The abnormal genetic segregation of more than 6,000 TDNA insertional transgenic rice lines(selectable marker gene hygromycin B phosphotransferase,hpt) was analyzed. 216 of them showed 1∶1 segregation, and 57 mutant lines were initially identified as male gametophytic sterile candidate mutants through test cross. Further multiple genetic analysis and pollen cytological observation indicated the blackdyed pollen rates of one of the 38 candidate mutations were about 50%, TDNA abnormal separation of selfing progeny due to the semisterility of pollen of transgenic plants, TDNA could be transferred only by the female gametophyte. In addition, expression levels of cry1Ac (Bt) genes tested by Enzyme Linked Immunosorbent Assay (ELISA) demonstrated their pollen sterility and foreign gene expression had no direct relationship, indiating that the abortion of the 38 male gametophytic sterile mutants was mainly attributed to endogenous gene variation caused by TDNA insertion. Flanking sequences of 22 TDNA insertional male gametophytic sterile mutants were obtained by TAILPCR. According to BLAST search, 15 different insertion sites were located, of which 12 were located in the gene or gene regulatory region.
CHEN Rui, YU Fake, LIU Huaqing, YANG Shaohua, WANG Feng*
. Characterization of Male Gametophytic Sterile Mutant Collections in Rice by TDNA Insertion[J]. Chinese Journal OF Rice Science, 2012
, 26(2)
: 173
-181
.
DOI: 10.3969/j.issn.10017216.2012.02.006
\[1\]McCormick S. Control of male gametophyte development. Plant Cell, 2004, 16: S142S153.
\[2\]Borg M, Brownfield L, Twell D. Male gametophyte development: A molecular perspective. J Exp Bot, 2009, 60(5):14651478.
\[3\]Liu J J, Qu L J. Meiotic and mitotic cell cycle mutants involved in gametophyte development in Arabidopsis. Mol Plant, 2008, 1(4): 564574.
\[4\]陈睿, 李清贤, 杨绍华．水稻花粉发育相关基因的研究进展．福建农业学报, 2010, 25(3): 274281.
\[5\]Chen Y C, McCormick S. Sidecar pollen, an Arabidopsis thaliana male gametophytic mutant with aberrant cell divisions during pollen development. Development, 1996, 122: 32433253.
\[6\]Park S K, Howden R, Twell D. The Arabidopsis thaliana gametophytic mutation gemini pollen 1 disrupts microspore polarity, division asymmetry and pollen cell fate. Development, 1998, 125（19）: 37893799.
\[7\]Lalanne E, Twell D. Genetic control of male germ unit organization in Arabidopsis. Plant Physiol, 2002, 129(2): 865875.
\[8\]Durbarry A, Vizir I, Twell D. Male germ line development in Arabidopsis duo pollen mutants reveal gametophytic regulators of generative cell cycle progression. Plant Physiol, 2005, 137(1): 297307.
\[9\]Johnson S A, McCormick S. Pollen germinates precociously in the anthers of raringtogo, an Arabidopsis gametophytic mutant. Plant Physiol, 2001， 126(2): 685695.
\[10\]Feldmann K A, Coury D A, Christianson M L. Exceptional segregation of a selectable marker (KanR) in Arabidopsis identifies genes important for gametophytic growth and development. Genetics, 1997, 147(3): 14111422.
\[11\]Howden R, Park S K , Moore J M, et al. Selection of TDNAtagged male and female gametophytic mutants by segregation distortion in Arabidopsis. Genetics, 1998, 149(2): 621631.
\[12\]Bonhomme S, Horlow C, Vezon D, et al. TDNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone. Mol Gen Genet, 1998, 260(5): 444452.
\[13\]Lalanne E, Michaelidis C, Moore J M, et al. Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis. Genetics, 2004, 167(4): 19751986.
\[14\]Boavida L C, Shuai B, Yu H J, et al. A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana. Genetics, 2009, 181(4): 13691385.
\[15\]Jeong D H, An S, Kang H G, et al. TDNA insertional mutagenesis for activation tagging in rice. Plant Physiol, 2002, 130(4): 16361644.
\[16\]Enoki H, Izawa T, Kawahara M, et al. Ac as a tool for the functional genomics of rice. Plant J, 1999, 19(5): 605613.
\[17\]Moritoh S, Miki D, Akiyama M, et al. RNAimediated silencing of OsGENL (OsGENlike), a new member of the RAD2/XPG nuclease family, causes male sterility by defect of microspore development in rice. Plant Cell Physiol, 2005, 46(5): 699715.
\[18\]Jiang S Y, Cai M, Ramachandran S. The Oryza sativa no pollen (Osnop) gene plays a role in male gametophyte development and most likely encodes a C2GRAM domaincontaining protein. Plant Mol Biol, 2005, 57(6): 835853.
\[19\]Murra M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8(19): 43214326.
\[20\]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of proteindye binding. Anal Biochem, 1976, 72: 248254.
\[21\]秦崇涛, 陈在杰, 苏军, 等. 3种ELISA法定量检测转cry1Ac基因水稻crylAc蛋白的比较研究. 福建农业学报, 2003, 18(4): 258263.
\[22\]Liu Y G, Whittier R F. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert endfragments from P1 and YAC clones for chromosome walking. Genomics, 1995, 25(3): 674681.
\[23\]Vogler D W, Peretz S, Stephenson A G. Floral plasticity in an iteroparous plant: The interactive effects of genotype, environment, and ontogeny in Campanula rapunculoides (Campanulaceae). Am J Bot, 1999, 86: 482.
\[24\]McCormick S. Male gametophyte development. Plant Cell, 1993, 5: 12651275.
