| Peanut (Arachis hypogaea L.) is the world's fourth largest oil crop grown worldwide in subtropical and tropical regions. China is the largest country of peanut production and comsumption in the world. Seed is the most important organ for peanut genomics studies and the early embryo development status is a key to yield and quality formation in peanut. For many years, deficient calcium in soil has been a general problem existing in peanut production areas in the red and yellow sandy soil regions of south China, which results in barepods and fall in yield. To know the molecular mechanism and to disclose functional genome relating to embryo development, we successfully constructed two SSH libraries with low and high calcium-treated embryos, respectively, and a mixed full-length cDNA library with both treatment embryos, after finishing comparison observation of biological and agronomic characters. The full-length cDNA library was sequenced by one read for ESTs on a small scale and gene annotation and function categories were carried afterwards. Using clony hybridization technology, SSH library of deficient calcium treated embryo was also screened and differential genes were identified by RT-PCR. The results were obtained as follows:(1)Embryo development status of deficiency and abundance calcium peanut were observed at podding stage and correlated biological characters were examined at harvest. The results showed that calcium deficiency peanut plants were blooming more than calcium abundance ones with dark purple on stem. Pods of calcium deficiency peanut were mostly empty or rotted, and calcium abudance peanut pods were full with kernal. The molecular mechanism underneath early abortive embryo and the prevention tactics were discussed in the paper, which could provide benifit to peanut production.(2)Forward and reverse suppression subtractive hybridization(SSH) cDNA libraries were constructed with early peanut embryos of deficiency and abundance calcium treatment. 93.2% of the colonies were white in calcium deficiency SSH cDNA library and 91.2% of the colonies were white in calcium abundance SSH cDNA library. The positive recombinants were confirmed by PCR, and about 85.3% and 86.9% clones tested were single band. The size of inserts ranged from 200bp to 800bp. (3)A full-length cDNA library of mixed early embryos from deficient and sufficient calcium treated plants was constructed by SMART technique, with a titer of 1.867×10~6pfu/mL and 95.8% of recombinant clones in unamplified library. The length of inserts ranges from 500bp to 2000bp. Twenty clones were picked randomly from the library for sequencing from both sides of inserts and the results indicated that 75% of the inserts were full-length cDNA, so the library should meet all the requirements as a standard library. Two hundred and ninty-eight clones from the library were sequenced from 5'end and 288 ESTs were got with an average length of 671bp. Among the 288 ESTs, 46 ESTs can not be found a homologue on web, and 242 ESTs showed homology to genes in the nr database,, among them only 53 ESTs had higher homology to genes of known function and 189 unknown function. The known-function genes were further grouped into six categories according to their putative function which included protein synthesis or storage genes, metabolism genes, disease/defence genes, transcription, cell growth and division genes, and cell structure.(4)Fourteen differential expression genes were screened from calcium deficiency early embryo SSH cDNA library by colony hybridization method, using deficient and abundant calcium treated early embryo cDNA labled with DIG as probes. These genes named SSHQ-1,SSHQ-12,SSHQ-13,SSHQ-14 were sequenced and then analyzed by Semi-quantitave RT-PCR in calcium deficiency and abudance peanut early embryo. The results showed that these four genes expressed much higher in calcium deficiency peanut early embryo than in calcium abudance peanut early embryo.We presumed that calcium played important roles during peanut early embryo growth and development.The obtained results offered a plateform for further study on functional genomics of peanut embryo development, and would suply with basis for geocarpy and molecular mechanism of embro development in peanut.
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