【作者】 覃庆锋；

【导师】 贺伟； 张星耀；

【作者基本信息】 北京林业大学 ， 森林保护， 2005， 硕士

【摘要】 基因控制技术是森林有害生物可持续控制策略实现的技术体系之一,发现自主产权的功能基因资源可以为我国森林保护工作者更好地利用这一控制技术打下良好的基础。巯基蛋白酶抑制剂(cysteine proteinase inhibitor(cystatin),CPI)是一类广泛存在于动植物和微生物体内的蛋白超家族。根据它的蛋白特性植物CPI已经被归入一个新的CPI家族(Phytocystatin)。由于CPI可以通过抑制昆虫中肠中作为消化酶的巯基蛋白水解酶作用达到杀虫目的,它作为一种重要的抗虫因子已经被广泛用于转基因工程。本研究主要在毛白杨CPI基因的克隆、测序、鉴定以及其基因序列分析方面开展了一些工作。取得的初步成果有: 1.根据生物信息学相关原理,通过分析软件,获得毛白杨CPI的氨基酸保守序列区段,根据此保守区段首先设计合成了用于CPI保守序列区段扩增的正向及反向简并引物,通过对毛白杨形成层RNA的特异RT-PCR扩增,获得大小为165bp的单一片段条带,鉴定为毛白杨CPI基因的保守序列区段。 2.利用RT-PCR技术从毛白杨形成层提取的RNA中扩增出一个大小为696bp的的单一片段条带。将其克隆到pGEM-T Easy载体上,获得了重组质粒。通过对该重组质粒进行PCR扩增鉴定以及序列测序分析,证明插入到载体上的片段即是毛白杨CPI基因cDNA序列片段,包含完整的开放阅读框(ORF)。 3.通过相同的引物利用PCR技术从毛白杨形成层提取的基因组DNA中扩增出一个大小为1950bp的单一片段条带,将其克隆到pGEM-T Easy载体上,通过对该重组质粒进行了PCR扩增以及测序分析,证明插入到载体上的片段即是毛白杨CPI基因DNA片段。通过和毛白杨CPI基因cDNA片段的比较进行基因分析,获得基因编码区中内含子和外显子具体定位。更多还原

【Abstract】 The technology of gene control is one of the technological systems of sustainable pests management in forest (SPMF). Finding self-determination property right functional gene can do better for our country’s forestry protection workers to use this technology. The cysteine proteinase inhibitor (cystatin) (CPI) is a protein super family and exists comprehensively in animal and plant. The plant CPI has been regarded as a new family of the CPI super family (Phytocystatin). Because it can kill insects by inhibiting the digestive function of the cysteine proteinase in gut, it has been used in introducing gene engineering comprehensively as an important insect-resistant factor. This study focuses on the molecular cloning, sequencing and identification of the CPI gene of Populus tomentosa and the analysis of this gene. The major results of this dissertation were as follows:According the conserved amino acid motifs of the Phytocystatin, we had designed the special primers and used them to amplify the conserved sequence fragment of the CPI gene from the cambium of Populus tomentosa. The amplified fragment was about 165 base pairs as we expected. This indicated that our designation of the premiers was adequate.A cDNA fragment was obtained from the RNA of the cambium of Populus tomentos through RT-PCR. This fragment was cloned into pGEM-Teasy vector. The insert was identified by PCR amplification and sequencing. The sequence of the amplified cDNA fragment was 696 base pairs. Alignment with all phytocystatins in Genbank showed that they were identical greatly. Therefore it is believed that the fragment was amplified fromthe CPI gene.Using the same premiers a DNA fragment from the genome DNA of Populus tomentosa was obtained with the PCR amplification. It was 1950bp in length. After it was cloned in pGEM-Teasy vector, the insert was identified by PCR amplification and sequencing. The alignment with CPI cDNA could identify the concrete location of the extron and intron.更多还原