The Construction And Identification Of Dualmembrane Yeast Two-hybrid CDNA Library In Rice And Os VDAC5Bait Vectors

VDACs (voltage-dependent anion channels) is a class of eukaryotic porin in themitochondrial outer membrane. VDACs mainly localized in the mitochondrial outermembrane forming channels for the transport of mitochondrion's metabolites whichpass between mitochondrial intermembrane space and the plasmid of mitochondria.VDACs have many important biological functions.VDACs might have located inmitochondrial outer membrane, cytomembrane, endoplasmic reticulum membrane,Golgi apparatus membrane and other inner memebrane system too. The former studiesof our laboratory and prediction from bioinformatics shows that most of the eightOsVDACs are locating in cellular inner membrane system. For the further study of thefunction of OsVDAC5, my study construct a full-length cDNA library throughDualmembrane system from young panicle in rice YTB and four different OsVDAC5Bait vectors. We currently have gotten the following results:1. We have construct a cDNA library through Dualmembrane system from youngpanicle in rice YTB and inspected quality of the cDNA library. The parameters for theprimary cDNA library transformation were calculated that independent clones of finalcDNA library were up to2.4×106. The sizes of inserts digested with Sfi I were between400bp and2200bp, and the average of insert sizes turned out to be approximately1.0kb.136randomly selected clones were sequenced to test the representation of differenttranscripts of cDNA library. There are132chones were inserted with differentfragments. The identity of these genes was confirmed by sequence blast and alignments,and the results showed that the redundancy of the clones was very low down to4.6%.120(91%) sequences shared homology with known functional genes.12(9%)sequences shared significant homology with unknown proteins. The analysis showedthat the cDNA library was highly qualified in according to complexity, redundancy,efficiency and the length of the cDNAs, and met almost all requirements of a successfulcDNA library. These results confirmed that the library we constructed could be used for screening of interactors of OsVDACs in rice.2. According to the transmembrane form of OsVDAC5, four different OsVDAC5bait vectors were constructed, two of which are full-length OsVDAC5(pBT3-N-osvdac5ORF and pBT3-SUC-osvdac5ORF), the other two have removed13amino acids(42bases) of OsVDAC5(pBT3-N-osvdac542and pBT3-SUC-osvdac542). Using Dualmembrane system, four vectors were separately transformed into yeaststrain NMY51to select the bait vector which can be used for screening of interactors ofOsVDAC5. Results show that full-length OsVDAC5bait vectors are not suitable forscreening of interactors of OsVDAC5, most likely because N-terminal and C-terminalof the OsVDAC5are not in cytoplasm.13-animo acid-removed OsVDAC5bait vectorsare also not suitable for screening of interactors of OsVDAC5. More different deletionbait vectors should be constructed for screening of interactors of OsVDAC5.