【作者】 夏宗伟；

【导师】 王文兵； 谭小力；

【作者基本信息】 江苏大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 油菜角果开裂给油菜的生产带来了许多不利的影响,彻底弄清角果开裂过程及其潜在的遗传调节机制能为油菜抗裂角品种的培育及挑选提供新的途径。前期对模式植物拟南芥的研究,鉴定出几个转录调节因子通过调节裂区不同细胞形态的分化形成参与了拟南芥角果的开裂过程。这几个转录因子分别是:FRUITFULL(FUL)、SHATTERPROOF1(SHP1)、SHATTERPROOF2(SHP2)、INDEHISCENT(IND)、ALCATRAZ(ALC)。为了寻找油菜中以上几个基因的同源基因,本文运用BLAST手段在油菜cDNA库中搜寻到与这几个基因相似性比较高的一些EST序列和cDNA序列。通过拼接、克隆以及RACE方法,获得了几条与FUL、SHP1、SHP2、IND和ALC基因序列同源性比较高的cDNA序列,一个为已知基因BnSHP1,其它的暂命名为甘蓝型油菜的候选基因BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND和BnALC。RT-PCR结果显示,BnFUL-b、BnALC、BnSHP2和BnSHP1基因在花和角果中都有显著的表达,此外,BnFUL-b、BnALC、BnSHP2还在其它部位有表达。Southern杂交技术鉴定了BnSHP2基因在甘蓝型油菜基因组中至少存在两个拷贝。为了鉴定这几个预测基因的功能,本文构建了BnFUL-b基因的过量表达载体和BnALC基因的抑制表达载体,然后通过农杆菌介导转化甘蓝型油菜,通过PCR鉴定了转基因阳性植株。将BnALC、BnFUL-b、BnIND基因去除了终止密码的ORF片段构建到携带sGFP(S65T)荧光蛋白基因的瞬时表达载体上,通过基因枪轰击洋葱表皮细胞后,在荧光显微镜下观察洋葱表皮细胞发现细胞核内有强烈的荧光产生,表明这几个基因应该定位在细胞核内。更多还原

【Abstract】 Fruit dehiscence of oilseed rape bring many disadvantages for breeding, therefore,to ravel the dehiscence process and genetic mechanisms underlying the regulation of fruit dehiscence in oilseed rape could suggest new approach to produce new dehiscence tolerant varieties of oilseed rape.Recent studies on Arabidopsis have identified several transcriptional factors involved in fruit dehiscence by regulating the proper differentiation of cell types in dehiscence zone.These transcriptional factors are FRUITFULL(FUL),SHATTERPROOF1(SHP1),SHATTERPROOF2(SHP2), INDEHISCENT(IND),ALCATRAZ(ALC).To obtain these genes list above in oilseed rape,BLAST procedure was carried out in similarity search in oilseed rape database using FUL,SHP1,SHP2,IND and ALC sequeces as queries respectively,thus some ESTs and cDNAs sequences sharing hingh similarity to queries respectively were obtained.BnSHP1 has been reported and the other candidate genes in Brassica napus containing BnFUL-a、BnFUL-b、BnFUL-c、BnFUL-d、BnSHP2、BnIND and BnALC were finally deduced by sequence assembly,subsequently cloning and RACE method.The results of RT-PCR showed that BnFUL-b,BnALC,BnSHP1 and BnSHP2 were all expressed in flower and fruit,moreover,BnFUL-b,BnALC and BnSHP2 were also expressed in other vegetative tissues.The southern bloting result showed that there are two duplication of BnSHP2 at least in genome of Brassica napus.The recombinant plasmid for overexpression of BnFUL-b and the recombinant plasmid for suppressed expression of BnALC were successfully transformed into Brassica napus by Agrobacterium Tumefaciens respectively for further identifying their functions.The ORF sequences without termination codons of BnFUL-b,BnIND and BnALC were inserted in the position between 35S promoter and sGFP(S65T)gene of the sGFP(S65T)vector respectively,so three transient-expression vectors were constructed and subsequently transferred into onion epidermal cell with particle bombardment.The expressiont of sGFP(S65T)were mainly restricted into the nucleus observed under fluorescence microscope,suggesting that BnFUL-b,BnIND and BnALC proteins were mainly located in the cell nucleus.更多还原