Cloning And Expression Analysis Of PowderyMildew Resistance Relative Genes In Momordica Charantia L.

Powdery mildew is one of the most serious diseases in bitter gourd.Chemical control,introduction of disease-resistant varieties and breeding of new broad-spectrum disease-resistant varieties are the main means to control powdery mildew.However,chemical methods can also lead to food safety issue and environmental pollution.Resistant materials were introduced by detecting the physiological races of powdery mildew fungi,but they were no longer resistant because of the change of dominant races.Exploring broad-spectrum resistant materials may be the most effective way to solve the rapid succession of local physiological races in a short time and accelerate the breeding of broad-spectrum resistant varieties.Therefore,exploring the most effective time and mode of chemical methods and accelerating the breeding of disease-resistant varieties have become the top priority of the current work.To study the powdery mildew pathogen,physiological races and inheritance of powdery mildew resistance of bitter gourd in Hainan,and the genes related to powdery mildew resistance of balsam pear were excavated,the morphological and molecular identification methods were used to analyze the disease-producing germs and physiological races of powdery mildew,microscopes was used to observe infection process of powdery mildew,mixed major-gene plus polygenes inheritance model analysis was used to inquire into main genetic rules of powdery mildew resistance of bitter gourd.The MLO genes related to powdery mildew in bitter gourd were cloned and analyzed by bioinformatics.The tissue expression specificity of MLO genes and the genes expression in various periods under powdery mildew induction,environmental stress and hormone treatment were detected by real-time quantitative PCR.1.The results showed that the bitter gourd powdery mildew disease was caused by Sphaerotheca fuliginea which physiological races was 2F.Microscopic observation showed that there were four key stages when the pathogen infected the leaves of bitter gourd:conidia germination peak at 4 h post infection,appressorium formation peak(8 hpi),secondary hyphal formation peak(16-24 hpi)and conidiophore formation peak at 5 d post infection.The powdery mildew resistance fitted two major genes with additive-dominance-epistasis major genes and plus-dominant poly genes model,and the resistance of bitter gourd to powdery mildew disease was controlled by main genes and poly genes,in which major genes were dominate and affected by environmental variation in the meantime.2.Eight McMLO genes were cloned from leaves of bitter gourd cultivar 25 with high susceptibility to powdery mildew,named McMLOl,McML02,McMLO3,McMLO4?McML05,McML06,McML07 and McML08,respectively.The OFR of these gene were 1539 bp,1584 bp,1791 bp,1737bp,1713 bp,1719 bp,1662 bp,1620 bp in length,contained 512,527,596,578,572,553,539 amino acids,respectively.3.Prediction and analysis of McMLO gene sequences and amino acid sequences in bitter gourd:The results showed that all the McMLO proteins were composed of 22 amino acids,of which McMLOl and McMLO5 proteins were stable proteins.Alpha helix and Random coil were the main components of the secondary structure of the protein.They all contained more protein phosphorylation sites.Among them,only McML04 and MMLOIO proteins had signal peptide and its amino acid sequence was conservative and contained 6-8 transmembrane domains.There were 13-16 exons and 12-15 introns.The promoter regions contained multiple cis-acting elements that responded to different environment stress and plant hormone stress.According to phylogenetic analysis,McMLO3 and McML08 may be related to powdery mildew susceptibility.4.We detected the expression of eight McMLO genes in different tissues(tender leaves,old leaves,stems,tendrils,flowers,fruits,roots)of bitter gourd during its growth period by real-time quantitative PCR.However,eight McMLO genes in bitter gourd were expressed in all the above tissues,but these were found to differ considerably among McMLO genes,and be relatively low expressed in tender leaves.McMLOl,McML04,McML05,McML07 and McML08 were found to be the hightest expressed in roots,while McML02 in tendrils,McML03 in flowers and McML06 in tendrils.5.We detected that McMLOl and McML03 genes were expression level was significantly increased in response to powdery mildew infection,followed by McML08 by real-time quantitative PCR.This result indicated that McMLOl and McML03 may concerned in regulating powdery mildew defense in bitter gourd cultivar 25 with high susceptibility to powdery mildew.6.The results showed that the expression patterns of McMLO genes were different under stress.McML02 gene can rapidly respond to 4? low temperature stress.McML05 gene expression increased significantly at the 4 th hour of NaCl stress.McMLOl and McML05 showed significant response under mannitol treatment.McMLOl and McML07 responded slowly under SA treatment,and gene expression increased significantly at the 4 d or 7 d of SA treatment.McML03 expression increased significantly at the 7 d of JA treatment.ABA treatment at 24 th hour,the expression of McML05 and MCML04 genes were significantly up-regulated.In this study,the pathogen and race of powdery mildew in Hainan bitter gourd were identified.It indicated that the first two days after inoculation was the best time to control powdery mildew.According to the inheritance rule of resistance,F2 generation had the highest heritability and was least affected by environment,and the early generation F2 was used as the effective selection period in powdery mildew resistance breeding of bitter gourd.The McMLO family genes of bitter gourd,presume that there are differences in gene functions among different members.Some genes are involved in powdery mildew induction,and some may respond to different environmental stresses and plant hormone stress.This study provided the best period for chemical control of powdery mildew in bitter gourd,and provided a basis for further study of resistance gene and McMLO protein function of bitter gourd,and played a certain role in promoting the breeding of powdery mildew resistant varieties by using McMLO gene.