BAC Library Construction For The Inbreed Line 87-1 Line Andscreening The BAC Including The Restoring Gene Rf4 For Cms-C In Maize.

Maize plays an important role for social development as one of the three major cereal crops in worldwide. And the increasing of maize yield greatly depends on the use of heterosis. Strikingly, the utilization of cytoplasmic male sterility (CMS) is an effective pathway for heterosis exploitation in practices and functions on decreasing seed production cost and improving seed purity.?After the first sterile line was discovered in maize, many researchers have majored on the molecular mechanism of male sterile fertility restoration (Rf). And at least five restored genes were identified controlling the fertility restoration of male sterile lines. Rf1 was located on the short arm of chromosome 3 (Duvick et al.,1961); Rf2 was located near the Wx locus on chromosome 9 (Snyder et al.,1969) and cloned by transposon tagging (Cui et al.,1996); Rf3 was located on the long arm of chromosome 2 (Langhnan et al.,1978); Rf4 was located on chromosome 8 (Josephson et al.,1978) and Rf5 was located on chromosome 5 (personal communication). Although the importance of these genes, none of them was cloned but Rf2. Based on our previous works, Rf4 gene was fine mapped between X-21-1 and X-33 markers in a physical distance about 0.05 MB.To finally clone Rf4 gene, the inbred line 87-1 with this gene was used as test material. Genomic DNA was extracted from the etiolated seedlings using Zhang HB’s method (Zhang H B et al.,2001) and partial digested by restriction enzyme HindIII. 100kb fragment was recovered and connected in pIndigoBAC-5 vector which was transferred into E. coli DH10B competent cell by the electroporation method. 500-5,000 BAC clones were retrieved for each transformation. Then the final BAC library with 214,300 clones was constructed and stored in mixture. All BAC clones on each dish were collected to form the sub-pool. 1,296 sub-pools were produced and each sub-pool contained about 100-500 BAC monoclonal. Then every 81 sub-pools were mixed into a super-pool and 16 super-pools were obtained. 100 random BAC clones were selected to evaluate the effect of BAC library. The results showed that the length of average insertion was about 100kb covering about 10 times of maize genome with 5% of empty clone. Subsequently, X-21-1 and X-33 markers were used as probes to screen the 87-1 BAC library and one BAC covering complete region of Rf4 gene was identified.The 87-1 BAC library constructed in this study can not only supply favorable basis for map-based cloning and biological research of Rf4 but also for genomic sequencing, physical mapping and gene cloning of other important genes.To study maize male sterility could be further clear the abortion mechanism of male sterility, clear interaction between nuclear and cytoplasmic of cytoplasmic male sterile lines and proven cytoplasmic male sterility’s mechanism of the nuclear restorer gene, provide theoretical support of breeding good CMS, the corresponding maintainer lines and restorer lines corresponding.