Differential Expression And Screening Application Of Hypoglycemic Polypeptide-P Of Bitter Melon

Bitter melon (Momordica charantia L.) is the most extensively used as food crop for diabetes mellitus, its hypoglycemic and antihyperglycemic fruits has been universally accepted and proved by clinical experiments. Active hpyerglycemic ingredient of bitter melon contains saponins and polypeptides. Polypeptide-P has been reported and proved in1980s. In this thesis, the differential expression levels of polypeptide-P in different kinds of bitter melons were studied at molecular level.Firstly, we optimized the total RNA extraction methods and obtained high-quality RNA, then we compared three methods that are used for eliminating the DNA pollution of the RNA and ascertained the most effective method. Achieving the cDNA products through reverse transcription reaction,which included polypeptide-P gene.Secondly, we designed β-actin primers according to cucurbitaceous plant conserved β-actin sequence and acquired a length of 329bp bitter melon P-actin sequence, which contained 88 bp intron-construction. β-actin could be the refer gene in the bitter melon after stability tests in different varieties of balsam pears. Except that, we found out 18S rRNA partial sequence and assured it possibility to be another refer gene at NCBI.Analyzing the β-actin and polypeptide-P primers by means of the Melt curve and Standard curve, the primers can be applied in the Real-time PCR finally. Selected eight kinds of bitter melons were extracted for the total RNA by improved Trizol and gained the equal quality reverse-transfer products. Analyzed these eight kinds of cDNA by Real-time PCR and confirmed the bitter melon with high-expression polypeptide-P.