| Holotrichia parallela Motschulsky is an important pest of agriculture and forestry, which is difficult to control. Its larva seriously damages many crops such as peanut, soybean, potato, wheat, and so on. Bacillus thuringiensis(Bt), is one of the world’s most widely used microbial insecticide. Bt can produce parasporals which conclude insecticidal Crystal Proteins, Some researches show that these proteins have some specific insecticidal function for more than two hundreds specises of insects, especially for Lepidoptera, Coleoptera, Diptera and nematodes. Cadherin-like protein is one of transmembrane glycoproteins relying on the calcium ion. It not only plays an efficient role on cell recognition, signal transduction and keeps cell morphological structure, but also is the most significant receptor of Bt. Unfortunately, these researches almost concentrated on Lepidoptera, so Coleoptera took a very poor study, especially for underground pest.In this paper, the cadherin-like gene of H. parallela larva was cloned using transcriptome, reverse transcription PCR(RT-PCR), rapid-amplification of cDNA ends(RACE), and the expression was analyzed through real-time PCR. The main results are as follows:1. Three kinds Bt proteins were used, there were diluted to different concentrations to feed five days old H. parallela larvae under 25 by the new ℃ measuring system with agarose gel particles. After 14 days, we checked the death larvae and calculated the median lethal concentration(LC50). The results showed that all three proteins had some effect on the larvae, for the protein Cry8 E, Cry8 G, Cry8 C the LC50 were 126.537, 304.890 and 2933.042(the unit is ug per 1 gram dried potatoes).2. Using the high throughput sequencing technology, the transcriptome data-base of H. parallela third larvae midgut was obtained, which contained 38,445 Unigenes after a series of assembling such as SOAP denovo. By blasting with NCBI database, 24,885 Unigenes can match to the known proteins, which account for 64.73 percent Unigenes, and the vast genes of sequences with Tribolium castaneum had high homology(account for 58.93%).3.The cadherin-like gene was cloned by using of RT-PCR and RACE. The full-length sequence was named HarpCadl contained 4 833bp(GenBank accession number: KM892868), and the open reading frame(ORF) of this gene was 4 503 bp in length, encoding 1501 amino acid residues, with a calculated molecular weight of 167.967 ku and the theoretical point of 4.11. It had a typical structure with other cadherin proteins, from N to C were a signal sequence, a proprotein region, five cadherin repeats, a membrane-proximal, a transmembrane and a cytoplasmic. Multiple sequence alignment by MEGA5.0 revealed that deduced amino acid sequence of HarpCadl had a relative high degree of identity to HpCadl from other insect species, such as Diabrotica virgifera virgifera, T. castaneum, but comparing with other Lepidoptera insects,they had a low degree of identity, for example Mythimna separata, Heliothis armigera, and Spodoptera litura.4. The expression of HarpCadl in different instars and different parts of the third instar larvae was analyzed by using real-time PCR. The result showed that the transcripts of HarpCadl were detected in all stages and all parts, while the expression levels were different, the expression level reach the highest point in third instar larvae, which was significant higher than the other instars( p < 0.01). Compared among foregut, midgut, hindgut and other parts of the body, the expression of HarpCadl was fluctuated, and the other parts except for intestinal had the lowest expression level while the midgut expressed the highest, which was about 3 times of foregut and about 175 times of other parts. |
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