水稻T-DNA插入雄配子不育突变体的创建

Characterization of Male Gametophytic Sterile Mutant Collections in Rice by T-DNA Insertion

对6000多个转基因T-DNA插入水稻株系进行异常遗传分离分析(选择标记基因为潮霉素磷酸转移酶基因hpt),发现216个转基因株系的T-DNA呈现1∶1分离。接着利用测交方法检测T-DNA的遗传规律,初步确定其中57个候选株系为雄配子不育候选突变体。随后对候选突变株进行多代遗传分析及花粉细胞学观察,结果表明其中的38个突变体花粉黑染率约为50%,自交后代T-DNA的异常分离是由于转基因植株的花粉半不育所致,T-DNA的传递只能通过雌配子体。另外,利用ELISA定量测定突变体中cry1Ac(Bt)基因编码蛋白的表达量,发现花粉的这种不育性与外源基因的表达量之间没有直接关系,推测这38个雄配子突变体败育的原因主要是T-DNA插入引起内源基因变异。TAIL-PCR获得了22个水稻雄配子不育T-DNA插入突变体的侧翼序列,通过BLAST检索,定位了15个不同的插入位点,其中12个插入位点位于基因区或基因调控区。

The abnormal genetic segregation of more than 6,000 T-DNA insertional transgenic rice lines （selectable marker gene hygromycin B phosphotransferase,hpt） was analyzed. 216 of them showed 1 ： 1 segregation, and 57 mu- tant lines were initially identified as male gametophytlc sterile candidate mutants through test cross. Further multiple genetic analysis and pollen cytological observation indicated the black-dyed pollen rates of one of the 38 candidate mutations were about 50%, T-DNA abnormal separation of selflng progeny due to the semi sterility of pollen of transgenic plants, T-DNA could be transferred only by the female gametophyte. In addition, expression levels of crylAc （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 T-DNA insertion. Flanking sequences of 22 T-DNA insertional male gametophytic sterile mutants were obtained by TAIL-PCR. According to BLAST search, 15 different insertion sites were located, of which 12 were located in the gene or gene regulatory region.