Mining And Functional Identification Of Alkaloid Synthetic Genes From Lotus Leaves

Nelumbo nucifera is an important economic crop both for its nutritional and medicinal value.China has abundant lotus resources from its wide cultivation area of approximately 10 million mu.Alkaloids are the most economically valuable bioactive components in lotus,but their content in the leaves of the main cultivated variety in China is generally lower.The genetic mechanism of alkaloid synthesis in lotus leaves has been studied,and new varieties with high alkaloid content have been developed to undoubtedly helping and increase the added value of lotus,and to promote the sustainable and healthy development of lotus industry in China.Therefore,in this study,the differences of alkaloid content in leaves of different lotus varieties and the accumulation characteristics of alkaloids in different developmental stages of leaves were analyzed.Two lotus varieties Luming and WD40 with significant differences in alkaloid content were selected for comparative transcriptome studies in the leaf.The aim of the study was to mining structural genes and important transcriptional regulators controlling genes of the alkaloid biosynthesis pathway in lotus leaves,to elucidate its regulation mechanism in lotus,and to provide both theoretical and molecular basis for developing lotus varieties with high alkaloid content.The main results are as follows:1.Characteristic analysis of alkaloid components and contents in lotus leaves of different cultivarsHigh performance liquid chromatography(HPLC)was used to detect the alkaloid components and content in the leaves of 38 lotus varieties for construction of a leaf alkaloid phenotype database.The lotus leaves mainly accumulate benzylisoquinoline alkaloids(BIAs),and contain five components: N-nornuciferine,O-nornuciferine,anonaine,nuciferine and roemerine,of which the nuciferine is the dominant alkaloid in most lotus varieties.At the same time,it was found that there were significant differences in leaf alkaloid content among different varieties.The dynamic changes of alkaloid accumulation during whole leaf development in Luming and WD40 varieties with high and low BIAs,respectively were analyzed.A gradual increase in alkaloid content was detected during leaf growth,while a small drop occurred during leaf senescence.In addition,two major BIAs components nuciferine and Onornuciferine,showed a great difference in concentration between the leaves of Luming and WD40 varieties.The remaining three alkaloids components N-nornuciferine,anonaine and roemerine showed a low concentration in the lotus leaf throughout the whole developmental stages with a less significant variation between the two cultivars.2.Mining and functional analysis biosynthetic pathway and transcriptional regulation genes involved BIA in lotusThere have been few reports on the biosynthesis and regulation of lotus BIA.In this study,two cultivars leaf samples of Luming and WD40 were selected as the test materials.The bioinformatics analysis of the two stages by comparative transcriptomics in the early stage of leaf development revealed the structural genes of the biosynthetic pathway of lotus BIA,two of the tandem clusters of CYP80 structural genes NnCYP80 A and NnCYP80 G have undergone functional differentiation,which may be involved the biosynthesis of bis-BIAs and aporphine-type BIAs.At the same time,five alkaloid synthesis transcriptional regulators including three MYB genes,NnMYB12,NnMYB6,and NnMYB113,an ethylene-response factors(ERF)NnRAV1 and a basic helix-loop-helix(bHLH)NnbHLH1 gene were mining.The results of dual luciferase reporter assay showed a good activation effect of these regulatory genes on the structural genes of lotus alkaloid synthesis.Meanwhile,we found that NnbHLH1 interacts with NnRAV1 and showed a strong activation on the NnNCS1 promoter.In summary,the above studies revealed both biosynthesis pathway and transcriptional regulatory mechanism of BIA in lotus,which not only has important theoretical significance for deeper understanding the biosynthesis of plant BIA,but also provides technical support for the development of new lotus varieties with high alkaloids content.