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Cloning and Expression of Cold Resistance Related Genes in Dendrobium Officinale under Low Temperature Stress

Author: LiDongBin
Tutor: SiJinPing
School: Zhejiang Forestry University
Course: Tree Genetics and Breeding
Keywords: Dendrobium officinale Low Temperature Stress physiological index SCoT Actin HSP70 expression analysis
CLC: S567.239
Type: Master's thesis
Year: 2013
Downloads: 60
Quote: 0
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Dendrobium officinale Kimura et Migo is a rare precious traditional Chinese herbal medicine,which has a special efficacy of tonifying stomach and promoting fluid and nourishing yin clearingheat and so on. Because of excessive excavation and cultivation environment destroy, wild D.officinale resources have been endangered. Since21st century, D.officinale on ecological cultivationhas made breakthrough progress, however the ability of fight cold is the main bottleneck restrictingfor the ecological cultivation. In order to study the D. officinale’s molecular mechanism of coldresistance and breed cold resistance varieties to adapt to the original ecological growth environment,this article had carried on the cloning and expression of cold resistance related genes in D.officinaleunder low temperature stress. Main research results are as follows:The cold resistance ability of the D.officinale were determined through the measurement of leafrelative electrical conductivity and activity of leaf protective enzyme under low temperature stress,the results should that the cold resistance ability order was: E>D>C>B>A, which is consistent withthe conclusion by calculation semilethal temperature of the leaf relative electrical conductivity.Compared with the effects of several kinds of RNA extraction methods in D.officinale, the method ofCTAB+LiCl+RNAiso Plus Can be used for the extraction of total RNA from D.officinale, and willprovide the beneficial reference for extracting total RNA from medicinal plants with highconcentrations of Polysaccharides.11transcripts derived fragments were got by SCoT differential expression of cold resistance relatedgenes in D.officinale Under Low Temperature Stress. The sequence analysis revealed that thefragments were significantly homologous in nucleotide sequence with membrane-associated proteins,Osmotic regulation protein, Transcriptional factor, Resistance protein. The other2gene segmentsfunctions were still unknown, which may be related to the cold resistant gene expression inD.officinale.The full-length cDNA sequence of Actin was successfully cloned from D.officinale by Actin genedegenerate primers from other species and RACE technology. The D.officinale Actin and other plantsActin genes’s nucleotide sequences and encoding amino acid sequences were highly conservative andhomology. HSP70gene full length cDNA was cloned by RACE on the basis of HSP70gene fragment sequenceswhich we got from SCoT differential expression. The full length of HSP70gene cDNA was2296bpcontaining a1944bp open reading frame (ORF) that encoded a protein of647amino acids; Its aminoacids sequence has typical of HSP70characteristics and has a high homology with other plants’sHSP70; Cold stress expression analysis showed that expression of the HSP70gene could be inducedunder low temperature.

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