Mass genetic transformation of rice with Maize B73 BIBAC clones and analyses of rice transgenic mutants
Yafei Wang, Lin Liu, Wentao Xiong, Wei Peng, Haiyang Zeng, Xue Shi, Meizhong Luo*
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
*Corresponding author: Meizhong Luo: email@example.com
BIBAC system has been developed not only for structural genomics research as a BAC library, but also for functional genomics studies through Agrobacterium-mediated transformation of large genomic DNA fragments into plants. We previously modified BIBAC and BAC vectors (Shi et al., 2011, Plant Methods 7:33) that can largely facilitate BAC/BIBAC library construction and exchange of intact large insert DNA fragment between the BIBAC vector and the BAC vector. A maize B73 BIBAC library was constructed using our modified BIBAC vector. Stability test showed that BIBAC clones with different insert sizes from 40 kb to >160 kb were all stable in Agrobacterium EHA105 after cultured for at least 96 h (4 days), the time length that are needed to complete co-inoculation with rice callus in rice transformation. We propose to construct transgenic rice lines with tiling maize B73 BIBACs. To date, more than 1000 B73 BIBAC clones were end-sequenced and more than 2000 BIBAC clones were used for rice transformation. We have demonstrated that an 164-kb maize B73 genomic DNA fragment with 88.1% repeat sequences and containing 5 filtered gene models was completely integrated into rice genome and the maize gene models were expressed in the transgenic rice plants. Some BIBAC transgenic rice plants with changed phenotypes possibly from gain of function of maize genes or loss of function of rice genes have been obtained and are being further analyzed. More information can be obtained through our website http://GResource.hzau.edu.cn.