The Clone Of ClpP Relating To Virescent Mutant In Cucumber And The Function Analysis

Object:(1) The ClpP gene from leaves of the natural type cucumber (xintai dense thorn) and its virescent mutant type cucumber (xintai dense thorn) has been Screened and its gene structures has been analyzed. (2) The part of ClpP gene was expressed in tobacco by plant express system. Then we analyzed ClpP gene function by observe physical trait.Methods:(1) The total RNA was extracted from the leaves of the natural type cucumber and its virescent mutant type cucumber, and cDNA was synthesized by reverse transcription. Then the ClpP was obtained by PCR with specific primer pairs. The PCR product was linked to the pGEM-T vector and suquenced. Then the sequence was analysed depend on bioinformatical softwares and GenBank.(2)The recombinant of the target DNA fragments and the vector of EhA105 agrobacterium were constructed. After callus sprouting, we observe its physiology trait and analysed its function.Results:(1) The overall length of the ClpP is 494bp. And it codings the total sequence of ClpP to 164 amino acids. It contains a conserved domain S14_ClpP₂ between 1 to 132 amino acids,but have no signal peptide.So it maybe not a ecretes protein by peculating its. The total sequence of ClpP which we obtained only have two exons but not three exosn like predicted in NCBI. The amino acids of this sequence was compares with other seven plants'amino acids of ClpP After constructing homologous tree, we know that cucumber and Ranunculus macranthus have close kin,the next is Arabidopsis thaliana (2) After agrobacterium-mediated genetic transformation for tobacco,the obvious virescent phenomenon did not appear in recombinant tobacco of ClpPZ and ClpPY.Conclusion:(1) the natural type cucumber and its virescent mutants have the same ClpP gene including S14_ClpP₂ highly conserved sequence. ClpP may play a role in regulating level of the buds yellow phenomenon in the cucumber. (2) The experiment of genetic transformation with agrobacterium-mediatedcarrying ClpP genes from the natural cucumber and its virescent mutant shows none obvious virescent phenomenon. Perhaps:1. The ClpP of cucumbers in tobacco failed to express; 2. The protein of ClpP involved in regulate and control or signal transduction, but failed to play a role in tobacco; 3. The ClpP genes got nothing to do with virescent trait of cucumber. The specific reason need further study. To get complete gene sequences by amplification the ClpP gene, its related functions can be researched further.