MRNA Differential Expression Of Longan Under The Stress Of The Acid Rain & Cloning Of DHAR From Longan

Acid rain pollution is a major ecological problem internationally and contemporarily.Adult Wulongling longan trees were used to study differential gene expressions of Longan leaves under the stress of pH 2.5,3.5,5.6(control)of simulated acid rain.The RNA extraction methods of Longan leaves were explored,as a template on the optimizing of important parameters which impact of DDRT-PCR amplification, and established differential display analysis system of longan leaves,the best system: 25μl system RNA input 3.0μg,anchored primer concentration of 0.8μmol/L, random primer concentration of 0.8μmol/L,dNTP concentration of 200μmol/L, 1.25 U Taq enzyme concentration,and annealing temperature of 40℃.The mRNA differential display conditions were optimized to find an EST sequence related with acid rain stress.By logging on to Genbank with the account number ofFC860625,the results of BLASTN nucleotide sequence comparison showed that the this fragment had 75%homology with the EST of polar leaves under drought stress.In addition,by using the BLASTX protein homology,the results showed that the potential encoding products had about 66%similarity with an unnamed protein product of grapes,had 59%similarity with a hypothetical transcription initiation factor of rice and 39% similarity with ATP-binding factor/RNA polymerase transcription factor of Arabidopsis.We can speculate this difference fragment is the transcription factor which activated by the second messenger substances.Longan percepted of acid rain signal,then carried out the second messenger substances.The second messenger substances activated transcription factor,cause acid rain corresponding gene expression.This difference fragment may also be carried out under the conditions of anaerobic respiration,energy consumption acceleratedBased on the conservative sequence of DHAR homologous gene cloned in apples, tomatoes,Arabidopsis,wheat,degenerate primers were designed to obtain DHAR fragment with the length of 437bp from Longan trees by cloning;Based on the DHAR cDNA sequence obtained from the experiment,RACE method was adopted in designing 3'-terminal specific primers and obtaining 3'-terminal sequences of this gene by cloning.Speculate ascorbic acid metabolism is a viable means of Longan to improve the regulation and control of acid rain.Through the promotion of excessive expression enzyme activity such as ascorbic acid regeneration enzyme DHAR,can improve the content of ascorbic acid in longan,longan resistance to acid rain may also be a certain degree of increase.