Cloning Of Calmodulin Gene And Rearches On In Vitro Embryo Development Regulated By Calcium In Peanut (Arachis Hypogaea L.)

Peanut (Arachis hypogaea L.) growing on drought sandy soil usually occurs embryo abortion due to calcium shortage in the soil in South China, which is an important product problem. To know the molecular mechanism, CaM genes in peanut were cloned from both genomic DNA and cDNA using two primers designed according to the CaM conservative sequences. Meanwhile, expression vectors were constructed with sence and antisence of PCaM-1 cDNA. In addition, the effects of calcium concentration in media were also studied during based on the build-up isolated culture systems. The results are as followings.Four genes, PCaM-1, PCaM-2 ,PGCaM-1and PGCaM-1 were cloned and registered in Genebank as AY517930, AY517931, AY517933 and AY572856 respectively. The analysis showed that the four different genes were all composed of 447bp encoding 148 amino acids with a stop codon. These four genes are all highly homologous with each other and also shared high homology with other known plants, and they blonged to EF-Hand superfamily. PGCaM-1 gene had a 973bp intron behind the 25th amino acid and the whole length was 1425bp.There were highly homologous sequences among these four genes. The sequence identity between PGCaM-1 and PCaM-1, PCaM-2, PGCaM-3 were 97%, 95%, 96% for nucleic acids, and 98%, 96%, 98% for amino acids, respectively. The sequence identity between PGCaM-3 and PCaM-1, PCaM-2 were 97%, 85% for nucleotides and 98% 96% identical for amino acids, respectively. PCaM-1 and PCaM-2 also showed varation in nucleotides sequence with 84% identity and amino acids sequence with 96% identity.The sence and antisence cDNA of PCaM-1 were integrated rightly into PC General and pEGAD expression vector,respctively.Meanwhile, the effects of calcium concentration on embryo development from immature to mature one in vitro were studied in the paper. Under calcium deficiency circumstances the explants became browning rapidly to death without expansion but those isolated embryos expanded to a different extent.There was no sharply differences in the growth pattern between cultivars. Under the same calcium concentration the embryos showed little contamination, less browning and growed more rapidly. The whole ovule had no expansion while the embryo could grow from the cut ovule with more deeply browning around the placenta and cut site. Besides, it was seen that the isolated would not become browning even calcium deficiency in liquid culture mediums. The results showed that peanut young embryos can be induced to be a mature one in vitro culture.