Abstract:

A high quality of RNA was essential for construction of yeast two hybrid library. In this work, four methods were performed to extract total RNA from samples of 9dpa (day post anthesis) and 19dpa cotton fibers. RNA integrity and purity was detected by using agarose gel and spectrophotometer. As a result, the hot borate method worked well for 9 and 19dpa cotton fibers, whereas CTAB-PVP was applicable only for 9dpa fibers. As a comparison, both guanidinium thiocyanate and high ion-density methods were not suitable for the samples with high proteins and polysaccharides. The total RNA was extracted by hot borate extraction, the purified mRNA was reverse transcripted to cDNA. Then, the amplified cDNAs were inserted into pGADT7-Rec vector through highly efficient recombination system. Hence, the titers of the 9 and 19dpa fibers cDNA library could respectively reach to 1.01×107 cfu/mL and 8×106 cfu/mL, and the cDNA insertions varied from 250 to 2500 bp. The two libraries with high quality could be used for the research of cellulose synthesis and regulation.