All above maybe show the factors why cell wall have compact structure and it become smaller.