Masson Pine Multicone Transcriptome Analysis And Flowering-related Gene Cloning

Pinus massoniana Lamb,belonging to the pine family and it's monoecious,is one of the important tree species of afforestation and timber in China.Usually 2-5 megastrobili of P.massoniana are clustered on top of new shoots.Occasionally,there are dozens or even hundreds of clusters formed by the sexual reversal of microspores,named polycone.This phenomenon has high genetic stability and high potential application value in improving seed yield.We studied and classified the normal megastrobilli and microstrobilli before and after the sexual reversal in P.massoniana by transcriptome approach that excavated the related genes in the formation process of polycone and enriched transcriptome database of P.massoniana.By RT-PCR and RACE cloning technique,we cloned and started expression analysis of flowering-related genes of P.massoniana,which provides a reference for reproductive regulation of polycone phenomenon.The main research work and acheviments are as follow:(1)The normal megastrobilli and microstrobilli before and after the sexual reversal in P.massoniana,were studied and classified by transcriptome approach.In the analysis,a total of 190,023 unigenes were obtained with an average length of 595 bp.The annotated unigenes were thus divided into 56 functional groups and 130 metabolic pathways involved in the physiological and biochemical processes related to ribosome,carbon metabolism,and the biosynthesis of amino acids.There were 4,758 differentially expressed genes(DEGs)between the mega-and microstrobili from the polycone twig.The DEGs between the mega-and microstrobili from the normal twig were 5,550.In the polycone twig,1,188 DEGs were yielded between the microstrobili and the sexually reversed megastrobili.The process of microstrobili and the sexually reversed megastrobili were 69 unique DEGs that probably associated with factors of reversal process.Transcript comparison found there are DEGs in the Gibberellin Pathway and Photoperiodic Pathway of sexually reversing megastrobili,and the DEGs probably involved in the development of polycone.(2)CONSTANS homologous gene was cloned from P.massoniana using RACE technology,named PmCO1.This sequence length is 1448 bp,ORF is 1329 bp,encoding 448 amino acids.Expression analysis showed that PmCO1 had expressions in different developmental condition and tissues of strobiles in P.massoniana.That is,its expression level first increased and then decreased in the whole development stage of polycone,indicating that PmCO1 gene is involved in regulating sex differentiation of P.massoniana.(3)Based on transcriptome data,we cloned PmCO2 and PmGID1 genes using PCR techniques.Their sequences full-length are 1461 bp and 1189 bp,ORF are 1164 bp and 1062 bp,encoding 448 and 353 amino acids respectively.Expression analysis of PmCO2 gene showed that PmCO2 was expressed and varied in different developmental stages of polycones and normal cones,indicating that PmCO2 is involved in regulating the growth process of polycone and normal cones from vegetative growth to reproductive growth.But the expression patterns of PmCO1 and PmCO2 was different.It is speculated that PmCO1 and PmCO2 gene have different regulatory mechanisms.Whatsmore,the successful of PmCO2 indicateed that there are many CO homologous genes in P.massoniana.Expression analysis of PmGID1 gene showed that PmGID1 was expressed in different developmental stages of polycones and normal cones,indicating that PmGID1 is involved in controlling the formation of polycone.The highest expression of stem and the lowest of leaves were found in different tissues,and the difference between them was 13.73 times.It indicated that PmGID1 have tissue specificity.