| Seed storage proteins is one important protein source for human beings .They provide large quantities of amino acids to human , some of which are essential ones. Genarally, seed proteins lack some kind of essential amino acids. Hence , increasing the essential amino acid content in seed proteins through gene engineering technique and enhancing their nutritional quality are very important to human' s nutrition and health . Peanut seed proteins is a good source of essential amino acids for human consumption .But some of them are allergens to people. That fact makes the study of the structures, functions and regulations of peanut seed proteins more significant.In this study the total RNA of peanut immature seeds was isolated using improved Guanidine Thiocyanate method , and the ratio of OD260/OD280 was 1.98. The mRNA was transcribed into the first strand cDNA with the oligo(dT) primers and double cD.NA strands were synthesized using strand replacement method . The auto radiography showed that thelongest synthesized fragment could be 5kb .The two ends of the cDNAs were ligated with a adaptor each and then inserted into @ gt10 vectors . The ligation mixture was packed in vitro and transducted into the host strain C600hfl . A cDNA library was constructed.The unamplified cDNA library contained about 3#106 clones .The titer of amplified library was about 1010pfu/ml. The recombinant clones in the library accounted to 91% of the total. Twenty-six randomly selected clones were amplified and the average size of inserts was about 1. 2kb. Two of the PCR products were sequenced . One was an unknown gene and the other was similar in sequence to the actin gene of pea.Total RNA was isolated from the immature soybean seeds, and RT-PCR was performed using primers designed on a published sequence of soybean Glycinin gene, resulting a product of 455bp , which was consisted with the expected size. The 455bp cDNA fragment was inserted into a T-vector and sequenced . It proved to be the cDNA fragment of soybean Glycinin gene. We screened the library primarily using the radiolabeled cDNA fragment of soybean' s glycinin gene as probe and get two positive clones. |
