Cloning Of Antheraea Pernyi Arylphorin

Antheraea pernyi belongs to the Lepidoptera saturniidae, is a kind of complete metamorphosis insect, its lifetime experiences form structure and physiology function respectively different four insects scheduled time, which are ovum , larva (silkworm) , pupa , imago (moth) scheduled time. The insect absorbs food only in larva scheduled time, accumulates nutrient substance for the need of imago's development. Insect's storage protein is a kind of peculiarity protein. Such protein is synthesized in fatbody of feeding larvae or nymphs period in vivo and then released into the hemolymph. This kind of protein is uptaken by the fat body in pupation time of stopped feeding and storaged in the form of protein particle, so it was called the storage protein. Storage protein, such as arylphorin exists in Lepidoptera and Diptera insects, is the main protein of these insects'hemolymph, before the pupae period the fat body would storage the storage protein in the form of protein crystal storage. Storage protein as the store of amino acids, the main function of it is to provide the necessary amino acids when the larvae changes into adults.Arylphorin is rich in tyrosine and phenylalanine, which provides the aromatic amino acids for the metamorphosis. Arylphorin in antheraea pernyi early age of 5 is the lowest, then followed with the growth and development of silkworm gradually increased, peaked at the end 5 ages. Silkworm physical can synthesis a large number of protein in this relatively short period, indicating that the protein has a strong promoter, and this kind of protein provides aromatic amino acids for the period of adult, and has little impact on the larvae .So we can use antheraea pernyi arylphorin promoter to construct transgenic antheraea pernyi bioreactor. The experiment can be completed in phases, firstly to gain antheraea pernyi arylphorin DNA sequences and by analysis the gene sequence. Then build up the 5 'regulation region sequence +GFP+3' sequence carrier. Then use restriction endonuclease to cut it into different 5 'end of the linear vector and were transfected into insect cell lines to determine the function region of the promoter. Finally, connect the core promoter region and GFP reporter gene and the 3 'end sequence,then was transfected into worms'ovarian, produce generations with GFP transgenic antheraea pernyi, by screening next year we'll get transgenic antheraea pernyi. This stage will complete the sequencing of antheraea pernyi arylphorin DNA sequence and the construction of the 5 'regulation region sequence +GFP+3' sequence carrier to determine the core function region of promoter.The RNA sequence of antheraea pernyi arylphorin gene has been sequenced and some features of it have been explained. But the study of DNA sequence characteristics is little, only part of DNA sequence between 3 and 4 exon has been sequenced, for the rest of the structure and regulation region sequences are not detected.Through the analysis of DNA sequence of Bombyx mori SP2 protein (which is a kind of aromatic protein rich in aromatic amino acids) and Tobacco hornworm arylphorin, I found that they have 704 amino acids, equal to the length of antheraea pernyi Arylphorin, the number of exon of Bombyx mori and Tobacco hornworm is five and each exon's length is equal, it is estimated that antheraea pernyi arylphorin has five exons, and on the basis of separated fragments to design primers, by the PCR method I got the destination fragment and then connected it to the T-vector, after sequencing I obtained the DNA sequence of the structure gene. Based on sequence analysis and found that the sequence has eukaryotic gene characteristics which has a very short conservative sequence in connection site. Each of intron 5 'end sequence is consistent with the GT and 3' end contains the same sequence AG.For the 5 'end sequence, first through the analysis of antheraea pernyi first exon sequence to design the reverse PCR primers, through the reverse PCR,I obtain a fragment of 800bp.By the analysis of this fragment, I found that this segment is mostly the first exon sequence and the first intron sequence, and this fragment is the same with the structure gene sequence on NCBI. By aromatic protein sequence comparison of Silkworm, Tobacco hornworm, and the blow fly, designing degenerate primers, through the first exon to design specific primers, by thermal asymmetry PCR ,I gained the 5 'end regulatory sequence of the antheraea pernyi Arylphorin. These fragments were connected to the PMD-18T vector and undertook the sequencing. Detected by the software, this sequence has the TATA box, GC box of the promoter feature.To determine its core function domain of the promoter, put the 5 'end sequence including the signal peptide sequence behind the green fluorescent protein and then connect a fragment of 3 'end of the structure gene sequence after the green fluorescent protein. In this way we can construct the vector of T + antheraea pernyi arylphorin 5 'end +GFP+3' end. Then use restriction enzymes to cut this vector into different length of the 5 'end of the linear carrier for the future use of liposomal transfection kit to transfect different insect cell lines such as sf9, SC, GV-1 and so on. By comparing the fluorescence intensity to determine the core promoter regions, to establish the foundation of the research about transgenic antheraea pernyi bioreaction .