Cloning and Functional Analysis of TCAP3 Gene in Taxus Chinensis var. mairei
Abstract
Taxus Chinensis var. mairei is a valuable plant species for timber and taxoids isolated from this species are very important compounds that are used for cancer treatment. Although chemical investigation on T. chinensis var. mairei are popular, functional identification of genes isolated from this species is rare. In this investigation, we have isolated TCAP 3 gene and analyzed its expression pattern in different tissue and developmental stages through Real time -PCR; then we transformed this gene into Arabidopsis and analyzed its function. Our results demonstrated that its cDNA contains 846 bp bases (coding 197 amino acids) constituted by four typical domains, M, I, K, C with conserved motif, Phylogenetic analysis showed that TCAP3 is more ancient than angiosperm B class genes. Alignment of protein sequence demonstrated the conserved motifs, which illustrated that TCAP3 belongs to gymnosperm Gymno B class MADS -box genes with PI -derived, on C-teminal, which is similar structure to the Gymno B class MADS -box genes that they share the same B class gene specific conserved motif. Expression analysis of TCAP3 in different tissue showed that it only expression in male strobilus, not in leaf, bud and female strobilus at different developmental stages. We divided the stages according to paraffin sections of male strobilus. The results indicated that TCAP3 expresses dynamically along with the male strobilus. Heterologous expression of TCAP3 in Arabidopsis demonstrated that TCAP3 was involved in flower, especially the filaments morphological development.
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Introduction
Taxus Chinensis var. mairei is a typical cretaceous precious tree species scattered distributed in wild and cultivated habitats in eastern, south central, southwest of China. T. Chinensis var. mairei is a dioecism that has low setting percentage of fruits because of the desynchronized flowering between male and female strobilus. T. Chinensis var. mairei has low seeds germination rate due to long cycle dormancy. Its natural populations have been endangered or extinct in some original habitats. There are some initial studies on morphology development of male strobilus, but not in molecular level. In this investigation, we have cloned a MADS-box gene that is related to the development of male strobilus from T. Chinensis var. mairei.
MADS-box genes encode transcription factors that play essential roles in cell signaling and development processes including flowering induction, flower organ identity, and embryo development. The heterodimer formation between B-class MADSbox proteins plays a core role for petal formation through protein-protein interactions in transgenic plants. For example, transgenic gentian ectopically expressing GsPI2 produced an elongated tubular structure that consisted of an elongated petaloid organ in the first whorl and stunted inner floral organs, suggesting that B-class MADS-box proteins might be important for the complete development of petal organs (Nakatsuka, et al. 2016). MADS-box genes in grapevine can be crucial for development of central cell, endosperm (Grimplet, et al. 2016). Hormone-related transcription changes were associated with regulation of MADS-box transcription factor expression in grapevine inflorescence fruit set (Domingos, et al. 2016).
Phylogenetic analysis of MADS-box proteins suggests functional conservation in floral signal integration and meristem determination pathways that may help in devising strategies to improve important traits in apple (Kumar, et al. 2016). MADS-box transcription factors play central role in peach endodormancy regulation and transitions (Wells, et al. 2015). In sesame (Sesamum indicum L.), MADS-box genes were identified from 14 linkage groups of the sesame genome and motif distribution analysis indicated that type II sesame MADS-box genes had more complex structures (Wei, et al. 2015). MADSbox transcription factors play a key role in controlling lateral root development through nitrate signal in Arabidopsis and are positive regulator control lateral and primary root development in rice (Yu, et al. 2015). MADS-box genes are transiently expressed in small numbers of cells in the floral apex in Nicotiana tabacum (Mandel, et al. 1994). MADS box genes are involved in proper ovule development in petunia (Angenent, et al. 1995) and regulating floral meristem and floral organ identity (Colombo, et al. 1995). Chromosomal map positions of MADS-box genes were determined in recombinant inbred lines of maize (Zea mays ssp. mays). It appears that MADS-box genes are scattered throughout the maize genome (Fischer, et al. 1995).
Studies on distantly related dicot plant species have identified MADS-box genes that specify floral meristem identity and determine the fate of floral organ primordial, providing the basis for further studies into the regulation of floral organ morphogenesis among the grasses (Mena, et al. 1995). In T. Chinensis var. mairei, investigating the development process of ovulate strobilus and microstrobil for understanding the molecular mechanism of development regulation will not only help us to know sexual reproduction process, but also having important significance in conservation genetics that would help us to generate strategies in the improvement of the seed setting rate and breeding measurements. Meanwhile, It also can provide theoretical support and protection and update of the new way of thinking for the breeding and protection of Taxus Chinensis var. mairei’s population from the molecular level.
In this investigation, we have isolated TCAP3 and sequenced it; analyzed its expression pattern in different tissue and developmental stages through real time-PCR; then transformed the gene into Arabidopsis mutants to discover its function. This is the first report of cloning and functional analysis of TCAP3 gene in Taxus Chinensis var. mairei.
