【作者】 杨玲玲；

【导师】 赵德刚； 赵懿琛；

【作者基本信息】 贵州大学 ， 生物学， 2018， 硕士

【摘要】 NaD1(Nicotiana alata Defensin 1)蛋白是一种在花烟草(Nicotiana alata)花中发现的植物防御素。其对多种植物病原菌及人类病源性真菌具有抗性,同时还具有撕裂癌细胞的功能,在植物学和医学研究中均具有重要的价值。本研究以花烟草为材料克隆NaD1基因并进行深入研究,取得如下结果:1.NaD1基因克隆及结构分析根据已经报道的NaD1基因序列,提取mRNA反转cDNA,利用同源克隆法克隆花烟草NaD1基因编码序列进行克隆,分析表明该序列长为318 bp,与已知的NaD1基因编码序列的同源性为100%。该基因共编码105个氨基酸,其中丙氨酸(A)含量最高(12.4%),谷氨酸(E)含量次之(10.7%),其他氨基酸含量相对较低。分析发现NaD1为不稳定的疏水性蛋白,相对分子质量为11.7kD,理论等电点pI为6.56,具有信号肽(第4-21位氨基酸)、跨膜结构及多个磷酸化位点;其二级结构中3个ɑ-螺旋及1个β-折叠,符合植物防御素的结构特征。利用同源克隆技术,以花烟草基因组DNA为模板,克隆了NaD1基因的基因组DNA序列,分析表明其大小为514 bp,包含2个外显子和1个内含子,内含子边界序列符合GT-AG原则。分析发现NaD1基因内含子属于U12-型内含子,其序列富含AT,含量为76.02%,具有非编码区AT含量高的特点。利用热不对称交错PCR,以花烟草基因组DNA为模板,成功地克隆NaD1基因的5’端长为644 bp的序列。分析表明,该序列中位置538-588处为基础启动子区域,转录起始位点为A;该序列中存在启动子相关的多个保守区,其中包含启动子基本顺式作用元件TATA-box(序列为TATA)和CAAT-box(序列为CAAAT)。软件分析显示该序列还存在如MYB蛋白结合位点MBS(序列为TAACTG)、抗逆反应顺式作用元件TC-rich repeats(序列为ATTTTTTTCA)、光反应元件BOXΙ(序列为TTTCAAA)、乙烯反应元件ERE(序列为ATTTCAAA)以及真菌诱导响应元件TGACG-motif(序列为TGACG)等多个转录因子结合区域。为进一步分析该基因表达调控提供了线索。2.NaD1基因启动子转录活性研究将NaD1基因启动子片段A(ATG的上游644 bp片段)及片段B(ATG的上游310 bp片段),分别与质粒pCAMBIA1391z连接构建驱动GUS基因表达的植物表达载体pCAMBIA1391z-PNaD1-644::GUS及pCAMBIA1391z-PNaD1-310::GUS。通过农杆菌介导的愈伤组织遗传转化法,分别遗传转化花烟草,对再生芽进行GUS组织化学染色分析,结果显示片段B具有转录活性。3.花期花烟草各器官中NaD1基因的表达分析利用荧光定量PCR研究NaD1基因在花烟草根、顶端嫩茎、嫩叶及花蕾中的表达量,发现NaD1基因在花中的表达量是根中的988倍,是顶端嫩茎中的15倍,是嫩叶中的10倍。更多还原

【Abstract】 The NaD1(Nicotiana alata Defensin 1)protein is a plant defensin extracted from the flowers of Nicotiana alata.As a cationic antibacterial protein,NaD1 protein not only has significant resistance to various plant pathogens and human pathogenic fungi,but also has the function of tearing cancer cells.Therefore,NaD1 protein has important research value in botany and medical research.In this study,NaD1 gene was cloned from Nicotiana alata,and the results are as follows:1.Molecular cloning and structural analysis of NaD1geneThe homologous cloning technology was used to clone the coding sequence of NaD1 gene from Nicotiana alata.The analysis showed that the sequence length was318 bp.The homology with the known NaD1 gene coding sequence was 100%.According to the bioinformatics analysis,NaD1 gene encoded a hydrophobic protein of 105 amino acids,whose relative molecular mass was 11.7kDa and isoelectric point was 6.56.It was predicted that this protein contains signal peptide,transmembrane region and 5 phosphorylation sites.The secondary structure of NaD1 protein included 3 α-helix,1 β-sheet,which confirmed the characteristics of the plant defensin.Using homologous cloning technology,the NaD1 gene DNA sequence was successfully cloned from the genome of Nicotiana alata.The DNA sequence is514 bp in length and contains two introns and one intron.The cleavage of exons and introns conforms to the GT-AG principle.The intron of NaD1 gene was analyzed and it was found that the intron belongs to U12-type intron.Its sequence is rich in ATand its content is 76.02%,which is consistent with the high content of AT in non-coding region.Using thermal asymmetric interlaced PCR,the 5’-end regulatory sequence of the full-length 644 bp NaD1 gene was successfully cloned.Analysis shows that,In this sequence,the position of 538-588 is the basal promoter region.The transcription initiation site is A.The analysis showed that there are multiple conserved regions associated with the promoter in this sequence.It contains the promoter basic cis-acting elements TATA-box and CAAT-box.It also contains a 5’UTR py-rich stretch,a highly efficient transcriptional element,and cis-acting elements that respond to factors such as light,methyl jasmonate,and ethylene.2.Transcriptional activity analysis of prompter of NaD1 geneThe prompter fragments A(644 bp)and the prompter fragments B(310 bp)of NaD1 gene were each ligated to the plasmid pCAMBIA1391 z toconstruct the plant expression vectors pCAMBIA1391z-PNaD1-644::GUS and pCAMBIA1391z-PNaD1-310::GUS,which drive the expression of the GUS gene.The Agrobacterium-mediated callus genetic transformation method was used to transform Nicotiana alata with this tow recombinant plasmids,respectively.The positive transgenic plants containing GUS gene have been detected by GUS histochemical assay.The results showed that the fragment B had transcriptional activity.3.Expression of NaD1 gene in different tissues of Nicotiana alataThe expression of NaD1 in Nicotiana alata(roots,shoots,leaves and buds)was analyzed by fluorescent quantitative PCR.It was found that the expression level of NaD1 gene in flowers was 988 times that in roots,15 times that in the top shoots,and 10 times that in the leaves.更多还原