Cloning And Functional Study Of NtLEA1Gene In Tobacco

LEA proteins （late embryogenesis abundant proteins） are widely distributed in thebiosphere and highly aggregated in the seed late embryo stage. These proteins areclosely related to plant defense. LEA gene can be induced by many environmentalfactors such as high salt, drought, cold damage, abscisic acid （ABA） and other planthormones. Overexpression of LEA gene can improve the drought or salt tolerance oftransgenic plants. LEA genes have been investigated in many plants except tobacco.In this study, a LEA gene was cloned from tobacco leave, named NtLEA1gene,with a high similarity （80%） compared with SlLEA.The length of open reading flame（ORF） is501bp, coding166amino acid. Bioinformatic analysis shows molecularweight of NtLEA1protein is17.3551kD, PI is8.62, instability coefficient is15.71, andGRAVY is-1.103. This protein contains seven tandem repeats of11-conserved aminoacid motif, belonging to the third group of LEA family. Promoter Sequence analysisshows that it includes one endosperm expression required element （Skn-1motif）, twoABA response elements （ABRE）, one MeJA response element （CGTCA-motif）, onesalicylic acid responsive element（TCA-element）, one high-temperature stress responseelement （HSE）, one drought response element （MBS） and two light regulatory elements（Box4）.Semi-quantitative PCR analysis shows that NtLEA1gene can be induced by PEG、ABA、salt stress with different expression patterns, of which salt stress has the moststrongly inducement. In order to explore the function of NtLEA1, pCXSN-NtLEA1recombinant vector was construted and transducted to tobacco leaf disc mediated byAgrobacterium tumefaciens strain LBA4404. Four NtLEA1over expression lines（OEX-L1^L4） have been identified by PCR amplification of CaMV35S promoter andhpt gene, aslo by fluorescence quantitative PCR analysis. Salt resistance analysis of T₀transgenic tobacco plants shows: after1.5%NaCl cultured for12days, the leaves of thetransgenic lines（OEX-L1^L4） were still fresh and green, while the control leaves startedatrophying and being yellow;120mM/L NaCl stress incubated for about20days,rooting rate of the transgenic lines（OEX-L2）（80%） was higher than the wide typeplants （60%）; after3d salt stress, transgenic and control plants had less content ofchlorophyll in leaf than before, but the reduction of transgenic plants（16.3%） is less thanthe control（28.6%）. Hence, the results indicate that NtLEA1protein can improve the salt tolerance of transgenic tobacco plants.