黄酮类化合物广泛存在于动植物中，具有多种生理活性，黄酮合酶Ⅰ基因可以催化多种黄酮类化合物的生物合成。根据NCBI已登录的其他物种黄酮合酶Ⅰ基因的氨基酸序列，与家蚕基因组和EST数据库进行电子克隆获得家蚕的同源基因BmFNS Ⅰ。克隆测序结果表明该基因长1451 bp，7个外显子。根据基因序列推测其编码蛋白由345个氨基酸构成，179～295间氨基酸为一2-氧戊二酸和Fe(Ⅱ)依赖性的加氧酶家族成员的结构域。RT-PCR检测该基因在本试验所调查的家蚕中肠有高表达。最后将该基因片段亚克隆至具有2个反向T7启动子可以用于体外诱导合成dsRNA的L4440载体。
Flavone is widely existing in animals and plants.They have various physiological activity and the synthesization of them can be catalyzed by flavone synthase I gene.Using the reported amino acid sequences of other organisms and the scaffold and EST sequences of silkworm,silkworm flavone synthase I gene,BmFNS I,was obtained by electronic clone.The gene is 1451 bp in length and 7 exons in it by cloning and sequencing.The deduced protein contains 345 amino acids and a structural domain of 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily located in amino acids 179 and 295.It is shown by RT-PCR experiment that the gene expressed mostly in midgut in all the tissues tested.Then gene was subcloned to L4440,which has two T7 polymerase promoter in opposite orientation to synthesize dsRNA in vitro.
[1] 白风梅,蔡同一.类黄酮生物活性及其机理的研究进展[J].食品科学,1999,20(8):11-13.
[2] Hegg E L,Que L J R.The 2-His-1-carboxylate facial triad an emerging structural motif in mononuclear non-heme iron(II) enzymes[J].Eur J Biochem,1997,250:625-629.
[3] J Zhi-shen,T Meng-cheng,W Jian-ming.The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals[J].Food Chemistry,1999,64:555-559.
[4] 金洁,刘淑梅,时连根.家蚕幼虫体中黄酮类化合物含量的变化规律[J].蚕业科学,2005,31(2):141-144.
[5] Fujimoto N,Hayashiya K,Nakajima K.Studies on the pigments of cocoon.(IV) The formation and translocation of the pigments of green cocoon in silkworm larvae[J].J Seric Sci Jpn,1959,28:30-32.
[6] Hayashiya K,Sugimoto S,Fujimoto N.Studies on the pigments of cocoon.(III) The qualitative test of the pigments of green cocoon[J].J Seric Sci Jpn,1959,28:27-29.
[7] Kurioka A,Yamazaki M.Purification and identification of flavonoids from the yellow green cocoon shell (Sasamayu) of the silkworm,Bombyx mori[J].Biosci Biotech & Biochem,2002,66:1396-1399.
[8] Hirayama C,Ono H,Tamura Y.C-prolinylquercetins from the yellow cocoon shell of the silkworm,Bombyx mori[J].Phytochemistry,2006,67:579-583.
[9] Tamura Y,Nakajima K I.Flavonoid 5-glucosides from the cocoon shell of the silkworm,Bombyx mori[J].Phytochemistry,2002,59:275-278.
[10] Hirayama C,Ono H,Tamura Y,et al.Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm,Bombyx mori[J].Phytochemistry,2008,69:1141-1149.
[11] 程道军,夏庆友,周泽扬,等.家蚕cDNA文库构建及大规模EST测序[J].蚕业科学,2003,29(4):335-339.
[12] Mita K,Morimyo M,Okano K,et al.The construction of an EST database for Bombyx mori and its application[J].PNAS,2003,100(24):14121-14126.
[13] Xia Q Y,Zhou Z Y,Lu C,et al.A draft sequence for the genome of the domesticated silkworm (Bombyx mori)[J].Science,2004,306:1937-1940.
[14] Quan G X,Kanda T,Tamura T.Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white gene[J].Insect Molecular Biology,2002,11(3):217-222.
[15] Tabunoki H,Higurashi S,Ninagi O,et al.A carotenoid-binding protein(CBP)plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae[J].FEBS Letters,2004,567(2-3):175-178.
[16] Kamath R S,Ahringer J.Genome-wide RNAi screening in Caenorhabditis elegans[J].Methods,2003,30:313-321.
[17] Tenllado F,Martinez-gacia B,Vargas M,et al.Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections[J].BMC Biotechnology,2003,3:1-11.
