RESUMO
OBJECTIVE: To study the serological characteristics of ABO*A2.08 subtype and explore its genetic molecular mechanism. METHODS: ABO blood group identification was performed on proband and her family members by routine serological methods. ABO genotyping and sequence analysis were performed by polymerase chain reaction-sequence specific primer (PCR-SSP), and direct sequencing of PCR products from exons 6 and 7 of ABO gene were directly sequenced and analyzed. The effect of gene mutation in A2.08 subtype on structural stability of GTA protein was investigated by homologous protein conserved analysis, 3D molecular modeling and protein stability prediction. RESULTS: The proband's serological test results showed subtype Ax, and ABO genotyping confirmed that the proband's genotype was ABO*A207/08. Gene sequencing of the proband's father confirmed the characteristic variation of c.539G>C in the 7th exon of ABO gene, leading to the replacement of polypeptide chain p.Arg180Pro (R180P). 3D protein molecular modeling and analysis suggested that the number of hydrogen bonds of local amino acids in the protein structure was changed after the mutation, and protein stability prediction showed that the mutation had a great influence on the protein structure stability. CONCLUSION: The mutation of the 7th exon c.539G>C of ABO gene leads to the substitution of polypeptide chain amino acid, which affects the structural stability of GTA protein and leads to the change of enzyme activity, resulting in the A2.08 phenotype. The mutated gene can be stably inherited.
Assuntos
Peptídeos , Humanos , Recém-Nascido , Feminino , Alelos , Sequência de Bases , Genótipo , FenótipoRESUMO
OBJECTIVE: To explore the molecular biology of D variant blood group with RHD*95A genotype and the genetic mechanism of its generation. METHODS: A total of 6 samples from 3 generations of a family were analyzed. RHD blood group was identified by saline test tube and microcolumn gel card method. 10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and analyzed by direct sequencing. Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein. RESULTS: The proband was identified as D variant by serological identification, RHD gene sequencing directly detected a c. 95 c > A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr (T) into aspartic acid Asn (N), the rest of the exon sequences were normal compared with the normal RHD*01 gene. In the family, the proband's father, grandmather and uncle were all carried the same RHD*95A allele. Protein modeling results suggested that the hydrogen chain connected to the 32nd amino acid residue was changed after p.T32N mutation, which affected the structural stability of RHD protein. CONCLUSION: The first genetic lineage of the RHD*95A gene was identified in a Chinese population. The c.95C>A mutation in RHD gene was found in the family, which resulted in reduced expression of RHD antigen and showed D variant, the mutation could be stably inheritable. Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.
Assuntos
Antígenos de Grupos Sanguíneos , HumanosRESUMO
Understanding the dynamics of phosphate-solubilizing and N2-fixing bacteria on soil nutrient and related enzyme activity under different organic fertilizer proportions (OFP) could provide references for screening appropriate inoculant type, OFP, and fertilization period. Here, we set four OFP levels (mass ratio: 0%, 4%, 8%, 12%) and inoculated two phosphate-solubilizing bacteria (Bacillus megaterium, Pseudomonas fluorescens) and two N2-fixing bacteria (Azotobacter chroococcum, Azospirillum brasilence) in the subtropical yellow-brown barren soil. After a 60-day soil incubation under controlled conditions (28 â, darkness), we examined the impacts of single/mixed applications of beneficial bacteria on soil available nutrients and related enzyme activities at different OFP levels and different sampling times (3rd, 8th, 16th, 30th, 45th, 60th day). The results showed that soil available nutrient contents increased with the elevated OFP levels, and exhibited as 12%>8%>4%>0%. With the extension of culture time, soil nutrient contents in all treatments first increased and then decreased. Compared with the single application of organic fertilizer, combined application of organic fertilizer and bacterial inoculants resulted in higher and longer improvement of soil nutrient contents and enzyme activities. The effects of inoculants on soil nutrient properties varied across four OFP levels. When the OFP was low (0-4%), inoculation significantly increased soil available nutrient contents, with no the differences between inoculants at the initial stage. However, with the extension of the culture time and the elevation of OFP, phosphate-solubilizing bacteria (especially for B. megaterium) significantly increased available phosphorus content while N2-fixing bacteria (especially for A. brasilence) significantly increased available nitrogen content. The mixed inoculant with four strains showed phosphate-solubilizing effect on soil and performed better than the single application of phosphate-solubilizing bacteria, but without prominent effect on nitrogen fixation. Soil nutrient contents were positively correlated with enzyme activity, which was affected by both cultural time and carbon-nitrogen ratio. Bacterial inoculations could significantly increase nutrient contents in the short term, but the specific functions of beneficial bacteria on soil were highly dependent on organic carbon input and carbon-nitrogen ratio. Coupled application of inoculants and organic fertilizer at an appropriate OFP level (8%-12%) could increase and extend the soil-remediating effects, while the inoculation should be conducted with an interval of 45-60 days to ensure the survival rate and the consecutive effect on soil.