| The pine wood nematode (B. xylophilus) is a kind of quarantine invaded pest to coniferous trees, which has caused huge economic losses in many countries and regions. Affected area in our country has been broaden to15provinces and cities, and has caused a great loss. Now there are many researches about B. xylophilus, but it is still hard to be contoled. In this research, Bt-06A is desired to transform it into Botrytis cinerea which causes lethal effect after being fed with the B.cinerea which can express Bt-06A crystallin in order to control B.xylophilus.In this research, two overexpression vectors, pBI-GOH-BT06A and pBI-GOH-sGFP, were constructed and transformed into Agrobacterium. B. xylophilus were fed by B.cinerea which was successfully transformed by Agrobacterium to test the lethal effect. The main results are as follows:1. The T-DNA region in vector pBI-121was substituted by complex segment including multiple clone site and hygromycin resistance gene segment. Then the vector pBI-GOH was successfully constructed by sequencing.2. pBI-GOH is used to construct the overexpression vectors, pBI-GOH-Bt06A and pBI-GOH-sGFP. Both Bt-06A gene and sGFP gene were separately linked to pBI-GOH.3. Bt-06A gene and sGFP gene were transformed into Agrobacterium using overexpression vectors pBI-GOH-Bt06A and pBI-GOH-sGFP. Then the Bt-06A gene and sGFP gene were transformed into B.cinerea using Agrobacterium which cover Bt-06A gene and sGFP gene.4. B. xylophilus were fed using B.cinerea which covers Bt-06A gene and sGFP gene to test the lethal effect. Based on comparison of wild type B.cinerea and B.cinerea with sGFP gene in growth rate and death volume. The results showed that B.cinerea which cover Bt-06A gene has a great difference in growth rate and death volume with wild type B.cinerea and B.cinerea with sGFP gene. It shows that Bt-06A gene can cause the death of B.xylophilus. |
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