| Freesia is one of the ten cut flowers in the world.Its reproduction and commodity circulation mainly depend on seed balls.The quality of seed balls directly affects its ornamental value and commercial value.Many studies have shown that bulb quality is closely related to endogenous hormones,sugar metabolism and regulation.Based on the observation of bulb development of Freesia hybrida ‘SN Huangjin’,the dynamic changes of endogenous hormones and sugar metabolism with bulb development were analyzed.The endogenous hormones and sugar metabolism pathways closely related to bulb development were identified by transcriptome analysis,and the differential expression profiles of bulb development were established.At the same time,the expression characteristics of key differential genes in main metabolic pathways were analyzed,and the regulation mechanism of bulb growth and development was revealed from the molecular level.The aim is to provide scientific guidance for the practice of bulb production and regulation.The main results are as follows:.1.Firstly,the bulb formation process of Freesia was divided into four stages by periodic observation,namely: new globular formation stage(50-90 DAP),the above-ground plant stage was five-leaf stage to eight-leaf stage;the above-ground plant stage was budding stage to flowering stage,including two stages: 90-120 DAP was the initial growth stage,120-140 DAP was the rapid expansion stage;and finally,the mature stage of bulb(140-190 DAP),the aboveground plants showed from the late flowering stage to the seedling withering stage.2.Indirect enzyme-linked immunosorbent assay(ELISA)was used to determine the endogenous hormones in Freesia bulb regularly.The results showed that GA3 content showed a double-peak "M" trend,with two peaks appearing in the early stage of bulb expansion and the early stage of bulb maturity respectively;ABA level showed a downward trend as a whole,with the highest value appearing in the early stage of new sphere formation,and then rose again after falling to the lowest value during budding,and then decreased again;IAA change trend was contrary to ABA,the whole trend was upward,and IAA wave appeared in the bulb formation period.The changes of ZR content showed a multi-peak "M" trend,with three peaks appearing in the early stage of bulb formation,the late stage of bulb expansion and the full maturity of bulb.The correlation analysis showed that all the other indicators were significantly correlated except ZR,which indicated that the levels of GA3,IAA,ABA and other endogenous hormones as well as the dynamic balance between hormones were closely related to the development of bulbs.3.The content of sucrose and starch was positively correlated with the development of Freesia bulb.The content of sucrose and starch increased with the bulb expanding.Sucrose phosphate synthase(SPS),the key enzyme in sucrose metabolism pathway,showed a trend of first increasing,then decreasing and then rising,sucrose synthase(Su Sy)showed a trend of first decreasing,then rising and then declining,while invertase(INV)showed a trend of first rising and then declining on the whole.The dynamic changes of three key enzymes in starch metabolic pathway showed that amylase increased first,then decreased and then increased,while the activities of Q enzyme and ADPG pyrophosphorylase continued to increase on the whole,showing a positive correlation with the changes of starch content.4.Twelve c DNA libraries were constructed from the bulbs of Freesia at different developmental stages.A total of 82.54 Gb Clean Data was obtained by sequencing.The percentage of Q30 base was higher than 89%,and the percentage of GC base was about 48%.After assembly,100,999 unigenes were obtained,with N50 of 1,507 and average length of 772 nt.The assembly integrity was high.44,405 articles were annotated in NR,Pfam and other databases,accounting for 44% of the total unigene.Through functional analysis of COG and GO databases,it was found that the annotated genes involved in carbohydrate metabolism and secondary metabolic processes were concentrated.Six types of SSR were identified in Freesia’Shangnong Gold’bulb by using MISA software.9,200 SSR loci were obtained by analyzing Unigene over 1 kb.Among them,single base duplication was the most common,accounting for 48.5% of the total.5.Internal reference genes have great influence on the accuracy of realtime quantitative fluorescence PCR analysis of target gene expression.Eight traditional housekeeping genes were selected from the above transcripts to screen the internal reference genes,and the stability of their expression was analyzed by Best Keeper,Norm Finder and ge Norm.The results showed that the expression of QCR,Actin and TUB was the most stable in the four developmental stages of Freesia bulb,suitable as internal reference gene,CYP and 18 sr RNA were the most unstable,while Actin and 18 sr RNA were the most suitable internal reference genes in six tissues and organs.The stability of GAPDH,beta-Tubulin and SAMDC are different in different software analysis results.The stability of GAPDH is moderate,while EF-1beta is the least unstable.6.Digital gene expression profiles of differentially expressed genes(DEG)in the development of Freesia bulb were constructed,and the expression patterns of key differentially expressed genes were further studied by q PCR.By comparing the distribution of the expressed genes,it was found that DEG was the most common and mainly downregulated gene in the rapid expansion period compared with the mature period,which indicated that these two periods were the most active periods for the development of Freesia bulb.KEGG-based pathway analysis showed that there were more than 20 metabolic pathways involved in DEG related to bulb development in Freesia,and sucrose-starch metabolism and hormone metabolism were active.Functional enrichment analysis showed that DEG was mainly concentrated in metabolic and developmental processes.In addition to the function of basic life activities,carbohydrate metabolism ranked second during bulb development,and metabolic activities were very active.According to the FPKM value of key enzymes,the main regulation patterns of endogenous hormones and sugar metabolism in Freesia bulb were constructed.The expression patterns of key enzymes in hormone and glucose metabolism were analyzed by q PCR.It was found that the expression of alpha-glucosidase gene increased sharply at the mature stage of bulb.The expression of INV and Su Sy genes reached the highest level at the rapid expansion stage.The expression of Su Sy was relatively high at the initial formation stage and the early growth stage,and then decreased.The expression of Q enzyme was low in the first three stages of bulb development,but high in mature stage;the expression of ADPG pyrophosphorylase increased with bulb development;the expression of beta amylase increased sharply in the first three stages of bulb development,but decreased in mature stage.The expression of cytokinin trans-hydroxylase was the highest in ZR synthesis pathway at the early stage of growth;the expression of abscisic acid 8’-hydroxylase was the lowest in ABA metabolism pathway at maturity,while the expression of 9-ciscyclooxycarotenoid dioxygenase was the highest in early stage of ZR synthesis pathway;gibberellin-20-oxidase and cytokinin dehydrogenase were the key enzymes in GA and IAA metabolism pathway.The highest expression was found at the beginning of bulb formation.It is speculated that ZR,GA and IAA promote the morphological formation of Freesia bulb,while ABA may play an active role in promoting bulb maturation. |
PDF Full Text Request