Identification Of Key Genes Involved In Metabolic Pathway Of Lotus Leaf Alkaloids

Lotus (Nelumbo) is a traditional Chinese medical herb. Alkaloids, the main bioactive constituent in lotus leaves, have been paid much attention because of their remarkable curative effects on obesity, hyperlipidemia and hypertension. Currently, although some pharmcological effects of lotus leaf alkaloids have been uncovered, the genetic and molecular mechanisms underlying alkaloids biosynthesis are still unknow. In this research, the key genes involved in the biosythesis of lotus leaf alkaloids were explored through two methods----QTL mapping and gene cloning, based on a constructed high-density genetic map and the lotus reference genome information. The main results were as follows:1. In this study, five main alkaloids content in mature leaves of the F1 population and their parents’Chinese Antique Lotus’(low alkaloids content) and America Lotus ’AL1’ (high alkaloids content) were measured by HPLC-UV/MS. Based on a well constructed high-desity genetic linkage map of lotus and the alkaloids content data detected in lotus leaves for three years (2012,2013,2014),12 significant QTLs were detected for total alkaloids content and individual alkaloids profiles in lotus leaves using IM mapping method, and then were integrated into four consensus QTLs which supposed to be controlling more than one main alkaloids biosynthesis. Four consensus QTLs are---- Con-qtl-C2-1 which controls the content of O-nornuciferne, Nuciferine and total alkaloids; Con-qtl-C2-2 which controls the content of O-nornuciferne, Nuciferine, Anonaine and total alkaloids; Con-qtl-C2-3 which controls the content of N-nornuciferne, Nuciferine and total alkaloids; Con-qtl-C4 which controls the content of O-nornuciferne, Nuciferine. Based on the lotus reference genome information,588 annotated genes belonging to the four conensus QTLs were identified as candidate genes, including 17 plant resistance relative protein genes and a CYP80B similar gene which plays key roles in the biosynthesis of benzylisoquinline alkaloids.2. Using the methods of RT-PCR and 5’RACE, three types of key genes which were supposed to play key roles in lotus leaf alkaloids biosythesis were cloned from lotus. Three NCS gene, NnNCSl, NnNCS2 and NnNCS3, were cloned, with the ORFs length of 486 bp,489bp, and 468 bp. Three CYP80B genes, NnCYP80B1, NnCYP80B2 and NnCYP80B3, contain 1458 bp,1521 bp and 1491 bp ORE NnCYP80G, a key enzyme controling benzylisoquinoline pahway flowing into amphine alkaloids, was cloned from lotus as well with 1461 bp ORE Content of five main alkaloids from six developmental leaves and four tissues of’Luminglian’and’10-48’were independently measured. Meanwhile, the expression levels of the three types of genes in all those samples were detected. Analysing the realtionships between alkaloids contents and expression levels of the genes, we got the following reuslts:(1)Leave was the main alkaloids biosynthetic tissue of the cultivar ’Luminglian’, which is regareded as ’High-alkaloids’ cultivar. Flower was the main alkaloids biosynthetic tissue of’Low-alkaloids’cultivar’10-48’, so that it was leaves contain a little alkaloids. (2)NnNCS1, NnNCS2, NnCYP80B1, NnCYP80B3 and NnCYP80G probably mainly participate in the biosynthesis of N-nornuciferine and Nuciferine, which were two major alkaloids in leaf. While NnNCS3 and CYP80B2 might only control the biosythesis of some low content alkaloids. The different expression of NnNCSl, NnNCS2, NnCYP80B1, NnCYP80B3 and NnCYP80G in ’Luminglian’ and’10-48’might cause the different alkaloids content in the two lotus cultivars. (3)NnNCSl, NnNCS2 and NnNCS3 might be the main genes that contributed to the uneven distribution of alkaloids in different tissues of both two lotus cultivars. NnCYP80B1, NnCYP80B2 may have similar influence only in’Luminglian’ and NnCYP80G may play roles in the differential distribution of different tissues in’10-48’.The key genes involved in the metabolic pathways of lotus leaf alkaloids were explored by QTL mapping and gene cloning. It is the first time to study the genetic and molecular mechnisms of lotus leaf alkaloids biosythesis, which will provide useful information for molecular breeding of lotus cultivars with high alkaloids content.