Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.
Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.
