juvenile early flowering trifoliate orange mutant subtracted cDNA library

Vector: PGEM-T; The subtracted library was obtained by the juvenile wild type mRNAs as tester and cDNA from juvenile mutant mRNAs as driver. In January of 2005, the seeds of precocious trifoliate orange and wild type trifoliate orange were planted in 20 cm pots containing potting mix of a commercial medium and perlite at a ratio of 3:1. These juvenile trees were watered regularly with nutrient solution. Shoot meristem of the juvenile mutant and wild type trees were collected in March, June, September, and December, respectively. To prepare a representative sample of total RNA from the juvenile mutant and wild type in SSH process, different developmental stages of plant organs from roughly equal numbers of the juvenile mutant and wild type material were pooled, respectively. SSH library was performed using PCR Select cDNA Subtraction kit (Clontech, USA) according to the manufacturers instructions. The final PCR products were purified using QIAquick PCR purification kit (Qiagen). The fragments obtained were cloned into the pGEM-T vector (Promega, USA) and used for the transformation of Escherichia coli DH5a. Individual clones from the library were randomly picked and stored in 384-well plates. A total of 3840 clones were collected in the forward subtracted and the reverse subtracted library. The inserts were amplified with the solution of 1ul single clone bacterial culture as PCR template, 0.5ul primer T7 (10umol/ul), 0.5ul primer SP6 (10umol/ul), 0.25 ul Taq DNA polymerase (Shanghai Sangon, China), 2.5 ul 10 x DNA polymerase buffer, 0.5 ul dNTPs (10mmo/ul) and 1.5 ul MgCl2 (25 mmol/ul), and added ddH2o to 25 ul. The PCR program is: 94 oC for 5 min; 94 oC for 30 s, 58 oC for 35s, 72 oC for 1.5 min, 30 cycles followed by a 5 min final extension at 72 oC.