Molecular Identification And Functional Study Of Bursicon In Helicoverpa Armisera (Lepidoptera:Noctuidae)

Bursicon, a kind of neuropeptitde hormone, consists of two cystine knot proteins (burs and pburs) which form a heterodimer. Bursicon mainly acts on the newly emerged adults and regulates the cuticle sclerotization (tanning and hardening), wing expansion, even the immune response. Firstly, Bursicon binds with its receptor LGR2 and then activates the cAMP/PKA signal transduction, thus promotes the phosphorylation of the tyrosine hydroxylase (TH), the actived tyrosine hydroxylase would convert the tyrosine into DOPA, and consequently regulate the cuticle sclerotization; At the same time, Bursicon can also induce the apoptosis of the wing epidermal cells, followed by promoting the mature and expansion of the insect wing. In addition, the homodimer formed by burs or pburs can mediate the immune response via the IMD pathway by activating the transcription factor relish. The present study uses the cotton bollworm as a model to study bursicon actions. The full-length cDNA sequence of burs and pburs subunit was amplified from the cotton bollworm by PCR, RT-PCR and RACE methods. The spatial and temporal expression profiles of burs, pburs and the bursicon receptor rickerts were analyzed by using real-time quantitative PCR. The differentially expressed genes were analyzed by using the transcriptome sequencing during embryonic development of cotton bollworm, The nucleotide sequence of tyrosine hydroxylase was spliced from transcriptome sequencing result, The spatial expression profile of tyrosine hydroxylase was also analyzed by using real-time quantitative PCR. The double-stranded RNA of the molting hormone (20-hydroxyecdysone) receptor, EcR, was synthesized, after the interfernence of EcR, the effect of ecdysone on tyrosine hydroxylase expression were further studied using real-time quantitative PCR. At the same time, the actived burs homodimer protein, pburs homodimer protein and burs/pburs heterodimer protein were successfully expressed in 293T cells which was verfied by western blot method. The fat body of the cotton bollworms was treated with the actived burs homodimer protein, pburs homodimer protein, burs/pburs heterodimer protein and cAMP, followed by detecting the activity of tyrosine hydroxylase. Afterwards, the effects of recombinant proteins on the expression of the immune-related genes in the fat body of the cotton bollworm was studied in vitro tissue culture. The double-stranded RNA of the subunit pburs was synthesized, and then effect of pburs on the expression of the immune-related genes was studied after the interference of pburs subunit. The following is the main conclusions:(1) The full-length of burs cDNA sequence is 694 bp, The open reading frame is 480bp with 159 amino acid residues. The molecular weight and isoeletric point are 17.706KD and 8.04 respectively. The full-length of pburs cDNA sequence is 779 bp, The open reading frame is 420bp, encoding 139 amino acid residues. The molecular weight and isoeletric point are 15.876KD and 4.51 respectively. The results of the amino acid sequence alignment show amino acis sequence of burs in cotton bollworm share more than 60.19% identity with that of bur homologes in other insect species. Especially amino acid sequence of H.armigera burs shares the highest identity (75.93%) with that of bur in tobacco hornworm Manduca sexta. Amino acid sequence of H.armigera pburs shares more than 50.22% amino acid identity with that of.pburs in other insects. Especially amino acid sequence of pburs in H.armigera shares the highest identity (71.43%) with that of M.sexta pburs.(2) Burs and pburs express richly in the thoracic ganalia of cotton bollworms and arrive the highest expression level in the second thoracic ganalia followed by the suboesophageal ganglia, however burs and pburs less express in brain and abdominal ganglion, which suggested that bursicon is mainly synthesized in the thoracic ganalia.(3) The bursicon receptor rickerts gene has a significantly higher expression level in sex pheromone glands of the female cotton bollworms, but is lower expressed in the hairpencile of the male cotton bollworms which is the equal tissue with the female sex pheromone glands, suggesting that bursicon may paly an important role in the development of sex pheromone glands; In larvae stage of the cotton bollworm, the bursicon receptor rickerts gene has a higher expression level in the tissue of the fat body, epidermis and midgut, indicating that bursicon act in these tissues as targets.(4) The higher expressional level of burs, pburs and the bursicon receptor rickerts genes are found in the embryonic and pupa period. The expression level of the three genes are all lower from the 3rd instar to pupation, however in larval molting stages, the expressional level of three genes also slight increase. The expressional level of pburs is significantly higher than that of burs, which suggested that pburs probably plays other roles except that the function in bursicon heterodimer.(5)A lot of insect cuticle darkening and immune related genes were filtered out by comparing the transcriptome results between E1、E2、E3 and EL, including Toll protein、Toll receptor、lysozyme、lectin、tyrosine hydroxylase、 Dopa decarboxylase and so on. The full-length of the cDNA sequence of tyrosine hydroxylase is obtained by transcriptome sequencing result of cotton bollworm eggs. The result revealed that the open reading frame of tyrosine hydroxylase is 1686 bp, encoding 561 amino acid residues. The result of the amino acid sequence alignment shows that the amino acid sequence of tyrosine hydroxylase in cotton bollworm share more than 88.80% identity with its insect homologes. Especially the amino acid sequence of tyrosine hydroxylase in H.armigera share the highest identity (97.86%) with that of beet armyworm Spodoptera exigua homolog.(6) The higher expression level of tyrosine hydroxylase are found in embryon, larval molting period, pupa period and newly emerged adult, suggesting that tyrosine hydroxylase play important roles in the period of embryon, early larvae, pupa and newly emerged adult.(7) The double-stranded RNA of the ecdysone receptor, EcR was synthesized and injected into the fifth instar 24h larvas of cotton bollworm with gfp as control. After successful reduction in the expression of EcR, ecdysone was injected into the larvas. The result revealed that expression of tyrosine hydroxylase is significantly inhibited, which suggested that ecdysone regulate the transcript level of the tyrosine hydroxylase.(8) Fat body of H.armigera were dissected and treated with burs homodimer protein, pburs homodimer protein and burs/pburs heterodimer protein that were successfully expressed in 293T cells. The results of tyrosine hydroxylase activity in these treated samples manifested that the activated burs homodimer protein, burs/pburs heterodimer protein and cAMP can rapidly active the tyrosine hydroxylase activity in a relativly short time, however pburs homodimer fail to activate tyrosine hydroxylase activity in a short time, which indicating that the pburs homodimer protein couldn’t use cAMP as a second messenger, However the detailed mechanism is requied to further explore.(9) After burs homodimer protein, pburs homodimer protein and burs/pburs heterodimer protein were expressed in 293T cells, the effect of these recombant protein on immune were further studed. Results revealed that these recombinant proteins significantly induce the expression of the nine antimicrobial peptide genes (cecropin1、cecropin2、cecropin3、cecropinD、attacin、gloverin、defensin、moricin、lebocin) and the eight lectin genes (lectin1、lectin2、lectin3、lectin4、lectin5、lectin6、lectin7、lectin8) using real-time quantitative PCR... Especially the pburs homodimer, the expression of these immune-associated genes arrived the highest level 30min after treatment,(10) The five instar larva was injected with double-stranded RNA of pburs, the effect of pburs on the expression of the immune-related proteins was detected 72 hours later after dsRNA injection. Results manifested that the expression level of the six antimicrobial peptide genes(cecropinl、cecropin2、cecropin3、gloverin、attacin、defensin)are down-regulate significantly. Theses results further demonstrated that pburs homodimer protein could regulate the immunologic mechanism in cotton bollworm.