The Detection On The Foreign Gene And Expression Of The Transgenic Triploid Of Chinese White Poplar

Triploid of Chinese white poplar is one of growing rapidly trees that are popularized in our country in recent years.With the enlargement of area of planting triploids of Chinese white poplar, many kinds of danger from the pest which eating leaves are more and more serious . It is important to cultivate poplar that can resist insect.Transgenic technology has become main means to improve the quality of insect-resistant of present plant by creating new varieties. The triploid of Chinese white poplar have been transformed genetically by the two insect-resistant genes (BtCrylAc gene and arrowhead protease inhibitor gene API) constructed in the same carrier in the Forestry biology technology lab of the Agricultural university of Hebei. This research was mainly to get the best insect-resistance clones of the triploid of Chinese white poplar through the test of molecule biology detection, the expression of foreign gene detection, the expression rule and relationship of foreign gene and the security of the Agrobacterium transformed gene.By the PCR analysis, 80% of all the 42 clones of the triploid of Chinese white poplar there are 33 clones have been tested the Bt-API genes. The Bt gene has been successfully conformed to the transgenic triploid of Chinese white poplar and the foreign gene of all the clones were one copy detected by Southern hybridization .By the primary insect-resistant experiment, 11 clones(39.3% of the all) took on the best quality of insect-resistant, 7clones(25% of the all) were better and 10 clones (35.7% of the all)were badly through feeding the young tender bladeseses of gene-transformed triploid of Chinese white poplar to Gypsy moth(Lmantria dispar L.) and Clostera anachoreta (Fabricius). The quality of insect-resistant of all the clones were accordant to the above two insects. The killing effect of insect-resistant was mainly to kill the lower instar larvae mostly and restrain the growth of the higher instar larvae survived to make them grow badly or even be killed. Analysis indicated that the weight, body length, frass weight of the larvae of the clones which took on the best or better killing effect were distinctly below the normal triploid of Chinese white, but the clones which killing effect was badly that index was similar to CK. The analysis of the body weight and frass weight of the larvae through feeding by the clone 5, 6 and 47 showed that these three clones hadhigher killing effect and the growth of the larvae was restrained to some extent with days. By two years'(2002-2003) experiments, the effect of insect-resistance of the clones were steady. It indicated the Bt-API genes in the transgenic plant were steady , the two kinds of insects did not show any resistence. Comparing the result of ELISA with that of insect-resistant showed that expression of the Bt poison protien of the higher insect-resistant clones were also higher than that of the lower insect-resistant clones.The Kanamycin(km') election for the transgenic tissue culture seedlings can not reflect the plant resistance really as a method to detect the foreign gene, so it can not reflect the final result of killing effect. Electing the transgenic plants by using of km' different consistency maked out primarily that km' election in the field could be as a quick method to filter foreign gene of the triploid of Chinese white poplar , the proper consistency was 500mg/L and high temperature, low humidity and funny weather would be better.It was founded that in the glass of the tissue culture seedlings of the clone 5,33,37 there were Agrobacterium had been identified by PCR, It demonstrated it was necessary to detect the Agrobacterium transformed gene in the glass of the tissue culture seedlings before transfer plant.