同源序列法克隆甘蓝型油菜核不育基因BnMS1及其功能研究
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摘要
雄性不育是指植物在有性繁殖过程中不能产生正常的花药、花粉或雄配子而雌蕊功能正常的现象。雄性不育广泛应用于杂交育种中,图位克隆核不育基因费时费力。目前为止,虽已对许多与细胞核雄性不育相关基因进行分子标记与定位,但尚未有图位克隆到甘蓝型油菜不育基因的报道。
油菜与拟南芥同属十字花科芸薹属植物,比较基因组学研究证明油菜与拟南芥基因组同源性很高,这意味着可以利用拟南芥育性相关基因克隆油菜的雄性不育基因以及开展育性相关性的研究。目前已分离的拟南芥育性相关基因有：MS2基因、MS5基因、Coll基因、spl基因、MS1基因、eMS1基因、dde2-2基因和mmd1基因等。其中拟南芥MS1只在小孢子释放时期的绒毡层组织中低丰度表达,拟南芥MS1突变体能导致花粉败育而对其它花器官的生长发育没有影响,这在生产上有较大的应用价值。
因此本研究围绕拟南芥核不育基因MS1开展了以下工作：
1.克隆甘蓝型油菜核不育基因BnMs1及生物信息学分析：根据拟南芥MS1基因保守区域序列设计特异性引物,PCR扩增甘蓝型油菜超油二号基因组,将PCR产物克隆测序,并将所得序列拼接。根据所得序列两端设计引物,利用PCRWalking技术克隆该基因全长。将克隆的基因序列全长与拟南芥MS1基因全长序列进行同源性比对及生物信息学分析。结果：BnMs1基因全长3424bp,BnMS1与MS1全长序列同源性60.3%；BnMs1与MS1编码序列同源性89.5%；BnMS1基因外显子分别位于521bp-+821bp、1189bp-+1471bp、1876bp-+3296bp；内含子分别位于822bp-+1188bp、1472bp-+1875bp；开放阅读框长2004bp,编码667个氨基酸,蛋白分子量为76.5KD,等电点为8.01；根据BnMS1基因序列推测的蛋白质氨基酸序列与已知拟南芥MS1基因蛋白质氨基酸序列同源性达到93%。
2.构建BnMS1基因反义表达载体转化拟南芥。克隆BnMS1基因第三个外显子2402-2919bp序列命名为antiMS,反向插入pFGC5941植物表达载体中,并转化入农杆菌中。T1代种子播种,种子萌发3-5d后喷施除草剂筛选抗性植株,后期提取DNA进行PCR鉴定,提取花RNA进行RT-PCR,并观察植株生长情况,对花粉进行取样,碘、碘化钾染色,在显微镜下观察。结果：转anti-MS拟南芥共筛选到14株,PCR阳性植株11株,植株表型、花粉粒数目活性与野生型无区别,RT-PCR均扩出条带且与野生型无明差异。
3.构建拟南芥MS1基因人工小RNA表达载体转化拟南芥。根据Schwab的方法构建拟南芥MS1基因amiRNA载体,并与pFGC5941表达载体连接,并转入农杆菌中。利用花絮浸润法转化拟南芥,转化种子播种,种子萌发3-5d后喷施除草剂筛选抗性植株,后期提取DNA进行PCR鉴定,提取花RNA进行RT-PCR,并观察植株生长情况,对花粉进行取样,碘、碘化钾染色,在显微镜下观察。结果：转amiRNA载体拟南芥共筛选到27株转化株,PCR阳性植株25株,其中8株转基因植株表现出中间型雄性不育的性状：植株部分荚短小不育,在早期花中观察到花粉囊中含有数目极少的花粉粒,后期与野生型并无明显区别,其余转基因植株与野生型无区别。
4.构建BnMS1启动子序列与pGUS-eGFP双标记连接的植物表达载体。PCR克隆BnMS1启动子序列,将启动子序列与pGUS-eGFP双标记连接的植物表达载体连接,并转化入农杆菌中,以待转化拟南芥,验证启动子是否具有功能。对重组农杆菌的PCR结果表明,启动子序列已转入农杆菌中,载体构建成功。
Male srerility is a phenomenon that in the process of sexual reproduction in plants, anthers, pollen and male gametes are abnormal but the function of the female bud is normal.Male srerility of rapeseed is wildly used in domestic and international hybridization.because of the genome of brassica napus is complicated,so far there is no report on map-based cloning of male sterility gene on brassica napus.
Rapeseed and Arabidopsis belong to the Brassica species of Cruciferae plant.Comparative genomics studies have shown that the rape and Arabidopsis thaliana share highly homologous genome,which means that we can make use of fertility-related genes in Arabidopsis thaliana cloning male sterile gene in Rape and conducting research on male sterility.So far there are many fertility-related genes separated and cloned from Arabidopsis thaliana, such as MS2,MS5,Coll, spl, MS1, eMSl,dde2-2,mmdl and so on. The gene of MS1 expresses during the period of spores release in tapetal tissues,and the expression is low in abundance.The pollen of MSI mutation is abortion and the development of the other floral organs are not affected, which can be have a great use in production.
In this research,we carried some works on MS1 gene,as follows:
1.The cloning of male sterile gene BnMS1 in Brassica napus and its bioinformatics analysis.According to the coding sequences of Arabidopsis MS1 gene,a series of primers were designed to clone the homologous sequences in Brassica napus. PCR Walking method was used to clone the full sequence of MS1 gene of Brassica napus. The total lengh of the BnMSl gene cloned here is 3424bp,and we deduce there are three exons located at 521bp-+821 bp,1189bp-+1471 bp and1876bp-+3296 bp;while introns located at 822bp-+1188 bp,and 1472bp-+1875 bp.The MS1 from Brassica napus shared a similarity of 89.5% in encoding sequence and 60.3% in full sequence with that from Arabidopsis thaliana. According to the results we deduced that it has an open reading frame(ORF) of 2004bp;encoding a protein of 667 amino acid, with protein molecμlar weight of 26kD and isoelectric point of 8.01.The similarity between the MS1 from Brassica napus and that from Arabidopsis thaliana is 93% in amino acid sequence.
2.A plant expressed vector with AntiMS was constructed. Cloning the third exon sequence between 2402-+2919bp of BnMSl gene and liagased with pFGC5941 Vector reversed. Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip transformation method to invert the expressed vector with Anti-MS into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and use RT-PCR amplification to analysis the expression of MS1 gene.Stain and Observ the pollens under the microscope. The results:14 transgenic plants have been selected,11 of them are positive and there are no significant difference between positive transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana on Phenotype,pollen activity and expression of MS1 gene.
3.A plant expressed vector with amiRNA of MS1 was constructed. Clone the amiRNA of MS1 then liagsed with pFGC5941 vector.Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip method to invert the expressed vector with amiRNA into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and using RT-PCR to analysis the expression of MSI gene.Stain and Observe the pollens under the microscope. The results:we have selected 27 transgenic plants,25 of them are positive. Some strains of transgenic Arabidopsis showed intermediate male sterility,and the Pods are shorter and the number of pollen grains in early flower anthers are fewer than col-0 Arabidopsis.there are no significant difference between the other transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana.
4.Construct a expressed vector with the promoter of BnMSl.Using PCR to amplified the promoter sequence of BnMSl then connecting the promoter sequence with the pGUS-eGFP double-labeled plant expression vector, at last it was transferred to agrobacterium tumefaciens for future study. Identification of the transgenic Agrobacterium tumefaciens by PCR amplification.the results showed that the promoter sequence was transferred into Agrobacterium,so the vector was successfully constructed.
油菜与拟南芥同属十字花科芸薹属植物,比较基因组学研究证明油菜与拟南芥基因组同源性很高,这意味着可以利用拟南芥育性相关基因克隆油菜的雄性不育基因以及开展育性相关性的研究。目前已分离的拟南芥育性相关基因有：MS2基因、MS5基因、Coll基因、spl基因、MS1基因、eMS1基因、dde2-2基因和mmd1基因等。其中拟南芥MS1只在小孢子释放时期的绒毡层组织中低丰度表达,拟南芥MS1突变体能导致花粉败育而对其它花器官的生长发育没有影响,这在生产上有较大的应用价值。
因此本研究围绕拟南芥核不育基因MS1开展了以下工作：
1.克隆甘蓝型油菜核不育基因BnMs1及生物信息学分析：根据拟南芥MS1基因保守区域序列设计特异性引物,PCR扩增甘蓝型油菜超油二号基因组,将PCR产物克隆测序,并将所得序列拼接。根据所得序列两端设计引物,利用PCRWalking技术克隆该基因全长。将克隆的基因序列全长与拟南芥MS1基因全长序列进行同源性比对及生物信息学分析。结果：BnMs1基因全长3424bp,BnMS1与MS1全长序列同源性60.3%；BnMs1与MS1编码序列同源性89.5%；BnMS1基因外显子分别位于521bp-+821bp、1189bp-+1471bp、1876bp-+3296bp；内含子分别位于822bp-+1188bp、1472bp-+1875bp；开放阅读框长2004bp,编码667个氨基酸,蛋白分子量为76.5KD,等电点为8.01；根据BnMS1基因序列推测的蛋白质氨基酸序列与已知拟南芥MS1基因蛋白质氨基酸序列同源性达到93%。
2.构建BnMS1基因反义表达载体转化拟南芥。克隆BnMS1基因第三个外显子2402-2919bp序列命名为antiMS,反向插入pFGC5941植物表达载体中,并转化入农杆菌中。T1代种子播种,种子萌发3-5d后喷施除草剂筛选抗性植株,后期提取DNA进行PCR鉴定,提取花RNA进行RT-PCR,并观察植株生长情况,对花粉进行取样,碘、碘化钾染色,在显微镜下观察。结果：转anti-MS拟南芥共筛选到14株,PCR阳性植株11株,植株表型、花粉粒数目活性与野生型无区别,RT-PCR均扩出条带且与野生型无明差异。
3.构建拟南芥MS1基因人工小RNA表达载体转化拟南芥。根据Schwab的方法构建拟南芥MS1基因amiRNA载体,并与pFGC5941表达载体连接,并转入农杆菌中。利用花絮浸润法转化拟南芥,转化种子播种,种子萌发3-5d后喷施除草剂筛选抗性植株,后期提取DNA进行PCR鉴定,提取花RNA进行RT-PCR,并观察植株生长情况,对花粉进行取样,碘、碘化钾染色,在显微镜下观察。结果：转amiRNA载体拟南芥共筛选到27株转化株,PCR阳性植株25株,其中8株转基因植株表现出中间型雄性不育的性状：植株部分荚短小不育,在早期花中观察到花粉囊中含有数目极少的花粉粒,后期与野生型并无明显区别,其余转基因植株与野生型无区别。
4.构建BnMS1启动子序列与pGUS-eGFP双标记连接的植物表达载体。PCR克隆BnMS1启动子序列,将启动子序列与pGUS-eGFP双标记连接的植物表达载体连接,并转化入农杆菌中,以待转化拟南芥,验证启动子是否具有功能。对重组农杆菌的PCR结果表明,启动子序列已转入农杆菌中,载体构建成功。
Male srerility is a phenomenon that in the process of sexual reproduction in plants, anthers, pollen and male gametes are abnormal but the function of the female bud is normal.Male srerility of rapeseed is wildly used in domestic and international hybridization.because of the genome of brassica napus is complicated,so far there is no report on map-based cloning of male sterility gene on brassica napus.
Rapeseed and Arabidopsis belong to the Brassica species of Cruciferae plant.Comparative genomics studies have shown that the rape and Arabidopsis thaliana share highly homologous genome,which means that we can make use of fertility-related genes in Arabidopsis thaliana cloning male sterile gene in Rape and conducting research on male sterility.So far there are many fertility-related genes separated and cloned from Arabidopsis thaliana, such as MS2,MS5,Coll, spl, MS1, eMSl,dde2-2,mmdl and so on. The gene of MS1 expresses during the period of spores release in tapetal tissues,and the expression is low in abundance.The pollen of MSI mutation is abortion and the development of the other floral organs are not affected, which can be have a great use in production.
In this research,we carried some works on MS1 gene,as follows:
1.The cloning of male sterile gene BnMS1 in Brassica napus and its bioinformatics analysis.According to the coding sequences of Arabidopsis MS1 gene,a series of primers were designed to clone the homologous sequences in Brassica napus. PCR Walking method was used to clone the full sequence of MS1 gene of Brassica napus. The total lengh of the BnMSl gene cloned here is 3424bp,and we deduce there are three exons located at 521bp-+821 bp,1189bp-+1471 bp and1876bp-+3296 bp;while introns located at 822bp-+1188 bp,and 1472bp-+1875 bp.The MS1 from Brassica napus shared a similarity of 89.5% in encoding sequence and 60.3% in full sequence with that from Arabidopsis thaliana. According to the results we deduced that it has an open reading frame(ORF) of 2004bp;encoding a protein of 667 amino acid, with protein molecμlar weight of 26kD and isoelectric point of 8.01.The similarity between the MS1 from Brassica napus and that from Arabidopsis thaliana is 93% in amino acid sequence.
2.A plant expressed vector with AntiMS was constructed. Cloning the third exon sequence between 2402-+2919bp of BnMSl gene and liagased with pFGC5941 Vector reversed. Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip transformation method to invert the expressed vector with Anti-MS into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and use RT-PCR amplification to analysis the expression of MS1 gene.Stain and Observ the pollens under the microscope. The results:14 transgenic plants have been selected,11 of them are positive and there are no significant difference between positive transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana on Phenotype,pollen activity and expression of MS1 gene.
3.A plant expressed vector with amiRNA of MS1 was constructed. Clone the amiRNA of MS1 then liagsed with pFGC5941 vector.Recombinant plasmid was transformed into Agrobacterium tumefaciens.Using Floral-Dip method to invert the expressed vector with amiRNA into Arabidopsis thaliana. Identificat of the transgenic plants by PCR amplification and using RT-PCR to analysis the expression of MSI gene.Stain and Observe the pollens under the microscope. The results:we have selected 27 transgenic plants,25 of them are positive. Some strains of transgenic Arabidopsis showed intermediate male sterility,and the Pods are shorter and the number of pollen grains in early flower anthers are fewer than col-0 Arabidopsis.there are no significant difference between the other transgenic Arabidopsis thaliana and col-o Arabidopsis thaliana.
4.Construct a expressed vector with the promoter of BnMSl.Using PCR to amplified the promoter sequence of BnMSl then connecting the promoter sequence with the pGUS-eGFP double-labeled plant expression vector, at last it was transferred to agrobacterium tumefaciens for future study. Identification of the transgenic Agrobacterium tumefaciens by PCR amplification.the results showed that the promoter sequence was transferred into Agrobacterium,so the vector was successfully constructed.
引文
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