不同启动子驱动下马铃薯蛋白酶抑制剂PinⅡ转基因水稻的遗传、表达和对粘虫抗性分析

Inheritance,Expression and Armyworm Resistance of Protease InhibitorⅡ Gene(PinⅡ) Driven by Different Promoters in Transgenic Rice

以不同启动子驱动的马铃薯蛋白酶抑制剂Ⅱ基因(PinⅡ-2x,PinⅡ-4x)转基因水稻为材料,经潮霉素抗性、PCR和Southern blot等检测对转基因水稻后代进行了遗传分析。结果显示:外源基因在68.4%的转基因植株中符合孟德尔遗传模式,单拷贝植株率为63.6%。转基因植株后代的PinⅡ蛋白活性测定结果表明:ActI和Ubi驱动的PinⅡ-2x转基因水稻植株中,每克鲜叶片的PinⅡ蛋白含量为160μg和176μg,而由PIN5′驱动的PinⅡ-4x为104μg,对照水稻仅为20μg。ActI和Ubi驱动的PinⅡ表达产物对胰蛋白酶活性抑制程度分别达到37.7%和43.1%,明显高于PinⅡ自身启动子PIN5′(29.2%)。叶片饲养粘虫幼虫的实验表明:转基因植株叶片对粘虫有抗性,但抗性达不到显著水平,且启动子效率、PinⅡ表达量与抗粘虫性之间也没有相关性。

The inheritance of rice lines transformed by protease inhibitor Ⅱ gene under control of different promoters was investigated by analysis of hygromycin resistance , PCR and Southern blot. For segregation patterns of foreign gene, 68.4% of the transgenic rice plants were conformed to a Mendelian ratio and in which the rate of transgenic plants with single copy was 63.6 %. Quantitative analysis of PinⅡ protein expressed in transgenic rice plants showed that PinⅡ protein in fresh leaves was 160 μg/g for Act-Pin Ⅱ -2x, 176 μg/g for Ubi- Pin Ⅱ -2x, and 104 μg/g for PIN5＇- Pin Ⅱ -4x separately while in control rice plants was only 20 μg/g. The inhibitory activity against tryspin of Pin Ⅱ gena driven by Actl and Ubi promoter reached 37.7% and 43. 1% , much higher than that driven by PIN5＇ （29.2%）. Bioassay for insect resistance to armyworm （ Pseudaletia separato Walker） revealed that transgenic plants had increased their resistance to the pest but there was not significantly different from controls, and also there was no correlation between insect resistance to armyworm and quantity of Pin Ⅱprotein as well as promoters in transgenic