| The Prunus campanulata Maxim.(P. campanulata), which belongs to the Rosaceae family, blooms early, around the Chinese Spring Festival; its flowering period can be as long as50d, the bright colour of the particular campanulate flowers confers an extremely high ornamental value on P. campanulata, and its strong resistance and wide adaptability also contribute to it being an excellent native ornamental plant. This species originated in Fujian, Zhejiang, Guangdong, Guangxi, Taiwan in subtropical regions in which the threat of low-temperature damage is not present. However, this species will suffer frost damage if introduced from origination area to the Yangtze River basin and northern areas. Low temperature is one of the most important abiotic factors limiting the growth, development and distribution of plants. Low temperature is also one of the most important limiting factors affecting P. campanulata when it is planted at higher latitudes.After exposing the one-year-old P. campanulata seedling to low temperature stress, physiological and biochemical indexes were measured in leaves to detect the boundary temperature and time of stress. From the physiological and biochemical point of view, the mechanism of P. campanulata response to low temperature stress is clarified, based on physiological and biochemical tests, A full-length cDNA library is established using the young leaf of P. campanulata treated with1℃3d and sequencing a large number of ESTs. Cold-related genes were screened through ESTs sequencing and bioinformatic analysis and analyzed by real-time quantitative PCR. The main purpose of this study is to investigate the cold-resistance mechanisms and lay a foundation for resistance breeding of P. campanulata. The specific findings are as follows.1. The relative chlorophyll content and chlorophyll fluorescence parameters were measured in leaves to detect the influence of temperature on the photosynthetic activity. Compared to the control, P. campanulata exhibited a continuous increase in initial fluorescence (Fo) and decrease in chlorophyll content, maximum photochemical efficiency (Fv/Fm) and photochemical quenching coefficient (qP) with decreases in temperature. The actual photochemical quantum (yield) and electron transfer rate (ETR) in the15℃treatment were significantly higher than those in the control, while they declined under the6℃and1℃ treatment over time. The non-photochemical quenching coefficient (qN) increased under the15℃and6℃treatments and decreased under the1℃treatment. The most significant changes in the parameters occurred under the1℃treatment, indicating that P. campanulata exhibited low-temperature photoinhibition. The results indicated that P. campanulata could protect the photosynthetic apparatus through the increased dissipation of excess light only within a certain range of temperature and stress time.2. After Exposuring of P. campanulata seedlings to different temperature, electrical conductivity, the activities of superoxide dismutase (SOD), peroxidase(POD), catalase(CAT), the content of malondialdehyde (MDA), soluble protein, soluble sugar and free proline were measured in leaves to detect the influence of temperatures on the physiological and biochemical characteristics. The results showed that the content of eight indexes have a signifcant difference with control. Electrical conductivity and the content of MDA exhibited a upward trend under different temperature over time; other indexes sustained a slight increase under the10℃treatment and firstly rose then went down under the6℃and1℃treatment over treatment time. The highest values of SOD, soluble protein, soluble sugar and free proline occurred on day3of1℃treatment. At the beginning of the stress, P.campanulata could reduce the harm through self-regulation mechanism, however, it will cause metabolic disorders and irreversible damage if the protection of antioxidant enzymes and osmolytes protective on membrane systems and regulatory mechanisms exceed a certain limit.3. The content of GA3(Gibberellic acid), IAA (indoleacetic acid), ABA (Abseisic acid) were measured in leaves to detect the influence of temperatures on the endogenous hormones. Compared to the control, P. campanulata exhibited higher contents in GA3、IAA and no significant difference in ABA in the10℃treatment, while GA3、IAA content were lower and ABA、ABA/GA3were higher significantly than control under the6℃and1℃treatment. GA3、IAA content exhibited a continuous decrease and ABA、ABA/GA3firstly rose then went down over treatment time under the6℃and1℃treatment. Under low temperature, P. campanulata can adjust the balance of endogenous hormones in plants to cope with low temperatures.4.19physiological and biochemical indexes were analyzed by factor analysis, as a result, comprehensive maximum efficiency of P. campanulata was the treatment of1℃for3days according to composite score, the growth of P. campanulata seedlings would be inhibited over this node.5. Based on SMART(switching mechanism at5end of RNA transcript) technology, a full-length cDNA library was constructed form the tissue of young leaves of Prunus campanulata Maxim, treated with1℃3d. The results showed:the titer of the cDNA library was1.2×106cfu·mL-1, the average size of inserted cDNA fragment was1.3kb, the recombination rate was94%using blue-white screening, however, it was100%detecting by PCR. To evaluate EST information,834clones were single-pass sequenced and667valid sequences were generated, in which574singlets and93contigs were obtained after initial assembly with SeqMan program. Based on the results of Blast analysis,475unigenes were found to significantly match the proteins with known and putative function. With Blast2GO annotation,475unigenes were found to cover most of the biological functions and express a specific period information (low temperature stress). On the basis of similarity search and GO annotation,84unigenes were found responsive to biotic and abiotic stresses. Homology blast results show that the ESTs encoded amino acid sequence of catalase (CAT), allene oxide cyclase (OsAOC), heat shock protein (HSP), dehydration response proteins (DRP), lipoxygenase (LOX), zinc finger protein, calcium-dependent protein kinase, MYB transcription factor and other defence responses related proteins have a more high homology.475unigenes with known and putative function were distributed into functional categories with Blast2GO and generated1597GO terms (3categories)."Biological Process" was the major functional category, containing750GO terms (46.93). Second larger functional category was "Cellular Component", constituted by620GO terms (38.8%),227GO terms (14.27%) were classified into the "Molecular Function" category.6. Based on the ESTs analysis,12putative cold-related genes of P. campanulata were chosen to measure the expression pattern by qRT-PCR with gene specific primers designed with primer premier software (version5), the following is the results. Compared to the control, the expression levels of DRP, MYB, HSP, DRH56, GPX, GA20-ox, CAB, LOX were obviously induced by treatment temperature, while the transcript level of AOC, TIL, CDPK were lower in the treatment group than in the control group, the expression of CAT gene was induced only in6℃treatment. On one hand, the results indicated that cold related genes of P.campanulata involved in signal transduction, active oxygen scavenging process, transcription, stress and defense process, while plant photosynthesis, water, ion transport, energy transfer processes were verified to be effected by low temperature; on the other hand, these cold related genes may play different roles in low temperature stress. |
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