Constraction Of Plant Expression Vector Of Lectin Gene And Transformation Into Sugarcane

Sugarcane (Saccharum officinarum L.) is the most important sugar crops and an important energy cash crop in China. Sugarcane cottony aphid and sugarcane mealybug (Homoptera) are the main pests on sugarcane, which not only lead to sugarcane yields losses and reducing sucrose content, but also they can transmit virus, which brings on worse loss than pests. So, breeding insect-resistant sugarcane varieties by using plant genetic engineering techniques is the most effective ways to resist pests.The galanthus nivalis agglutinin (gna), one kind of plant mannose combine agglutinin, has been widely used to control plants pests. It can integrate on the glycoprotein acceptor of insect enteron epithelial cell, and produces partial or system poisons to the insect and suppresses its growth. It has effective toxicity on insects in Homoptera, such as aphid and cicadellidae, and partial toxicity on insects in lepidoptera order and coleoptera order. Therefore, transferring the gna gene into sugarcane and expressing effectively can enhance the ability of vermin-resistance in sugarcane.Intermediate expression vector pUN and the plant expression vector pUNG, harboring gna gene drivered by Ubi promoter was constructed and introduced into sugarcane by Agrobatorium-mediated transformation, and 227 transforments were obtained after successive PPT resistance selection.124 in 183 ubi driven gna PPT-resistant plants showed positive by PCR detection. In addition,54 Rbcs driven gna PPT-resistant plants were obtained and 35 plants showed positive by PCR detection. It preliminarily proved that gna had integrated into sugarcane genome.Moreover, six ubi-gna PCR positive plants were selected in random to carry on RT-PCR detection, and each plant expressed gna on transcription level. Then the biological activity of raw proteins extracted form leaves of these plants were measured by red blood cell agglutination bioassay. It showed that GNA expressed in these transformed plants were of normal biological activity. While two rbcs driven gna PCR positive plants were detected by RT-PCR, and only one was positive. These results indicated that gna were expressed on transcription level.