两个百合雄性不育相关基因的克隆和功能初步分析
发布时间：2018-10-19 09:50
【摘要】：百合一直以其花姿优雅,气度不凡具有极高的观赏价值而著称。其在颜色,图案和香味方面的吸引力而被顾客高度重视,在世界范围的切花行业中占有极高的市场份额。然而它具有巨大的花药,同时伴有大量的花粉,如果花药不能及时去除,则花粉会污染花瓣和周围表面,沾染衣物甚至会导致人类对花粉过敏等症状。因为消费者不愿购买有污染的百合花,且人工摘除花药又费时费力。因此,控制花药的发育和减少花粉量的生产,同时又不影响百合花的美观一直是百合的分子育种的焦点。本实验选取不产花粉的"无粉白"百合雄蕊和同一时期有粉百合雄蕊为实验材料,结合之前转录组分析数据通过荧光定量筛选鉴定基因,RT-PCR克隆得到C2基因的ORF序列,随即克隆其5'端序列,并在其启动子序列中找到与花药特异性相关的表达元件。还通过同源克隆的方法克隆得到C4基因,该基因与控制雄性不育基因MMD1具有较高的同源性,并对该基因进行一系列的生物信息学分析。具体研究结果如下:1.通过荧光定量PCR分析鉴定,发现C2基因在无粉百合雄蕊中的表达明显迟于其在有粉百合雄蕊中的表达,且表达量很低。结合其在百合不同器官中表达量分析,C2基因在花药中表达最高。这说明C2基因主要的作用位点在花药中。2.在有粉可育雄蕊与无粉不可育雄蕊中分别通过RT-PCR克隆到C2基因ORF区,全长为774 bp,通过序列比对分析发现其ORF区没有突变,推测可能其发挥作用的区域在启动子部分。3.在有粉可育雄蕊与无粉不可育雄蕊中分别通过染色体步移的方法克隆其启动子序列,且有粉可育材料中克隆得到序列619 bp,无粉不可育雄蕊中克隆得到片段395 bp。通过启动子分析软件NewPlace预测发现启动子序列中含有TATA-box以及CAAT-box,其中TATA-box分别位于转录起始位点"A"位于起始密码子上游-53 bp和-54 bp处,CAAT-box位于起始密码子上游-132 bp和-167 bp处。在该序列中还含有大量的顺式作用元件,如花特异性表达元件如:GTGA、AGAAA和TGTGG及其它顺势作用元件。4.通过同源克隆方法得到C4基因,结合转录组数据分析发现,该基因与已报道的控制雄性不育基因PHD-锌指蛋白白 MMD1基因具有较高的同源性,经过一系列的生物信息学分析发现,C4基因与凤仙花MMD1同源性最高为57%,该基因氨基酸序列不存在跨膜区域,且不存在信号肽,亚细胞定位推测其在细胞核上,其结构域含有PHD-锌指蛋白结构域,且所有的活性位点都位于这个区域,具有锌指蛋白酶的活性,与之前预测的相符,为后续研究此基因奠定基础。
[Abstract]:Lily has always been famous for its elegant flower posture, extraordinary demeanor with extremely high ornamental value. Its appeal in color, design and fragrance is highly valued by customers and has a high market share in the world-wide cut-flower industry. However, it has a huge anther, accompanied by a large number of pollen, if the anther can not be removed in time, the pollen will contaminate the petals and the surrounding surface, and even cause human allergies to pollen symptoms. Because consumers do not want to buy contaminated lilies, and manual extraction of anthers is time-consuming and laborious. Therefore, controlling anther development and reducing pollen production without affecting the beauty of lilies has always been the focus of molecular breeding of lily. In this experiment, the pollen free "powdery white" lily stamen and the pollen lily stamen at the same time were selected as the experimental materials. The ORF sequence of C2 gene was cloned by RT-PCR by fluorescence quantitative screening and identification according to the previous transcriptome analysis data. The 5 '-terminal sequence was cloned and the anther specific expression elements were found in the promoter sequence. The C4 gene was cloned by homologous cloning, which had high homology with the male sterility control gene MMD1, and was analyzed by a series of bioinformatics. The results are as follows: 1. Fluorescence quantitative PCR analysis showed that the expression of C2 gene in the stamen of non-pollen lily was significantly later than that in the stamen of the pollen lily, and the expression of C2 gene was very low. Combined with the analysis of the expression of C2 gene in different organs of lily, C2 gene expression was the highest in anthers. This indicates that C2 gene is mainly located in anthers. 2. The C2 gene ORF region was cloned from the sterile and sterile stamens by RT-PCR. The total length of the C2 gene was 774 bp,. No mutation in the ORF region was found by sequence alignment analysis. It was speculated that the region that might play a role was in the promoter part. 3. The promoter sequence was cloned by chromosome step method in the sterile and sterile stamens, and the sequence 619 bp, was cloned from the pollen fertile material. The fragment 395 bp. was cloned from the sterile stamen and the sterile unfertile stamen. Promoter analysis software NewPlace predicted that the promoter sequence contained TATA-box and CAAT-box, where TATA-box was located at -53 bp and -54 bp upstream of the initiation codon, and CAAT-box was located at -132 bp and -167 bp upstream of the start codon, respectively. The sequence also contains a large number of cis-acting elements, such as floral specific expression elements such as: GTGA,AGAAA and TGTGG and other homeotropic elements. 4. C4 gene was obtained by homologous cloning method. Combined with transcriptome data analysis, it was found that this gene had high homology with PHD- zinc finger protein white MMD1 gene, which controlled male sterility gene. A series of bioinformatics analysis showed that the highest homology of C4 gene and MMD1 was 57. The amino acid sequence of C4 gene had no transmembrane region and no signal peptide. The subcellular localization suggested that C4 gene was located in the nucleus. The domain contains PHD- zinc finger protein domain, and all the active sites are located in this region, which has the activity of zinc finger protease, which is consistent with the previous prediction, which lays the foundation for further study of this gene.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.29;Q943.2
，
本文编号：2280775
[Abstract]:Lily has always been famous for its elegant flower posture, extraordinary demeanor with extremely high ornamental value. Its appeal in color, design and fragrance is highly valued by customers and has a high market share in the world-wide cut-flower industry. However, it has a huge anther, accompanied by a large number of pollen, if the anther can not be removed in time, the pollen will contaminate the petals and the surrounding surface, and even cause human allergies to pollen symptoms. Because consumers do not want to buy contaminated lilies, and manual extraction of anthers is time-consuming and laborious. Therefore, controlling anther development and reducing pollen production without affecting the beauty of lilies has always been the focus of molecular breeding of lily. In this experiment, the pollen free "powdery white" lily stamen and the pollen lily stamen at the same time were selected as the experimental materials. The ORF sequence of C2 gene was cloned by RT-PCR by fluorescence quantitative screening and identification according to the previous transcriptome analysis data. The 5 '-terminal sequence was cloned and the anther specific expression elements were found in the promoter sequence. The C4 gene was cloned by homologous cloning, which had high homology with the male sterility control gene MMD1, and was analyzed by a series of bioinformatics. The results are as follows: 1. Fluorescence quantitative PCR analysis showed that the expression of C2 gene in the stamen of non-pollen lily was significantly later than that in the stamen of the pollen lily, and the expression of C2 gene was very low. Combined with the analysis of the expression of C2 gene in different organs of lily, C2 gene expression was the highest in anthers. This indicates that C2 gene is mainly located in anthers. 2. The C2 gene ORF region was cloned from the sterile and sterile stamens by RT-PCR. The total length of the C2 gene was 774 bp,. No mutation in the ORF region was found by sequence alignment analysis. It was speculated that the region that might play a role was in the promoter part. 3. The promoter sequence was cloned by chromosome step method in the sterile and sterile stamens, and the sequence 619 bp, was cloned from the pollen fertile material. The fragment 395 bp. was cloned from the sterile stamen and the sterile unfertile stamen. Promoter analysis software NewPlace predicted that the promoter sequence contained TATA-box and CAAT-box, where TATA-box was located at -53 bp and -54 bp upstream of the initiation codon, and CAAT-box was located at -132 bp and -167 bp upstream of the start codon, respectively. The sequence also contains a large number of cis-acting elements, such as floral specific expression elements such as: GTGA,AGAAA and TGTGG and other homeotropic elements. 4. C4 gene was obtained by homologous cloning method. Combined with transcriptome data analysis, it was found that this gene had high homology with PHD- zinc finger protein white MMD1 gene, which controlled male sterility gene. A series of bioinformatics analysis showed that the highest homology of C4 gene and MMD1 was 57. The amino acid sequence of C4 gene had no transmembrane region and no signal peptide. The subcellular localization suggested that C4 gene was located in the nucleus. The domain contains PHD- zinc finger protein domain, and all the active sites are located in this region, which has the activity of zinc finger protease, which is consistent with the previous prediction, which lays the foundation for further study of this gene.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S682.29;Q943.2
，
本文编号：2280775
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2280775.html
最近更新
教材专著
