甘蓝型油菜细胞核雄性不育恢复基因BnMs5~α的克隆
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摘要
Rs1046AB是派生于宜3A的细胞核雄性不育系,遗传分析表明Rs1046AB的育性受一个复等位基因位点控制,它们的显隐性关系为恢复基因型(BnMs5a)>不育基因型(BnMs5b)>保持基因型(BnMs5c)(洪登峰,2006)。利用Rs1046AB中的不育株Rs1046A与其恢复系19514A杂交构建的一个F2作图群体,本课题组已将BnMs5位点定位于标记SCD8和SC6之间1.1cM的遗传区间内。并以BnMs5两侧最近标记SCD8和SC6为探针筛选了甘蓝型油菜Tapidor(在BnMs5位点基因型为BnMs5c) BAC文库,获得了可能覆盖目标基因区段的29个阳性克隆。为了克隆该不育基因位点并解析其育性遗传的分子机理,本研究对上述阳性克隆分组和测序,开发出一个与BnMs5紧密连锁的标记,从而将BnMs5定位在一个BAC克隆上约21-kb的物理区段内,并最终通过遗传互补实验证实其中一个候选基因即为BnMs5位点的目标基因。其主要结果如下：
1. BnMs5位点起源。利用12个与BnMs5连锁标记的序列比对白菜和甘蓝全基因组序列,发现绝大多数标记能够在白菜MF2亚基因组及甘蓝subgenome Ⅱ亚基因组的鉴定出高度同源区,且这些同源区在白菜或甘蓝基因组上的排列顺序与上述标记在BnMs5附近的排列顺序基本一致。与BnMs5连锁的标记SCD2亦可定位在甘蓝型油菜TNDH群体(Long et al,2007)的A8染色体上。因此,推测BnMs5位点起源于白菜MF2亚基因组。
2.目标BAC克隆鉴定。利用7组位于候选基因区段的亚基因组特异或保守的PCR引物将可能包含BnMs5位点的29个阳性克隆分为6组。在获得第1组中编号为JBnB007P02的BAC克隆的插入片段序列后,开发出BnMs5一侧最近且在F2群体中呈共显性遗传的标记BE10。利用BE10重新分析上述6组BAC克隆后确认目标BAC克隆位于第1II组中,而第1组克隆中的JBnB007P02则是和目标区段高度同源的拷贝。
3.候选基因预测。以白菜已测序BAC克隆KBrB111O21的序列为参考,从第1II组编号为JBnB034L06的BAC克隆中分离了两侧最近标记SCD8-2和BE10之间的完整序列。基因预测显示该21-kb的候选基因区段包含8个潜在的开放读码框。除ORF2和ORF3为反转录转座子相关基因外,其余6个开放读码框都可能为BnMs5的候选基因。
4.亲本中候选基因序列分离。参考JBnB034L06的可利用信息,从定位群体亲本Rs1046A和19514A中分离出候选区段内的大部分序列及部分目标拷贝特异的标记。利用上述标记锚定的长片段PCR(以预测的全长基因为单位)成功的从两亲本中分别克隆到包含ORF4、ORF5、ORF6、ORF7和ORF8完整基因的序列。
5.候选基因的遗传转化与功能互补。将上述分离的各候选基因片段分别克隆到表达载体pFGC5941中构建互补载体,采用农杆菌介导的方法转化相应的受体材料(全不育系或临保系)。对T0代阳性单株的表型鉴定表明,从恢复系亲本19514A中分离到的一个候选基因ORFna能够恢复全不育系的育性；阳性单株自交的T1代育性观察发现该转基因与育性恢复共分离,证实ORFna就是BnMs5位点的恢复基因BnMs5a。
The rapeseed (Brassica napus L.) line Rs1046AB carries the genetic male sterility locus derived from a spontaneous mutation in Yi-3A. Systematic genetic analyses have indicated that the sterility of Rs1046AB is controlled by a triallelic locus, with the dominance relationship of BnMs5a (the restorer type)□BnMs5b (the male-sterile type)□BnMs5c (the normal male-fertile type). According to the triallelic model, the BnMs5locus has been delimited to a1.1cM genetic fragment between marker SCD8and SC6via the (Rs1046A×19514A) F2mapping population. Furthermore, JBnB BAC library has been screened by using the probes of SCD8and SC6, and29positive BAC clones have been identified, In order to isolate the BnMs5locus and elucidate the molecular mechanism involved in fertility control, we grouped the above positive BAC clones and sequenced one of them. A codominant marker linked with BnMs5was successfully developed based on the BAC sequence, which further narrows the candidate genomic region to a21-kb region. We finally validated that one of the candidate ORFs from19514A could restore the fertility of the all-sterile lines in T0and T1positively transformed plants. The main results were displayed as follows:
1. Determination of the BnMs5-originated subgenome. The sequences of12BnMs5-linked markers were used to query the rough genome sequences of B. rapa and B. oleracea, respectively. Most of the markers could identify homologues with highest similarity both from the MF2subgenome of B. rapa and subgenome Ⅱ of B. oleracea. Moreover, the genetical arrangement of markers around BnMs5was consistent with the physical distribution of those homologues on B. rapa or B. oleracea subgenome. In combination with the localization of a BnMs5-associated maker SCD2on the TNDH A8chromosome, we speculated that the ancestor of BnMs5should evolve from the B. rapa MF2subgenome.
2. Characterization of the target BAC clones. We designed a series of PCR primers according to the genomic sequences of B. rapa MF2subgenome and B. oleracea subgenome II genes delimited by flanking markers SCD8-2and SC6. Empolyment of these primers enabled us to effectively group the29BAC clones into six groups. One BAC clone JBnB007P02, from Group-I was initially shotgun-sequenced. A co-dominant marker BE10developed from this sequence was then mapped as the closest marker on one side of BnMs5. Further comparision of the PCR products from all the clones suggested that the BnMs5locus could be anchored to the Group-Ⅲ BAC clones, while the Group-I clone JBnB007P02is a highly homologous copy of the target one.
3. Prediction of the candidate genes. By using the sequence of a B. rapa BAC clone KBrB111O21as a reference, we obtained the complete sequence between BnMs5-flanking closest markers from a Group-Ⅲ BAC clone JBnB034L06. Gene prediction of this21-kb DNA fragment showed that it contains8putative open reading frames (ORF). Except for the two ORFs (ORF2and ORF3) individually annotated as a reverse transcriptase and a RNase-H protein, the other six ORFs could not be excluded as the candidate of BnMs5according to the available annotation information.
4. Isolation of the candidate sequences from two mapping parents. Most of the candidate sequences were respectively amplified from Rs1046A and19514A with the assistance of a long-PCR method by using the known genomic sequence of JBnB034L06as a reference. Comparison of the multiple orthologues enabled to generate several target copy-specific markers, which were further used to specifically anchor the complete genomic sequences encompassing each candidates, such as ORF4, ORF5, ORF6, ORF7and ORF8.
5. Function complementation test of the candidates. The target genomic sequences from each candidate were cloned into the binary expression plasmid pFGC5941. Agrobacterium-mediated genetic transformations of the candidate restorer allele of BnMs5a into all-sterile plants and the male-sterile allele of BnMs5b into temporary maintainer plants, produced some independent transformants. Among them, the restorer allele from one candidate could restore the fertility of male-sterile plants in positive To plants and T1families, therefore demonstrating that it is the restorer allele of the BnMs5locus.
1. BnMs5位点起源。利用12个与BnMs5连锁标记的序列比对白菜和甘蓝全基因组序列,发现绝大多数标记能够在白菜MF2亚基因组及甘蓝subgenome Ⅱ亚基因组的鉴定出高度同源区,且这些同源区在白菜或甘蓝基因组上的排列顺序与上述标记在BnMs5附近的排列顺序基本一致。与BnMs5连锁的标记SCD2亦可定位在甘蓝型油菜TNDH群体(Long et al,2007)的A8染色体上。因此,推测BnMs5位点起源于白菜MF2亚基因组。
2.目标BAC克隆鉴定。利用7组位于候选基因区段的亚基因组特异或保守的PCR引物将可能包含BnMs5位点的29个阳性克隆分为6组。在获得第1组中编号为JBnB007P02的BAC克隆的插入片段序列后,开发出BnMs5一侧最近且在F2群体中呈共显性遗传的标记BE10。利用BE10重新分析上述6组BAC克隆后确认目标BAC克隆位于第1II组中,而第1组克隆中的JBnB007P02则是和目标区段高度同源的拷贝。
3.候选基因预测。以白菜已测序BAC克隆KBrB111O21的序列为参考,从第1II组编号为JBnB034L06的BAC克隆中分离了两侧最近标记SCD8-2和BE10之间的完整序列。基因预测显示该21-kb的候选基因区段包含8个潜在的开放读码框。除ORF2和ORF3为反转录转座子相关基因外,其余6个开放读码框都可能为BnMs5的候选基因。
4.亲本中候选基因序列分离。参考JBnB034L06的可利用信息,从定位群体亲本Rs1046A和19514A中分离出候选区段内的大部分序列及部分目标拷贝特异的标记。利用上述标记锚定的长片段PCR(以预测的全长基因为单位)成功的从两亲本中分别克隆到包含ORF4、ORF5、ORF6、ORF7和ORF8完整基因的序列。
5.候选基因的遗传转化与功能互补。将上述分离的各候选基因片段分别克隆到表达载体pFGC5941中构建互补载体,采用农杆菌介导的方法转化相应的受体材料(全不育系或临保系)。对T0代阳性单株的表型鉴定表明,从恢复系亲本19514A中分离到的一个候选基因ORFna能够恢复全不育系的育性；阳性单株自交的T1代育性观察发现该转基因与育性恢复共分离,证实ORFna就是BnMs5位点的恢复基因BnMs5a。
The rapeseed (Brassica napus L.) line Rs1046AB carries the genetic male sterility locus derived from a spontaneous mutation in Yi-3A. Systematic genetic analyses have indicated that the sterility of Rs1046AB is controlled by a triallelic locus, with the dominance relationship of BnMs5a (the restorer type)□BnMs5b (the male-sterile type)□BnMs5c (the normal male-fertile type). According to the triallelic model, the BnMs5locus has been delimited to a1.1cM genetic fragment between marker SCD8and SC6via the (Rs1046A×19514A) F2mapping population. Furthermore, JBnB BAC library has been screened by using the probes of SCD8and SC6, and29positive BAC clones have been identified, In order to isolate the BnMs5locus and elucidate the molecular mechanism involved in fertility control, we grouped the above positive BAC clones and sequenced one of them. A codominant marker linked with BnMs5was successfully developed based on the BAC sequence, which further narrows the candidate genomic region to a21-kb region. We finally validated that one of the candidate ORFs from19514A could restore the fertility of the all-sterile lines in T0and T1positively transformed plants. The main results were displayed as follows:
1. Determination of the BnMs5-originated subgenome. The sequences of12BnMs5-linked markers were used to query the rough genome sequences of B. rapa and B. oleracea, respectively. Most of the markers could identify homologues with highest similarity both from the MF2subgenome of B. rapa and subgenome Ⅱ of B. oleracea. Moreover, the genetical arrangement of markers around BnMs5was consistent with the physical distribution of those homologues on B. rapa or B. oleracea subgenome. In combination with the localization of a BnMs5-associated maker SCD2on the TNDH A8chromosome, we speculated that the ancestor of BnMs5should evolve from the B. rapa MF2subgenome.
2. Characterization of the target BAC clones. We designed a series of PCR primers according to the genomic sequences of B. rapa MF2subgenome and B. oleracea subgenome II genes delimited by flanking markers SCD8-2and SC6. Empolyment of these primers enabled us to effectively group the29BAC clones into six groups. One BAC clone JBnB007P02, from Group-I was initially shotgun-sequenced. A co-dominant marker BE10developed from this sequence was then mapped as the closest marker on one side of BnMs5. Further comparision of the PCR products from all the clones suggested that the BnMs5locus could be anchored to the Group-Ⅲ BAC clones, while the Group-I clone JBnB007P02is a highly homologous copy of the target one.
3. Prediction of the candidate genes. By using the sequence of a B. rapa BAC clone KBrB111O21as a reference, we obtained the complete sequence between BnMs5-flanking closest markers from a Group-Ⅲ BAC clone JBnB034L06. Gene prediction of this21-kb DNA fragment showed that it contains8putative open reading frames (ORF). Except for the two ORFs (ORF2and ORF3) individually annotated as a reverse transcriptase and a RNase-H protein, the other six ORFs could not be excluded as the candidate of BnMs5according to the available annotation information.
4. Isolation of the candidate sequences from two mapping parents. Most of the candidate sequences were respectively amplified from Rs1046A and19514A with the assistance of a long-PCR method by using the known genomic sequence of JBnB034L06as a reference. Comparison of the multiple orthologues enabled to generate several target copy-specific markers, which were further used to specifically anchor the complete genomic sequences encompassing each candidates, such as ORF4, ORF5, ORF6, ORF7and ORF8.
5. Function complementation test of the candidates. The target genomic sequences from each candidate were cloned into the binary expression plasmid pFGC5941. Agrobacterium-mediated genetic transformations of the candidate restorer allele of BnMs5a into all-sterile plants and the male-sterile allele of BnMs5b into temporary maintainer plants, produced some independent transformants. Among them, the restorer allele from one candidate could restore the fertility of male-sterile plants in positive To plants and T1families, therefore demonstrating that it is the restorer allele of the BnMs5locus.
引文
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