猕猴桃基因组DNA文库的构建

采用改良CTAB法提取美味猕猴桃(Actinidiadeliciosacv.Bruno)基因组DNA,经CsCl密度梯度离心纯化后用Sau3AI部分酶切,通过透析袋电泳的方法分级回收,剔除14kb以下的片段,回收长为14～23kb的酶切片段。部分酶切片段经dATP和dGTP部分补平后与LambdaFIXII载体连接,连接产物用GigapackⅢPackagingExtract进行包装,最后得到含有1.05×106pfu的基因组文库,扩增后文库滴度为2.5×109pfu/mL。利用6对根据植物基因保守区或猕猴桃基因序列设计的专一引物对基因组文库和基因组DNA进行PCR扩增,均得到与预计长度相符的目的基因片段。RR

Construction and Application of A Genomic DNA Library of Actinidia deliciosa

Genomic DNA was extracted from Actinidia deliciosa with a modified CTAB method, and purified by CsCl density gradient centrifugation. The purified DNA was then digested with Sau3A I. In order to remove the fragments less than 14 kb, the partially digested DNA was further size-fractioned by preparative gel electrophoresis and then the fragments between 14 to 23 kb were recovered from the gel by electroelution. The fragments were filled in with dATP and dGTP before ligated to Lambda FIX II vector, and the ligation products were incubated with "Gigapack III Gold" (Stratagene) packaging extracts. As a result,we constructed a genomic DNA library with a primary titer of 1.05×106 and the titer of amplified library is 2.5×109 pfu/mL. To evaluate the representation of the constructed library, the phages in the library were served as PCR template as well as genomic DNA with 6 pairs of specific primers designed from either reserved regions of plant genes or kiwifruit genes. Fragments of expected size were amplified for both templates.