| Lecanicillium lecanii is an important entomogenous fungus, which has a wide range of host. It is one kind of effective biological control fungi which can be used in controlling aphids, whiteflies, horned toads, thrips, mites and other agricultural pests. However, Lecanicillium lecanii is very sensitive to the commonly used fungicides, which makes it difficult to play a stable role in controlling crop pests. Here in this research, with the expectation to solve this problem, we screened plant pathogenic fungus which have resistance to benomyl, and cloned its resistant gene. Benomyl resistant gene was transformed into Lecanicillium lecanii via Agrobacterium-mediated method, so that Lecanicillium lecanii have resistantace to benomyl. Therefore, we can coordinate biological control and chemical control.The outcome and conclusions of this study as follows:1. Screening of the isolate to benomyl resistance: we collect plant pathogenic fungi from the area which long-term use of benzimidazole fungicides, Beijing agricultural product market, and research institutes. Separated, purificated and dentificated the plant pathogenic fungi were 3 genus 5 species (about 35 isolate). The benomyl sensitive were evaluate by measure the strain's colony diameter after different benomyl level. A lot of benomyl sensitive strains were screened, and their EC50 were between 0.0158 and 0.0384μg/mL. Two benomyl resistance strains were screened, and the higher EC50 was 936.26μg/mL.2. Cloning of the resistance gene: In this experiment, Botrytis cinerea strain SD2 is the starting material. BenA gene was cloned by the primers designed by Botrytis cinerea's benA sequence and cDNA of benomyl resisence fungi. The sequence of BenA gene cloned and the sequence of BenA gene from NCBI was 99% homology. The BenA gene was benomyl resisence gene, because the 198th amino acid was mutated to Val compare to sensitive strain which was Glu.3. Construction of the binary vector pBHt2-TB: The vector pBHt2-TB was constructed on the backbone of pBHt2. The benomyl resistance gene (BenA) and the Aspergillus nidulansTrpC promoter were ligated by overlapping PCR."TrpC-BenA"and liner pGM-T were ligated by T4 DNA ligase, is pGM-TB. The pGM-TB was digested by XhoI and BstXI, and the vector pBHt2 was digested by XhoI and BstX too, so"TrpC-BenA"and liner pBHt2 can be t ligated by T4 DNA ligase, and that is pBHt2-TB. The vector pBHt2-TB was transform to AGL-1.4. Construction of transformation system of Lecanicillium lecanii by Agrobacterium-mediated: In this experiment, the benomyl resistance gene (BenA) was transform to Lecanicillium lecanii CA-14 by Agrobacterium-mediated. Transformants were screened by hygromycin marker. After optimization,AS concentration was 200μM in induce medium and co-culture medium, induce time was 6h, the final concentration of Agrobacterium OD600 value was 0.6, co-culture time was 48h, the co-culture temperature was 24-26℃, spore concentration of Lecanicillium lecanii was 106 spores / mL.the transformation efficiency by Agrobacterium-mediated in this experiment was 24 transformants per 106 spores. 5. Verification and analysis of transformants: Forty-seven hypothesize transformants were randomly selected from a large number of transformants. False positive transformants were the percentage of 6.38% by PCR test. The percentage of 90.91% transformants can remain mitotically stable, and maintain the hygromycin resistance, when cultured on PDA medium without hygromycin after 5 generations. Benomyl resistance transformants were screened by 5μg/mL benomyl.The transcription of BenA was normal by RT-PCR test. The maximum of EC50 of the benomyl resistance of tansformants was 61.86μg/mL, which was 170 times to the original strain. |
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