此篇是本人的碩士畢業(yè)論文《殺蟲(chóng)蛋白基因cry8Fa2的克隆及在Bt無(wú)晶體突變株中的表達(dá)
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摘 要
蘇云金芽孢桿菌（Bacillus thuringiensis，簡(jiǎn)稱(chēng)Bt）是目前研究最深入的殺蟲(chóng)微生物，其殺蟲(chóng)蛋白基因也是應(yīng)用最廣泛和最有發(fā)展?jié)摿Φ目瓜x(chóng)基因，因此，深入發(fā)掘Bt新菌株、分離克隆新型的殺蟲(chóng)基因?qū)οx(chóng)的防治具有重要意義。近年來(lái)，鞘翅目害蟲(chóng)尤其是金龜甲科害蟲(chóng)的危害日趨嚴(yán)重，由于其幼蟲(chóng)（俗稱(chēng)蠐螬）生活在土壤中，給防治帶來(lái)很大的困難，本研究從本實(shí)驗(yàn)室保存的菌株中篩選高毒力野生菌株，并從中分離克隆對(duì)金龜甲科害蟲(chóng)有效的新型殺蟲(chóng)蛋白基因，研究該基因的表達(dá)和殺蟲(chóng)活性，為構(gòu)建高效廣譜的Bt工程菌和培育轉(zhuǎn)基因抗蟲(chóng)植物提供新型基因資源。本研究的主要內(nèi)容和結(jié)果如下：
以本實(shí)驗(yàn)室分離的Bt菌株B-DLL質(zhì)粒DNA為模板，利用特異性引物JJX5和JJX3擴(kuò)增出3.5 kb大小的片段，將該片段插入克隆載體pMD 18-T中，篩選獲得一個(gè)含新基因的克隆pMD18-cry8new。序列測(cè)定表明，該基因編碼區(qū)為3525 bps，編碼的蛋白質(zhì)由1174個(gè)氨基酸殘基組成，理論分子量為133.05 kD，等電點(diǎn)為pH4.69，為弱酸性蛋白。該基因核苷酸序列已在GenBank登錄（Accession number：HQ174208），編碼的氨基酸序列與Cry8Fa1的同源性最高達(dá)99.8%，被國(guó)際Bt δ-內(nèi)毒素命名委員會(huì)正式命名為Cry8Fa2。將cry8Fa2基因插入Bt表達(dá)載體pSXY422b中，電擊導(dǎo)入Bt無(wú)晶體突變株HD-73－中，獲得重組Bt工程菌HD73-Cry8Fa2，該工程菌能形成方形的伴孢晶體，并表達(dá)130 kD的蛋白。生物測(cè)定結(jié)果表明，該基因表達(dá)產(chǎn)物對(duì)暗黑鰓金龜和華北大黑鰓金龜均不具有殺蟲(chóng)活性。
關(guān)鍵詞：Bt；cry8Fa2基因；基因克?。坏鞍妆磉_(dá)

Abstract
Bacillus thuringiensis（Bt）is a Gram-positive, soil-dwelling bacterium. As an important insect microbial pathogen, its insecticidal crystal proteins are the most widely and potentially used against insect pests in agriculture and forestry. Therefore, selection of new Bt strains and cloning of novel insecticidal protein genes for control of insect pests are of great significance. In recent years, the damage of coleopteran pest Scarabaeoid beetle is serious increasingly especially, because it is very difficult to control with larvae living in the soil. This study mainly includes screening of Bt strains, identification of new cry gene-type and cloning, expression and insecticidal activity of novel insecticidal protein gene that is toxic to Scarabaeoid beetle in order to provide new genetic resources for constructing genetically-engineered Bt strain and cultivatingtransgenic crops. The main contents of this study are as follows:
The 3.5 kb fragments was amplified by PCR using a pair of Bt cry8-type genes special primers, JJX5 and JJX3, and inserted into vector pMD 18-T, the new recombinant plasmid, pMD18-cry8new, was isolated and obtained. Nucleic acid sequence analysis showed that this gene was 3525 base pairs encoding 1174 amino acids, which were homolog of 99.8% compared with Cry8Fal, the molecular weight of the protein was 133.05 kD with isoelectric point pH 4.69. This gene sequence had been registered in GenBank (accession number was HQ174208), and named Cry8Fa2 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The cry8Fa2 gene could be formed cubelike crystal and expressed as a 130 kD protein in Bt acrystalliferous mutant strain HD-73－. Bioassay result showed the expression product of cry8Fa2 gene was not toxic to the larvae of Holotrichia parallela and H. oblita.
Key words：Bacillus thuringiensis; cry8Fa2 gene; Cloning; Protein expression