缺铁玉米根cDNA文库的构建及蛋白和cDNA差异比较

Construction of cDNA Library from Iron |deficiency Induced Maize Roots and Comparing the Difference of Protein and cDNA between Iron |deficient and Iron |sufficient Condition

采用异硫氰酸胍酚氯仿法分离铁胁迫玉米根总ＲＮＡ，然后采用寡聚ｄＴ 纤维素层析柱法纯化Ｐｏｌｙ（Ａ） ＋ ＲＮＡ．用含有ＸｈｏＩ位点的ｌｉｎｋｅｒｐｒｉｍｅｒ 锚定合成ｃＤＮＡ 第一条链后，采用替代法合成第二条链，接着连接上ＥｃｏＲＩＡｄａｐｔｅｒ 以便定向重组到载体上．ｃＤＮＡ 经过ＸｈｏＩ消化和分级分离后，带有ＸｈｏＩ和ＥｃｏＲＩ粘性末端的ｃＤＮＡ 定向重组到λＺＡＰ表达载体的ＸｈｏＩ和ＥｃｏＲＩ位点上．经体外包装，重组的噬菌体转导宿主菌ＸＬ１Ｂｌｕｅ ＭＲＦ，最终获得滴度为４－５ ×１０５ ｐｆｕ／ｕｇ 的λＺＡＰ 表达ｃＤＮＡ 文库．通过差异筛选法和质膜双向电泳验证了缺铁和加铁条件下ｃＤＮＡ

Total RNA from Fe |deficient maize roots was isolated by the guanidinium thiocyanate |phen |chloroform extraction method,and then poly(A) ++RNA was purified by column chromatography with oligo(dT) cellulose.After double |stranded cDNA with XhoI linker was synthesized,EcoRI adapters was ligated with cDNA.By XhoI digestion and size fractionation,cDNA with XhoI and EcoRI cohensived ends was recombined into XhoI and EcoRI site of λZAP Express vector.Packaged in vitro ,recombinant phages were transducted into XL1 |BLue MRF host strain.At last the λZAP Express cDNA library of 4 ^5×10 +5 pfu ug was constructed.It was demonstrated that cDNA clones and some PM proteins were different between iron |deficient and iron |sufficient condition by the differential screening and two |dimensional electrophoresis.