| Plants without the capacity to move have evolved mechanisms to survive ever changing biotic and abiotic environmental stresses. One of such mechanisms by which plants cope with these stresses is through the production of defensive secondary metabolites. Over the past two decades, glucosinolates(GLSs) present almost exclusively in the order Brassicales, including model plant Arabidopsis thaliana, have been used as a model system for the study of secondary metabolite diversity in plants. GLS are sulphur-rich, nitrogen-containing, amino acid-derived compounds that are involved in aiding the plant to resist a myriad of attacking herbivores and pathogens and cancer prevention in humans. The diversity of stresses has led to an equal diversification of GLSs with a huge number of lineage and species specific compounds, which are also influenced by other defense compounds. As a complex metabolic pathway, the regulation of GLS biosynthesis is still largely unkown. Brassica napus is one of the most important oilseed crop, which is an allotetraploid and shared a common ancestor with Arabidopsis thaliana. Most of GLS biosynthetic genes‘ function have not been well studied in B. napus. In this study, we carried out follow researches: 1. We found two conserved transcription factors(TFs), MYB115 and MYB118, control expression of the newly evolved benzoyloxy GLS pathway in Arabidopsis thaliana. 2. Analyze the functions of Bn MAM1, Bn CYP83A1 and Bn UGT74B1 involved in the GLS biosynthesis within B. napus. 3. Identify the resistance of Bn MAM1-, Bn CYP83A1- and Bn UGT74B1-overexpressing lines to Sclerotinia sclerotiorum and Botrytis cinerea. The main results of this study are as follows:1. Using a comprehensive strategy of co-expression analysis, we found that two conserved TFs, MYB115 and MYB118, were highly co-expressed with BZ-GLS-related genes(AOP3, BZO1 and SCPL17). We further validated the potential regulatory roles of MYB115 and MYB118 in the aliphatic GLS biosynthetic pathway by yeast one-hybrid study and q RT-PCR analysis. The GLS profiles of these two TFs knockouts showed that they influenced all AOP3/BZO1 related GLSs(Methylsulfinylalkyl, hydroxyalkyl and benzoyloxy GLS) accumulation within seed. MYB118 and MYB115 also can alter the accumulation of aliphatic GLS within the leaf even with low residual transcript accumulation. And all flower organs of myb115 and myb118 mutants had increased levels of short chain MS-GLSs. Interestingly, we did not find any changes in indolic GLSs in organs of seed, leaf and flower. Complementation study confirmed their roles in regulating the aliphatic GLS biosynthesis. These results thus strongly support the roles of MYB115 and MYB118 in regulating aliphatic GLS biosynthesis.2. There was a statistically significant effect of the myb115myb118 double mutant on some aliphatic GLSs, and MYB115 had a repressive activity on these related genes that was redundant with MYB118. MYB115 and MYB118 also appear to lead to different shifts in GLS partitioning when abolishing parts of the BZ-GSL pathway. A myb118aop3 double mutation results in a large amount of accumulation of 4MSOB GLS but no corresponding increases in 4MTB whereas the myb115aop3 pushed the GLS to the 4MTB form. The accumulation of the short-chain OH-GSLs in myb118bzo1 double mutant increased at the expense of the MT and MS-GSLs, while there was only a minor difference in glucosinolate contents examined between myb115bzo1 double mutant and bzo1 single mutantAll these results indicated that MYB115 and MYB118 epistatically interacted to modulate the expression of the respective GLS genes and their corresponding metabolites in Arabidopsis seeds. MYB115 and MYB118 are conserved regulators in Arabidopsis genome and involved in the regulation of newly evolved biosynthetic pathways of glucosinolates.3. We cloned three GLS biosynthesis genes in B. napus, including Bn MAM1 and Bn CYP83 A which are involved in aliphatic GLS biosynthesis, and Bn UGT74B1, which takes part in both aliphatic and indolic GLS biosynthesis. We detect the expression of the three genes in response to pathogen infection by q RT-PCR. The results show that both Bn MAM1 and Bn CYP83A1 transcription were induced in the early stage of S. sclerotiorum infection while Bn UGT74B1 was up-regulated in whole stages of infection. After inoculation with B. cinerea, the expression of Bn MAM1 and Bn CYP83A1 was continuously down-regulated from the beginning, conversely, the expression of Bn UGT74B1 was up-regulated from the beginning to the end. These results show that the response models of aliphatic and indolic GLS biosynthesis may be different when the plants resist against the infection of these two necrotrophic pathogens, S. sclerotiorum and B. cinerea. Compared with wild type, the contents of two aliphatic GLSs(2OH3B and 3-Butenyl GLS) were increased in all the three gene overexpressing lines, in addition, the Bn UGT74B1-overexpressing lines showed increased content of indolic GLS(I3M GLS). These results further describe the functions of these three genes for GLS biosynthesis in B. napus.4. The results of plant inoculation demonstrated that more than 90% of wild type control leaves were infected as early as 16 h post-inoculation(hpi)(S. sclerotiorum) and 32 hpi(B. cinerea), while necrosis were observed in less than 12% of Bn UGT74B1-overexpressing plants at the same time. And the Bn UGT74B1-overexpressing lines also developed less severe disease symptoms and less tissue damage than the control at 48 hpi(S. sclerotiorum) and 96 hpi(B. cinerea). But the Bn MAM1 or Bn CYP83A1-overexpressing lines showed no significant difference from the control on lesion development and tissue damage after inoculation with the pathogens.These results suggest that the resistance to S. sclerotiorum and B. cinerea in B.napus could be enhanced through tailoring the GLS profiles by transgenic approaches or molecular breeding, which provide useful information to create resistant breeding materials and design improved breeding strategies. |
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