Polyamines Changes After Inoculation With Powdery Mildew And The Functional Identification Of CmSAMDC Gene In Melon

Melon (Cucumis melo L.) belongs to the genus of cucumis and the family ofcucurbitacea. It is an annual vine herbaceous plant and an important type of fruit vegetablecrops widely cultivated in our country and around the whole world. However, with theexpansion of its planting area, powdery mildew has become one of its main obstacle inplanting, and influence the yield and quality of melon very seriously. Meanwhile，thefungicide was extensively used which could result in the drug resistance of pathogens, as wellas the environment pollution. Therefore, it is a practical method to control this disease byexcavating and utilizing the resistance genes of the resistant germplasm to breed new varieties.The previous studies have shown that the Chinese wild melon ‘Yuntian930’ was a resistantvariety to Podosphaera xanthii2F., and lots of different transcriptions were discoverd in itsinteraction with powdery mildew by cDNA-amplified fragment length polymorphism(cDNA-AFLP) method. Their functions were involved in the transcription regulation,polyamine synthesis and metabolism, signal transduction, etc. However, it remained limitedin understanding the resistance mechanism of the resistant germplasm, especially in realizingthe mechanism of physiological and biochemical systematicly in the molecular level.In present study, we firstly made a comparative observation in leaf structure between thedifferent resistant melon materials. Meanwhile, after the treatment with exogenousspermidine(Spd) and dicyclohexylamine(DCHA) to the different resistant melonmaterials(resistant cultivar ‘Yuntian930’ and susceptible cultivar ‘0544’), we also discussedthe disease index, photosynthetic characteristics, antioxidant system, polyamines metabolismand their genes expression, explored the roles of leaf structure, activities of antioxidantenzymes, endogenous polyamine contents and function of exogenous polyamine in melonresisting to powdery mildew. And then we systematically analysed the differential expressiongenes in ‘Yuntian930’ at different times after inoculation with powdery mildew by digitalgene expression profiling(DGE) method. Following this, the CmSAMDC gene was clonedfrom ‘Yuntian930’, and then it was heterogenously expressed in Arabidopsis thaliana toverify its role in resistance to powdery mildew. The main results were as follows:1. By the observation of leaf structure, it was shown that the density of the stings, leaf tightness, the ratio between palisade tissue and spongy tissue were higher in resistant cultivars‘Yuntian930’ and ‘0426’ than in susceptible cultivars ‘0544’ and ‘066’, with the verysignificant level in former one and significant level in latter two indexes. The wax contents ofthe resistant cultivars were also higher than those of the susceptible cultivars. However, therewas no obvious regularity in the thickness of epidermal cells, the thickness of palisade andspongy tissues, the size and density of stomas between the different resistant cultivars. Thisindicated that the sting, palisade tissue and waxy of melon leaf could act as a kind of structurebarrier to prevent the powdery mildew invasion, and the leaf tightness, ratio of palisade tissueand spongy tissue, the shape and density of leaf sting could be used as auxiliary appraisalindex in melon resistance breeding process.2. After inoculation with the Podosphaera xanthii, the activities of polyphenoloxidase(PPO), peroxidase(POD), catalase(CAT) enzymes and the contents of hydrogenperoxide(H2O2), as well as the up-regulated expression of S-adenosylmethioninedecarboxylase(SAMDC), ornithine decarboxylase(ODC), arginine decarboxylase(ADC),polyamine oxidase(PAO) genes were higher and earlier rise in ‘Yuntian930’ than in ‘0544’,and the putrescine(Put), spermidine(Spd) and spermine(Spm) contents were in the same trend.This demonstrated that the resistant cultivar could better coordinate the gene expression ofpolyamines and the contents of endogenous polyamines, and motivate the defense enzymesactivities in response to pathogen infection.3. The application of exogenous Spd could induce the genes related to polyamineup-regulated expression and the contents of Put, Spd and Spm increased in melon, especiallythe contents of Spd and Put increased obviously. When infected by powdery mildew, themelon seedlings could eliminate the excess reactive oxygen by increasing the activities ofPPO, POD, superoxide dismutase(SOD) enzymes, and generate the disease-resistant materialsto defense the powdery mildew infection. More interestingly,1mM concentration of Spdcould enhance this resistance reaction, and then alleviated the disease symptoms in melonseedlings.4. The application of5mM concentration of DCHA in melon resulted in the decrease ofSpd contents within48hours, and the Put contents did not change, but promoted theaccumulations of Spm. After48hours, with the increase of genes expression related topolyamines, the three kinds of polyamines all increased to some different extent. Theexogenous DCHA also induced the increase of POD, SOD and PPO enzyme activities andreduced the disease index of infected plants.5. After inoculation with powdery mildew, there were219differential expression genes at24h, then increased to1784at48h, and top to2371at72h, in which the up-regulatedexpression genes were more than the down-regulated expression genes. The differentialexpression genes were involved in photosynthetic system, protein and nucleic acidmetabolism, secondary substances metabolism, carbon metabolism, cell signal transduction,etc. We also found there were five genes related to polyamine biosynthesis and one generelated to polyamine metabolism were up-regulated significantly at48h after inoculation.This suggested that the polyamine synthesis and metabolism pathways were involved in theresponse process of melon to powdery mildew infection.6. The SAMDC gene was cloned from melon, with an intact open reading frame of1095bp and364deduced amino acids sequence. It contained two obvious conservative domainstructures of this kind of genes. In addition, it showed seven bases difference in nucleotidesequence coding three different amino acids beyond their conservative domains. The SAMDCgene of ‘Yuntian930’ was renamed as CmSAMDC. It was subcellular localized in thecytoplasm cell, nucleus and membrane, and it was successfully expressed in E. coli strainBL21(DE3). Then the35S:: CmSAMDC over-expression vector was constructed andheterologously expressed in Arabidopsis thaliana. Interestingly, the transgenic plants withCmSAMDC gene increased the resistance to powdery mildew as well as salt stress, and theincrease of hormones were also discovered.