Study On Stress And The Growth Of Dendrobium Candidium And Its Related Genetic Expression And Gene Clone

With Bio+X disciplines developing, the combination of physics and botany is being paid attention by the academe of inland and outland. It was found by scholars both at home and abroad that man-made mechanical stress had obvious effect on regulation of growth and division of plant cells, and plant cells had priority to grow elongately in vertical direction to the stress vector under proper loading stress. However, there were only some sporadic experimental results about the study on reception of plant cells to signals of mechanical stress and mechanism of signal transduction, as well as the effect of stress on growth, metabolism and regulation of gene expression of medicinal plants without explanation of their systematic and intact approach.On the base of working foundation and researching methods obtained in our lab, in this dissertation, we used Dendrobium candidium, a precious medicinal plant, as experimental object, and made use of researching means and advancing methods of multiple disciplines, e.g. sound-wave stress uploading, biochemical detecting, gene screening and cloning, and bio-information predicting on function genes, etc., to study the relative anti-oxidase system, the contents of metabolite, the amplification and expression of some enzyme genes fragments, the screening and cloning of especially expressed genes, and their function prediction on bio-informatics. The results provided some elemental materials and firsthand molecular evidences of the regulative mechanism on plant growth and metabolism under physical stress stimulation, pretended a new method attempt to promote metabolizing products contents of medicine plant, processed preliminary works to search the functional genes and proteins in plant induced by the stress. The main results of this research are as follows:Firstly, on the influence of sound wave stimulation on D. candidium growth, the results showed that the in vitro-cultured seedlings would get the most obvious promotion under appropriate sound wave stress (the frequency 1000Hz, the intensity 100dB) and beyond some range the promotion would reduce, that is inhibited. Those are consistent with the experimental results achieved by our lab from model plants, Chrysanthemum and Arabidopsis thaliana. By determining the activities of anti-oxidases in different growing organs of leaf, stem and root on D. candidium under sound wave stimulation, it showed that the activities of several main anti-oxidases, APX, CAT, SOD, and POD, were elevating at large. For example, the activities of APX in leaf, stem and root increased by 15.7%, 29.5% and 21.5% respectively, those of CAT by 12.3%, 8.5% and 26.6%. It also showed that the type of different enzyme responding to the stress stimulation was not same, and the responding character on the same enzyme in different growth organs was various. For example, the activity of POD in stem was more sensitive than that in root, the activity of SOD in root was not more sensitive than that in stem. The results illuminated that it made active oxygen species (AOS) abrupt in plant cell under sound wave stress, stimulated plant cells proliferation, and promoted plant growth. Hence, we confer that AOS is a possible important signal molecular in the course of the stress signal transduction.Secondly, researches had been done on the influence of some metabolite contents in D. candidium under the sound wave stimulation. The results showed that by contrast with the control groups, the stimulated groups would get obvious increasing in the contents of polysaccharides, on average by 8.8%; total dendrobine elevated by 0.022%; soluble proteins advanced by 14.4%, which change existed the trend of first increasing and after a long time decreasing gradually; the variety of the content of free proline was not large due to taking on the trend alike wave; MDA was decreasing by 4.9%. The results indicated that the effects of metabolites in D. candidium responding to sound wave stimulation were prominent. The stimulation was in favor of accumulation of polysaccharides and enhanced soluble proteins contents in D. candidium. The sound wave stress improved probably the activities of key enzymes of metabolites, promoted or inhibited the contents of relative metabolite, so made plant adapt to the stress stimulation.Thirdly, the effects of gene expression on POD isoenzymes were detected responding to sound wave stress. New enzymatic band induced by the sound stress was not emerging, which were consistent to the test of molecular hybridization. POD genes expression could be enhanced in a long time in effect of the sound wave stress, but it might be analyzed about the sound wave acting as promotion or inhibition as to a special POD gene.Fourthly, the differentially expressed genes were screened, cloned and sequenced on D. candidium responding to sound wave stress using mRNA differentially displayed reverse transcription-polymerase chain reaction (DDRT-PCR), polyacrylamide gel electrophoresis (PAGE), silver stain and reverse Northern dot hybridization technology. The results showed that the content of total RNA in the stimulated groups contrast to that in the control elevated by 3%. Four differential gene bands appearing stable repetition, SA8, SA5, SG6, and CC7, were obtained, of which length were 552bp, 390bp, 408bp, and 496bp respectively. In the four gene fragments, SA8 was a dominantly expressive band reduced by the sound wave stress, SA5 and SG6 differentially expressive, CC7 a repressively expressive.Fifthly, the four gene sequences above were analyzed primarily on bio- informatics. The results are following, (i). In Clone-SA8 gene sequence, 18-527 nucleotide acids are coding 169 amino acids, which including an iniation codon ATG and a termination codon TAA, and being considered a short full polypeptide. Clone-SA8 protein, according to the identity of protein in GenBank by Blastp program, has highly similar, compared with the related proteins of cell division cycle protein of many animals. In the amino acid level, it has 31% identities and 50% positives with the cell cycle switch protein CCS52A (GI: AAY58271.1) of Lotus corniculatus var. japonicus, and has 31% identities and 50% positives with the Srw1-like protein (GI: CAB44330.1, CAB78235.1) of Arabidopsis thaliana when aligning protein sequence indentities. (ii). Clone-SA5 gene sequence has high identities with part sequences of some genes in alpha-tubulin gene family of multiple plants. In the gene sequence, 96-390 nucleotide acids are coding 98 amino acids, which including an iniation codon ATG and not finding termination codon. (iii). Clone-SG6 sequence has high identities with part sequences of some genes in H+-ATPase gene family of multiple plants, in which 4-408 nucleotide acids are coding 136 amino acids, including an iniation codon ATG and not finding termination codon. (iv). Clone-CC7 sequence has not been searched for any identities.Sixthly, above the amino acid sequences of the cloned fragments were analyzed and predicted on function by bio-informatics. (i). By multiple sequences alignment analyzing, Clone-SA5 and Clone-SG6 gene in D. candidium have high identities with many species and are extremely conservative in evolution. (ii). By pairwise alignment of Clone-SA8 and L. corniculatus var. japonicus CCS52A, A. thaliana Srw1-like analyzing respectively, the identities are not high, the homology of part sequences relatively high, but some breakings existing in the middle. Clone-SA8 protein includes 14 possible structural domains or functional domains, which belongs to five functional types. As to Clone-SA8 protein, the molecular weight 18.932 KD, the theoretical isoelctric point (pI) 9.67, the formula C810H1313N253O265S3, the half-life 30 hours, the instability index 68.49, the aliphatic index 62.90, the grand average of hydropathicity–1.070 which is showing a hydrophile protein. Its secondary structure is commixed, mainly consisting ofα-helix and circularity or turn. Signal peptide probability is zero. Non-secretory protein is predicted. No probable transmembrane protein is found. An appropriate coiling helix is not predicted. Six O-glycosylation sites exceeded the threshold are found out.Finally, the discussions about the correlation between stress and plant growth, metabolism and gene expression were primarily made.