Construction Of CDNA Library Of Pear Fruits

The development of Human Genome Project (HGP) not only promoted the revolution of technology 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 come into functional genome research from structural genome research. To the genome research of pear, should make full use of the chance and technology makes progress both on structural genome and functional genome at the same time.In this study, some methods , including Improved Guanidine Thiocyanate method , hot borate method, Modified CTAB and Qiagen Rneasy Plant RNA kit, were evaluated about their availabilities for isolating total RNA from pear fruits tissue by comparing UV absorbance ratio and gel electrophoresis. The result showed that the total RNAs obtained by Improved Guanidine Thiocyanate method were highly pure and undegraded. The ratio of ranged between 1.7-2.0, and theOD260/OD230 higher than 2.0. Whereas the ratio OD260/OD280 were lower than 1.7 and the OD260/OD230 were below 2.0 for all other extracted RNA, indicating that the contaminations were present of polysaccharides, polyphenols and proteins.The single strand cDNA was synthesized from mRNA with M-MLV reverse transcriptase, and then double strand cDNA was synthesized by using PCR. Double strand cDNA size fractionation was completed by SPIN-400Column, so the low molecular weight cDNA fragments shorter than 500bp could be removed out.The two end of the cDNAs were ligated with a adaptor each and inserted into A. gtll vectors. Gigapack Gold III Packaging Extract was provided by Stratagene. So a cDNA library of pear fruits link was constructed for the first time in the world. The cDNA library has been constructed and amplified through transducting Y1088. The constructed cDNA library has the titer of 1.192 X 106 and the clone number is 1.151 x 106. While the average insert sizes of the library are 1 kb with the recombination efficiency more than 93%. Therefore, a cDNA library of pear fruits has been successfully constructed.