| The axillary buds of plants are released from dormancy to develop branches,which is an important protective mechanism for plants to avoid the loss of terminal buds and other injuries,and it is also an important aspect for plants to adapt to the environment throughout their life cycle.There is a fleshy axillary bud in a dormant state in the short branch(coniferous bundle)growing on the annual branch of Pinus massoniana,after the truncation of the long branch,the dormancy will be released and further developed into a branch.In this study,The axillary buds of short branches on the annual growth branches of Pinus massoniana were studied,The external morphology and internal tissue development of axillary buds of short shoots after dormancy was observed systematically,on this basis,the key stages of axillary bud development of short shoots of Pinus massoniana were analyzed by transcriptome analysis combined with fulllength transcriptome of the third generation and high-throughput sequencing technology of the second generation,Molecular regulation mechanism during dormancy release and development of axillary buds of branchlets.The results are as follows:1.It was found that a fleshy axillary bud with a diameter of about 1/5 of the width of needles and complete meristematic tissues was formed on the 1A shoot of Pinus massoniana,which maintained the dormant state of growth stagnation.Morphological and structural changes of axillary buds of Pinus massoniana were studied by means of stereomicroscope photography and paraffin section preparation.The results showed that the dormancy of axillary buds was relieved,meristem meristematic characteristics were activated and axillary buds began to grow and develop after long branch truncation.After 12 days,new scaly leaves were differentiated;at 24 days,the scaly leaves were non-fleshy,covered with full axillary buds,and differentiated into coniferous primordia at axillary axilla of scaly leaves under axillary buds;at about 32 days,axillary buds had developed completely,and then began to shoot,similar to the development of long branch buds of long branches.2.Three generations of full-length transcriptome sequencing technology based on PacBio Sequel platform combined with biological information analysis method.In this study,83671 full-length Unigenes sequences were obtained which participated in the development of axillary buds of Pinus massoniana.81103 Unigene sequences were successfully annotated,with the annotation rate as high as 96.93%.77656 Unigenes were successfully annotated in NR database,with the annotation rate of 92.71%.Spruce is the most closely related to phylogenetic evolution.GO database annotations show that most Unigenes are involved in metabolic processes,cell structure and protein binding,catalytic activity and other biological processes.In KEGG classification,most Unigenes participate in cell processes,signal transduction,carbohydrate metabolism and other pathways.3.Based on high-throughput transcriptome sequencing data,and the results of fulllength transcriptome sequencing were compared and analyzed.RNA-seq analysis showed that 398 genes were differentially expressed before and 4 days after truncation.GO enrichment results showed that most of these genes were involved in single organism and redox process.KEGG enrichment results showed that most of these genes involved in flavonoid biosynthesis,terpenoid biosynthesis,circadian rhythm and other pathways.Some key genes of flavonoid biosynthesis,such as CHS,C4 H,were found.At the same time,GA1,the key gene of gibberellin biosynthesis,was annotated.GA1 is an important gene for promoting plant growth and development.It is speculated that the possible differential expression of these genes may be closely related to the mechanism of axillary bud dormancy release and the growth and development regulatory network.There were 444 differentially expressed genes after 4 days of truncation and 20 days of truncation.GO enrichment results showed that most of these gene products were involved in biological regulation and cell regulation.KEGG enrichment results showed that these genes were mostly involved in the differential expression of plant hormone signal transduction,fatty acid metabolism and arginine biosynthesis.Especially,the differential expression of SAUR gene in plant hormone signal transduction pathway was the key gene for auxin synthesis.It is related to the synthesis of plant hormones in axillary buds and promotes the growth and germination of axillary buds.There were 63 differentially expressed genes after 20 days of truncation and 32 days of truncation.The results of GO enrichment showed that most of these gene products were involved in transportation and location.KEGG enrichment results showed that these genes were mainly involved in the differential expression of fatty acid biosynthesis,other types of O-glycan biosynthesis and fatty acid metabolism.FATB,the key gene of fat acid synthesis was annotated,the development of axillary buds has already possessed the morphological structure of branches and started the process of lipid biosynthesis.The results of qRT-PCR showed that the expression profiles of Unigenes randomly selected were similar to those of RNA-Seq,indicating that the results of transcriptome data analysis were reliable. |
