Clone The Gene Of Peanut Proteinase Inhibitor And Optimize The Peanut Regenesis System

Proteinase inhibitors (PI) are proteins that inhibited the catalytic activity of proteinases. They are important in a large variety of physiologic processes including coagulation, digestion, and immunity. Now, more and more PI has been purified from the bean plant seed. PI not only play a good part in the nourishment of crops, but also is the important factor of the plant defense system. This experiment cloned the gene of PI successfully by the genetic engineering technology. The size of the gene is 208bp. We used the tool of Blast in NCBI to obtain more message about this sequence. Blast search uncovered that: the nucleotide sequence shows 100% identity with a trypsin inhibitor cDNA (Yang et al, 2004) which was one of the Bowman-Birk super-family; 97% identity with a serine proteinase inhibitor cDNA (Drame et al 2005). Compared with other PI gene sequences, they have the same conserved domain: TGCACAAGGTCT CAGTGCCGTTG.This experiment has further discussed the condition of peanut regeneration system to establish a more highly effective regeneration system. The different section of leave, epicotyl, hypocotyls and cotyledon were used as the explants. The experiment obtains the following conclusion:1) Different section of leave has different induction ability. The percentage of calli of the complete leaf was the lowest, the percentage of calli of the middle cutting leaf was the highest. 2) The induction effect of hypocotyls was better than leave, and the induction effect of epicotyl was better than hypocotyls. 3) In the identical adventitious shoot bubs induction culture medium, laying aside way of the cotyledonary axis has significant influence to the adventitious shoot bubs induction. For example, when epicotyl oriented with their apical ends protruding from the medium, the induction percentage of adventitious shoot bubs reached 85.0%; when epicotyl was placed horizontally, the induction percentage of adventitious shoot buds reached only 22.2%. 4).The cotyledon explants from mature seeds of peanut were cultured on adventitious shoot buds induce media(MS+2,4-D lmg/L+6-BA 4mg/L) produced the highest frequency(77.1%) of multiple adventitious shoot buds(the cotyledons were split in vertical halves). 5) High concentration of 2, 4-D was disadvantageous to induce the callus tissue of explants. When the concentration of 2, 4-D was higher than 15mg/L, the most of explants turn brown. When the concentration of 2, 4-D was higher than 20mg/L, there was no callus tissue to be induced, all the explants turn brown.