Cloning And Characterization Of Rd22 Gene In Polygonum Sibiricum

In this study, a dehydration-responsive protein gene rd22 (GenBank accessionno.DQ836050) is cloned from a halophyte called Polygonum sibiricum by using RACE method.Sequence analysis shows that the rd22 gene is 1 302bp in length, including 1 218bp of openreading frame (ORF), and encodes a protein of 405 amino acids. The RD22 protein is analkaline secretory protein with a predicted molecular mass of 43.0kD and pI 9.47. The N-terminus of the RD22 protein has a signal peptide structure at the first 21 amino acids region,which indicates that RD22 function as a secretory protein may be transferred to a cellularspecific place or secreted out of cell. Homology analysis shows the C-terminus of the RD22protein contains a conserved BURP domain, and RD22 is the member of BURP domain proteinfamily. The BURP domain proteins consist of four modules: (1) an N-terminal hydrophobicdomain; (2) a short conserved segment; (3) an optional segment consisting of repeated units; (4)the C-terminal BURP domain.Characteration of rd22 from Polygonum sibiricum by Northern blotting shows that there isno expression in rhizome; and the transcription level remains high in stem, but it does not varywith the treatment time goes up; and in leaf, the expression is induced by NaHCO₃.The amino acid sequence encoded by rd22 gene contains a conserved BURP domain, butthe function of this domain is unknown. For understanding whether the rd22 gene and thenucleotide sequence of BURP domain has saline tolerance, we construct two plant expressionvectors named pROK-rd22 and pROK-burp. The two vectors are separately transformed intotobacco by using Agrobacterium mediated transformation method. 84 kanamycin-resistantseedlings are obtained via kanamycin-resistant selection. Some of the transgenic lines areconfirmed by PCR and Northern blotting analysis. The results indicate that exogenous gene hasintegrated into tobacco genome, and all exogenous genes have been expressed except onetransgenic line. Salt and alkaline tolerance experiments show that root growth condition oftransgenic plants is better than that of wild-type tobacco under 200mmol·L^-1 NaCl. and thedifference of root growth condition between wild-type and transgenic plants are not significantunder 5mmol·L^-1 NaHCO₃ stress. So, the resistance to salt of transgenic plants is better thanthat to alkaline. Determination of MDA content and SOD,POD activity indicate that under200mmol·L^-1 NaCl stress the MDA content of transgenic plants is lower than wild-type, andthe POD activity is higher than wild-type, but the SOD activity is not significant. Under5mmol·L^-1 NaHCO₃ stress, MDA content and SOD,POD activity are not significant betweenwild-type and transgenic plants. All of these indicate that the insertion of exogenous gene canmore or less improve the resistance to salt, but not significantly to alkaline stress. Therefore,the resistance of transgenic tissue culture seedlings to salt and alkaline stress is not improvedby the insertion of exogenous gene. rd22 gene and the nucleotide sequence of BURP domainfrom Polygonum sibiricum may not be functional genes for salt tolerance, and may not take part in the response of salt stress directly.