油菜BnROP1同源基因沉默导致拟南芥雄性不育

基于油菜杂合双显性雄性核不育系和拟南芥全基因组芯片的表达谱分析结果,选择了一个油菜差异表达基因,其对应的拟南芥同源基因为At3G51300（AtROP1）,采用荧光定量PCR方法,对油菜中该基因（BnROP1）在根、茎、叶、花、雌蕊、雄蕊、角果中的表达模式进行了分析,在此基础上构建了AtROP1基因的microRNA干扰载体,通过农杆菌介导的拟南芥转化,获得12株转基因苗。T1转基因植株大多出现角果数量减少或不结实、无花粉、花药萎缩以及小孢子数量大大减少的表型。对转基因T2多个株系的荧光定量PCR分析说明,目标基因的干扰是有效的,靶基因的表达水平都显著下调。说明转基因植株的雄性不育表型和ROP1基因的表达沉默有关,ROP1基因不仅控制了花粉管的极性和伸长,还和小孢子的正常发育密切相关。

Silencing of BnROP1 homologue gene leads to male sterility in Arabidopsis

Based on previous analysis of microarray expression on Brassica napus homozygous pairs of dominant male sterile line,a differentially expressed Brassica napus gene（BnROP1） which is homologous to At3G51300（AtROP1） was selected.Method of fluorescent quantitative PCR was used to analyze the expression pattern of BnROP1 in different tissues of B.napus,including root,stem,leaves,flower,pistil,stamen and silique.The results indicated that BnROP1 was highly expressed in fertile flower and stamen.On this basis,microRNA interference vector was constructed and then transformed into Arabidopsis thaliana by Agrobacterium,obtaining 12 transgenic seedlings.The phenotype of T1 transgenic generation was observed during florescence.Most transgenic seedlings showed reduction or disappearance of siliques,no pollen,short filaments,deteriorated anther and microspore.These phenomena indicated that these seedlings were fully-sterile or semi-sterile.The fluorescence quantitative PCR experiments on T2 generations of transgenic lines showed that the expression level of target gene decreased significantly.The correlation of transgenic male sterile phenotype and the silencing of ROP1 expressing indicated that ROP1 was not only promoting tip growth and maintaining growth polarity in pollen tubes but was also essential for plant fertility.