| Carboxylie transport protein gene is a new gene isolated in recent years, which may be related to energy metabolism involved in fungal penetration of insect integument, presuming it to be a virulence factor for entomogenous fungi. In order to ascertain its function in Beauveria bassiana, the gene BbJEN1 was silenced by RNA interference.In the present study, two interference vectors were constructed:①pRNAi: According to BbJEN1 mRNA sequence in Genbank, a pair of 64-nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. 0ligonucleotides were annealed and ligated with linearized pRNA-U6.1-Hygro with T4DNA ligase. The plasmid was transformed into E.coli DH5αand finally identified by colony PCR and sequencing.②pbarGPE1-RNAi: Based on BbJEN1 gene sequence in Genbank, two pairs of specific primers containing special restriction enzyme sites were designed. The target fragments forward and reverse, were amplified and inserted into the plasmid pbarGPE1. The recombinant plasmid was transformed into E.coli DH5αand finally identified by colony PCR and sequencing.After the construction of two interference vectors, the optimal inhibitory concentration for Hygromycin B selected by drug resistance assays with conidia was 600μg/mL. Transformants were obtained by two different genetic transformation methods: protoplast transformation and blastospore transformation. The blastospore transformation frequency was 7 transformants/μg DNA, while that of protoplast transformation was 0.9 transformants/μg DNA. The results of RT-PCR indicated that constructed interference vectors inhibited BbJEN1 expression in B. bassiana obviously. Three interference vectors resulted in good silencing effect, with silencing efficiency at 71%, 75% and 82%, respectively.Biological characteristics such as hyphal growth, sporulation, conidium germination and stress resistance of the transformants and the original strain were tested. The result revealed that growth rate and sporulation between the transformants and the original strain were not significantly different, suggesting BbJEN1 is not associated to hyphal growth and sporulation, while germination of the transformants was significantly retarded. Median germination time of the original strain was 14.24±0.58h, while those of the transformants with PRNAi-1, PRNAi-2, PRNAi-3 and pbarGPE1-RNAi were 14.45±0.79h, 16.22±1.02h, 16.13±1.66h and 16.00±0.41h, respectively. Stress resistance results showed that conidial survival rates of the transformants were lower than that of the original strain. Among them, the rates of the silenced isolates transformed with PRNAi-2, PRNAi-3 and pbarGPE1- RNAi were significantly lower, while that transformed with PRNAi-1 not significaantly lower. This suggested that BbJEN1 gene is related to stress resistance.Bioassay was run with the transformants and the original strain for their virulence on the caterpillar. The results showed that accumulated corrected mortality of Bb13 by the lowest dosage was 14.68±5.25%, and that by the highest dosage was 87.07±7.67%. While those of the transformant Bb13-S were 6.92±4.98% and 71.56±4.96%, respectively, lower than those of the original wild type strain. LT50 of Bb13 were 6.49±0.39d and 5.57±0.28d at two concentrations, 2.5×107 and 1.25×108conidia mm-2, while those of Bb13-S, the silenced strain, were 8.09±0.41d and 7.17±0.36d, respectively, both 1.6d longer than the original strain at respective concentration, with killing rate reduced by 26.65% averagely. LC50 and LD50 of the original strain were 2.79×106±0.21×106 conidia mL-1 and 84.12±4.11 conidiamm-2, respectively, while those of the transformant were 1.27×107±0.19×107conidia mL-1 and 382.92±24.32conidia mm-2 respectively. Spores number needed was averagely 3.6-folds more than that of the wild type, indicating a significantly declined virulence. It suggests that BbJEN1 gene is related to virulence.
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