甘蓝SRK激酶结构域编码区的克隆及其与MLPK的双色FISH定位研究
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摘要
芸薹属自交不亲和性由S位点所控制。SRK基因是决定自交不亲和性反应的关键因子,前人已经从芸薹属中的甘蓝、甘蓝型油菜等物种中克隆出SRK基因。SRK基因的变异和表达量都会影响自交不亲和性。SRK基因的胞外域有着较大的多态性,变异常常发生在此编码区。相对于胞外域,激酶结构域的保守型较强。本文专门对SRK基因激酶结构域进行了克隆分析,以期从进化的角度探讨激酶结构域对于自交不亲和性的作用。除此之外,MLPK被证明也是SI反应中必不可少的一个因子,作为SRK下游元件起作用。本文针对这两个基因在SI中的重要性,利用FISH技术将SRK和MLPK进行物理定位,从而探索其在甘蓝基因组中的分布及其拷贝数。得到的主要研究结果如下：
1.以结球甘蓝263、羽衣甘蓝AY627和甘蓝型油菜S11为材料,采用PCR、RT-PCR等技术,分别对SRK激酶结构域基因组DNA和cDNA进行了扩增、测序和序列分析,并构建了分子进化树。分别获得了1694bp和1307bp的片段、1705bp和1229bp的片段和1606bp和1214bp的片段,分别编码433,407和402个氨基酸。对获得的片段进行比对发现,结球甘蓝263包括5个外显子和4个内含子,羽衣甘蓝AY627和甘蓝型油菜S11包括6个外显子和5个内含子。并且发现结球甘蓝263的内含子少1个,并且有一段70bp左右的插入片段。对已报道的21种SRK基因和本文所克隆的3种材料的序列构建分子进化树,发现三者都与S单倍型的Ⅰ类SRK基因聚在一起,Ⅱ类基因与Ⅰ类基因分为两支,拟南芥与Ⅱ类聚在一起,而萝卜属未形成单独的一支。推导甘蓝26370bp片段的插入有可能是内含子转化而来并导致了自交不亲和性的产生。羽衣甘蓝AY627与甘蓝型油菜S11所获得的片段与预期一致,激酶结构域没有发生片段的插入或缺失,推导导致甘蓝型油菜S11自交不亲和性的原因没有发生在激酶结构域部分。通过进化树飞分析推导出萝卜属与芸薹属的分离晚于SRKⅠ类和Ⅱ类的分离。
2.在FISH过程中,对甘蓝根尖前中期染色体和DNA纤维的制片进行了一些条件的改变,试图提高FISH效果。在前中期染色体制片中,本文对冷处理时间和酶解时间进行了改变,将冷处理时间定在20h,酶解时间定在90min,制得的染色体图片前中期分裂相较多,并且粘连程度较低,背景也较干净。在DNA纤维制片中,细胞核的提取过程中,控制研磨力度和加入TritonX-100的量进行了改变；DNA纤维制片时,改变了细胞核裂解液的裂解时间。通过以上改变,降低了纤维的缠绕程度。
3.用经过以上改变条件制得的片子进行FISH。通过对前中期染色体的FISH结果表明,能够分别检测出两种颜色的信号,但是其信号检测率不高,推导其原因是除染色体本身的质量外,探针的单拷贝和小片段特点也是影响其效果的原因,所以本实验只能将SRK和MLPK初步定义在第l号染色体和第3号染色体上。通过对DNA纤维时期的FISH结果,未能检测出明显信号,推测其原因,在制片的过程中,不能从其理化性质来判断其制片的好坏,导致了FISH不甚理想。
Self-incompatibility (SI) is controlled by S-locus in Brassica. SRK gene is the key factor for SI, and was cloned from Brassica oleracea and Brassica napus. The mutation and expression of SRK gene would affect the self-incompatibility. S-domain is polymorphism and often occurs in the coding region. Compared to the S-domain, kinase domain is more conservative. Kinase domain of SRK gene was cloned and analyzed first time, in order to investigate the kinase domain for the role of SI from an evolutionary of view. MLPK was also proved an essential factor in SI and was the role of downstream component of SRK. In the paper, we located the SRK and MLPK genes associated with SI of Brassica oleracea on different extend chromosomes by dual-color FISH. The result showed that:
1. By PCR and RT-PCR, The gDNA and cDNA fragments of the gene encoding S-locus receptor kinase(SRK) kinase-domain was amplified from Brassica oleracea 263, Brassica oleracea AY627 and Brassica napus. And we constructed a phylogenetic tree of SRK. The lengths of gDNA an d cDNA were 1694 bp and 1307 bp,1705 bp and 1229 bp,1606 bp and 1214 bp, respectively. Sequence analysis indicated that the genomic DNA of Brassica oleracea 263 contained five exons and four introns, encoding 433 amino acids, and both the genomic DNA of Brassica oleracea AY627 and B.napus contained six exons and five introns, encoding 407 and 402 amino acids, respectively. Based both three nucleotide sequences cloned in this study and SRK sequences of twenty-first species released by NCBI, we constructed a phylogenetic tree of SRK. The three sequences were in the same group with classⅠ, and classⅠand classⅡwas divided into two groups. Arabidopsis thaliana and the classⅡwere in the same group, but Raphanus sativus was't formed a single group. By comparing of the three cDNA fragments, we found that there is a 70 bp of DNA insertion in Brassica oleracea 263, which may be one of the cause leading to the phenotype of the SC. We also found there were not any insertion and deletion in Brassica oleracea AY627 and Brassica napus, indicating that the SC of Brassica napus may not be caused by the kinase domain. The analysis of phylogenetic tree indicate that differentiation between Brassica and Raphanus sativus was later than the differentiation between classⅠand classⅡof SI in Brassica.
2. In the FISH process, we changed some conditions between prometaphase chromosome and DNA fibers. In the prometaphase chromosome. Our research have acquired the most suitable time of cold temperature treatment, which was 20 hours. We use enzyme mixture to treat the prometaphase chromosome for 90min, And we obtained the best cytological target slide without background and with better stretched state. We also changed some conditions to obtain the DNA fibers. Through improved method, such as the different treatment with TritonX-100, we obtained Nuclei with symmetrical. Through these changes,we obtained extended DNA fibers.
3. Two color hybridization signals were detected on a homologous chromosome, respectively. But the detection rate was low. One hand, the reason may be that the quality of homologous chromosome was not so well. Another hand, the single-copy and small fragments of probe affected the FISH. And SRK and MLPK gene was only located on Chromosome 1 and Chromosome 3, respectly. We couldn't detect the signal on DNA fiber. And the reason may be that the physical and chemical characteristics was undermined.
1.以结球甘蓝263、羽衣甘蓝AY627和甘蓝型油菜S11为材料,采用PCR、RT-PCR等技术,分别对SRK激酶结构域基因组DNA和cDNA进行了扩增、测序和序列分析,并构建了分子进化树。分别获得了1694bp和1307bp的片段、1705bp和1229bp的片段和1606bp和1214bp的片段,分别编码433,407和402个氨基酸。对获得的片段进行比对发现,结球甘蓝263包括5个外显子和4个内含子,羽衣甘蓝AY627和甘蓝型油菜S11包括6个外显子和5个内含子。并且发现结球甘蓝263的内含子少1个,并且有一段70bp左右的插入片段。对已报道的21种SRK基因和本文所克隆的3种材料的序列构建分子进化树,发现三者都与S单倍型的Ⅰ类SRK基因聚在一起,Ⅱ类基因与Ⅰ类基因分为两支,拟南芥与Ⅱ类聚在一起,而萝卜属未形成单独的一支。推导甘蓝26370bp片段的插入有可能是内含子转化而来并导致了自交不亲和性的产生。羽衣甘蓝AY627与甘蓝型油菜S11所获得的片段与预期一致,激酶结构域没有发生片段的插入或缺失,推导导致甘蓝型油菜S11自交不亲和性的原因没有发生在激酶结构域部分。通过进化树飞分析推导出萝卜属与芸薹属的分离晚于SRKⅠ类和Ⅱ类的分离。
2.在FISH过程中,对甘蓝根尖前中期染色体和DNA纤维的制片进行了一些条件的改变,试图提高FISH效果。在前中期染色体制片中,本文对冷处理时间和酶解时间进行了改变,将冷处理时间定在20h,酶解时间定在90min,制得的染色体图片前中期分裂相较多,并且粘连程度较低,背景也较干净。在DNA纤维制片中,细胞核的提取过程中,控制研磨力度和加入TritonX-100的量进行了改变；DNA纤维制片时,改变了细胞核裂解液的裂解时间。通过以上改变,降低了纤维的缠绕程度。
3.用经过以上改变条件制得的片子进行FISH。通过对前中期染色体的FISH结果表明,能够分别检测出两种颜色的信号,但是其信号检测率不高,推导其原因是除染色体本身的质量外,探针的单拷贝和小片段特点也是影响其效果的原因,所以本实验只能将SRK和MLPK初步定义在第l号染色体和第3号染色体上。通过对DNA纤维时期的FISH结果,未能检测出明显信号,推测其原因,在制片的过程中,不能从其理化性质来判断其制片的好坏,导致了FISH不甚理想。
Self-incompatibility (SI) is controlled by S-locus in Brassica. SRK gene is the key factor for SI, and was cloned from Brassica oleracea and Brassica napus. The mutation and expression of SRK gene would affect the self-incompatibility. S-domain is polymorphism and often occurs in the coding region. Compared to the S-domain, kinase domain is more conservative. Kinase domain of SRK gene was cloned and analyzed first time, in order to investigate the kinase domain for the role of SI from an evolutionary of view. MLPK was also proved an essential factor in SI and was the role of downstream component of SRK. In the paper, we located the SRK and MLPK genes associated with SI of Brassica oleracea on different extend chromosomes by dual-color FISH. The result showed that:
1. By PCR and RT-PCR, The gDNA and cDNA fragments of the gene encoding S-locus receptor kinase(SRK) kinase-domain was amplified from Brassica oleracea 263, Brassica oleracea AY627 and Brassica napus. And we constructed a phylogenetic tree of SRK. The lengths of gDNA an d cDNA were 1694 bp and 1307 bp,1705 bp and 1229 bp,1606 bp and 1214 bp, respectively. Sequence analysis indicated that the genomic DNA of Brassica oleracea 263 contained five exons and four introns, encoding 433 amino acids, and both the genomic DNA of Brassica oleracea AY627 and B.napus contained six exons and five introns, encoding 407 and 402 amino acids, respectively. Based both three nucleotide sequences cloned in this study and SRK sequences of twenty-first species released by NCBI, we constructed a phylogenetic tree of SRK. The three sequences were in the same group with classⅠ, and classⅠand classⅡwas divided into two groups. Arabidopsis thaliana and the classⅡwere in the same group, but Raphanus sativus was't formed a single group. By comparing of the three cDNA fragments, we found that there is a 70 bp of DNA insertion in Brassica oleracea 263, which may be one of the cause leading to the phenotype of the SC. We also found there were not any insertion and deletion in Brassica oleracea AY627 and Brassica napus, indicating that the SC of Brassica napus may not be caused by the kinase domain. The analysis of phylogenetic tree indicate that differentiation between Brassica and Raphanus sativus was later than the differentiation between classⅠand classⅡof SI in Brassica.
2. In the FISH process, we changed some conditions between prometaphase chromosome and DNA fibers. In the prometaphase chromosome. Our research have acquired the most suitable time of cold temperature treatment, which was 20 hours. We use enzyme mixture to treat the prometaphase chromosome for 90min, And we obtained the best cytological target slide without background and with better stretched state. We also changed some conditions to obtain the DNA fibers. Through improved method, such as the different treatment with TritonX-100, we obtained Nuclei with symmetrical. Through these changes,we obtained extended DNA fibers.
3. Two color hybridization signals were detected on a homologous chromosome, respectively. But the detection rate was low. One hand, the reason may be that the quality of homologous chromosome was not so well. Another hand, the single-copy and small fragments of probe affected the FISH. And SRK and MLPK gene was only located on Chromosome 1 and Chromosome 3, respectly. We couldn't detect the signal on DNA fiber. And the reason may be that the physical and chemical characteristics was undermined.
引文
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