Enhancement of Vitamin E Level in Oilseeds through Metabolic Engineering.

(1)从芝麻中克隆了三个编码生育酚生物合成途径酶的cdna，分别为HPT（均质植基转移酶）、MPBQMT （MPBQ甲基转移酶）和TC（生育酚环化酶）。因此，我们已经获得了芝麻中催化从均质酸到维生素E （α-生育酚）连续反应步骤的酶的cDNA克隆，因为我们之前分离了芝麻γ- tmt （γ-生育酚甲基转移酶）cDNA。从芝麻中分离出γ-TMT的基因组DNA克隆，并确定了其核苷酸序列。(2)已知脂肪酸去饱和酶或储存蛋白的几个基因在芝麻种子发育过程中表达强烈和特异性。我们试图分离包含这些基因的5'-上游调控区域的基因组dna，并成功分离了Δ9和微粒体ω-6的去饱和酶基因，以及油蛋白和2S白蛋白的基因。对这些5'上游区域进行测序，并通过Capsite作图方法检测其转录本中的第一个核苷酸。(3)将芝麻γ-TMT cDNA分别连接到从芝麻分离的Δ9去饱和酶、油蛋白和2S白蛋白基因的5′上游区域，或连接到大豆衍生的G1甘氨酸基因的5′上游区域。利用MAT载体系统，通过农杆菌侵染，将这些嵌合体基因分别导入野生芝麻（s . schrzzianum）幼苗。对于大豆G1 glycine:: γ-TMT，所有转基因植株都产生了很少发芽的未成熟种子。对于芝麻油蛋白：：γ-TMT和芝麻2S白蛋白：：γ-TMT，转基因植株目前处于营养生长阶段，对于sesameΔ9去饱和酶：：γ-TMT，目前在幼苗上观察到高效的冠瘿形成。

(1)Three cDNAs encoding respective enzymes in the tocopherol biosynthetic pathway, namely HPT (homogentisate phytyltransferase), MPBQMT (MPBQ methyltransferase) and TC (tocopherol cyclase), were cloned and characterized from sesame, S.indicum. We, thus, have obtained each of the cDNA clones cording for enzymes which catalyze the successive reaction steps from homogentisic acid to vitamin E (α-tocopherol) in sesame because of our previous isolation of sesameγ-TMT (γ-tocopherol methyltransferase) cDNA. A genomic DNA clone for γ-TMT was also isolated from sesame and its nucleotide sequence was determined.(2)Several genes for fatty-acid desaturases or storage proteins are known to express strongly and specifically during seed development in sesame. We tried to isolate genomic DNAs containg 5'-upstream regulatory regions of such genes, and succeeded for desaturase genes of Δ9 and a microsomal ω-6, and genes of oleosin and 2S albumin. These 5'-upstream regions were sequenced, and the first nucleotides in their transcripts were detected by the Capsite mapping mehtod.(3)The sesame γ-TMT cDNA was ligated to each of the 5'-upstream regions of Δ9 desaturase, oleosin and 2S albumin genes isolated from sesame, or to that of G1 glycinin gene derived from soybean. Such chimera genes were respectively introduced to seedlings of a wild sesame, S.schrxizianum by infection of Agrobacterium with the MAT vector system. For soybean G1 glycinin :: γ-TMT, all the transgenic plants produced immature seeds which seldom germinated. For sesame oleosin :: γ-TMT and sesame 2S albumin :: γ-TMT, the transgenic plants are now during vegetative growth, and for sesameΔ9 desaturase :: γ-TMT, efficient formation of crown galls are observed on the seedlings at present.