琯溪蜜柚BAC文库的构建和汁胞粒化相关基因的筛选

Construction of a BAC Library of 'Guanximiyou' and Identification of Clone Related to Granulation

【目的】构建琯溪蜜柚BAC文库,利用该文库筛选与汁胞粒化相关的基因。【方法】用温和的物理方法获得高分子量DNA,部分酶切后进行回收、连接、转化,超低温保存阳性克隆。构建DNA样品混合样,PCR法筛选文库,生物信息学分析DNA序列。【结果】改进了适合琯溪蜜柚BAC文库构建的方法;构建的琯溪蜜柚BAC文库含有26112个单克隆,空载率小于1%,叶绿体DNA的污染率不超过1%,插入片段平均大小大约120kb,覆盖8倍的琯溪蜜柚基因组。利用与琯溪蜜柚汁胞粒化相关的EST序列设计PCR引物筛选文库,得到一段大小为1088 bp的DNA序列,该序列含有两段大小分别为122bp和172bp的内含子,经同源比对发现,该序列中部分序列与蓖麻(Ricinus communis)、杨树(Populus trichocarpa)多铜氧化酶(multicopper oxidase)cDNA序列分别有85%和71%的同源性;与毛叶番荔枝(Annona cherimola)和拟南芥(Arabidopsis thaliana)果胶酯酶(pectinesterase)cDNA序列分别有76%和73%的同源性。【结论】本研究构建的BAC文库适用于琯溪蜜柚功能基因组的研究,从BAC文库筛选获得的DNA序列与汁胞粒化相关的基因有高度同源性。

Objective This study aims to construct a BAC library of ＇Guanximiyou＇,and screen the library based on EST sequence related to granulation.Method High molecular weight DNA was obtained by gentle physical homogenization.The DNA was partial digested by restriction endonucleases,and cloned to BAC vector.The positive clones were conserved.BAC clones were pooled for PCR screening.The sequence of positive clone was analyzed.Result Method of BAC library construction was improved.The library consists of 26 112 clones.A random sample of 100 clones indicated an average insert size of 120 kb,corresponding to 8 genome equivalents.Less than 1% of the clones did not contain inserts.Screening of BAC library with chloroplast DNA probes indicated that less than 1% of the clones contained organelle-derived DNA.The BAC library was screened with a pair of primer deprived from EST related to granulation.A 1 088 bp DNA sequence was amplified by PCR.It contained two introns with the length 122 bp and 172 bp,respectively.The sequence has 85% and 71% identities with multicopper oxidase cDNA sequences of Ricinus communis and Populus trichocarpa,furthermore,it has 76% and 73% identities with pectinesterase cDNA sequence of Annona cherimola and Arabidopsis thaliana,respectively.Conclusion According to the characterization of BAC clones and the results of screening,the library is suitable for functional genome research on ＇Guanximiyou＇.Two genes related to granulation were predicted.