Cloning Of Arachin CDNA And Studies Of Arachin Expression Pattern

Legume seed storage proteins constitute one of the three largest sources of dietary proteins on Earth. They are of particular importance as a nutritional source in developing countries that lack ample supplies of animal protein. The legume globulins are nutritionally deficient in some essential amino acids especially the sulfer-containing Met and Cys. In addition to their importance to the developing plants many globulins have been identified as allergens. Improving the nutritional quality of storage proteins through genetic engineering has been proved as an effective method.A peanut immature seed cDNA library was constructed to clone the major storage protein Arachin genes. Using specific PCR primers corresponding to the highly conserved region of Glycinin A2BJa , a novel Arachin cDNA sequence , 462 bp in length , was identified and cloned into pGEM-T vector. Sequence analysis shows that it has more than 90% identity with Glycinin genes. The cDNA library was screened using this sequence as a specific probe under stringent conditions. The insert of the positive clone was amplified and subcloned into pGEM-T vector. The complete sequence consists of 1886 nucleotides with one long reading frame to encode a protein of 536 amino acids. Analysis of the coding region of this cDNA indicates that it codes for an acidic subunit corresponding to 331 amino acids, with a calculated molecular weight of 38.5 KD, and a basic subunit corresponding to 185 amino acids, with a calculated molecular weight of 20.5 KD. It is quite evident that two subunits are synthesized as a precursor and then undergoes post-translational processing to form the specific acidic and basic subunit. This is in agreement with that of pea legumin andglycinin. The deduced amino acid sequence contains two "cupin" domains, which play an important role in permitting sufficient immunologically active fragments to pass down the gastrointestinal tract and resisting both thermal denaturation and prototysis.The expression pattern of Arachin was also studied by Northern blotting and hybridization. The mRNA of Arachin is weakly detected in the first period of development. Then the transcription level remains high till the last period. But its expression in peanut leaves and pericarps can not be detected. This shows that the expression of Arachin was developmentally regulated and tissue specific and was regulated mainly at transcription level.Peanut was widely planted in China, especially in Shandong province, with the yield among the highest in the world. Generally, seed storage proteins lack some kind of essential amino acids. To improve the quality of peanut seed storage protein by genetic engineering has been proved an effective method. We cloned an Arachin complete cDNA sequence, adding a new member to the peanut storage protein gene family. Sequence analysis shows that the post- translational process was similar to that of glycinin and other globulin proteins, so the mechanism of the post-translational process of this kind of proteins was highly conserved.