Construction of yeast two-hybrid cDNA library in cambium tissue of Hevea brasiliensis and screening of HbHDA6 interacting proteins

Abstract

The secondary laticifer is the position for synthesis and storage of natural rubber (NR), which is differentiated from the vascular cambium cells of bark in stem of rubber trees (Hevea brasiliensis). The quantity of secondary laticifer is depended on the frequency of the secondary laticifer differentiation from cambia, which is the main index of yield breeding of rubber tree. In previous studies, we found trichostatin A (TSA), an inhibitor of histone deacetylase (HDA), can also induce laticifer differentiation, and the histone deacetylase gene (HbHDA6) is a participator in laticifer differentiation. Because of the molecular mechanism of secondary laticifer differentiation regulated by histone acetylation has not been clarified. Therefore, we construct a yeast two-hybrid cDNA library used the vascular cambium tissues treatment by coronatine (COR), and screening the yeast two-hybrid library by HbHDA6 gene as the bait, for determining the proteins interacting with HbHDA6. The results were as follows: (1) The homogenized yeast two-hybrid cDNA library of vascular cambium was constructed by the technology of Gateway. The capacity of the primary library was 6.34 × 106 CFU·mL-1, the total number of clones was 1.27 × 107, and the capacity of secondary library was 7.72 × 106 CFU·mL-1, the total number of clones was 1.54 × 107, and the recombination rates of two libraries were 100%. The average length of inserted fragments was 1.1 kb and 1.2 kb in primary and secondary library, respectively. (2) The bait vector of pGBKT7-HbHDA6 for screening the proteins interacting with HbHDA6 was successfully constructed and confirmed no self-activation activity. (3) The pGBKT7-HbHDA6 bait vector was used to screen the constructed yeast two-hybrid cDNA library, and 22 proteins interacting with HbHDA6 were obtained by NCBI_BLAST comparison and removing duplicates, including CLP1, ERF3, ERF4, HSP82, LARP6a, APT5, PP2A, APT5, FBA6, etc. The results provide a theoretical basis for analyzing the molecular regulatory network of the secondary laticifer differentiation of rubber tree, and provide candidate genes for the rubber production potential of genetically modified and a new clue for the genetic improvement and breeding of high-performance NR.

Keywords

• hevea brasiliensis
• secondary laticifer differentiation
• vascular cambium
• yeast two-hybrid
• hbhda6