CDNA Library Construction Of Cunninghamia Lanceolata (Lamb.) Hook Using Green Stem

The development of Humen Genome Project (HOP) not only promoted the revolution of technology in molecular biology, but also helped forward the study of other organisms. Now many model biology had been sequenced. With the sequencing completion of human and model biology, their study had came into functional genome research from structural genome research. To the genome research of Cunninghamia Lanceolata, should make full use of the chance and technology make progress both on the structural genome and functional genome at the same time.cDNA library is the bridge connecting the functional genome research and structural genome research. We use the green stem from a clone No." Ju 0"of Cunninghamia Lanceolata , which grew out in March, as the material for RNA preparing. To ensure the quality of cDNA library constructed, we used the Concert?Plant RNA Reagent to isolate the general RNA from the Cunninghamia Lanceolata to ensure the integration and purity of general RNA isolated from Cunninghamia Lanceolata; Poly(A)+ mRNA isolate kit was used to isolate mRNA from the general RNA and got rid of tRNA and rRNA. A ZAP Express cDNA Synthesis Kit and A ZAP Express cDNA Gigapack III Gold Cloning Kit were used to construct cDNA library, which was very convinence and effective as to construct cDNA library. The results manifested that the cDNA library construction of Cunninghamia Lanceolata was very successful. The capacity of primary cDNA was 3 × 105. The liter of amplified cDNA library was 0.15 × 109 pfu/ml. The cDNA fraction inserted into the A ZAP Express vector was more than 500bp on average by the verification of Polymerase Chain Reaction(PCR) and two enzymes (Ecor I and Xho I) cutting reaction .This research provided the fundamental basis for structural genome research and functional genome research and made good preparation for the study in Cunninghamia Lanceolata ' s genome next stage.