Cloning And Single Nucleotide Polymorphisms Analysis Of Resilience-related Gene In Populus Simonii Carr

Populus simonii Carr, indigenous to China, is one of key forest resources for shelterbelt with a lot of characteristics, While P. simonii has excellent resources with tolerance resistant genes, mechanisms of molecular genetics involved in traits of tolerance resistance to adaptability have not been deeply studied. So, it is necessary to explore the bases of molecular genetics of traits with stress resistance, in particularly, to clone the genes controlling these traits and to analyze their nucleotide diversity.This paper, materials for Populus simonii, The PCR products of DREB1D,Hsf gene were separated by agarose gel running, and then purified, ligated with PGEMT-Vector. Ligated products were transformed to Escherichia coli with DH5É‘, and positive clones were selected for sequencing. Gene sequence analysis, research results are as follows:1. The PsHSFB gene are 2884 bp in length with corresponding open reading frames (ORFs) which are capable of encoding the protein of 466 amino acids (aa). The deduced aa sequence of the PsHsf protein shares 63% and 78% identity with the other recently isolated A. thaliana, and Vitis vinifera Hsf1, respectively. The genomic sequences of PsHsf in 16 individuals were aligned, compared and analyzed using the software MEGA3.1 and DNASP4.0. A total of 107 SNPs were detected and the diversity of them is 1/27 bp, in which 42 are common SNPs and 65 are rare SNPs. There are 71 transition and 36 transversion of mutation types. In total, 52 SNPs were detected in the coding regions of PsHsf, of which 15 are nonsense mutation and 37 are missense mutation.2. The PsDREB1D gene are 800 bp in length with corresponding open reading frames (ORFs) which are capable of encoding the protein of 240 amino acids (aa) , respectively. Their molecular weight (MW) of proteins are 33KDa, and PI are 4.85, The phylogenetic tree of DREB1D protein of 16 species was constructed, and result shows that the relativeness of P. simonii has the highest identity with P. trichocarpa for DREB1D gene. Nucleotide diversity and linkage disequilibrium (LD) of DREB1D gene was analyzed using DNASP4.0 software. The result showed that nucleotide diversity of DREB1D gene is 0.04375 in P. simonii, of which 5 common SNPs and 30 rare SNPs were detected, respectively. A total of 24 missense mutations and 11 silent mutations were detected in coding regions of DREB1D gene, Analysis of LD showed that LD declines rapidly within candidate gene of DREB1D in P. simonii.The results, therefore, provided the important foundation for association genetics of resistance gene and gene-assisted breeding of new germplasms with drought and high temperature tolerance in P. simonii.