Study On Wing Expansion Regulation Function Of Bursicon Gene In Sogatella Furcifera (Horváth) (Hemiptera: Delphacidae)

The white-ba Controled planthopper（Sogatella furcifera Horváth）is an important migration pest of rice.The wings of S.furcifera is an important functional structure that facilitates its migration and contributes to the extensive damage to rice production.Therefore,genes regulating wing expansion may be potential targets for pest Control.The wing expansion of S.furcifera is affected by environmental factors and regulated by genes.However,the related genes of S.furcifera wing expansion and how they regulate wing expansion are not known.It has been shown that wing expansion is regulated by the Bursicon gene in Drosophila,Tribolium castaneum,Bombyx mori and other insects.The Bursicon may also be a key gene regulating S.furcifera wing expansion.In this study,the Bursicon gene of S.furcifera was cloned,its spatiotemporal expression pattern was analysed,and its function in regulating the wing expansion was verified using RNAi technology.Key genes of the signaling pathway of S.furcifera wing expansion were then selected from transcriptome data,and their function and relationship with Bursicon in relation to S.furcifera wing expansion regulation were investigated.Knowledge gained facilitates the understanding of molecular mechanisms of Bursicon in regulating wing expansion of S.furcifera.The main results are as follows:1 Sequence composition,expression patterns and function of S.furcifera Bursicon（1）The Sf Burs-αand Sf Burs-βgenes of S.furcifera were cloned.Their ORF sequences were 474 bp and 417 bp,which encoded 159 and 138 amino acids,respectively.More than 85%sequence composition of Sf Burs-αand Sf Burs-βof S.furcifera overlaps with those of N.lugens.Phylogenetic tree showed that Sf Burs-αand Sf Burs-βgenes were firstly clustered with N.lugens as a clade which was grouped into a large group with Halyomorpha halys.These results suggest that the Bursicon gene of S.furcifera was highly conservative during evolution.（2）The analysis of spatiotemporal expression pattern showed that the expression of Sf Burs-βand Sf Burs-αpeaked at 40 min after eclosion,followed by 3-day-old 5^thinstar nymphs,with lowest expression at the egg stage.The order of expression levels was consistent across different stages with head>thorax>abdomen>ovary>fat body>gut>epidermal>wing>foot.My results showed that the Bursicon of S.furcifera,similar to other insects or arthropods,mainly secreted from the nervous system of head,throax,and abdomen,and also expressed in other tissues.（3）Treated 1-day-old nymphs of the 5^th instar with different concentrations of ds RNA.The mortality rates of S.furcifera increased with the increase of ds RNA concentrations and were under 14%,however,the differences were not significant compared with the Controls.The wings of emerged adutls were normal.When treating1-day-old nymphs of the 3rd instar with 4000ng/μL ds RNA（ds A,ds B and ds AB）,the the lethal rate was over 59.00%in treated groups,and wing deformity rate was over 74%for the survived adults.Mortality rates were all below 14%in the Control groups and there was no phenotype of atrophy or weak expansion observed in the adults after eclosion.These suggest that the 3^rd instar nymph is the critical stage for Bursicon gene to regulate wing expansion.2 Function verification of key genes in wing expansion signal pathway of S.furciferaThe genes Sf Hh,Sf Dpp,SfNotch,Sf Hippo and Sf Sal were cloned,their ds RNAs were synthesized in vitro,and their functions in wing expansion regulation were verified by injecting the ds RNAs into nymphs of S.furcifera.（1）The mortality and eclosion rates were 48.8%and 51.3%,respectively,after being treated with ds SfNotch.The emerged adults did not show phenotypes of folded and curled wings,but the wings of some adults were incomplete at the end region of wings.（2）Over 70%of the emerged adults showed wing deformity phenotypes such as complete wing folds,curled wings at end regions or bent wings after being treated with ds Sf Hh and ds Sf Dpp.（3）Only 8.7%adults had abnormal wings after being treated with ds Sf Hippo,and68.9%adults had abnormal wings after being treated with ds Sf Sal.3 Functional analysis of other wing expansion related genes in S.furciferaThe Sf ITP,Sf HOW and Sf Ap genes were cloned,their ds RNA synthesized in vitro,and their functions in wing expansion regulation were verified by injecting the ds RNA into nymphs of S.furcifera.（1）After being treated with ds Sf ITP,the mortality and eclosion rates were 54.8%and 45.6%,respectively,and 78.2%of the emerged adults showed wing deformities such as folded and curled wings.（2）After being treated with ds Sf HOW,the mortality and eclosion rates were 66.8%and 33.2%,respectively,and 62.0%of the emerged adults showed phenotypes of wing deformities such as folded and curled wings.（3）After being treated with dsSfAp,the mortality and eclosion rates were 48.8%and 51.25%,respectively,and 81.22%of the emerged adults showed phenotypes of wing deformities such as folded and curled wings.Moreover,some individuals did not complete molting and eclosion with the epidermis covering their abdomens.4 Molecular mechanism of Bursicon gene in regulating wing expansion of S.furciferaThe relationship between the expression of Bursicon gene and the expression of key genes related to wing expansion in S.furcifera was investigated using RT-q PCR.（1）After S.furcifera Bursicon RNAi,the expression of SfNotch,Sf Hippo,Sf Sal,Sf Hh,Sf Dpp,Sf HOW,Sf Tre1and Sf Tre2 genes significantly decreased,while the expression of Sf ITP gene significantly increased.（2）The interference of 5 key genes of 3 different signaling pathways affected the expression of S.furcifera Bursicon differently:When Sf Hippo gene was interfered,the expression of Sf Burs-αand Sf Burs-βgenes were significantly higher than those of the Control.When Sf Hh,Sf Dpp and Sf Ap genes were interfered,the expression of Sf Burs-αand Sf Burs-βgenes were significantly inhibited.When SfNotch was interfered,the expression of Sf Burs-αgene was significantly decreased,while the expression of Sf Burs-βgene was similar to that of the Control group.These results suggest that Sf Hippo gene may have a compensatory relationship with Bursicon;and the Sf Hh,Sf Dpp and Sf Ap genes and the Bursicon gene can be mutually regulated.（3）The interference of Sf ITP,Sf HOW and Sf Ap genes significantly inhibited the expression of the Sf Burs-αand Sf Burs-βgenes.Suggesting that Sf ITP gene may have a compensatory relationship with S.furcifera Bursicon,and the Sf HOW and Sf Ap genes and the S.furcifera Bursicon gene can be mutually regulateded.In summary,the critical period for the Bursicon gene to regulate S.furcifera wing expansion was the 3^rd instar nymph stage.The molecular mechanism of Bursicon gene in regulating wing expansion of S.furcifera was preliminarily investigated.The Bursicon gene can affect the transcription of growth factor Dpp through the regulation of the key wing genes expression in Notch,Hedgehog,and Hippo signaling pathways,and thus regulate the wing expansion of S.furcifera.