Production of Useful Compounds by Genetic Transformation

(1)类黄酮生物合成基因类黄酮类化合物的生物合成基因受环境刺激的诱导。这种诱导是在转录水平上调节的。本课题主要研究查尔酮合酶(CHS)和与查尔酮合酶协同作用的还原酶(CHR)基因。从葛根cDNA文库中克隆CHS基因。并用聚合酶链式反应克隆了还原酶基因的全长。以各自的cDNA为探针，从基因组DNA文库中克隆出CHS和ChR的基因组基因。(2)萜类生物合成基因在植物氧化鱼腥烯的环化过程中，对三萜类和甾醇的生物合成途径进行了剖析。环青蒿醇环化酶由氧化苦参烯生成环青蒿醇，进而生成甾醇。另一方面，三萜环化酶中的一种β-香兰素环化酶，环化氧化橙烯为β-香兰素，并导致三萜类化合物的生成。本研究以豌豆为原料，对环芳烃环化酶基因和β-芳香蛋白环化酶基因进行了分离纯化，并测定了几种氨基酸序列。在这些序列的基础上，合成了寡核苷酸，并将其作为聚合酶链式反应的引物。获得部分长度的cDNA，并以此为探针筛选文库。外源基因导入及启动子分析通过构建缺失克隆，对编码大豆种子贮藏蛋白的β-伴大豆球蛋白基因的启动子区域进行了分析。“CATGCAT”序列是种子特异表达所必需的，在外源基因导入植物的研究中，在相邻序列效应、转移位置效应和双重转化效应等方面取得了一些新的研究成果。

(1)The Genes of Flavonoids biosynthesisThe genes of flavonoids biosynthesis are induced by environmental stimuli. This induction was regulated at transcriptional level. In this project we focussed on the genes of chalcone synthase(CHS)and reductase(CHR)co-acting with chalcone synthase.The cDNA of CHS was cloned from Pueraria lobata cDNA library. And the cDNA of reductase was cloned by PCR. The genome genes of CHS and CHR were cloned from genome DNA library using the each cDNA as probe.(2)The Genes of Terpenoids BiosynthesisThe biosynthesis pathway of triterpene and sterol is dissected at cyclization of oxidosqualene in plant. Cycloartenol cyclase produces cycloartenol from oxidosqualene and leads to sterols. On the other hand, beta-amyrin cyclase, one of triterpene cyclases, cyclase oxidosqualene to beta-amyrin and leads to triterpines. In this project we focussed on the genes of cycloartenol cyclase and beta-amyrin cyclase.These two enzymes were purified from pea, and several amino acid sequences were determined. On the basis of these sequences, the oligo nucleotides were synthesized and used as primer of PCR. The partial length cDNA was obtained, and cDNA library was screened using the obtained partial length cDNA as probe. The full length cDNA of beta-amyrin cyclase was obtained, but cDNA of cycloartenol cyclase has not been obtained full length cDNA yet.(3)Introduction of Foreign Genes and Analysis of PromotersThe promoter region of beta-conglycinin gene which is coding the storage protein of soybean seeds is analyzed by construction of deletion clones. The sequence of "CATGCAT" is indispensable for specific expression in seeds.In the research of introduction of foreign genes into plant, some new results about effects of adjacent sequences, effects of transferred location and effects of double transformation, were obtained.