| Magnolia officinalis difference subtracted cDNA library construction and sequence analysis can effectively identify the main cold resistance related genes and analysis of metabolic pathways. Then the plants which have close relationship with Magnolia officinalis genetic resistance to low temperature characteristics of genetics and breeding to provide theoretical basis, and help us to study the natural cultivation of plants. The experiment was done three times in order to reduce the single proportion of non cold resistance related genes in the library. Specific research results are as follows:1、Magnolia officinalis in different temperature conductivity were measured using logistic equation Y=K/(1+aebx) were fitted to the data processing, the regression equation is:y=0.7526/ 1+18.925e-0.372x.Half lethal temperature is-7.93℃.2、Take two pots of Magnolia officinalis seedlings each 25℃ artificial climate incubator cultured for 7 days, seven days will be a pot of Magnolia officinalis stay in an artificial climate culture box, as a driver.3 days in the artificial climate incubator which was transferred from-8 to C in the growth stage, and was used as the Tester group. The total RNA was extracted by Trizol method, and the total length of cDNA was synthesized by SMART method. After two rounds of subtractive hybridization and two rounds of suppression amplification were obtained. The average length of the inserted fragment was 0.65 KB, and the recombination rate was 99.9%.3、After three repeated experiments sequencing received a total of 837 strip can be used for subsequent analysis of the EST sequences, the 837 EST sequences respectively for clustering splicing 286 unigene-1, unigene-1 in the NR database were compared, selected collection of genes that unigene-1 alignment results are the same or similar 171 unigene-2, unigene-2 second stitching of 60 unigene-3.4、Take unigene-3 blast in NCBI. Annotation results show that there are many related proteins such as zinc finger family protein, zinc finger protein, ethylene response transcription factor, peroxidase and so on.5、Take unigene-3 blast in blast2GO of functional classification, the results were obtained 144 GO terms these GO terms classified into three categories:the biological process GO terms most (67), accounted for 46.52%, followed by molecular function (42), and cell components (35), respectively, accounting for 24.31% and 29.17%. Species similarity was the highest with the lotus grapes, followed by jatropha, citrus, plum, pepper, Populus, palm oil and other.144 GO term directly involved in six of the defence reaction to external stimuli, such as:the stimulus response, signal, stimulate cell reaction and nucleic acid binding transcription factor activity, oxidoreductase activity, phosphatase activity, accounting for 4.16%.6、After 60 unigene-3 of the information processing. According to results of the NCBI database comparison results and the go functional classification, and selection of the 11 most likely genes involved in cold stress defense of FLS gene, F-box gene, LHCB1 gene, WRKY gene and HSP70 gene and APX genes and PP2C and TRX gene, ERF gene, DREB gene and GAPDH Gene.7、Fluorescence quantitative PCR showed that the F-box, APX, TRX, ERF, DREB gene are with the increasing temperature of the lower expression, LHCB, WRKY, HSP, PP2C, GAPDH and-8 degrees Celsius temperature due to temperature too low metabolism is inhibited the expression of decline. The gene expression of ERF increased with the decreasing of the temperature, and the correlation between the two genes and the temperature was proved to have a strong correlation with the temperature. But when the temperature is too low, the plant metabolism is blocked, the expression of gene is inhibited, which is due to the low temperature damage the normal metabolism of the gene.8、Eventually concluded the F-box, APX, TRX, ERF, DREB and LHCB, WRKY, HSP, PP2C, GAPDH in Magnolia officinalis by low temperature injury may exhibit the defense mechanism. |
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