Cloning And Expression Of Lignin Genes In Citrus Maxima (Burm.)Merr

Pummelo [Citrus maxima (Burm.) Merr.] belongs to rutaceae, which is the greatest sort of citrus fruits, and very popular in consumers. Juice sac granulation seriously hinders commodity value of pummelo fruit. In this study,’Guanximiyou’ was used as material. Genes involved in lignin biosynthesis were cloned by RT-PCR and RACE techniques. In order to explore the relationship between lignin biosynthesis and Juice sac granulation, expresstion levels in different developmental stages and parts of pomelo fruit were analyzed by Real Time PCR. The main results were as follows:A total of11lignin related genes from pummelo leaves and one from juice sac were obtained by homology cloning method. Two genes including cmC4H and cm4CL were full-length cDNAs. Five genes including cmCCoAOMT1, cmCCR, cmC3H, cmF5H and cmCAD1were partial cDNAs with ORF, and cmPAL, cmHCT, cmCCoAOMT2, cmCOMT(from leaf) as well as cmCAD2were cDNA fragments.The sequence of cmPAL contained1318base pairs encoding436amino acids. Blast result showed that the similarity with Citrus limon, Ricinus communis, Rhus chinensis, Manihot esculenta, Populus trichocarpa and Theobroma cacao was over91%. Prediction result indicated that cmPAL belongs to transmembrane protein located in extracellular probability.The cDNA of cmC4H contained1650base pairs with an open reading frame1518base pairs encoding519amino acids. Blast result showed that the similarity with Citrus sinensis, Canarium album, Camellia chekiangoleosa, Populus trichocarpa and Gossypium arboretum was over84%. Prediction result indicated that cmC4H belongs to transmembrane protein located in endoplasmic reticulum probability.The cDNA of cm4CL contained1390base pairs with an open reading frame1041base pairs encoding384amino acids. Blast result showed that the similarity with Citrus sinensis, Theobroma cacao, Salix arbutifolia, Populus trichocarpa and Populus tomentosasimilarity was over78%. Prediction result indicated that cm4CL belongs to transmembrane protein located in plasmalemma probability.The sequence of cmHCT contained1018base pairs encoding334amino acids. Blast result showed that the similarity with Populus nigra, Populus tomentosa, Hibiscus cannabinus and Gossypium hirsutum similarity was over84%. Prediction result indicated that cmHCT belongs to transmembrane protein located in plasmalemma probability.The sequence of cmC3H contained1527base pairs encoding508amino acids. Blast result showed that the similarity with Ruta graveolens, Populus tomentosa, Caragana korshinskii and Populus nigrasimilarity was over87%. Prediction result indicated that cmC3H belongs to transmembrane protein located in plasmalemma probability.The sequence of cmCCoAOMT1contained947base pairs with an open reading frame744base pairs encoding247amino acids. The sequence of cmCCoAOMT2contained729base pairs encoding192amino acids. Blast result showed that the similarity with Eriobotrya japonica, Pyrus x bretschneideri, Gossypium hirsutum, Betula luminifera and Dimocarpus longan similarity was over91%. Prediction result indicated that cmCCoAOMTl belongs to transmembrane protein located in extracellular probability; cmCCoAOMT2belongs to transmembrane protein located in endoplasmic reticulum probabilityThe sequence of cmCCR contained1224base pairs encoding339amino acids. Blast result showed that the similarity with Gossypium hirsutum, Vaccinium corymbosum, Capsicum annuum, Populus trichocarpa and Populus nigra similarity was over86%. Prediction result indicated that cmCCR belongs to transmembrane protein located in endoplasmic reticulum probability.The sequence of cmF5H contained1545base pairs encoding514amino acids. Blast result showed that the similarity with Theobroma cacao, Populus trichocarpa, Populus trichocarpa, Camptotheca acuminata and Liquidambar styraciflua similarity was over80%. Prediction result indicated that cmF5H belongs to transmembrane protein located in extracellular probability.The sequence of cmCOMT contained394base pairs encoding130amino acids. Blast result showed that the similarity with Citrus aurantium, Gossypium hirsutum, Hibiscus cannabinus and Populus tomentosa similarity was over82%. Prediction result indicated that cmCOMT belongs to transmembrane protein located in Plasma Membranes probability.The sequence of cmCADl contained984base pairs with an open reading frame984base pairs encoding327amino acids. The sequence of cmCAD2contained554base pairs which was capable of encoding130amino acids. Blast result showed that the similarity with Populus trichocarpa, Malus domestica, Populus trichocarpa, Prunus mume and Rosa rugosa similarity was over81%. Prediction result indicated that cmCADl belongs to transmembrane protein, located in endoplasmic reticulum probability; cmCAD2belongs to transmembrane protein located in extracellular probability.Using β-Tubulin as internal control gene, the quantitative analysis of10lignin genes were studied by Fluorescence quota PCR(qPCR). This study indicates the expression quantity of3genes—cmCCR, cm4CL and cmCAD1—in juice sac near the column were higher than in juice sac away from the column.7genes have the highest relative expression levels in211d after flowering:cmPAL, cmC4H, cmHCT, cmC3H, cmCCoAOMT1, cmCCR and cmCAD1, cmCOMT cDNA was obtained only in leaf but Juice sac, the result predicted that COMT expressed in leaf and particularity expressed in Juice sac. This data indicated the expression of lignin genes is closely related to Juice sac granulation. However, cmCAD2wasn’t obvious regularity, and cmCCoAOMT2almost had no expression during granulation, it was revealed that these two genes should not be invole in Juice sac granulation.