Wakasa Yuhya, Ozawa Kenjirou, Takaiwa Fumio
Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki 305-8604, Japan.
J Biosci Bioeng. 2009 Jan;107(1):78-83. doi: 10.1016/j.jbiosc.2008.09.002.
Transgenic rice plants that accumulate the hypocholesterolemic pentapeptide lactostatin (IIAEK) as a fusion protein with the endosperm seed storage protein glutelin were developed. Plants were selected from Agrobacterium-mediated transformants using a mutated-rice acetolactate synthase (mALS) marker protein expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS). Lactostatin accumulation levels were compared in mature seeds of 78 independent transgenic rice lines selected for either the CSP:mALS gene cassette or hygromycin phosphotransferase (HPT) under the control of the CaMV 35S promoter (35S:HPT). Transgenic rice seeds harboring a CSP:mALS gene cassette accumulated more (approximately 120-200% on average) lactostatin than those with a 35S:HPT gene cassette, and accumulation was largely independent of transgene copy number. Furthermore, transgenic rice seeds that were selected by HPT under the control of CSP (CSP:HPT) also accumulated more lactostatin (approximately 160% on average) than transgenic rice seeds with the 35S:HPT gene cassette. These results indicate that high-value bioactive peptides such as lactostatin can be produced at higher levels using callus-specific selection than by conventional constitutive selection. Specific expression of a marker gene at the selection stage may thus result in increased target peptide accumulation levels in seeds.
开发了转基因水稻植株，这些植株积累了具有降胆固醇作用的五肽乳抑素（IIAEK），它与胚乳种子贮藏蛋白谷蛋白形成融合蛋白。利用在水稻愈伤组织特异性启动子（CSP）（CSP:mALS）控制下表达的突变型水稻乙酰乳酸合酶（mALS）标记蛋白，从农杆菌介导的转化体中筛选植株。比较了78个独立转基因水稻株系成熟种子中乳抑素的积累水平，这些株系分别选择了CSP:mALS基因盒或在花椰菜花叶病毒35S启动子（35S:HPT）控制下的潮霉素磷酸转移酶（HPT）。携带CSP:mALS基因盒的转基因水稻种子积累的乳抑素比携带35S:HPT基因盒的种子更多（平均约多120 - 200%），且积累量在很大程度上与转基因拷贝数无关。此外，在CSP（CSP:HPT）控制下通过HPT筛选的转基因水稻种子积累的乳抑素也比携带35S:HPT基因盒的转基因水稻种子更多（平均约多160%）。这些结果表明，与传统的组成型选择相比，使用愈伤组织特异性选择可以更高水平地生产高价值生物活性肽，如乳抑素。因此，在选择阶段标记基因的特异性表达可能会导致种子中目标肽积累水平的提高。
