| Bud dormancy is a crucial adaptive strategy for avoiding injury in unsuitable growing environment,playing important roles in annual growth cycle of perennial plants.Most tea planting area in China experience long-term winter.The formation of bud dormancy is related to the safety overwintering of tea plants,and the timing of dormancy release affects the time of buds sprouting in spring.Therefore,the mechanism analysis of key factors involved in tea bud dormancy regulation is helpful for clarifing the dormancy mechanism,which is also of great significance to promote the application of molecular assisted breeding on tea plant.DAM is initially identified as an important gene in evergrowing peach mutant which directly involved in dormancy regulation.Previous studies have shown that DAM may also play an important role in regulating the dormancy formation and release of tea overwintering buds.In this paper,the gene structures,tissue-specificity,sequence polymorphism analyses of two CsDAM and cloning and expression analysis of their possible downstream key genes were performed to reveal the dormancy regulation mechanism of tea plants.The main results are as follows:1.CsDAM1 and CsDAM2 were cloned and their gene structure,subcellular localization and tissue-specific expression were studied.The results showed that they were both composed of 8 exons and 7 introns,but there were significant differences in gene length and sequence similarity.Promoter sequence analysis suggested that the two CsDAM genes might be mainly regulated by light signals,hormones,and stress stimuli.Proteins they both encoded were localized in the nucleus.CsDAM1 and CsDAM2 showed highest expression abundance in leaves,axillary buds and roots,followed by apical buds and stems,and the lowest expression in flowers.2.A differential expression gene,AIL,which plays an important role in growth and development regulation of plants,was identified from CsDAM1 over-expressed transgenic poplar based on RNA-seq.The CsAIL was screened out of tea plant transcriptome by homology analysis.The CsAIL was cloned from tea plant.The open reading frame length of CsAIL was 1 872 bp.CsAIL had relatively high expression level in apical buds,axillary buds and stems,had low expression level in leaves,and had no expression in flowers.Promoter of CsAIL may be regulated by light,hormones and abiotic stresses.The CsAIL and its possible downstream target gene CsCYCD3.2 and CsCYCD6.1 had the lowest expression level in the physiological dormancy period of overwintering buds,had the highest expression level in the ecological dormancy period of overwintering buds,and had relatively high expression level in the initiation period of overwintering buds.There were significant positive correlation(P < 0.01)in the expression between CsAIL and CsCYCD3.2,CsCYCD6.1 in buds and leaves,which showed that CsAIL may be a crucial gene involved in the tea dormancy regulation and plays an important role in the dormancy-growth cycle of tea plants together with CsCYCDs.3.Transcriptional level of CsDAM1 gene has a close relationship with the dormancy release time of tea buds,which can be used as an early identification index for sprouting phenology of tea plants.Gene expression analysis of different phenological varieties revealed that,the expression of CsDAM1 increased to the highest level firstly and then decreased rapidly during dormancy formation and release.The time of CsDAM1 expression down-regulation in early sprouting tea varieties was early than that in late ones,while CsDAM2 had no such correlation.Under dormancy induction in climate chamber,there were no significant difference in the expression levels of CsDAM1 and CsDAM2 between eight tea varieties.There were single-base mutations at multiple sites and the insertion and deletion of some small fragments of CsDAM1 among different sprouting phenological tea cultivars.Above all,CsDAM1 may play a more important role in the regulation of dormancy formation and release in different germinating phenological tea plants. |
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