| The soil insect grubs is one of the most serious hazards in the process of planting peanuts. For a long time, the prevention and cure of soil pests is main use of chemical pesticides, but chemical pesticides pollute the environment, and the cost of prevention is high, and it is easy to produce drug resistance, especially the increased levels of pesticides in peanuts because of the pods of peanut is in touch with pesticide, and it is harm to human health directly. Therefore, the method of biocontrol for controlling grubs has become the main trend. Metarhizium is a kind of important entomopathogenic fungi. More and more attention has been paid to it, there is advangtage of strong target, persistence length, produce drug resistance hardly, environmental safety, et al. But in the field application, it exposed the disadvangtage of low toxicity, long lethal time, et al. Thus, it is the current trends to use genetic engineering methods constructing efficient insecticide strain of Metarhizium anisopliae, wich is inherited stably. And it is the inevitable choice of conforming the current agricultural green plant protection and the theme of governance cycle.The main results obtained in this study are as follows:(1) We constructed vector pCAMBIA1300-ben-8e harboring marker gene benA3, and target gene cry8E, which exhibited high virulence to the larvae of soil pests Holotrichia parallela. The vector was verified by restriction and sequencing. and integrated it respectively into Agrobactrium tumefaciens EHA105and LBA4404, then integrated it into M. anisopliae by the method of Agrobacterium-mediated transformation. The positive transformants were verified by PCR detection, southern hybridization was done to further confirmed that gene cry8E was a single copy inserted into the genome of M. anisopliae.(2) Using indigenous method, we tested the virulence of different transformants of Metarhizium strains against H. parallela, the results indicates that the virulence among transformants assayed was different, the estimates of LT50, corrected cumulative mortality were14.4806-21.2617d,64.5022%～92.8571%. The corrected cumulative mortality and LT50of transformants M202-31are significant to other strains, and its value is92.8571%and14.9264d, so the virulent of transformants M202-31is high, we won the engineered strain wich could inherited stably.(3) The activity of acid protease, neutral protease, alkaline protease, chitinase, phospholipase from six M. anisopliae positive transformants were tested and the correlation between various indicators of enzyme activity and virulence was analyzed. The results indicates that There was also a large difference among the enzyme activity, and the variation trend was different. Compared to the wild type strain, transformants M202-31has a significant difference of the acidic protease, chitinase and phospholipase activity assay. Regression analysis revealed that acid protease and chitinase were significantly correlated to the corrected cumulative mortality and LT50. To further determine the combined effects of acid protease and chitinase on virulence, binary regression analysis was conducted and revealed that only chitinase was significantly correlated to the corrected cumulative mortality and LT50-Therefore, we suggested that the difference of virulence among transformants may be mainly derived from the activity of chitinase and chitinase could be used as virulence index for early selection of candidate transformants in large quantity, but it could not completely replace the virulence measurement.(4) Using the third-instar nymphs of Locusta migratoria manilensis as the test insect, the virulence of6transformants were tested, wich are wild-type strain M202; strain Bt185; strain Bt185and M202were mixed; two positive transformants and the control handle. The results shows that:it is significant increase the virulence of wild-type strain M202, after strain Btl85mixed with M202; meanwhile, the virulence of wild-type strain was significantly increased, after inserting gene cry8E, the the estimates of corrected cumulative mortality and LT50were38.6542%and2.9180d.(5) We tested the activity changes of ESTs, AChE, PO, SOD, POD and CAT in L.migratoria manilensis for five consecutive days, wich treatments are CK, M202, transformants M202-12and M202-31. The results indicates that: cmpared to the wild-type strain M202, six activity indicators have the broadly consistent variation of transformants M202-12and M202-31. Among them, there is no significant difference on the POD for7days of3treatments; cmpared to the wild-type strain M202, the activity of ESTs and AChE mainly significant difference in3-4days; the activity of PO mainly significant difference in3-5days; but the activity of SOD and CAT mainly significantly different. in the6th days. |
PDF Full Text Request