| Pears as plants of Pyrus L. (Rosaceae, Seyako) are major fruit trees. The production of pear ranks the third after that of apple and citrus in China. Pyrus betulifolia Bge. as a rootstock is widely used in the central, western and northern areas due to the useful trait to tolerate extreme drought and poor soil. At present, soil salinization caused by osmotic stress and ion produced by high salt and oxidative stress is the main problem of agricultural production and ecological environment around the world, and direct restrict the normal growth and development of plants including pear. Calcineurin B-like proteins (CBLs), a plant calcium sensor, play a critical role in the regulation of plant growth and stress response process. CBLs genes exert specific function between different species. To understand the sequence and expression of the three CBLs genes in Pyrus betulaefolia Bunge Bge,this study uses its seedings as materials to conduct research on the respones of CBLs genes under adversity stress.The major research contents are as follows.(1) In this study, we isolated the cDNA, genomic DNA and its responding promoter sequences of PbCBL genes from birch-leaf pear seedlings by EST database mining, rapid amplification of cDNA ends (RACE) and genome walking approaches, we successfully isolated PbCBL2?PbCBL4and PbCBL10. The results have showed that PbCBL2 cDNA sequence contained a 681 bp open reading frame which encoded 226 amino acid residues. The length of genomic DNA sequence was 1927 bp which consists of 8 exons and 7 introns. PbCBL4cDNA sequence contained a 639bp open reading frame which encoded 212 amino acid residues. The length of genomic DNA sequence was1645 bp which consists of 8 exons and 7 introns. PbCBL10 cDNA sequence contained a 801 bp open reading frame which encoded 266 amino acid residues. The length of genomic DNA sequence was 1983 bp which consists of 9 exons and 8 introns. The deduced CBLs polypeptide had four EF-hand structure domains which was necessary for calcium-binding and one calcineurin A subunit binding sites, with the closest affinity AtCBL2, AtCBL4 and AtCBL10's respectively in the same clade.(2) PbCBL2, PbCBL4 and PbCBL10 were up-regulated by NaCl, PEG6000 and mannitol in roots, stem and leaf of Pyrus betulaefolia. However, the expression patterns of three genes were different. The expression of PbCBL2?PbCBL4 and PbCBL10 was induced by 20 ?mol·L-1 ABA, although the expression patterns were different among diverse tissues. The expression of PbCBL2 was activated in leaves and obtained the maximum at 16h after ABA-treatment. The expression of PbCBL4 was activated in leaves and the peak was at 24h after ABA-treatment. The expression of PbCBL10 was activated in leaves and stems but not in roots. The expression of three genes was not observed in roots, leaves and stems of control plants. Insum, PbCBL2?PbCBL4 and PbCBL10 was up-regulated by ABA. Consequently, we concluded that the signal pathway of Ca2+ mediated by PbCBL2?PbCBL4 and PbCBL10 might be ABA-dependent.(3) The growth curve of the E.coli strains BL21 (DE) with pET-22b(+), pET-22b(+)-PbCBL2, pET-22b(+)-PbCBL4 or pET-22b(+)-PbCBL10 had no significant difference. The growth of four E.coli strains BL21(DE3) was slowed in LB medium with NaCl, PEG6000, mannitol and ABA. The higher the concentration of stressor was, the more server inhibited growth of strains was. However, stressors exerted different inhibition effects on different strains. For example, the growth of the strains pET-22b(+)-PbCBL2 and pET-22b(+)-PbCBL4 was slightly suppressed, whereas, the growth of the strain pET-22b(+)-PbCBL10, which was similar to the growth of wild-type, was significantly repressed. The tolerant ability of the strain pET-22b(+)-PbCBL2 was higher than that of the strain pET-22b(+)-PbCBL4. Consequently, PbCBL2 and PbCBLA genes enhanced the tolerance of transgenic E.coli strains to abiotic stresses. |
PDF Full Text Request