| Rubber tree (Hevea brasiliensis) is an important economic woody-crop in tropical areas.Its latex is the unique source of crude rubber used in current industry .Because of its special and important use,the rubber tree has been extensively planted in tropical areas.Increase production is always the main target in rubber tree cultivation.Since the ethrel was applied in increasing latex production in 1968 for the first time as a chemical stimulant,not only the latex production had been increased largely,but also a new set of rubber tapping system had been established,leading to a series of economic benefit.Owing to ethrel' s extensive application,its side effects had been found more and more obviously,such as tapping dry,speeding up senescence,shortening the life span of rubber tree etc.In order to overcome the side effects and increase production more availably,for a long time,people had carried out lots of research work on cell level,membrane level,physiology and biochemistry of laticifer contents.But the mechanism why ethrel increased latex production was not yet understood completely.This study had cloned the ethylene receptor gene(efrl) from rubber tree,and researched the relationship between etrl expression in laticifers and ethrel stimulation on transcription level and protein translation level.The results were as follows:1. The special primers were designed and synthesized after the analysis to the nucleotide sequence homology of etrl recorded in GenBank.The total RNAextracted from latex was reverse-transcribed into first strain of cDNA.The special amplification products(cDNA fragment) were obtained from PCR when using the first strain of cDNA as template.2. The special amplification products were ligated with the pGEM-T Easy Vector and transferred into E. Coli DH5 a via Cohn Method. After screening and identification,the positive clones were obtained.3. The cloning cDNA fragment was extracted from positive clones and sequenced.The results showed that the cDNA fragment was 816bp in size,encoding a protein which included 272 amino acids.The sequence homology analysis was carried out via the software Blast 2.0 Network Service in the four large databases-GenBank,EMBL,DDBJ,PDB,which had recorded 1 337 978 nucleotide and protein sequences.The results of the analysis indicated that the nucleotide homologous rates between the rubber tree etr\ and 15 recorded etrl of other plants(mango,passion fruit,persia plum,strawberry,grape...etc) were 75%-80%;the protein homologous rates between the rubber tree etrl and these recorded etrl genes were 90%-95%.From the results mentioned above,we could confirm that the cDNA of rubber tree etrl had been cloned.4. The genomic DNA extracted from rubber tree PR107 strain was digested with EcoR. I and Hindlll respectively.After the digested products were run via agarose gel electrophoresis and transferred into nylon membrane,the Southern Blot was carried out using the cDNA of rubber tree etrl as probe.The result of the Southern Blot showed that a hybridization band(-3.0kb) turned up from the EcoR I digested product and another band(-4.8kb) turned up from the Hindi]! digested product.The digestion site analysis to the cDNA of rubber tree etrl indicated that there was no digestion site inside the cDNA.So,there was only one copy of etrl in rubber tree genome.The etrl was a low copy gene in rubber tree.5. The genomic DNA extracted from three rubber tree strains(PR107,RRIM600,GT1) was digested with HincKR.The Southern Blot was made with the same probe as above.The result showed that all the three strains turned up only one brand(--4.8kb).The three strains had the same inherent background.6. The differential expression analysis to the rubber tree etrl in transcription (mRNA) level was made via reverse-transcribed PCR(RT-PCR).The special amplification bands were obtained from all the six samples of the three strains(ethrel treatments and their controls).The bands contained clear differentiation in brilliance.From the brilliance,the relative value of each b...
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