Study On The Cloning And Expression Of Ha Caspase-1 And Ha Hsc70 From Helicoverpa Armigera

The cotton bollworm, Helicoverpa armigera, which belongs to Lepidoptera Noctuidae, distributes broadly in the world and harms a lot of crops including cotton, cereal, corn, potato, bean and tobacco leaf. The cotton bollworm is one of the major pests of the world agriculture, which often brings great lost in economy. At the present time, the majority chemical pesticides used to prevent cotton bollworm are harmful to human and environment, and there is controversy on the security of the transgene crops such as the Bt cotton. Therefore, development of the biological pesticides based on the theory studies is always the focus of the pest prevention.Apoptosis, or programmed cell death (PCD), a process of cell suicide, which eliminates superfluous, damaged or deleterious cells during development, cell differentiation, and disease to maintain homeostasis, is essential to the development of organisms. In this study, two genes, Ha caspase-1 and Ha hsc70, involved in apoptosis were cloned from H. armigera. Sequences of the genes and the deduced proteins were analyzed, and the expression pattern, induction expression and function of Ha caspase-1 were investigated. 1. Cloning and expression of Ha caspaase-1 from H. armigeraCaspase (cysteinylaspartate specific proteinase) is a group of cysteine proteinase with substrate specificity at aspartate, which is the key factor in apoptosis and has non-apoptotic functions in cell survival, proliferation and differentiation. The proteins of caspase family are evolutionally conserved, executing the similar function in apoptosis. The caspases involved in apoptosis are divided into two groups according to the sequences of their N terminal domain named prodomain. The caspases with long prodomain are the initiator caspases which transfer the signals of apoptosis and activate the downstream caspases, while the caspsaes with short prodomain are the effector caspases which cleavage the substrates in cell and are the direct executioners of apoptosis.In this study, a caspase gene named Ha caspase-1 was cloned from the cDNA library of metamorphically committed H. armigera using the strategy of homologous cloning. The full-length of Ha caspase-1 cDNA is 1350 bp, including an 885 bp open reading frame (ORF) that encodes 294 amino acids. The deduced protein has a computed molecular mass of 33.5 kDa and a predicted isoelectric point of 5.65. Sequence analysis of the domain and active site shows that the protein has two domains, caspase-p20 and caspase-p10, which are the large subunit and the small subunit, respectively. In addition, the predicted protein contains the typical residues His and Cys required for catalysis by caspases and the catalytic cysteine (C₁₇₆) located in a QACQG pentapeptide is an example. Sequence alignment of amino acids of Ha caspase-1 with other known caspases revealed that it has a short prodomian and is great homologous to Sf caspase-1of Spodoptera frugiperda and drICE of Drosophila melanogaster. The homology of Ha caspase-1 to human caspase-3 is even 32%. These results suggested that Ha caspase-1 is an effector caspase in downstream of caspase cascade.Northern blot analysis and semi-quantitative RT-PCR were used to investigate the expression pattern of Ha caspase-1 in different larval tissues. The result of Northern blot showed that the Ha caspase-1 mRNA was only detected in the feeding and wandering larval haemocytes, with the concentration in haemocytes of wandering larvae slightly higher than that in haemocytes of feeding ones. There was no positive signal detected in the epidermis, midgut, fat body from both feeding and wandering larvae, suggesting that either the expression of Ha caspase-1 mRNA was probably haemocyte-specific or its mRNA concentration in haemocytes was significantly higher than that of the other tissues. The result of RT-PCR showed that the signal detected in haemocytes from wandering larvae was much stronger than that in feeding ones and other tissues, in agreement with that of Northern blot. However, in the feeding larval fat body and the midgut and wandering larval fat body, Ha caspase-1 was also found at lower levels, which is different from the result of Northern blot and may be due to the higher sensitivity of PCR compared with Northern blot, or haemocytes attachment or migration into fat body and midgut.Northern blot analysis was applied to determine the developmental profiles of Ha caspase-1 mRNA levels. The results revealed that Ha caspase-1 was expressed in embryos, which is consistent with the notion that cell death is extensive during embryogenesis, but there is no Ha caspase-1 detected in younger instar larvae (lst³rd) by whole body preparation of mRNA, which may because Ha caspase-1 mRNA level is too low to be detected in these early developmental stages. However, in later instar larval haemocytes (4th—6th instar), Ha caspase-1 mRNA concentration was higher during 4th and 5th molting and wandering stages, which appears to be correlate with the two waves of ecdysone during the periods of last larva molting and metamoephosis.In situ hybridization was performed to locate the transcript of Ha caspaes-1 in haemocytes. Results indicated that the plasmatocytes and granulocytes in haemocytes from 6th instar wandering larvae released extensive Ha caspase-1 signal. Other cells, including prohaemocytes and spherulocytes, did not show obvious signal compared with the negative control cells. The expression of Ha caspase-1 in these two kinds of haemocytes suggested that Ha caspase-1 may be involved in the larval-pupal metamorphosis because the populations of both plasmatocytes and granulocytes increased in the late stage of the last instar larvae.Because Ha caspase-1 mRNA levels increased in haemocytes during molting and metamoephosis, it is inferred that Ha caspase-1 should be regulated by 20-hydroxyecdysone (20E). Therefore, the relationship between Ha caspase-1 and ecdysone was measured through Northern blot analysis and RT-PCR. Results showed that Ha caspase-1 expression increased in haemocytes in vivo after ecdysone agonist RH-2485 injection and Ha caspase-1 mRNA also went up in haemocytes in vitro after treated with 20E, suggesting that Ha caspase-1 expression during development correlates to the release of ecdysone.As an effector caspase, Ha caspase-1 should play a role in apoptosis. The morphological alterations, DNA degeneration and Ha caspase-1 expression was therefore observed in the cultured epidermis cells undergoing apoptosis induced by AcMNPV (Autographa Californica multiple nucleocapsid polyhedrovirus). After AcMNPV induction, the morphological changes on cells including chromatin condensation, membrane shrinkage and vacuole appearance and apoptotic bodies were observed. In addition, nuclear DNA presented the DNA ladder on agarose gel electrophoresis. The result of RT-PCR showed that Ha caspase-1 expression in cells induced by AcMNPV was much higher than that in the non-induced cells, indicating that Ha caspase-1 is a function factor involvd in apopotosis.In addition, a fragment containing ORF of Ha caspase-1 was subcloned into prokaryotic expression vector pET-30a(+) to construct recombined plasmid Ha casp/pET-30a(+), and recombinant protein was highly expressed in E. coli strain BL21(DE3), which are to be used for function study on Ha caspase-1.2. Cloning of Ha hsc70 from H. armigeraHeat shock protein is a group of greatly conserved proteins, which has a broad range of biochemical functions. Hsps not only play indispensable roles in the stress reaction to maintain the self-stabilization of cell and avoid the damage of cell, but also participate in apoptosis, immune regulation, tumor and infection. Hsps is a super family including a large number of members in which Hsp70 family is the most conserved and important.In this study, a hsc70 gene named Ha hsc70 was cloned from the SMART cDNA of epidermis from the metamorphically committedally committed H. armigera using the strategy of homologous cloning. The full-length Ha hsc70 cDNA is 2145 bp, including a 1965 bp ORF that encodes 654 amino acids. The deduced protein has a computed molecular mass of 71.6 kDa and a predicted isoelectric point of 5.24. Sequence analysis shows the protein has only one large HSP70 domain without signal peptide. Sequence alignment of amino acids of Ha Hsc70 with other known Hsc70 revealed it has the greatest homology to the Hsc70 of other insects, with an identity over 90%. The identity of Ha Hsc70 to mammal and human Hsc70s also reaches 80%. The identities of Ha Hsc70 to plant and prokaryotic Hsc70s (or Hsp70s) are even about 70% and 45%, implying that Hsc70 is greatly evolutionally conserved.In summary, we cloned a caspase gene from H. armigera , Ha caspase-1, which is a effector caspase resulted from the sequence analysis and alignment. The expression profiles of Ha caspase-1 during development and induced expression of Ha caspase-1 by RH-2485 in vivo and 20E in vitro imply that Ha caspase-lmay be up regulated by ecdysone and play a role during molting and metamorphosis. The high expression of Ha caspase-1 in the apoptotic cells approves of its function as an effector caspase. We also cloned a hsp70 gene from H. armigera, Ha hsc70, which is highly homologous to hsc70 of other species resulted from the sequence analysis and alignment. The expression and function of Ha hsc70 is under study now.Apoptosis plays an essential role in development, cell proliferation and differentiation. Caspases are the key regulator in the apoptotic pathways, Hsps also participate in apoptosis, and the interaction exists between caspases and Hsps. To develop biological pesticides with high security based on the study of the characterization, expression and function of both caspase and Hsps is important to the exploitation of new methods for pest control. On the other hand, to study the function and regulation of genes involved in apoptosis during insect development is in favor of better understanding the mechanism of apoptosis and insect development.