| Bacillus thuringiensis produces crystal proteins (Cry toxins) that have been used to kill the insects by interacting with 4 types of receptor proteins. Cry toxins are biopesticides which have many advantages. The toxins have been expressed in many plant species to control a broad spectrum of pests. In order to effectively control the cotton bollworm, Helicoverpa armigera, transgenic cottons expressing Cry1A have been widely planted in China. However, the beet armyworm Spodoptera exigua, which is relatively resistant to Cry1A, has become more and more serious in the cotton field.The S. exigua larvae are relatively susceptible to Cry1Ca, so Cry1Ca should be excellent for S. exigua control. However, it remains unproven whether Cry1Ca interacts with the same receptor proteins as Cry1A.In contrast, the mode of action of Cryl A is well understood. There are four types of receptor proteins of Cryl A:alkaline phosphatase (ALP), aminopeptidase N (APN), ATP-binding cassette transporter (ABCC2) and cadherin (Cad). ALP is a phosphomonoester hydrolase. and some of the membrane-bound ALPs have been proven to be the receptor proteins of Cry toxin. APNs are endopeptidases, and widely distribute in the insect gut. In the mode of action of Cryl A toxins, ALP and APN have been suggested redundantly interact with Cryl A. ABC transporter C2 (ABCC2) has been implicated to play a role in Cry intoxication, and the resistance to Cryl A toxins has been confirmed to link to mutations in the ABCC2 gene. The cadherin in insect midgut is a crucial receptor for Cry toxins. Until now, cadherins have been proven or suggested to interact with Cry toxins in many insects.In this dissertation, therefore, we intend to determine whether there are the same four types of receptor proteins (ALP, APN, ABCC2 and Cad) that mediate the toxicity of Cry1Ca to S. exigua larvae.1. Cloning of membrane-bound alkaline phosphatases and its interaction with Cry1Ca in Spodoptera exiguaUsing the RACE technique, full length cDNAs of five membrane-bound alkaline phosphatases (ALPs) were obtained from S. exigua. All the five SeALPs show the similar structural characters:a hydrophobic signal sequence in the N-terminal region,3 conserved residues involved in substrate-binding,10 conserved residues in metal binding sites and a GPI-anchor sequence in the C-terminal region. The five SeALP genes were highly expressed in the larvae stage. However, each of the five SeALP genes exhibited specific expression profiles in pupae and adults, and in different tissues. The five SeALP genes, lacking the secretion signal and GPI attachment sequences, were heterologously expressed in Escherichia coli, and their binding abilities to Cry1Ca and Cry 1 Ac were investigated. All the five SeALPs preferred binding with CrylCa, but not Cry1Ac. Continuous ingestion of double stranded RNA (dsRNA) of SeALP1, SeALP2, SeALP3, SeALP4 and SeALP5 for four days significantly reduced the corresponding target gene by 58.9%,40.0%,64.7%,48.3% and 68.7% respectively. When the dsSeALP-ingested larvae then fed on CrylCa-contained diet for 6 days, the larval mortalities were 49.1%,54.9%,65.3%,52.5% and 77.4% respectively. An ANOVA revealed that the larvae previously fed dsSeALP1-, dsSeALP2-, and dsSeALP4-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeALP2-and dsSeALP 5-overlaid diets. Thus, SeALP 1, SeALP2 and SeALP4 are candidate receptors for CrylCa in S. exigua.2. Cloning and RNAi-mediated characterization of aminopeptidases N in S. exiguaTwo novel SeAPNs were cloned from the midgut of Spodoptera exigua larvae. The two novel SeAPNs, along with other four published previously, could be classified into six classes according to an APN phylogenetic tree. All the SeAPNs share similar structural characters:a signal peptide in the N-terminus, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif HEXXH(X)18E, and a GPI-anchor sequence in the C-terminus. The six SeAPNs were highly expressed the larval stage, especially in the midgut, foregut and hindgut. Four days continuous ingestion of dsRNA of SeAPN1, SeAPN2, SeAPN3, SeAPN4, SeAPN5 and SeAPN6 significantly reduced the target genes by 55.6%,45.5%,43.2%, 56.8%,45.4% and 46.0% respectively. When the dsSeAPN ingested larvae then fed on Cry1Ca-contained diet for 6 days, the larval mortalities were 52.0%,77.2%,43.3%,62.0%, 65.4% and 53.8% respectively. An ANOVA revealed that the larvae previously fed dsSeAPN1-, dsSeAPN3-, and dsSeAPN6-overaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeAPN2-, dsSeAPN4- and dsSeAPN5-overlaid diets. Thus, SeAPN1, SeAPN3 and SeAPN6 can be considered as candidate receptors for Cry1Ca in S. exigua.3. Cloning of the ABCC2 and its interaction with Cry1Ca in Spodoptera exiguaWe isolated an ABC transporter gene (SeABCC2) from the midgut of S. exigua larvae by degenerate primer. SeABCC2 consists of two cytosolic nucleotide-binding domains (NBDs) and two integral transmembrane domains (TMDs). The gene was highly expressed in the larvae stage. However, it was scarcely detected in male and female pupae and male and female adults. The results of tissue specific expression profile indicated that SeABCC2 was highly abundant in the midgut and then in the foregut and hindgut. SeABCC2 was heterologously expressed in Sf9 cells. Addition of Cry1Ca to culture solution caused 28.5% of the SeABCC2-expressed cells to swell. In contrast, only 7.4% of the controls cells swelled in response to CrylCa addition. It seems that Sf9 cells expressing SeABCC2 are more susceptible to Cry1Ca. After continuous ingestion of bacterially expressed SeABCC2-dsRNA for 4 days, the amount of SeABCC2 transcript in the larvae was reduced-approximately 58.2%, the larval mortality was decreased from 79.4% to 49.0%. Thus, SeABCC2 is the potential receptor of Cry1Ca in S. exigua.4. The interaction of Spodoptera exigua cadherin and Cry1Ca toxinWe bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera containing the putative toxin-binding regions. Competitive binding assays showed that both CrylCa and Cryl Ac could bind to rSeCadlbp and rHaBtRp, and they did not compete with each other. SeCadlb was heterologously expressed in Sf9 cells. Addition of Cry1Ca to culture solution caused 47.1% of the SeABCC2-expressed cells to swell. In contrast, only 7.4% of the controls cells swelled in response to Cry1Ca addition. It seems that Sf9 cells expressing SeCad1b are more susceptible to Cry1Ca. Dietary introduction of SeCad1b-dsRNA declined approximately 80% of the target mRNA and the larval morality to Cry1Ca were reduced from 83.0% to 52.1%. Therefore, we provide some evidences to suggest that SeCadlb from 5. exigua is a potential receptor of Cry1Ca.Thus, the results of this thesis indicated that Cry1Ca interacts with the same 4 types of receptor proteins as Cry1A (ALP, APN, ABCC2 and Cad) in S. exigua larvae. The results of this thesis will aid in the development of novel Bt biopesticides with increased efficacy and in establishing resistance management strategies. |
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