Cloning And Functional Analysis Of Huamn Alpha-Salivary Gland Amylase Gene And Guangxi Bama Mini-Pig Parotid Secretory Protein Gene5’Regulatory Sequence

The phytate in the feed can not be degraded by monogastric animals. Therefore, high-phosphorus faeces from monogastric animals has become a major source of pollution in livestock production. The best way to solve the feed phosphorus pollution is to add phytase in animal feed. But the loss of phytase activity in the feed processing that makes this method difficult to obtain satisfactory results. Cultivation the transgenic pigs that can self-produce phytase by salivary glands will be a new research strategy to solve the phosphorus contamination problem in swine production. Salivary amylase and parotid secretary protein are both protein that have specific and very high abundance expression in saliva. In this study, the regulatory sequences of human alpha-amylase gene and Guangxi Bama miniature pig parotid secretary protein gene were cloned and its promoter activity was verified in vitro with attempt to lay the foundation for the research of phytase-transgenic pigs production. Primers used to amplify5’regulatory sequences of human alpha amylase and Guangxi Bama mini-pig parotid secretary protein were designed according to the sequence released in GenBank (AMY:M19339.1, PSP:AY197556). DNA used as template was extracted from blood of human and Guangxi Bama mini-pig, respectively. The human alpha amylase gene and Guangxi Bama mini-pig parotid secretary protein gene5’regulatory sequences amplified by PCR then were ligated with pMD18-T vector and transformed into E.coli DH5a for sequencing. The sequencing results revealed that two segments cloned from human alpha amylase gene5’regulatory sequences contained1057bp and769bp respectively, and one segment cloned from Guangxi Bama mini-pig parotid secretary protein gene5’regulatory sequences (PSP)only contained475bp. Comparing with the sequence in GenBank, the PSP sequences cloned from Guangxi Bama mini-pig showed100%homology to the reported sequence, however, human alpha amylase gene5’regulatory sequences present a162Aâ†'C transvertion, a675C insertion and136T and212G deletions, respectively.A segment named as AMY763(763bp) was cloned using the segment of human alpha amylase gene5’regulatory sequences (769bp) as template. AMY763and PSP were ligated into pEGFP-N1vector to replace CMV promoter, respectively, after digested by restricted enzyme Asel and EcoRI, and two recombinant plasmids designated as AMY763-pEGFP-N1and PSP-pEGFP-N1were constructed. EGFP-N1-AMY763and PSP-pEGFP-N1vectors were transferred into mouse parotid gland cells, respectively, mediated by lipofectamine2000.The PCR verification indicated that the two recombinant plasmids were successfully transferred into mouse parotid cells, but only the cells transferred with PSP-pEGFP-N1expressed pEGFP protein which confirmed that the PSP sequence from pig play a role as active promoter in mouse parotid gland cells. However, pEGFP can not expressed in the cells transferred with AMY763-pEGFP-N1indicated AMY763regulatory sequence (763bp) lost promoter activity in mouse-parotid-gland cells.Using testicular injection method, the recombinant plasmids AMY763-pEGFP-N1and PSP-pEGFP-N1were transfected into the sexually mature mouse’s testis, The injection was repeated for3times with a interval of2days, and then mate with the female to produce the transgenic F1generation. Five F1mice from parent transfected with AMY763-pEGFP-N1showed slow-growing hair located around the parotid-gland area, but PCR verification can not found they carried any AMY763sequences. Mouse transfected with PSP-pEGFP-N1produced9offerings, of which3mice were transgenic positive verified by PCR test and DNA sequencing.In summary, human salivary gland alpha-amylase gene and Guangxi Bama mini-pig parotid secretary protein5’regulatory sequences were successfully cloned in the present study. The in vitro transfection experiment confirmed that PSP sequence has promoter activity in salivary gland cells. The transgenic mice carried with pig’s PSP sequence also were obtained. The results from present study laid a solid foundation for carrying out the cultivation of phytase-transgenic pig in the further.