Production of Useful Compounds by Genetic Transformation

（1）黄酮类化合物生物合成的基因黄酮类化合物生物合成的基因受环境刺激的诱导。这种诱导在转录水平上受到调控。本研究从野葛cDNA文库中克隆了查耳酮合成酶（CHS）基因和与之共同作用的还原酶基因。用PCR方法克隆了还原酶的cDNA。以CHS和CHS的cDNA为探针，从基因组文库中克隆了CHS和CHS的基因组基因。（2）萜类化合物生物合成的相关基因研究以氧化角鲨烯环化反应为切入点，剖析了植物中三萜和甾醇的生物合成途径。环阿屯烯醇环化酶从氧化角鲨烯产生环阿屯烯醇并导致甾醇。另一方面，β-香树脂素环化酶，三萜环化酶之一，环化酶将角鲨烯氧化成β-香树脂素并导致三萜。本研究从豌豆中分离纯化了环阿屯醇环化酶和β-香树脂醇环化酶基因，并测定了其氨基酸序列。在此基础上，合成寡核苷酸，作为PCR引物。获得部分长度的cDNA，并使用获得的部分长度的cDNA作为探针筛选cDNA文库。已获得β-香树脂醇环化酶的全长cDNA，但尚未获得环阿屯醇环化酶的全长cDNA。（3）外源基因的导入及启动子的分析通过构建缺失克隆，对大豆种子贮藏蛋白基因β-伴大豆球蛋白的启动子区进行了分析。“CATGCAT”序列是种子中特异表达所必需的序列，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结果。

(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.