Analysis The Mechanism Of Polygonum Sibiricum To The Salt Tolerance By The Technology Of EST And Cloning The Relevant Gene To The Salt Tolerance

Polygonum sibiricum Laxm, a perennial herbage, can grow in the Saline-alkali spots andis the dominant population in the saline-alkali soil, which is suitable material for the study onthe tolerance mechanism of saline stress. Based on the previous study on the Polygonumsibiricum, the following aspects were engaged in this work:1.1977 tentative unique transcripts (TUTs) containing 206 contigs and 1771 singletonESTs were obtained on the analysis of the 2575 ESTs from the mixed cDNA library ofPolygonum sibiricum under the NaHCO₃ stress. The ESTs expressed in high abundance,moderate abundance and low abundance were confirmed respectively. There are 51 genesexpressed in high abundance, which amount to 2.6ï¼…in the total TUTs and represent 457 ESTs,accounted for 17.77ï¼…in all ESTs, the photosythesis ESTs are 54.92ï¼…in the ESTs expressedin high abundance. It indicated that photosynthesis is still the main physiological function ofleaf under the NaHCO₃ stress. There are 154 genes expressed in moderate abundance, whichamount to 7.8ï¼…in the total TUTs and represent 342 ESTs, accounted for 13.30ï¼…in all ESTs.There are 1771 single copy ESTs expressed in low abundance, which amount to 68.86ï¼…in thetotal ESTs. It indicated that the complexity of the cDNA library is enough.1977 TUTs were annotated by blastX, 720 in which are annotated with definite function,amounting to 36.42ï¼…in all TUTs. 109 TUTs are annotated with unknown protein, amountingto 5.51ï¼…in all TUTs. The others are considered as novel gene for little homology amounting to62.62ï¼…in all TUTs. According MIPS Functional Catalogue, the 720 ESTs are involved inmetabolism, energy, photosynthesis, transport facilitation, transcription, signal transduction,cell rescue defence and the other vital process. Among the above classification the ESTsinvolving metabolism, energy and photosynthesis are in the highest, amounting to 22.90ï¼…,12.25ï¼…and 11.86ï¼…in the all ESTs respectively, while those involving cell rescue defence arelittle and amount to 1.24ï¼…in all ESTs. The result indicated that under the NaHCO₃ stress, themain activities of leaf such as photosynthesis, metabolism and energy transformation, are stilldominant, which illustrated the tolerance ability of Polygonum sibiricum to the salt stress.2. Using PCR-Select^TMcDNA Subtraction Kit based on SMART strategy, the reverse andforward SSH library of the stem and leaf were constructed treated with NaHCO₃ for 48 hour,clones selected randomly were sequenced, 2282 valid sequence were obtained, which included598 ESTs in the stems forword SSH library and 490 ESTs in the stem reverse SSH library, 627ESTs in the leaf forword SSH library and 567 in the leaf reverse SSH library. According to thefunctional catalogue of MIPS and the comparison of the reverse and forward SSH library of thestem and leaf, the responses to NaHCO3 stress are different between leaf and stem, except for the same trend in cell rescue defence and transport facilitation, the trend in the metabolism,energy, photosynthesis, protein synthesis, transcription, and signal transduction is opposite. Thegenes involving transport facilitation, transcription and protein synthesis are down-regulatedunder NaHCO₃ stress in stem, but up-regulated in leaf. The genes involving photosynthesis,signal transduction, energy and metabolism are up-regulated under NaHCO₃ stress in stem, butdown-regulated in leaf.The result indicate that there are different mechanism in response to stress between leafand stem in Polygonum sibiricum. Combining with previous study, the conclusion is that thestem play an important role in salt-resistance in Polygonum sibiricum.3.17 cDNAs relevant to stress were cloned by the RACE, and were analyzed bybioinformatics, the result showed that the 17 cDNAs encode the water channel protein, lateembryogenesis abundant protein (LEA), glyoxalaseâ…¡, ozone-responsive stress-related protein,glutaredoxin (Grx), Mn-Superoxide dismutase (SOD), Cu-Zn SOD, universal stress protein(USP), cysteine protease inhibitor, copper chaperone, Calmodulin, glutathione s-transferase(GST), metallothionein(MT), cysteine synthase, glutamine synthetase, acireductonedioxygenase and ubiquitin-protein ligase/zinc ion binding respectively. Based on the presentstudy, the LEA, glyoxalaseâ…¡, Mn-SOD, Cu-Zn SOD, glutamine synthetase and MT havepotential function of salt-resistance, which are latent gene resource for genetic engineering ofsalt stress.