Construction Of Suppression Subtractive Hybridization EST Library And Isolation And Expression Analysis Under Stress Of CBF Genes From Eucalyptus Grandis

Eucalyptus is native to Australia. It is widely cultivated in the world due to its desirable traits suchas being fast-growing sources of wood, wide wood application and producing oil which has themedicinal functions. Now, Eucalyptus has been one of the most important commercial wood species inthe tropics and subtropics of China. In addition, Eucalyptus can also be used for the forestation ofcoastal shelter forest and landscaping species in the coastal area of Zhejiang province. However, mosteucalypts are sensitive to cold and low temperature has been one of vital factors which give damage toEucalyptus cultivation because of the present abnormal climate. To elucidate the mechanism ofEucalyptus cold acclimation is helpful to promote its breeding through the method of molecularmarker-assisted selection. In this study, a subtractive EST library of Eucalyptus grandis which wastreated by low temperature was constructed by suppression subtractive hybridization (SSH), thesequence from the library was analyzed and annotated. Moreover, two CBF genes were isolated fromEucalyptus grandis under low temperature with homologous cloning method. Quantitative real-timePCR was use to analyzed the expression of EgrCBF genes under different stress conditions. Thesestudies lay the theory foundation for Eucalyptus genetic improvement to cold resistant. The main resultsare as follows:Eucalyptus suppression subtractive hybridization EST library was constructed using RNA from theleaves of low temperature(4℃) in dark (tester) and normal temperature (25℃) in dark after2hourstreatment with the purpose of enriching differential expression genes. Finally,280high quality ESTswere obtained by sequencing. Genes induced under low temperature were analyzed by functionannotation based on sequence obtained, some genes related cold tolerance were included such as fattyacid reductase gene, Signal peptidase gene and zinc finger protein genes. Among them, two ESTs88(fatty acid reductase gene) and93(zinc finger protein gene) were induced obviously in low temperature,the expression of88and93were22and27times as high as control respectively. The results implied theeffectiveness of the library.Two CBF genes were isolated from Eucalyptus grandis seedlings under low temperature byhomologous cloning which were named EgrCBF1and EgrCBF2. Sequence analysis showed thatEgrCBF1cDNA was1062bp and EgrCBF2was1203bp. The two genes encoded proteins of220and 196amino acids separately and both contained one AP2domain. The results of homology comparisonshowed that EgrCBF1and EgrCBF2protein shared a91%and80%homology with EguCBF1a.The expression of EgrCBF1was mainly in leaves and roots while EgrCBF2expressed in leaves,stem and root with no significant difference. qRT-PCR result of EgrCBF1and EgrCBF2under differentcool temperatures showed that they were both induced by low temperatures. To the treatment ofdifferent time at4℃, the two genes were induced rapidly and then decreased. Changes of expression ofEgrCBF1and EgrCBF2under100μM ABA,200mM NaCl and drought were also analyzed, the resultsimplied EgrCBF1was up-regulated under drought sress and ABA treatment, while the expression ofEgrCBF2increased under drought and salt stress.