Studies On Different Chinese-fir Clones Under Salicylic Acid Stress Using DDRT-PCR Technique

Chinese fir recommended as one of the most important planting tree species in southern China,but now most of which grows on continuous plantation land.The long-term forestry production practice indicates that Chinese-fir continuous plantation can result in soil degeneration and productivity declining,which has become more and more evident along with the increasing times of pure plantation cultivation.The self-allelopathy is one of the most important reasons of continuous plantation obstacle.In this study,tissue culture and artificial stress tests were used to screen out three kinds of Chinese fir clones(including tolerant type,intermediate type and sensitive type),mRNA differential DDRT-PCR technology was taken to identify differentially expressed genes,the differentially expressed genes were cloned and sequenced,then compared with the GeneBank database.The results were as follows:1.All Chinese fir clones were planted on the MS culture medium which the exogenous salicylic acid had added into,measured Poison-index,the result showed that the inhibitory effect of the salicylic acid on FS02 was the strongest,FS01 middling,FS10 minimum,so that the FS02 clones was used as the sensitivity type,FS01 clones was used as the intermediate type,and FS10 clones was used as the tolerant type.2.By comparing the effect of one,two and three random primers on the result of PCR,the results showed that the effect of two random primers was the best.3.The orthogonal test were performed to find the optional conditions of DDRT-PCR by analyzing the five critical parameters of primer,dNTPs,MgCl₂,Taq DNA polymerase,annealing temperature.The results showed that a total volume of DDRT- PCR system consisted of,0.4μmol/L arbitrary primer,1.0μmol/L anchor primer,2.0 mM/L MgCl₂,1.5 U Taq DNA polymerase,0.20 mM/L dNTPs and annealed at 44℃.4.The autoxins of sensitive,intermediate and tolerant type of the Chinese fir clones were planted on the MS culture medium for 1,3,5 days which the exogenous phenolic acid had be added into,mRNA differential display analysis showed that the autoxins of FS02 sensitive clones were detected 14 differentially expressed genes,and different bands of the intermediate and tolerant type of the Chinese fir clones were relatively mroe,which were detected 31 and 38 of differentially expressed genes. 5.Three differentially displayed fragments were cloned by each type of Chinese fir clones,By both analysis of Bioinformatics,The sequence 1.1,one sequence of sensitive type of the Chinese fir clones FS02 had significant identity to Pinus koraiensis chloroplast complete genome partial sequence and picea chloroplast partial atpB gene,may be related to anti-Stress gene.The sequence 1.4,one sequence of intermediate type of the Chinese fir clones FS01 had significant identity to Gossypium hirsutum waterstress protect protein and LEA gene,wich was an important anti-Stress gene;The sequence 1.6 had significant identity to Taxodium distichum and Glyptostrobus pensilis chloroplast genome partial sequence,may be also related to anti-Stress gene.The sequence 1.7,one sequence of tolerant type of the Chinese fir clones FS10 had significant identity to Nicotiana tabacum,Arabidopsis thaliana chloroplast genome,may be also related to anti-Stress gene The sequence 1.2,1.3,sequence of sensitive type of the Chinese fir clones FS02,The sequence 1.5,sequence of intermediate type of the Chinese fir clones FS01,and the sequence 1.8,1.9,sequence of tolerant type of the Chinese fir clone FS10 had low similarity to known proteins,may be related to the new stress-resistant gene.