| Lousta migratoria manilensis is a major agricultural pest in China. Chemical insecticide is still the predominant type of locust control today. However, over-reliance on chemical pesticides has led to "3R" problems. Metarhizium acridum has been developed into a variety of formulations for pest control, but its long lethal time seriously restrict its application.Essential gene of host insect is potential target for the design and development of new, safe and effective insecticides. ATP synthase is an organism’s energy factories, which generates ATP by proton gradient, providing 90% of the energy of cells and organisms. Based on the importance of this enzymes to living organisms, it may be a potential target for pest control. In this study, we idenfied Lm-ATP5 A was essential for Locusta migratoria, and the dsRNA of Lm-ATP5 A was expressed in Metarhizium acridum to enhance its virulence against Locusta migratoria.Cloning and functional analysis the F1-ATP synthase α subunit of Lousta migratoria manilensis By sequence alignment and 3`-RACE, we have successfully cloned the full-length cDNA sequence of F1-ATP synthase α subunit(Lm-ATP5A) from L. migratoria. The total length of the sequence was 1888 bp, and its ORF was 1656 bp, encoding 551 amino acids protein. Design the 210 bp specific fragments of Lm-ATP5 A, synthesis dsRNA in vitro, injection 100 ng dsATP5 A per locusta, led to reduc the expression of its target, the activity of the enzyme was decreased, and the content of ATP in the cells was decreased, finally resulted the insects to death. Also, the number of blood cells in the blood reduced, the number of mitochondria in the blood cells was reduce and change the mitochondrial morphology and its membrane potential. The LT50 were 2.3 ± 0.1 and 4.2 ± 0.2 days for the 5th-instar larvae and adults. Based on the above results, considering the conservation of protein sequences and functions, and the specificity of nucleic acid sequences, we believe that Lm-ATP5 A may be a potential target for pest control.Expression dsRNA of host F1 ATP synthase gene in Metarhizium acridum increases the virulence against L. migratoria Metarhizium acridum CQMa102 was used as host strain, using Agrobacterium-mediated genetic transformation technology, transformed ATP5A-RNAi interference vector into the recipient strain, and successfully screened transformed strains expression dsATP5 A. Semi-quantitative RT-PCR analysis showed that the transgenic strains were successfully transcribed RNA. Wild type and transgenic strains were dropping infected fifth instar larvae of L. migratoria manilensis, the LT50 of transgenic strain 10.9% short than that of the wild type, quantitative PCR and Western blot were used to detect gene expression, the expression of host target gene in L. migratoria decreased after infected compared to wild type, prove that transgenic strains can reduce the expression of target gene in the host 5 days after infection, and the hypal bodies in locust hemolymph was 15.8 times than the wild type. These results demonstrated that the fungus can synthesis dsRNA of target host genes and interference its expression to increase the virulence of insecticide.The functional analysis of F1-ATP synthase alpha subunit gene in L. migratoria provides a theoretical basis for the use of this new insecticidal targets. Entomopathogenic fungus can synthesis dsRNA of target host genes and interfer the expression of the host genes thus increase the virulence of entomopathogenic fungus, providing a new stretagy to improve the killing speed of fungal pesticides. |
