Cloning,Sequence Analysis And Expression Vector Construction On Proteinase Inhibitor CDNA Fragments Of Buckwheat

Proteinase inhibitor (PI) exist in various sorts and varieties creature, It take part in the activity of proteinase, interreaction of signaling receptor and the responder of adversity. Most of Proteinase inhibitor are in form of polypeptide or protein, Which have the ability to inhibitor the activity of proteinase. PI exist in plants widespreadly, especially in the storage organ such as seed and corm. In many field crops such as wheat, barley, maize, tomato, potato, It's content usually reach to 1%-10% of total protein. If the blade of plant was injured or stimulate by chemical material, PI will be accumulated in bulk. There are four main type PI were found up to now, they are serine protease inhibitor family, sulfhydryl protease inhibitor family(cysteine protease inhibitor family), metallo-protease inhibitor family and acidity protease inhibitor family. The PI exist in plants are mainly serine protease inhibitor family, this type of PI is a high performance implement on the research about protein metabolism relation to medicine and biology, especially it's latent therapycapacity on control disease such as pancreatitis, emphysema and cancer. Biochemist use it as model system to search for the mechanism of action between enzyme and substrate, meanwhile, it was researched as a resist pest gene engineering element and have make a progress. The seed of plant is the food resource of mankind and livestocks, the existence of PI make the research on it's functional to dietetics fascinating too. At present, PI in soyabean cowpea potato tomato and rice have been extracted and get most of gene sequence of PI from plant. There are few report on gene sequence of PI from buckwheat except ShanXi university Wang Zhuanhua once reported the gene sequence of PI from ShouYang buckwheat.In domestic there are no report about PI gene sequence for Ukraine Buckwheat.By retrievaling NCBI database and analyzing the conserved amino acid sequence of PI have been reported from plant, We design a couple of degenerate primer by Primer version 5.0, at the same time, we extracted first cotyledon of buckwheat total RNA, by method of RT-PCR and RACE, and get the C terminal cDNA sequence of PI from Ukraine buckwheat first time, We named it BPI-2 and register this sequence in GenBank(the accession number is EF507857). Analysing the sequence by DNAman software we found that this fragement is 307bp long, It contain a 171bp long open reading frame(ORF), this ORF encode 57 amino acids, and contain a 136bp long 3' Untranslational region(3'UTR). We retrieve the amino acid sequence by BLAST software in NCBI server to compare the homology and found that in GenBank+EMBL+DDBJ+PDB there are 100 sequence have the homology with this sequence, these 100 sequence are all sequence of PI. Among this the Amaranthus hypochondriacus have the maximum homology, then the Cucurbita, Arabidopsis thaliana, Linum, Euphorbiaceae, etc. The correspond amino acid sequence homology is 50%-63%, on these grounds we can conclude that this fragement is cDNA of PI from buckwheat. This sequence compare to the sequence of PI from ShanXi ShouYang buckwheat once be reported by Zhang Zheng and others,the homology is 64%, From this we can conclude that this gene is a new member of PI from Fagopyrum.The phyletic evolution analyzing of this PI sequence and PI sequence from other plants by DNAman software, we found that buckwheat and Amaranthus hypochondriacus merge firstly, then merge with Momordica, Sambucus, Solanum, etc. Meeting with the phyletic evolution relation assess by morphology.Based on these we analysed the primary structure and secondary structure of the polypeptide chain encoded by this sequence and construct the procaryon expression vector of this gene C terminal sequence.In a word, we extracted first cotyledon of buckwheat total RNA, by method of RT-PCR and 3'RACE, get the C terminal cDNA sequence of PI from Ukraine buckwheat, and do some bioinformatics analyse on this cDNA sequence of PI from buckwheat, such as gene characteristics, expression mode. All of this will be help for the forward research on functional protein and high performance of procaryon expression or eukaryon expression.