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| Functional Identification of lin-41 Gene in Heterorhabditidoides chongmingensis |
| LI Peng-Ya, HUANG Jin, ZHANG Ke-Yun* |
| College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Entomopathogenic nematodes (EPNs) are a class of highly effective and safety biocides whose capacity of reproduction is an important factor which affect the yield and quality of these EPNs as products potential. According to the differentially expression genes (DEGs) profile of the rabditid-EPN Heterorhabditidoides chongmingensis constructed in previous study, a RNA-binding protein lin-41 coding gene (lin-41) involved in regulating the development of nematodes was selected. The role of lin-41 in the growth and development of H. chongmingensis was revealed by identifying the biological functions of this gene in the nematode. Conservative analysis results showed that the conserved region of lin-41 of H. chongmingensis contained the non Hodgkin's lymphoma (NHL) superfamily domain and multiple NHL repeats. The amino acid sequences of lin-41 of H. chongmingensis, Ancylostoma duodenale, Dictyocaulus viviparus, Caenorhabditis elegans, C. brenneri and other related nematodes were highly conserved. The conserved NHL superfamily domain of the functional region fragment of lin-41 was selected as interfering fragment, and then the double-stranded RNA (dsRNA) expression vector of the fragment was constructed by L4440 plasmid vector and transformed into Escherichia?coli HT115 (DE3). The expression of the target gene was disturbed in the H. chongmingensis by feeding with the transformed E. coli HT115 (DE3). The effects of RNAi on the expression level of lin-41 and the reproductive related G protein α subunit gene (goa-1) were detected by qRT-PCR. The biological characteristics changes of the H. chongmingensis nematodes after lin-41 RNAi were recorded. The relative expression of lin-41 in H. chongmingensis after fed with the transformed E. coli HT115 (DE3) inducted by isopropyl-beta-D-thiogalactopyranoside (IPTG) with 0.1 mmoL/L for 4 h was reduced by 72.08% than that of the L4440 group. The number of eggs laid by the lin-41 RANi group was significantly less than that of the L4440 group, with an average of 45 eggs reduced (P<0.05). However, there was no significant change in the hatching rate of eggs between the 2 groups. These results indicated that the decreased in the expression of lin-41 neither affected the normal development and the hatch of eggs nor the quality of eggs. There were no significant change in body length between the 2 groups, although the female rate of the lin-41 RNAi group was slightly higher than that of the control group. These results indicated that the reducing expression level of lin-41 in H. chongmingensi neither affected the growth nor the sex differentiation of the nematode. In addition, RANi of lin-41 significantly reduced the expression level of goa-1 (P<0.05), which indicated that lin-41 not only participated in the oogenesis of nematodes but also affected the spawning behavior of nematodes in the complex reproductive system of H. chongmingensis by influencing the expression level of other gene related to reproduction in nematodes. This result also suggested that the regulatory network of egg laying behaviors of H. chongmingensi including both lin-41 and goa-1. The biological function of lin-41 in H. chongmingensis was explored by RNAi method in present study, which involved in regulating the oogenesis of nematodes to affect the egg production of H. chongmingensis and in the egg laying behaviors of the nematode by regulating expression level of goa-1. This study provides important theoretical basis and technical support for the subsequent expansion, development and utilization of H. chongmingensis nematodes in agriculture.
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Received: 15 March 2019
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Corresponding Authors:
* keyunzhang@njau.edu.cn
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