Molecular Cloning And Functional Analysis Of PvPinAt In Phyllostachys Violascens

Pin1（peptidyl-prolyl cis/trans isomerase, PPIase）encodes peptide-prolyl cis-trans isomerase.In the study of Arabidopsis, Pin1At regulated flowering time and floral transition by promoting theSer/Thr-Pro of SOC1and AGL24which are two MADS-domain transcription factorsphosphorylate, and thus regulate flowering time.The cloning research of Pin1At has importantimplications for the flowering mechanism of bamboo plants because of the uncertain floweringtime and the long-nutrient cycles. The experiment hopes to further understand the mechanism ofbamboo flowering plants through the homologous gene cloning and functional analysis ofPhyllostachys violascens Pin1At. Flowering bamboo plants research laid the foundation of theoryand practice.This project designed primer based on the reverse transcription database of rice, corn andPhyllostachys violascens, making Phyllostachys violascens as plant material, using RACE(rapidamplification of cDNA ends, RACE), getting the sequence length of cDNA of PvPin1At whichwas369bp, including whole ORF coding region whose length was369bp, coding122amino acids.We used high fidelity enzyme to amplify the ORF region of Phyllostachys violascens, getting twoPin1At-like genes, called PvPin1At1and PvPin1At2. Use PvPin1At genes get to do the followingfindings:（1）By sequence comparison and phylogenetic analysis, we found PvPin1At gene in plantsis quite conservative, and the grasses can be clustered together.（2）The results of RT-PCR analysis showed PvPin1At gene had different expression levelsin different parts of flowering Phyllostachys violascens. The expression in the leaves is thehighest，followed by are stems and whip, then the expression in flower buds，old leaves，shoots arethe lowest. The PvPin1At gene expression in the leaves of the same branch of floweringPhyllostachys violascens gradually increased from3.15to3.22and after3.22the expression ofthat decreased.（3）Yeast two-hybrid found PvPin1At1, PvPin1At2and PvSOC1proteins have nointeraction.（4）The wild-type Arabidopsis Columbia was transfected by35S::PvPin1At1plant expression vector，the results showed that the transgenic plants flowered earlier6-7days than inwild-type Arabidopsis. The wild-type Arabidopsis Columbia was transfected by35S::PvPin1At2plant expression vector，the results showed that the transgenic plants and wild-type Arabidopsishave no phenotypic differences.（5）Constructioning prokaryotic expression vector of PvPin1At, we got the purpose solublePvPin1At protein whose size is31kDa.（6）In order to study the function of PvPin1At，we made the27th serine mutated to asparticacid，laying the foundation of the further research of the flowering mechanism of Phyllostachysviolascens.（7）Constructioning binary expression vector of PvPin1At plant, we transform it into rice byAgrobacterium EHA105getting4035S::PvPin1At1transgenic rice, proved PvPin1At havetransformed into rice successfully through DNA testing.