The Development And Transcriptome Regulation Of Secondary Trunk Of Ginkgo Biloba L.

Ginkgo?Ginkgo biloba L.?is the only surviving tree species in Ginkgoaceae after the Quaternary Glacier.It is called historical heritage and"living fossil"of plant.Ginkgo biloba is a peculiar tree species in China,among which there are many ancient Ginkgo biloba trees and they are widely distributed.From the young age to millennial ancient trees of Ginkgo biloba,there is a common phenomenon of“stemmed branches”?secondary trunk?.Most of the ancient Ginkgo biloba in China are clones of multiple generations of the same plant,which have plenty of secondary trunks.The phenomenon of"multi-generation of the same strain"is an important breeding and renewal strategy for Ginkgo biloba when it reaches its growth limit or maintains a life-sustaining system in order to withstand the adverse environment.It is the key that Ginkgo biloba can survive after the disaster.Through the research on the secondary trunk development of Ginkgo biloba,the mechanism of secondary trunk development can be further understanded.This research can lay a theoretical foundation for revealing the secret of longevity of ancient Ginkgo biloba and provide theoretical guidance for the reasonable and appropriate use of secondary trunk for reproduction in practical production.At present,most researches on secondary trunk of Ginkgo biloba focus on the geographical distribution,the significance of secondary trunk,the process of replacing the trunk with secondary trunk and utilization of secondary trunk.In this study,the growth indexes were measured and anatomical structure was observed to understand the growth characteristics of sencondary trunk and anatomical structure of originating of sencondary trunk of Ginkgo biloba.Through plant physiological research,the endogenous hormone content of secondary trunk and branches of the same plant was measured at two different developmental stages for clarifying the physiological mechanism of secondary trunk formation and its differences from branches.Finally,functioncal genes were selected effecting occurrence of secondary trunk and the molecular mechanism of the regulation of secondary trunk of Ginkgo biloba development was discussed,by the second generation high-throughput sequencing and bioinformatics analysis.The transcriptome sequencing experiment was carried out using two different developmental stages of secondary trunk of Ginkgo biloba and two undeveloped parts of the same tree trunk as controls.The main results are as follows:?1?The number of secondary trunk per plant,the incidence of secondary trunk,the height of secondary trunk and the basal diameter were significantly different at different seedling ages of Ginkgo biloba in the natural state and they increased with seedling age.The three treatments of stubble,transplanting and combination of stubble and transplanting all can promote the occurrence of secondary trunk of Ginkgo biloba.Among them,stubble played a major role and it increased obviously height and base diameter of secondary trunk.?2?Anatomical observation of secondary trunk of Ginkgo biloba showed that secondary trunk of Ginkgo biloba originate from latent buds in cortex of stem at junction of stem and root.The development process of secondary trunk can be divided into four stages:dormant stage of secondary trunk bud,differentiation stage,conducting tissue formation stage and germination stage.Dormant buds can germinate when stimulated by certain internal and external environment.?3?The results of endogenous hormone analysis showed that the content of hormones in secondary trunk was higher than that in lateral branches during germination and elongation growth of secondary trunk.The content of cytokinin and gibberellin during germination was significantly higher than that in lateral branches.They can break the dormancy of bud and promote the germination of secondary trunk.The content of gibberellin decreased significantly in the elongation growth period compared with the germination period,but the content of auxin and cytokinin was significantly higher than that of germination period.In both stages,the ratio of?IAA+ZA+GA₃?/ABA of secondary trunk and lateral branch was greater than 1 and this ratio of secondary trunk was greater than that of lateral branch,while the ratio of IAA/ZA was smaller than that of lateral branch.The ratio of?IAA+ZA+GA₃?/ABA of secondary trunk in the elongation period was lower than that in the germination period and the growth trend was slowed down,while this ratio of lateral branches increased slightly.?4?A total of 44,340,111 Clean Reads were obtained by transcriptome sequencing by comparing the two developmental stages of secondary trunk of Ginkgo biloba with the normal site without secondary trunk,averaging 97.56%of the original number of sequences.The comparison rate of cDNA library and reference genome of Ginkgo biloba in each sample was over 88%.Through differential gene screening,a total of 74 differential genes were obtained in the comparison group of secondary trunk during germination and control group.104differential genes were obtained in the comparison group of secondary trunk during germination and elongating growth periods.2151 differential genes were obtained in the comparison group of secondary trunk during elongating growth period and control group.GO functional annotation found that differential genes are mainly involved in metabolic processes,cellular processes,membrane part,catalytic and binding in biological processes.Combined with the analysis of endogenous hormones to further analyze the KEGG enrichment of differential gene,it was found that the differential genes were annotated to Plant hormone signal transduction,Phenylpropanoid biosynthesis,Phenylalanine metabolism,Glycolysis/Gluconeogenesis and many other pathways related to secondary trunk development,of which42 differential genes were enriched in the plant hormone signal transduction pathway.The significantly enriched genes in these pathways have important regulatory effects on the suppression of secondary trunk dormant buds and the secondary trunk development.The expression of key genes of auxin and gibberellin promoted the synthesis of hormones and up-regulated the expression of receptor genes,leading to germination of secondary trunk.