| Many genes can be induced by drought stress.Dehydrin gene(dehydrin gene,DHN) is one of them,it belongs to LEA D-Ⅱfamily group(late embryogenesis abundant proteins), which can be induced by low temperature exogenous ABA,drought,high salinity where cellular dehydration occurs.Dehydrins are one of the proteins induced by water-deficit,due to its high hydrophilic,it can combine with the lecithoid membrane thereby to prevent the water lose of the interior cell,maintain the hydration protective system of the membrane structure, avoid the decrease of the distance between the double molecular layer of the lecithoid membrane,farther to prevent the fuse of the membrane and the wreck of the biology membrane structure.In the present,we cloned ThDHN from Tamarix hispida,and studied the expression mode of ThDHN under stress.Nextly ThDHN gene was introduced into Betula playphylla through zygotic embryos and birch leaves respectively.Then transgenic birch were obtained successfully the Agrobacterium-mediated transformation of zygotic embryos can induced directly resistance bud formation,as well as a higher efficiency for genetic transformation,which is a new way to genetic transformation to B.playphylla.The results are as follows:1.ThDHN(FJ627947) from cDNA library ThDHN was analyzed by bioinformatics,the gene has a total length of 1159bp,the 5'non-coding region is 143 bp,3'non-coding region is 401 bp,the ORF(open reading frame) is 615 bp,encoding 204 amino acids.Its expected molecular weight is 22.76kD,theoretical isoelectric point is 5.61.The gene sequence has one S-fragment,and two K-fragments,belongs to SKn type.The homology of ThDHN is 36.6%～86.7%,and it has a near relationship with Citrus dehydration protein(AAP56259) and Coffea canephora dehydrin-like protein(ABC68275),the homology value is 86.7%,and it has a distant relationship with Pinus sylvestris dehydration protein(ACJ37787),the homology is only 36.6%.2.The expression pattern was studied by real-time PCR technology,results showed that, ThDHN gene may induced by NaCl,ABA,cold and heavy metal septum(Cd) stress,it was upregulated, particularly,ABA treatment can improved significantly the expression of ThDHN in T.hispida;while drought stress can suppressed the expression of ThDHN,the relative expression levels is lower than that of control,we speculated that the expression of ThDHN dependent on the participation of ABA.3.Mature zygotic embryos of Betula platyphylla Suk.as explants were disinfected and then used to inducing adventitious buds.Under WPM+2.0 mg·L-1 6-BA+0.2 mg·L-1 NAA conditions,the incision adventitious buds formed,and the rate of adventitious buds differentiation is 93.3%,with induction of adventitious buds is 8.7;Under WPM+4.0 mg·L-1 TDZ condition,the rate is 90.2%,and the induction number is 7.6.All these results indicate that WPM+2.0 mg L-1 6-BA+0.2 mg·L-1 NAA is better to differentiation of adventitious buds, and NAA can enhance the rooting of adventitious shoots,and the optimum concentration is 0.2 mg·L-1.4.Cephalosporin sodium is the optimized antibacterial antibiotics,it's effective concentration is 200 mg·L-1;kanamycin is the optimized antibiotics for the transformant selection,it's effective concentration is 20 mg·L-1;At the same time,after infected by Agrobacterium,the zygotic embryos can induced adventitious bud directly,and the cycle of transformants is short,eliminating the stage of callus induction and callus differentiation,this provides a new way to B.platyphylla.5.PCR identification,PCR-Southern blotting and Northern blotting were used to true transformants,and 7 independent plant lines were confirmed by PCR,All of these plantets produced the expected amplification product of 615 bp,while the non-transgenic plant lines is not;PCR-Southern blotting and Northern blotting confirmed that indicating that the transgenic lines were true transformants.This result indicated preliminary that the ThDHN gene had integrated into birch,and expression in transcription level.6.Salt-stress tests was done within non-transgenetic plants,0.3%NaCl(51.28mmol·L-1) is screeninged for the salt stress value.At that time,plants grow slowly,and the number of roots is less.Then,at this pressure,the main roots of 7 transgenic lines and non-transgenic lines was measured,results showed that,with the exception of BTD7,all of the main roots of transgenic lines were more than non-transgeneticlines,indicating that the damage of salt stress on transgenic lines is lighter than that on non-transgeneticlines,transgenic birch showed good salt tolerance.7.Under drought stress,2.5%-7.5%PEG6000 is screeninged for the drought stress value. After treated with PEG6000,the number of main root in all transgenic lines is more than nontransgenic lines,suggesting that the drought-resistance of transgenix birch is enhanced.
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