Studies On The Functions Of HbMlo Conferring Broad-spectrum Resistance To Powdery Mildew In Hevea Brasiliensis And Its Application

Growth and development of rubber tree is affected by multiple biotic and abiotic stresses, in which powdery mildew is an important leaf disease of rubber tree, which can lead to largely defoliate and cause huge economic losses to the growth and yield of the rubber tree. Mildew resistance locus o (Mlo) is a kind of plant-specific gene. Mlo gene can involve in a variety of cellular processes. The recessive allele mlo has a durable resistance to powdery mildew. But the Mlo gene and its functions are rarely reported in rubber tree. In order to investigation the structure and function of Mlo gene of rubber tree, this thesis identified rubber tree Mlo gene from the transcriptome database of rubber tree leaf by bioinformatic analysis tools. The HbMlos were Cloned,identified and analyzed their structure and function by molecular biology technique.Polymorphic analysis was performed by screening varieties and used to develop molecular markers related to the disease-resistant varieties. Analyzed the expression profiles of the cloned HbMlos gene under powdery mildew infection, stress and plant hormone treatments. Subcellular localization of HbMlol2 protein was determined by transient expression technique. The transgenic Arabidopsis plants of HbMlol2 gene were obtained by genetic transformation. The phenotype and development of transgenic plants and the responses to drought stress and powdery mildew infection were analyzed.The results are as follows:1 .Two new Mlo genes were cloned from rubber tree and may be involved in stress response process in rubber tree.HbMlo1-1 and HbMlo7 were hydrophobic proteins and consisted of 510 and 499 amino acids, respectively. They were predicted to locate in endoplasmic reticulum membrane. They had a conserved domain of Mlo family, and seven transmembrane domains. HbMlo1-1 and HbMlo7 were both expressed in stem, leaf, flower, latex and bark. After powdery mildew infection, the expression of HbMlol-1 was upregulated,but HbMlo7 expression was downregulated. The expression of HbMlo1-1 and HbMlo7 were inducd by wounding and drought stress as well as hydrogen peroxide, abscisic acid, ethephon and methyl jasmonate tratments. But they had no conserved domains which associated with Mlo proteins conferring powdery mildew resistance.2.Structural and expression analysis of HbMlos in rubber tree and Mlo of other species indicated that HbMlol2 contained two conserved domains and calmodulin-binding protein regions which associated with Mlo proteins conferring powdery mildew resistance. HbMlo12 and Mlo (SlMlo1、AtMlo2、AtMlo6、AtMlo12 and so on) which are known to have broad spectrum resistance, were clustered on the V group. The expression of HbMlo12 was upregulated after powdery mildew infection, suggesting that HbMlo12 was most likely the Mlo gene associated with powdery mildew resistance in rubber tree.3.The expression of HbMlos were different in diferent varieties of rubber tree. The expression of diferent HbMlos were also different in the same cultivars of rubber tree.Their expression was not correlated with the resistant or susceptible varieties of rubber tree.4.HbMlol2 was located on endoplasmic reticulum membrane, and the homozygous transgenic Arabidopsis plants overexpressing HbMlo12 was obtained. The growth of transgenic plants HbMlo12/Col-0 and HbMlo12/Atmlo2-9 was worse than that of wild plants Col-0. This result suggested that HbMlol2 effected the growth and development of transgenic Arabidopsis. Under drought stress, the bolting time of transgenic Arabidopsis was later than that of wild type. HbMlo12 enhanced the drought resistance of transgenic Arabidopsis at seedling stage to a certain extent. The susceptibility of four Arabidopsis strains under powdery mildew infection were as follow: Col-0 > HbMlo2/Col-0 > HbMlo2/Atmlo2-9 > Atmlo2-9. It can be deduced that HbMlo12 effected the affinity interaction between transgenic Arabidopsis and Oidium heveae.5.There were 47 SNP loci associated with powdery mildew resistance by comparing the sequence of HbMlo12 in different varieties. Among them 41 were single base mutations, multiple base-pair mutations were occurred at the 2762th, 2917th and 2922th loci, multiple base-pair insertions were present at the 2007th and 2781th loci,and multiple base-pair deletions were occurred at the 960th locus.Based on the above results, we concluded as follow:1.HbMlo1-1 and HbMlo7 were belong to the members of Mlo superfamily. They were associated with drought stress and wounding, and hormone stimulation.2.The Mlo family had functions in the growth of rubber tree, hormone stimulation and abiotic stress response, in which HbMlo12 was the Mlo gene associated with powdery mildew resistance in rubber tree.3.The study of transgenic Arabidopsis found that HbMlo12 was involved in the growth and drought stress response in transgenic Arabidopsis. HbMlo12 increase the susceptibility of transgenic Arabidopsis to rubber tree powdery mildew. The result suggested that HbMlo12 was a candidate gene for the broad-spectrum resistance of powdery mildew in rubber tree.4.Through the sequence analysis of HbMlo12 in different resistance or susceptible germplasms, 47 polymorphic locis were found and could be used to develop SNP markers to screen powdery mildew resistant germplasm.This study clarified the function of HbMlos gene family in the powdery mildew and stress rsponse in rubber tree. It provided a theoretical basis for studying the molecular mechanism of rubber tree resistance to powdery mildew,and a basis for screening and cultivating the germplasm with powdery mildew resistance in rubber tree.