葵百合β-1,3-葡聚醣酶基因之表現分析

Abstract

本實驗室在研究百合與灰黴病菌交互作用的過程中，分析自葵百合葉片回收之接種液蛋白質及進一步之分子選殖得到lpglu1 cDNA，解碼為一葡聚醣酶（β-1,3-glucanase），分子量為35.34 kDa，pI值為4.14，與植物酸性β-1,3-葡聚醣酶有高相似性。本研究以lpglu1專一性引子對進行PCR，可分別在雜交種鐵炮百合和台灣百合之基因體核酸增幅出大小類似於lpglu1的單一條帶核酸片段，最適增幅溫度各為54-57℃及55℃左右（葵百合lpglu1之最適黏合溫度為57-60℃）。進一步利用大腸桿菌XL1-Blue菌株表現LPGlu1蛋白質，可以pQE 30載體表現去掉訊息胜肽之LPGlu1 (His-lpg)，並可偵測到β-1,3-葡聚醣酶活性，7.34 U/mg。另一方面，以反轉錄聚合酶連鎖反應配合南方雜合分析偵測lpglu1在葵百合葉片之表現時間點及表現量，結果顯示以灰黴病菌接種及水楊酸、茉莉酸甲酯、離層酸或乙烯前驅物處理葵百合皆可以誘導lpglu1 mRNA之累積；此外，對葵百合植株施以傷口、低溫及高鹽處理亦皆可誘導lpglu1的表現。

In the study of the interaction between Botrytis elliptica and Lilium cv. Star Gazer, analysis of recovery of inoculation fluids and a subsequent cDNA cloning identified the LPGlu1 protein (35.34 kDa, pI 4.14), sharing high similarity to various plant acidic β-1,3-glucanases. Through PCR technique, specific primers to lpglu1 could be used to amplify relevant fragment from L. longiflorum (hybrid) and L. formosanum genome. The appropriate annealing temperature is between 54-57℃ and 55℃, and the annealing temperature of lpglu1 gene in Star Gazer is about 57-60℃. LPGlu1 without signal peptide could be expressed in Escherichia coli XL1-Blue strain by pQE 30 protein expression system. His-lpg displayed β-1,3-glucanase activity of 7.34 U/mg. The expression of lpglu1 in ‘Star Gazer’ leaves were investigated by RT-PCR and Southern hybridization. The results indicated that the accumulation of lpglu1 mRNA could be induced by B. elliptica, salicylic acid, methyl jasmonate, abscisic acid and 1-aminocyclopropane-1-carboxylic acid. In addition, wounding, cold and high salt could induce the expression of lpglu1 gene.