Preliminary Study of CsERF in Regulating Waterlogging Tolerance in Cucumis Sativus L
|Keywords||Cucumber waterlogging tolerance ethylene ERF ADH 1-MCP ethephon|
Cucumber (2n=14, Cucumis sativus L.) is a main kind of vegetable throughout the world. China is a big cucumber-producing country, and has a long history of cucumber cultivation, also generates obvious economy benefit.Cucumber is native to tropical regions, has the characteristics of liking wet soil but not resistant to waterlogging stress. Because of shallow root system and poor aerenchyma, cucumber is easily affected by waterlogging. The frequently occur seasonal rainfall and sudden burst of rainfall in the southern China along the downstream of Yangtze River not only affect open field cultivated cucumber, but also under facilities cultivated cucumber, which lead to yield loss and decline in quality. Hence, waterlogging tolerance improvement has become a burning question in cucumber breeding. Combining with the application of modern molecular biotechnology to innovate germplasm and breed waterlogging tolerant varieties, are effective ways to overcome the damage caused by waterlogging and produce high yield and good quality cucumbers. In this study, we take advantage of different stress treatment and combine with the using of promoter and inhibitor of ethylene to research the expression patterns of ERF and ADH under waterlogging condition in waterlogging tolerant line "Zaoer-N". The major research results were as follows:1.123pairs of ERF family genes were getted form cucumber genomic database, and divided into ten types by referring to classification method of Arabidopsis ERF family. In which, the7th class contain three genes, and were named as CsERF1, CsERF2and CsERF3, respectively. The overall sequences of the three genes were getted by homology-based cloning methods; all shared the100%similarity between "Zaoer-N" with sequences published in cucumber genomic database.2. The rates of ethylene-released quantity were determined, and the relative expression levels of ACS and ACO were analyzed in the root of Zaoer-N after12hours of waterlogging treatment by gas chromatograph, which plays an important role in controlling the ethylene synthesis. The results showed that:(1) the rates of ethylene release quantity in the root were obviously rise after12hours of waterlogging treatment when compared with CK;(2) the rates of ethylene release quantity in the root were obviously decreased when combining with ethylene synthesis inhibitor AgNOs;(3) the relative expressions of ACS were obviously up-regulated, and reach the peak level after12hours of waterlogging treatment, then gradual decline;(4) the relative expressions of ACO were gradually up-regulated, and reach the peak level after12hours of waterlogging treatment, and keep relatively stable during12and48hours. Up-regulated of these two genes accelerates the synthesis of ACS and ACO enzymes, which will in hence speed up the release of ethylene.3. The relative expression levels of CsERFl, CsERF2and CsERF3were analyzed by using RT-PCR methods after waterlogging treatment in Zaoer-N. The results showed that:(1) the relative expressions of CsERFl were obviously up-regulated under waterlogging condition, and reach the peak level after12hours of treatment, then gradual decline.(2) The relative expressions of CsERF2were without regularity.(3) The relative expressions of CsERF3were low and irregularly. The results indicate that CsERF1may involve in regulating watlerlogging response, while CsERF2and CsERF3are not involved in the response.4. The relative expression levels of CsERF were analyzed after4and12hours treatment of different concentration of agaropectin solutions. The results showed that:(1) the relative expression levels of CsERF1were obviously rise along with the rise of concentration of agaropectin solutions.(2) The relative expression levels of CsERF2and CsERF3were also low and irregularly. The results indicate that CsERFl is closely related to the DO content.5. The relative expression levels of waterlogging tolerant mark gene ADH was analyzed after waterlogging stress by using RT-PCR. The results showed that:(1) the relative expression levels of ADH is not obvious in controll material.(2) The relative expression ratio was increased significantly in Zaoer-N, and reaches the peak level at12hours, then gradual decline. The results indicate that ADH also involve in regulating watlerlogging response.6. The relative expression levels of CsERF1and ADH were analyzed after12hours waterlogging and pretreatment with1-MCP and ethephon. The results showed that:(1) Pretreatment with1-MCP can inhibit the expression of CsERFl and ADH under waterlogging condition.(2) Pretreatment with ethephon can promote the expression of CsERFl and ADH. Based on these experiments, we further confirmed that CsERFl is involved in waterlogging response, and may achieve the process by controlling of the expression of ADH.7. The relative expression levels of CsERFl, CsERF2, CsERF3and ADH were also analyzed under waterlogging condition in vegetative stage and flowering stage. The results showed that:(1) CsERF1and ADH share the same expression patterns under waterlogging condition in different growing stages.(2) The relative expression levels of CsERFl and ADH were:seeding stage<vegetative stage<flowering stage;(3) Both CsERF2and CsERF3were irregularly. The relative expressions of these two genes in flowering stage are both higher than vegetative stage, this may due to great energy and nutriment demand in the stage, and required more energy to maintain survive.