鐵皮石斛原球莖DoGAUT1和DoPGSIP6基因的克隆及其在不同晝夜溫差下的表達(dá)分析

張春　柳賴　鐘雄

摘 要 植物中的糖基轉(zhuǎn)移酶類能識(shí)別不同類型受體，最終生成多糖、復(fù)合糖、糖苷化合物等次生代謝產(chǎn)物。為了解其表達(dá)及調(diào)控機(jī)理，本研究利用同源克隆法從鐵皮石斛（Dendrobium officinale）原球莖中克隆了2個(gè)糖基轉(zhuǎn)移酶基因DoGAUT1和DoPGSIP6，分別為2 518、2 010 bp，分別編碼了705、549個(gè)氨基酸。生物信息學(xué)研究表明，它們同屬于糖基轉(zhuǎn)移酶家族8中GT-A類的糖基轉(zhuǎn)移酶，DoGAUT1和DoPGSIP6基因推定的氨基酸序列分別與二穗短柄草，大豆進(jìn)化關(guān)系最近。DoGAUT1為親水性跨膜蛋白，不含信號(hào)肽，亞細(xì)胞定位于高爾基體。DoPGSIP6為疏水性跨膜蛋白，含信號(hào)肽，亞細(xì)胞定位于質(zhì)膜。熒光定量PCR分析表明：DoGAUT1在一定晝夜溫差范圍內(nèi)，可響應(yīng)溫度調(diào)控，表現(xiàn)出上升趨勢，而DoPGSIP6在晝夜溫差為15 ℃時(shí)影響大。

關(guān)鍵詞 鐵皮石斛；DoGAUT1基因；DoPGSIP6基因；克??；晝夜溫差；qPCR

中圖分類號(hào) S682.31 文獻(xiàn)標(biāo)識(shí)碼 A

Abstract Glycosyltransferases（GTs）are able to recognize different kinds of receptors that finally produce secondary metabolites such as polysaccharides， complex carbohydrates and glycosyl compounds in plants. To further understand the biological function and the molecular mechanism， using protocorms as experimental materials， the glycosyltransferases genes of DoGAUT1 and DoPGSIP6 were cloned by homological cloning techniques in Dendrobium officinale. The full length sequences of cDNAs were 2 518 and 2 010 bp， encoding 705 amino acids and 549 amino acids， respectively. Bioinformatics analysis showed that they belonged to glycosyltransferases 8 with the GT-A fold. Amino acid sequence aligenment showed that DoGAUT1 and DoPGSIP6 displayed the closest evolutionary relationships to those of Brachypodium distachyon and Glycine max. DoGAUT1 protein was hydrophilic， without signal peptides and located in the Golgi. DoPGSIP6 protein was a high possibility of hydrophobic membrane protein with the existence of cleavage site for signal peptide in the plasma membrane. The qPCR analysis confirmed that DoGAUT1 gene was on the up trend in a certain day-and-night temperature difference， and DoPGSIP6 gene had the maximum effect with the day-and-night temperature difference of 15 ℃.

Key words Dendrobium officinale； DoGAUT1 gene； DoPGSIP6 gene； Cloning； Day-and-night temperature difference；qPCR

doi 10.3969/j.issn.1000-2561.2015.02.003

鐵皮石斛（Dendrobium officinale），屬蘭科石斛屬多年生草本植物，野生資源少，主要分布于東亞、東南亞等國家和地區(qū)[1]，福建主要集中分布于三明、德化、龍巖等地。鐵皮石斛含豐富的黏性物質(zhì)，其主要成分是水溶性多糖，具有降血糖、抗腫瘤、提高免疫力等功效[2-3]。因此，從分子水平上適度提高多糖的含量是當(dāng)前鐵皮石斛育種的一個(gè)重要方向。目前國內(nèi)外對(duì)鐵皮石斛多糖的研究主要集中在結(jié)構(gòu)功能研究、成分分析、遺傳標(biāo)記等方面[4-6]，而從分子水平上對(duì)多糖合成過程中相關(guān)酶基因的研究較少，對(duì)GAUT1和PGSIP6基因的研究集中在擬南芥的Galacturonosyltransferase 小基因家族上[7-9]。GAUT1（α-1，4-Galacturonosyltransferas）和PGSIP6（Glycogenin-like protein 6）分別是多糖（果膠）及葡糖醛酸木聚糖等糖類組分生物合成的關(guān)鍵酶[10-11]?！?br>