Cloning and functional analysis of HbTRXh9 gene in Hevea brasiliensis

【Objective】 This study aimed to clone the h-type thioredoxin （TRX） gene HbTRXh9 in Hevea brasiliensis，analyze its expression pattern，and verify its function in abiotic stress tolerance using transgenic yeast，thereby providing theoretical reference for abiotic stress tolerance and tapping panel dryness （TPD） tolerance in Hevea brasiliensis genetic improvement. 【Method】 The HbTRXh9 gene in Hevea brasiliensis was cloned by PCR， and real-time fluorescence quantitative PCR was used to detect the tissue-specific expression of HbTRXh9 gene and its expression patterns under abiotic stresses. The yeast expression vector pYES2-HbTRXh9 was constructed and transformed into yeast INVSc1 competent cells， and the growth of HbTRXh9 transgenic yeast and pYES2 empty vector-transformed yeast after abiotic stress treatments were compared. 【Result】 The coding sequence （CDS） of HbTRXh9 gene was 417 bp in length and encoded 138 amino acid residues， with the protein relative molecular mass of 15.52 kD and theoretical isoelectric point of 4.90. Subcellular localization was in cytoplasm， and the protein was classified as a non-secretory， hydrophilic protein containing a conserved TRX domain （pfam00085） and CGPC redox-active site. HbTRXh9 shared high amino acid sequence similarity with TRXh in Jatropha curcas and Manihot esculenta，reaching 84% and 80% respectively. These three proteins were in the same branch and clustered into the group III of h-type TRXs. The HbTRXh9 gene was expressed in different tissues， with distinct differences in relative expression， and the relative expression was significantly higher in latex than in other tissues （P<0.05，the same below）. The relative expression of TPD in the latex and bark of Hevea brasiliensis was significantly lower than that in healthy trees. Under drought and low temperature stresses， the relative expression of HbTRXh9 increased from 0-12 h， decreased significantly at 24 h， and then peaked at 48 h， which was significantly higher than that at other time. Under salt and oxidative stresses， the relative expression of HbTRXh9 exhibited an overall variation trend of first increasing and then decreasing， and the relative expression peaked at 12 h under salt stress and declined significantly subsequently； under oxidative stress， it significantly climbed to peak at 6 h， followed by a gradual reduction. After treated with oxidative and low-temperature stresses for different time， the colony number of HbTRXh9-transgenic yeast was higher than that of the pYES2 empty vector-transformed yeast under the same dilution condition； under salt stress at different treatment time， the colony number of HbTRXh9-transgenic yeast was lower than that of the pYES2 empty vector-transformed yeast. 【Conclusion】 The HbTRXh9 is involved in TPD occurrence and abiotic stress responses of Hevea brasiliensis， and expression of HbTRXh9 gene in yeast can improve oxidation tolerance and cold tolerance， but reduce salt tolerance.