Expression of porcine lactoferrin gene in transgenic rice

Abstract

Porcine lactoferrin is a glycoprotein, found mostly in milk, involving in iron absorption, antibacterial activity and immune response. A full-length lactoferrin cDNA fragment was isolated by PCR using plasmid PIGLTF as template. This lactoferrin cDNA was cloned into plant expression vectors under the control of CaMV35S or rice oleosin promoter. These expression vectors were then introduced into rice plants through agrobacterium-mediated transformation method. After selection, 25 hygromycin resistant transgenic plants were obtained. The existence of lactoferrin gene in antibiotic resistant plants was confirmed by PCR and Southern assays. Expressions of lactoferrin gene in various tissues were analyzed through Northern and Western analyses. For gene driven by 35S promoter, lactoferrin was detected in both leaves and seeds, whereas gene driven by rice oleosin promoter lactoferrin was observed only in seeds. These results suggested that rice oleosin promoter is a tissue-specific promoter. The amount of lactoferrin in rice plants is about 0.05 to 0.1% of total soluble protein. The expression pattern of lactoferrin is different in leaves and seeds. An apparent molecular weight of 75 kD was observed in leaves while two fragments with molecular weight of 70 kD and 60 kD were observed in seeds. These size differences in various tissues could be partly attributed to different degree of glycosylation or specific regulation of these proteins in different tissues. Several forms of lactoferrin were observed in protein samples prepared using non-reduced condition. Segregation of antibiotic resistance in T1 progeny and different degree of expression of lactoferrin in the antibiotic resistance T1 plants were observed. The characterizations of lactoferrin in T2 plants are underway.

豬乳鐵蛋白為豬乳汁中的醣蛋白，具有結合鐵離子，抑制細菌生長及增加免疫力的功能。本實驗以台灣養豬所分離到的豬乳鐵蛋白cDNA選殖體為模板，利用PCR分離得豬乳鐵蛋白基因之全長cDNA片段，做為本實驗之轉基因材料。將此基因構築於CaMV35S及水稻油體膜蛋白基因起動子後方，以農桿菌轉殖法﹪將其轉入水稻中，以探討二種起動子表現豬乳鐵蛋白的差異。經PCR及南方墨點分析確認共獲得25株轉基因植物，由北方墨點分析及西方墨點分析，觀察到含CaMV35S起動子的豬乳鐵蛋白表現在水稻的葉片及種子，而含水稻油體膜蛋白基因起動子的豬乳鐵蛋白則表現於種子而不表現於葉片中，初步證實水稻油體膜蛋白基因起動子具有組織專一性。而豬乳鐵蛋白的表現量則為水稻總蛋白量的0.05-0.1﹪。再觀察豬乳鐵蛋白於水稻中表現的形式，於水稻葉片中可見約75 kD的蛋白，在種子中則有兩個片段，分別為70 kD與60 kD，這種現象有部分原因來自於豬乳鐵蛋白在不同水稻組織中醣化程度不同，另外在non-reduced的條件下分析乳鐵蛋白，也可發現數種型式的蛋白。而在子代的分析結果上，經由抗生素篩選，得到抗性植株與感性植株的比例，於35S-LF株系為4.36 : 1，721-LF株系為1.26 : 1，在蛋白表現上，不同子代植株的表現量則略有差異，顯示不同子代間基因型也不盡相同。