Cloning and sequence analysis of inorganic pyrophosphatase gene from Citrus grandis var.shatianyu

Self-incompatibility( SI) is the prevalence of phenomenon in the process of plant reproduction,it is an intraspecific reproductive barrier adopted by angiosperms that allows the pistil to distinguish between self( genetically related) and non-self( genetically unrelated) pollen. Inorganic pyrophosphatase( IPPase) play important roles in regulating the growth and development in plants. In order to better understand the mechanism of IPP gene in the self-incompatibility of Citrus grandis var. shatianyu,the inorganic pyrophosphatase gene of C. grandis var. shatianyu was cloned and physicochemical properties of pyrophosphatase were analyzed. The total RNA was isolated from style of C. grandis var.shatianyu used the total RNA Purification System( Invitrogen) and following the manufacture's protocols. According to the EST sequence( internal fragment of inorganic pyrophosphatase gene) in suppression subtractive hybridization libraries of C. grandis var. shatianyu style,4 specific primers 5'-GSP1,5'-n GSP1,3'-GSP2 and 3'-n GSP2 were designed for amplifying 3-RACE and 5-RACE of the gene. The full-length sequences of c DNA of inorganic pyrophosphatase gene were obtained from suppression subtractive hybridization libraries of C. grandis var. shatianyu style by the SMART-RACE PCR method. A comparison of the similarity of the full-length c DNA sequence of the inorganic pyrophosphatase gene was performed in the Gen Bank database used the BLAST program. DNAman software was used for mino acid sequence and homology analysis. Prediction of molecular weight,isoeletric point( p I) and hydrophobicity were performed by using on line software Exp ASy and DNAman. A 150 bp band in 3-RACE,and a 900 bp band in 5-RACE were cloned by nested and non nested PCR method. For the full-length c DNA sequences,the middle sequence,3-RACE and 5-RACE sequence of the inorganic pyrophosphatase gene were spliced and formed the full-length c DNA sequences. As a result,the c DNA of inorganic pyrophosphatase was 1 136 bp in length containing an 654 bp open reading frame( ORF),which encoded a protein of 217 amino acids with a 170 bp 5 untranslated region( 5 UTR) and a 321 bp 3 UTR. The sequence of the cloned c DNA of the inorganic pyrophosphatase from C. grandis var. shatianyu was registered in Gen Bank under the accession No. KF990474. In addition,through the DNAman software analysis,the deduced molecular weight of encoded protein of the full-length c DNA sequence was 24. 4 k Da and theoretical p I value of 5. 96. Protein domain analysis showed that IPPase of C. grandis var. shatianyu and pyrophosphatase have the same conserved domain. Bioinformatics analysis showed that the IPPase gene was a hydrophilic protein without any signal peptide,has no winded helix structure and transmembrane domain. The full length inorganic pyrophosphatase c DNA from C. grandis var. shatianyu was cloned and characterized. Blastn search results showed that the sequence of IPP gene of C. grandis var. shatianyu was highly homologous with IPP gene of variety plants. The homology analysis indicated that C. grandis var. Shatianyu IPPase gene shared87% nucleotide sequence homology with Populus trichocarpa and Hevea brasiliensis IPPase,and 100% amino acid sequence homology with Citrus clementina IPPase. The research results would provide the basis for further exploration of the function of inorganic pyrophosphatase in self-incompatibility of C. grandis var. shatianyu.