| Helicoverpa armigera is a very serious agricultural pest,which brings huge economic losses to agriculture every year.In the case of the prominent disadvantages of traditional chemical pesticides,it is of high application value to use modern biotechnology for biological control of cotton bollworm.Therefore,it is urgent to use new prevention and control strategies for H.armigera.With the development of molecular biology and the improvement of genetic engineering technology,it is possible to use CRISPR/Cas9 gene editing technology to control H.armigera.CRISPR/Cas9 has been successfully used in a variety of model insects,but gene editing in Kr-h1 of H.armigera has not been reported.The aim of this study was to establish an effective gene knockout technology system for the study of gene function of H.armigera,and to provide a potential target for the control of cotton bollworm.The main findings are as follows:1.The temporal and spatial expression profiles of Kr-h1 gene of H.armigera were analyzed by qPCR.It was found that Kr-h1 peaked at the 36th hour,the 3rd day and the 6th day of the development of H.armigera larvae,and its expression was most abundant at the 36th hour.The expression of Kr-hl in different tissues of H.armigera larvae was significantly different,especially in the head tissue of the larva,but low in the epidermis and fat body.2.When dsKr-hl was used to knock down Kr-hl gene in H.armigera larvae,there was no significant difference in Kr-h1 expression between 72nd and 96th hour after dsRNA injection.At the 120th hour after dsRNA injection,the knockdown efficiency of RNAi was only 18.13%.3.The hardware conditions and injection methods suitable for egg microinjection of H.armigera were established.The microinjection system suitable for egg injection of H.armigera was assembled,and on this basis,the injection parameters and injection methods were explored and optimized.The sgRNA target sites of Kr-h1 gene of H.armigera were analyzed by online software,and CRISPR/Cas9-mediated genome editing was achieved by using RNP complex injected with sgRNA and Cas9 protein,and G0-generation gene knockout chimera H.armigera was obtained.The CRISPR/Cas9 technique system applicable to H.armigera eggs was successfully established.4.The phenotype,weight and survival rate of H.armigera larvae knocked with Kr-hl at different periods were analyzed.It was found that the chimera larvae of G0 generation stopped developing on the 6th day of development,and began to die on the 7th day of development.The mortality rate reached 96.31%on the 15th day of development.The knockout larvae maintained their three-instar morphology and body weight unchanged after stopping development.The expression of Kr-h1 gene in the knockout H.armigera at different developmental stages was analyzed.The expression trend of Kr-h1 in the knockout H.armigera at the third day of development was consistent with that of the wild type.On the 4th to 7th day of development,the expression trend of Kr-hl in H.armigera was different from that of the wild type,which might be due to the change of the expression trend of Kr-hl in larva after gene knockout,thus causing the death of individual larva.5.The juvenile hormone,ecdysone and insulin levels of Kr-h1 knockout larvae at different developmental stages were determined.The hormone levels of Kr-h1 knockout larvae and wild type maintained a consistent downward trend in the first 3 days,and the hormone levels of Kr-hl knockout larvae and wild type showed similar trends from 4 to 8 days,so Krh1 knockout had a lower effect on the changes of juvenile hormone,ecdysone and insulin.6.ATP and TG levels of Kr-h1 knockout larvae at different developmental stages were determined.The results showed that ATP content of Kr-h1 knockout larvae was significantly lower than that of the wild type.The TG content of the knockout larvae was not significantly different from that of the wild type in the first 6 days.On the 7th to 8th day,TG content of the knockout larvae was significantly different from that of the wild type.Therefore,the continuously low level of ATP and the decrease of TG content in the later stage of larval development may lead to the lack of nutrition and energy supply during the development of G0-generation chimera larvae,and eventually stop growing and die.In conclusion,the deletion mutant of Kr-h1 was constructed and its important role in the growth and development of H.armigera was analyzed,which will provide a useful target for gene function research and comprehensive control of H.armigera. |
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