Construction And Identification Of A Normalized Maize Ear CDNA Library Of Hybrid Y22

In this study the total RNAs of maize hybrid Y22' ears that are during the important period -ear development period-were isolated using Trizol Reagent, and synthesis the cDNA strands,then construct the normalized cDNA library,which is the basis for the study genes about heterosis.The RNA ratios of OD₂₆₀/OD₂₈₀ of four periods was 1.87, 1.86, 1.85 and 1.99 respectively。 The mRNAs were transcribed into the first strand cDNA with Not I primer-adapter and double cDNA strands were synthesized using strand replacement method. The results of 1% agrase gel showed that the longest synthesized fragment could be 5kb. The two ends of the cDNAs were ligated with a adaptor each, digested with Not I, column chromatography and ligated with Not I-Sal I-Cut vector. Four cDNA libraries were constructed. They contained about 2.7 × 10⁵, 6.4×10⁴, 3.1 × 10⁵ and 2.1 × 10⁴ respectively; the recombinant clones in the four libraries were 90%, 87%, 85% and 93 % respectively; and the average size of inserts were 1.2kb, 1.3 kb, 1.4 kb and 1.3 kb.Total DNA isolated from Y22 was digested with restriction enzymes EcoR I and BamW I, after first digested filled with Klenow Fragment and C-21-biotin-dUTP; combined with Dynabeads. The single-strand genomic DNA (5ug) fixed on Dynabeads was pre-hybridized, then add the mixed plasmids (40ug) from 4 individual cDNA libraries to hybrid. The hybridized plasmids DNA were washed and transformed. The normalized cDNA library was constructed. The library consisted of 30,000 clones. The 200 clones had an average insert length of 1.3kb. 150 clones random selected were sequenced, and showed, 81.6% of clones were unique ESTs;IM% of clones had repetitive frequency ranging from 2-3. These results suggested to some degree that the cDNA library was well normalized.