Evaluation Of Transgenic Tobacco With Cry8Ca Gene Resistance To Larvae Of Anomola Exoleta And A. Corpulenta (Coleoptera: Scarabaeoidea)

The white grubs(Scarab beetles) are serious pests throughout the world. They can damage through feeding to many plants such as crops, ornamentals, turfgrass. Scarab larvae are hidden within the soil and feed on subterranean organ causing the damage of the plant. It is very difficult to control the larvae with conventional measure because of the decreasing availability of chemical insecticides. Therefore it maybe effective against the larvae using of transgenic plants. The Cry8Ca protein from Bacillus thuringiensis strain HBF-1 has highly toxic to Anomola exoleta and A. corpulenta (Coleoptera: Scarabaeoidea). The cry8Ca gene had been cloned and transformed into the tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens. It was conformed that the foreign Cry8Ca protein had been correctly expressed in T1 transgenic plants. In this paper, the experiments were carried out to identify the resistance of transgenic tobacco and investigate the expression of pesticidal gene cry8Ca in the tested T1 transgenic tobacco plants with RNA dot blot and ELISA. The regulations were also formulated to identify the resistance of transgenic tobacco against Scarab larvae and it would be useful to the related research.The gene cry8Ca in T1 transgenic tobacco was detected by PCR and the 31 plants were positive from 88 transformed plants with pSmCN expressing vector in 110 test plants. The DNA probe was prepared by random primer DNA labeling with digoxigenin-dUTP and then the RNA was analyzed using dot blot hybirdization. The result indicated that the cry8Ca gene was hereditary expressed in the T1 generation transgenic tobacco.Enzyme linked immunosorbent assay is widely used to detect insecticidal protein in transgenic plant. The polyclonal antibody was prepared and purified, then labeled by HRP(horseradish peroxidase)with the sodium periodate method. It was determined using a square matrix that the density of anti-rabbit immune serum and the enzyme (HRP)-labeled antibody in ELISA reaction were 1:3200 and 1:400 respectively. The reaction conditions were optimized to detect cry8Ca protein in transgenic tobacco for Double-antibody Sandwich (DAS) ELISA as follows: NaHCO3- Na2CO3 (pH 9.6) was extracting buffer, PBS (pH7.4) was coating buffer, 0.5% Gelatin-PBST was confining liquid and TMB substrate action time was 15min.DAS—ELISA was used to detect Bt protein in transgenic tobacco with the optimization reaction conditions. The results showed that the highest content of Bt protein in the root of positive plants was 14.461μg per gram fresh weigh. It was about 0.057% of total protein. The content of Cry8Ca protein was higher observably in vegetative than one in reproductive organs of transgenic tobacco with pSmCN vector. The Cry protein content in the roots was the highest, secondly the stem base, thirdly the leaves and the lowest is in flower.The results showed that a life cycle for A. corpulenta was 272.3 days in the laboratory and it shortens 103.8 days than the natural population to complete a life cycle. It needs 230 days to complete a life cycle for A. exoleta. The results showed that the optimal condition was to inoculate A. corpulenta eggs into loam to bioassay the resistance of tabacco.The degree of damage to tobacco root was classified five grades, and then the resistance of transgenic tobacco against the scarab larva was evaluated according the damage index. The damage rates were 61.1%, 66.7% and damage indexes were 20.8, 23.6 after larvae of A. exoleta and A. corpulenta feed T1 tobacco plants transformed with pSmCN vector, while the damage rates were 100% and the damage indexes were 70.8, 65.3 for the pBI transformed plants and NC89 tobacoo feed. The difference was not obvious between the control tobacco NC89 and pBI transformed plants. The growth rates of characters of transgenic tobacco with cry8Ca were higher than control plants. It was demonstrated that injured degree of tobacco transformed with pSmCN vector was distinctly reduced than control pBI and NC89 plants. The T1 transgenic tobacco including cry8Ca gene exhibited highly resistance to Rutelidae larvae.