| Black locust(Robinia pseudoacacia Linn.),also known as locust tree,is a deciduous tree belonging to the genus Robinia,Leguminosae,which is native to the United States with strong adaptability.Tetraploid R.pseudoacacia,also called polyploid R.pseudoacacia,was an excellent tree species that was artificially induced by doubling the chromosomes of diploid R.pseudoacacia.Compared with the diploid,the tetraploid black locust is superior to the diploid in all indicators,with the characteristics of salt and alkali resistance,drought resistance,and strong resistance to pests and diseases.Lectins are a class of natural proteins widely present in animals,plants,and microorganisms,It regulates biological processes such as protection and defense.In this study,molecular biotechnology was used to clone a lectin gene TRpLl from salt-stressed tetraploid R.pseudoacacia leaf.A series of physiological,biochemical and molecular biological studies were performed,and the results were as follows:(1)Using bioinformatics methods to analyze the structural characteristics of TRpL1 protein,predict the signal peptides,domains,subcellular localization,modification sites,etc.,with the results indicating that TRpL1 is located in the cytoplasm and cell membrane.(2)The recombinant prokaryotic expression vector pQE-TRpL1 was constructed,and the expression conditions of pQE-TRpLl protein were optimized.The results,showed that the optimal induction conditions for expression of recombinant TRpLl protein was induced with 0.1 mM IPTG for 4 hours in 28℃.Under this condition,about 10.8 g of TRpL1 protein with a purity of about 95%was expressed and purified,and sent to a biotechnology company for preparation of the polycolonal antibody against TRpL1,which laid a foundation for further research on the protein expression profile of TRpL1 in transgenic plants under saline-alkali stress.(3)The prokaryotic expression model was used to investigate whether the expression of TRpL1 protein could increase the salt tolerance of E.coli.The salt tolerance of E.coli was investigated by measuring the growth amount(OD600 value)of M15 strain under different concentrations of NaCl(0,400,600,800,1000 mM)within 0-12 h,which carrying the pQE-TRpL1 plasmid and the pQE-30 empty vector respectively.The results indicated that under control conditions,the growth curves of pQE-TRpL1 and pQE-30 were basically the same;under NaCl stress,the growth of pQE-TRpLl was higher than that of pQE-30 empty vector.This might imply that the expression of TRpL1 protein can increase the tolerance of E.coli to salt stress.(4)The q-PCR method was used to analyze the expression profile of TRpL1 in leaves of R.pseudoacacia.The results showed that compared with the control,the expression level of TRpL1 was increased in the NaCl treated diploid and tetraploid black locust,except the expression level in the tetraploid was higher than that in the diploid,which might because and the TRpL1 in both diploid and tetraploid are respond to salt stress,yet TRpL1 in tetraploid responded to salt stress more sensitively than diploid.The reason for the difference in TRpLl expression pattern might related with the higher salt tolerance of tetraploid than that of diploid.The expression levels of TRpLl in diploid and tetaploid increased slightly,and not significantly different from that of control after 200 mM NaHCO3 stress.It was presumed that the response mechanism of plants to NaHCO3 stress and NaCl stress was different and TRpL1 does not respond significantly to NaHCO3 stress in diploid and tetraploids.(5)The plant over-expression vector 35S::TRpL1-GFP was constructed and arabidopsis transgenic plants were prepared,with the T3 transgenic plants were obtained,which provided the material basis for further exploration of the salt-alkali tolerant function of TRpL1 gene. |
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