Floral Bud Transcriptome Analysis And Research On Floral Development Regulation Mechanism Of Sapium Sebiferum

Sapium sebiferum (Linn.) Roxb (Chinese tallow tree), which belongs to Euphorbiaceae family, has attracted considerable recent attention due to its potential for biodiesel production, sightseeing, value of pharmaceutics, and its ability to grow in unproductive subtropical soils. In the subsequent years, agriculturally important characters such as fruit and oil yield, oil content and uses were evaluated. However, S. sebiferum is still an undomesticated plant in which many basic agronomic properties are not yet thoroughly understood. It has long life expectancy with bearing fruit after 3-5years, flourishing after ten years, which was considered as one of the major factors limiting yield in this species. It is important to accelerate flowering through reduction of the juvenile phase in order to facilitate the earliest possible production of fruit.In this paper, we use a new generation of high-throughput sequencing and bioinformaties analysis teehnology to study the S. sebiferum flowering bud transcriptome. The microscopic observation of floral organ development in S. sebiferum was performed for the first time. Flowering related genes of SsAP1 SsTFL SsAP2, SsCO and SsFUL were cloned. And early-flowering S. sebiferum induced by stress were analyzed in transcription and protein level. A reliable gene silencing method were established robust and in S. sebiferum. The main conclusions of this paper are as follows:1. The developmental anatomy study of S. sebiferum flowerThe flower bud differentiation process of S. sebiferum was studied with the method of paraffin section. According to the microstructure characteristics, the flower differentiation of S. sebiferum flower is divided into five phases: inflorescence primordium differentiation phase、staminate flower primordium differentiation phase、staminate pollen formation phase with pistillate flower primordium differentiation phase、staminate pollen maturation with pistillate flower ovary differentiation phase、pistillate flower ovary formation phase.2. The transcriptome sequencing analysis of S. sebiferum flower budIn this study, the first transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis,161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis.3. Cloning of flower development related gene from S. sebiferumBased on transcriptome of S. sebiferum, TFL1、LFY、AP1、CO、AP2 and FUL homologous gene were isolated from flower buds total RNA of S. sebiferum. Tissue specific-expression of these genes was analyzed. The ectopic expression of SsLFY in wild type Arabidopsis (Col-0) results in early-flowering, with high expression levels of exogenous SsLFY being observed in transgenic Arabidopsis. The virus-induced gene silencing of SsLFY in wild-type pea resulted in abnormal floral organs.4. Application of VIGS method in S. sebiferumVIGS provides a quick and convenient method for the identification of target gene function. Based on the different instructions gene PDS and AS1, we constructed pTRV virus expression vector by inserting a different section of the SsPDS gene (5’and 3’end) and different lengths of SsAS1 gene (493 bp and 323 bp). The constructed vector was transformed into Agrobacterium GV3101 to further inoculate to S. sebiferum plants. The infected S. sebiferum plants showed photobleaching symptom after 3 weeks, and the phenotype showed obviously after 6 weeks. Total RNA were isolated from the mutants, and detected TRV virus protein gene and SsPDS gene expression level by RT-PCR. These results demonstrated that the TRV virus was successful into S. sebiferum plants, and had transferred and replicated into S. sebiferum, the expression of SsPDS gene level had reduced significantly. The pTRV-SsASl infected S. sebiferum plants showed curled leaf after 3 weeks, and the phenotype showed obviously after 60 days. The detected TRV virus protein gene and SsAS1 gene expression level by RT-PCR. These results demonstrate that the TRV virus was successful into S. sebiferum plants, and had transferred and replicated into S. sebiferum, the expression of SsAS1 gene level had reduced significantly.5. The analysis of early-flowering S. sebiferum induced by stressIn order to explore the condition of stress induced early-flowering of S. sebiferum, the simulation pot experiment is carried out in greenhouse. And early-flowering S. sebiferum induced by stress were analyzed in transcription and protein level. After treated 3 months, we found that S. sebiferum plants showed obvious flowering phenotype in the 20 plants of treatment group. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, AG, GA1, SPY, SOC1, AP2, AP3, VRN2, RIP, CRY2, accorded with stress-related genes, such as GRX50435, GRX29400, CAT97419, SOD28463, PRX Ⅱ 25514, PRX Ⅱ 39562, PRX25913. In addition,10 proteins were identified successfully by total protein 2-D electrophoresis. In these proteins are mainly with the material and energy metabolism, photosynthesis, protein involved in oxidative stress response, stress response.Three is involved in glucose metabolism enzymes, including fructose diphosphate aldolase, glyceraldehyde 3-phosphoric acid dehydrogenase and ribulose phosphate kinase (19 and 16 points and 23), mainly in order to increase the synthesis of carbohydrates. The rest of seven proteins involved in other processes, oxygen-evolving enhancer protein 1, photosystem Ⅰ subunit Ⅶ, abscisic stress ripening-like protein, Osmotin, mta/sah nucleosidase, ascorbate peroxidase and peroxiredoxin.This paper provided functional genomics information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offered a useful database for the research of other Euphorbiaceae family plants.