Abstract: In order to investigate the key genes during wood formation, total RNA was extracted from the stem cambium tissue of twoyearold Populus simonii × P. nigra. The firststrand cDNA was synthesized using switching mechanism at 5′ end of RNA transcript (SMART) technology, and double strand cDNA was synthesized with LDPCR. Then double strand cDNA was cut by SfiⅠ restriction enzyme and ligated with pDNRLIB vector. The recombinant plasmid was transformed into Escherichia coli DH5α by electroporation. The library quality was evaluated, and the results showed that the titer of primary cDNA library and amplified cDNA library were 218×106 pfu/mL and 546×109 pfu/mL, respectively, with the recombination rate of 96%. The inserted fragments were distributed from 05 to 20 kb, and its average size was 112 kb. These results indicate that the P. simonii ×P. nigra cDNA library is constructed successfully. 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