Cloning, Identification Of The Anopheles Sinensis Defensins Gene And Preliminary Construction Of Its Transgenic System

Backgound: Mosquitoes transmit numerous human diseases. Both malaria and filariasis belong to "Ten Most Emphasis Prevention and Control Diseases" which announced by WHO. Especially malaria, which is one of the attacks that result in the highest infection ratio and mortality, taking a heavy toll on the human population in many parts of the world by infecting >300 million and killing>2 million people each year. Diseases caused by mosquito-borne viruses, especially Dengue fever, are reaching disastrous levels. In the U.S., the West Nile encephalitis virus is rapidly spreading westward. Lymphatic filariasis, a nematode-based disease transmitted by mosquitoes, affects millions of people in tropical regions of the world. The major reasons for this tragic situation are the unavailability of effective vaccines for malaria and other mosquito-borne diseases and the development of insecticide and drug resistance by the vectors and pathogens, respectively. Therefore, there is an urgent need to explore every possible avenue for developing novel control strategies against these mosquito-borne menacing diseases, and at the same time, drug resistance of malaria means that controlling of mosquito becomes the most effective and pragmatic way to relieve the burden caused by malaria.Over centuries this relationship between insect, parasite, and mammalian host has been finely tuned, enabling the parasite to partially evade both human and insect immune system, thus ensuring its own survival. Insects are the largest biological species all over the world, and they have been remarkably successful in evolution. Current estimates are that there are more than 10⁶ kinds and their population have attained 10¹⁸, they account for 80% of all extant animal species; with the exception of the seas, insects colonize all ecological niches. Consequently, they are confronted by an extremely large variety of potentially harmful microorganisms. These shows that insects have extremely strong ablity of adaption and defence. From researches, insects have no specific immune system like mammals, they are lack of not only B and T lymphoid cell series but also immunoglobulin and addiment. Because insects occupy such a dominant position, we can see that they have strikingly native or acquired immune faculty, and so did their defense system.Plenty of researches show that, in the circumstance of infected by bacteria, insects could quickly produce a large amount of antibacteria peptide to kill the bacteria, and then prevent bacteria from invading the host. Till now, a large amount of anti-bacteria peptides, anti-fungi peptides have been found, these peptides have a broad-spectrum anti-bacterial ability to defend bacteria, fungi, virus, parasites and cancer cells. Numerous inducible antibacterial peptides have been characterized and they can be classified broadly into four major groups: (1) cecropins, (2) defensins or sapecins, (3) attacin-like proteins, and (4) proline-rich peptides. Cecropins are able to lyse and kill many Gram-positive and Gram-negative bacteria. Attacin-like proteins block the synthesis of major outer-membrane proteins in E coli. The antibacterial mechanisms of proline-rich peptides, such as apidaecines and abaecins, are not well understood. Defensins, cationic antibacterial peptides, are the most widespread group of antimicrobial proteins in insects. When attacked by an extropathogeny, mosquitoes could produce an effective defense system by many kinds of ways to activate the expression of the defensin genes in their fat bodies. The first mosquito defensin gene was cloned from Aedes aegypti. In 2000, Eggleston P reported the structure of DNA and immune regulation of Anopheles gambiae defensin gene. In China, ZHI Guo-zhou, et al. cloned and analysed the defensin gene of Aedes aegypti, and Aedes albopictus at first in 2002. And then, LIU Xian-kai, et al. did further studies on Aedes aegypti, and Aedes albopictus defensin gene in 2002 and 2003. But the complete defensin gene(either genomic DNA or cDNA ) has not been reported in Anopheles sinensis, which is the most important vector of malaria in China, nor its relevant bioinformatics analysis.A new way to prevent and treat mosquito-borne diseases is genetic manipulation, it could lead to produce kinds of transgenic mosquitoes by regulating insects genomics. In our research, Anopheles sinensis defensin gene cDNA sequence and genomic DNA sequence were cloned and analysed in extro, and the cDNA fragment was ligased into the plasmid which have 3xP3-DsRed eye specific promoter, vitellogenin(Vg) promoter, and SV40 poly A tail, and the recombinant plasmid was injected into flesh mosquito eggs by microinjection. After blood meal, defensin gene of transgenic mosquitoes will be activated by Vg promoter, and kill pathgens invading the host. This kind of project may, at some extent, do benefits to prevent mosquito from transmitting diseases.Objective:To clone, identify and analyse Anopheles sinensis defensin gene complete cDNA sequence and genomic DNA sequence and construct pBac-Vg-Defensins-SV40 plasmid which contains a full regulation sequence, 3xP3-DsRed eye specific promoter, vitellogenin(Vg)promoter, defensin gene and SV40 poly A tail. To inject the plasmid pBac-Vg-Defensins-SV40 into flesh mosquito eggs by microinjection to produce transgenic mosquito, and then do preliminary qualitative analysis on it. Methods:1. Anopheles sinensis Genomic Library was constructed with U.S. Clonetech Laboratories Universal GenomeWalker Kit by molecular biology techniques.2. Complete genomic DNA sequence of Anopheles sinensis defensins gene was cloned from Anopheles sinensis Genomic Library by Nested-PCR.3. Based on pairs of primers, whole cDNA sequence of Anopheles sinensis defensin gene was cloned from Anopheles sinensis total RNA by RT-PCR.4. The complete sequence of genomic DNA and cDNA of Anopheles sinensis defensin gene were analysed by bioinformatics analysis software from Genbank and ExPasy in details.5. The plasmid which contain 3xP3-DsRed eye specific promoter. vitellogenin(Vg) promoter, defensin gene and SV40 poly A tail, was constructed by techniques of molecular cloning such as digestion and ligation methods with the base plasmids: pBac[3xP3-DsRed afm], pSLfa1180fa, pVg-SV40 and phsp-pBac which were kindly bestowed by Professor Alexander S. Raikhel in University of California, Riverside.6. recombinant plasmid was injected into fresh mosquito eggs by microinjection and transgenic mosquitoes were produced.7. After DNA microinjection, 16-h to 20-h-hold injected embryos were heat-shocked at 39℃for 60 min and then placed at 27℃, 85% humidity. G₀ adults mosquitoes were screened with eye DsRed fluorescent marker using a fluorescence microscope to conduct preliminary qualitative analysis.Result:1. Anopheles sinensis Genomic Library were constructed successfully.2. Complete genomic DNA sequence of Anopheles sinensis defensin gene was cloned in extro.3. The full length of cDNA sequence of Anopheles sinensis defensin gene was cloned in extro.4. The result of bioinformatics analysis shows that: the full length cDNA sequence of Anopheles sinensis defensins gene have 324 base pair, its Open Reading Frame codes for 107 amino acids, 1-28 residues are signal peptide, 29-68 residues are propeptide, 68-107 residues are mature peptide, and 36.45% areα-helix, 29.91% are random coils, 12.15% areβ-turn, 21.50% areβ-sheet. It is a secreted protein, containing a transmembrane supercoil. Its molecular weight is 11.1898 kD, pI is 6.05, the number of acidic amino acid residues are 9, basic amino acid residues are 10, lipid parameter is 88.82, hydrophilia(GRAVY) is 0.160. After being compared with as counterparts of other Diptera insects, Anopheles sinensis defensin gene encoding sequence has the same conservative region as arthropod defensin, and major differences are signal peptide and propeptide, while, have high identity in mature peptide, furthermore, they all have 6 conservative region of cysteine, C1-C4,C2-C5,C3-C6 disulfide bridge. Analysis of Anopheles sinensis defensin genomic DNA shows that it has 2256 base pair, two exons, encoding 107 amino acids, separated by an 85 base pair intron, and in its upstream, there are core promoter TATA Box, an arthropod initiator sequence TCAGT, and some other upstream immune response elements, such as NF-κB, GATA factor, NF-IL 6, ICRE, HNF-5 and SP1, et al.5. The plasmid which contain 3xP3-DsRed eye specific promoter, vitellogenin(Vg) promoter, defensins gene and SV40 poly A tail was constructed. Besides, Vg promoter is vitellogenin encoding sequence, which can activate defensin gene and get high expression after blood-meal. 6. Transgenic mosquitoes were screened with eye DsRed fluorescent marker by using a fluorescence microscope.Conclusion:7. Anopheles sinensis defensin cDNA sequence and genomic DNA sequence were cloned in extro, and analysed by bioinformatics method. The cDNA sequence were submit to GenBank(ID:002892). DNA microinjection was perform to produce transgenic mosquito by injection of the plasmid which contains 3xP3-DsRed eye specific promoter, vitellogenin (Vg) promoter, defensins gene and SV40 poly A tail. Transgenic mosquitoes were screened with eye DsRed fluorescent marker by using a fluorescence microscope.