Cloning And Expression Analysis Of Genes Related To The Waterlogging In Cherry

Cerasus serrulata Lindl.has very delicious tasty,beautiful posture and also has the high economic benefits which belongs to the deciduous trees,it is planted in large area in china as high-grade fruits.However,the shallow root system which is sensitive to environmental stress,is especially in waterlogging-tolerant.The uneven rainfall,poor drainage,and waterlogging occurs in the cherry cultivation process,which causes serious economic losses.Therefore,the modern molecular biology technologies were improved,to the benefit of genetic improvement and germplasm innovation in waterlogging-tolerant of cherry.In our study,the waterlogging'Yimeng',and sensitive'Aihua'germplasm were selected.By cloning,the PsERF and PsCIPK genes which were related to waterlogging tolerance in plants,and cDNA sequence of PsPDC,PsADH,PsSUS,PsRAMY and PsEXP genes were related to carbohydrate metabolism and root system.Expression level of the above genes in resistant/sensitive waterlogged cherry' leaf and root under submmerging and recovering treatnent were studied.These results showed that the expression level of PsERF,PsADH,PsSUS,PsRAMY and PsEXP genes were consistent by waterlogging stress.Taken together,all of results verified that the pathway which respond to waterlogging stress in cherry,and the molecular mechanism of waterlogging tolerance in different cherry germplasms was elucidated.These key genes were determined that regulating cherry waterlogging tolerance,lays the foundation for molecular genetic improvement and germplasm innovation by means of genetic engineering in Cherry waterlogging tolerance.The main results are as follows:1.The young leaves of 'Yimeng' was used to extract total RNA by the CTAB method,then the full cDNA sequence of PsERF and PsCIPK gene were cloned by RACE method.The results showed that the full length of PsERF is 1625bp with the start codon,which encodes 382 amino acid.and the full length of PsCIPK is 1846bp with the start codon,which encodes 459 amino acid;By the quantitative real-time RT-PCR,the results showed that expression level of PsERF gene was increased significantly in 'Yimeng'.The expression level of PsERF gene in flood-resistant 'Aihua' germplasm was significantly lower than ' Yimeng',and the whole forms was decreased slowly.Although the expression of PsCIPK gene was decreased in the first days of submergence,the gene expression in 'Yimeng' in the rapid rise in the amount,and in the'Aihua' roots continued to decline,indicating that PsERF and PsCIPK gene and closely was related to waterlogging tolerance of cherry.2.The young leaves of 'Yimeng' was used to extract total RNA by the CTAB method,then the full cDNA sequence of PsEXP gene were cloned by RACE method.The full length of PsEXP is 1290bp with the start codon,which encodes 278 amino acid;By the quantitative real-time RT-PCR analysis,these results showed that the expression of the overall resistance of PsEXP in leaf and root of the gene in 'Yimeng' waterlogging decreased under Submergence stress,which may reduce the loss of energy in the submergence period,by reducing the expression of PsEXP gene to inhibit the growth of plants.However,the expression of PsEXP gene in ' Aihua' root submergence rise sharply on the first day,then fell rapidly and remained at a relatively low level.It may be related to the sensitivity of root waterlogging stress response.But the expression of PsEXP gene significantly increased may consume a lot of ' Aihua ' in energy storage,leading to decreased ability of waterlogging.3.The young leaves of 'Yimeng' was used to extract total RNA by the CTAB method,then the full cDNA sequence of PsPDC?PsADH?PsSUS and PsRAMY gene were cloned by RACE method.The full length of PsPDC is 2070bp with the start codon;the full length of PsADH is 603bp with the start codon;the full length of PsSUS is 2737bp with the start codon;the full length of PsSUS is 1494 bp with the start codon;With the increasing expression of PsPDC and PsADH gene,ethanol fermentation pathway will account for a greater proportion,which leads to the sunsequence that the 'Yimeng' has higher anaerobic respiration rate under submerged condition in order to provide energy for sustaining the necessary life activities.In general,the expression of PsSUS gene is decreased,which could inhibit the elongation and growth of leaves for reducing the energy loss under the stress.In addition,the expression of PsRAMY gene in the process of submergence in Cherry plants is on the rise in aggregate.In 'Yimeng',the expression of the group with water flood treatment on the forth day is about 2.2 times higher than the control group,while in ' Aihua' the expression was about 3 times higher,which illustrates that starch consumption rate of 'Yimeng' is lower than ' Aihua' so that the starch 'Yimeng5 can be better prepared to deal with substained waterlogging.4.On the basis of the previous studies,our study proposed a possible mechanism for the flood tolerance in cherry.Under waterlogg'ing stress condition,the ethylene content was accumulated,which results in the expression of PsCIPK and PsERF gene increasing in waterlogging-tolerance cherry plants.In addition,the PsERF and PsCIPK genes promoted the expression level of PsPDC,PsADH and PsRAMY genes,while inhibited the PsSUS and PsErXP expression(Fig.5-1)and plant growth and energy consumption.However,with the extension of flooding time,the starch content in flood tolerant cherry was deficient,which led to PsRAMY gene expression in the leaves,and decreased in the root system.At the same time,ethanol fermentation related genes PsPDC,PsADH expression began to rise and the capacity of metabolism was declined.In addition,'Yimeng' began to anaerobic respiration which resists the damage caused by waterlogging.