The SsWRKY1 transcription factor of Saccharum spontaneum enhances drought tolerance in transgenic Arabidopsis thaliana and interacts with 21 potential proteins to regulate drought tolerance in S. spontaneum.

In this study, we characterized a WRKY family member gene, SsWRKY1, which is located in the nucleus and contains multiple stress-related cis-acting elements. In addition, constructed SsWRKY1-overexpressing Arabidopsis thaliana had higher antioxidant enzyme activity and proline content under drought stress conditions, with lower malondialdehyde content and reactive oxygen species (ROS) accumulation, and the expression levels of six stress-related genes were significantly upregulated. This indicates that the overexpression of SsWRKY1 in Arabidopsis thaliana improves resistance to drought stress. SsWRKY1 does not have transcriptional autoactivation activity in yeast cells. The yeast two-hybrid (Y2H) system and the S. spontaneum cDNA library were used to screen 21 potential proteins that interact with SsWRKY1, and the interaction between SsWRKY1 and ATAF2 was verified by GST pull-down assay. In summary, our results indicate that SsWRKY1 plays an important role in the response to drought stress and provide initial insights into the molecular mechanism of SsWRKY1 in response to drought stress.

Identification of a novel allele HLA-C*03:560N by next-generation sequencing in a hematopoietic stem cell recipient.

A frameshift because of a single nucleotide deletion results in a novel null allele, HLA-C*03:560N.

Association of HLA class II (-DRB1,-DQB1,-DPB1) alleles and haplotypes on susceptibility to aplastic anemia in northern Chinese Han.

The Human Leukocyte Antigen (HLA) genes, playing key roles in mediating the immune response, especially HLA class II alleles were suggested to play a role in the activation of autoreactive T-cells in aplastic anemia (AA). Previous studies in different ethnic groups have indicated that some of HLA-A,-B,-DRB1 alleles had a protective or susceptive association with the prevalence, pathogenesis and development of AA. HLA class II genes, especially HLA-DQB1 and -DPB1 alleles or haplotypes at high-resolution level associated with AA have not been fully identified in northern Chinese Han populations. The aim of this study was to identify association of the variations in HLA class II region with AA in northern Chinese Han population. A recent case-control study, including 96 AA patients and 824 healthy controls was performed. The high-resolution HLA genotyping was conducted by PCR-SBT, -SSO and NGS-ION S5TM platform. Based on genotypic data of the three loci, haplotype estimation was carried out. HLA-DRB1*15:01 (Pc = 2.87 × 10-3; OR = 2.11, 95% CI = 1.45-3.07) and HLA-DQB1*06:02 (Pc = 1.86 × 10-2; OR = 2.01, 95% CI = 1.32-3.06) were the risk and predisposition alleles to AA in northern Chinese Han after considering multiple testing. Moreover, the HLA-DRB1*15:01-DQB1*06:02 (Pc = 4.90 × 10-3; OR = 2.09, 95% CI = 1.37-3.19) and HLA-DRB1*14:05-DQB1*05:03 (Pc = 2.65 × 10-2; OR = 2.82, 95%CI = 1.45-5.50) haplotypes had direct strong relevance to AA and were the susceptible haplotypes. HLA-DPB1 alleles and 23 polymorphic amino acid residues spanning exon 2 ~ 4 of DPß1 molecules have showed no statistically significant associations between AA and controls. The present findings establish a novel link between inherited HLA-DRB1,-DQB1,-DPB1 risk alleles and haplotypes in northern Chinese Han with AA, and open new avenues for development of targeted therapies to prevent or redirect immunopathology in AA.

Utility of next-generation sequencing methods to identify the novel HLA alleles in potential stem cell donors from Chinese Marrow Donor Program.

The human leucocyte antigen (HLA) is the most polymorphic region of the human genome. Compared with Sanger-sequencing-based typing (SBT) methods, next-generation sequencing (NGS) has significantly higher throughput and depth sequencing characteristics, having dramatic impacts on HLA typing in clinical settings. Here, we performed NGS technology with Ion Torrent S5 platform to evaluate the potential four novel HLA alleles detected in five donors from Chinese Marrow Donor Program (CMDP, Shaanxi Province) during routine Sanger SBT testing. We also predicted the highest estimated relative frequency novel allele-bearing haplotypes according to their phenotypes and HaploStats database. NGS assays, as it provided the phase-defined and complete sequencing information, undoubtedly increase novel allele identification which will greatly enrich HLA database and provide more information for donor selection.

[Relationship between High-Resolution HLA-A,-B,-DRB1 Alleles and Haplotype Polymorphisms with Myeloid Leukemia of Han People in North China].

OBJECTIVE: To investigate the potential relationship between the high-resolution HLA-A,-B,-DRB1 alleles and haplotype polymorphism with actute myeloid leukemia (AML) and chronic myeloid leukemia (CML) of Han people in North China. METHODS: A total of 1241 healthy unrelated Han people's bone marrow donors in North China were used as a control group, 259 patients with myeloid leukemia were genotyped at high-resolution level by means of PCR-SBT, -SSO and -SSP typing methods for HLA-A,-B,-DRB1 loci. The frequencies of HLA allele and haplotype were calculated by software Arleguin 3.5.2. The different distribution of genes and haplotypes was analyzed by case control study, and the odd ratio (OR) of leukemia was also calculated. The structural difference of HLA alleles was analyzed 111by HLA three-dimensional structure modeling and software Swiss-PdbViewer v4.1. RESULTS: χ2 test and correction showed that an increased frequency of A*02:07 (8.47% vs 5.28%, P' =0.013), A*29:01 (1.85% vs 0.68%, P=0.044), B*07:02 (5.29% vs 3.10%, P=0.029), B*07:05:01G (1.85% vs 0.68%, P=0.044) and B*35:02 (1.06% vs 0.20%, P=0.023) were found in AML patients (n=189) as compared with controls, respectively; whereas A*02:03 was less frequent in AML as compared with controls (0.79% vs 3.10%, P=0.011). The frequency of B*46:01 was lower in CML patients (n=70) as compared with controls (2.86% vs 7.82%, P=0.031). However, the above-mentioned discrepancies were not statistically significant by Bonferroni correction. Through Fisher exact test and Bonferroni correction, the frequency of DRB1*11:28 and its haplotype A*24:02-B*15:01-DRB1*11:28 in CML group were very significantly higher than in controls (1.43% vs 0.00%, Pc=0.015; 1.43% vs 0.00%, P=0.003). Three-dimensional structure modeling of DRB1*11:28 and DRB1*11:01 presented significant structure differentiation (RMSD=0.09 nm) in peptide binding region of the backbone calculated by Swiss-PdbViewer v4.1. The haplotype A*03:01-B*50:01-DRB1*07:01 in AML and A*11:01-B*40:06-DRB1*09:01 in CML patients were significantly higher than that in controls (1.06% vs 0.00%, Pc=0.000; 2.86% vs 0.07%, Pc=0.000), and positively correlated with leukemia (OR=59.66, 95% CI=3.21-1110.39; OR=42.91, 95% CI=7.07-260.32). CONCLUSION: The relationship of HLA-A,-B,-DRB1 alleles and haplotype polymorphism with leukemia at high-resolution level were obtained and unique in north Chinese Han population. AML and CML patients in Northern Han people carry particular susceptible haplotypes. DRB1*11:28, which might not actively present bcr-abl peptide to CD4+ T cells, and is a susceptibile gene for CML patients of Northern Han people, especially in Shaanxi Province (OR=89.62, 95% CI=4.28-1875.87), as well as correlated with its particular haplotype.

Diversity study of 12 X-chromosomal STR loci in Hui ethnic from China.

X-chromosomal STRs (X-STRs) have been used as complements of autosomal STR application in recent years. In this work, we present population genetic data of 12 X-STRs including DXS101, DXS10159, DXS10162, DXS10164, DXS6789, DXS7133, DXS7423, DXS7424, DXS8378, DXS981, GATA165B12, and GATA31E08 loci in a sample of 231 unrelated healthy individuals from the Hui ethnic group in Ningxia Hui Autonomous Region, China. Allelic frequencies of the 12 X-STR loci and haplotypic frequencies of the reported linkage groups (DXS7424-DXS101 and DXS10159-DXS10164-DXS10162) were investigated in the group, respectively. No STR loci showed significant deviations from the Hardy-Weinberg equilibriums and no linkage disequilibriums of pairwise loci were found after Bonferroni correction, respectively. A combined power of discrimination in female individuals was 0.999999999985 and that in male individuals was 0.99999967, respectively. The combined mean exclusion chance in deficiency cases, normal trios and duo cases were 0.999934, 0.995754, and 0.999796, respectively. Significant differences were observed from 0 to 8 loci, when making comparisons between the data of Hui ethnic group and previously reported data from other 16 populations. The results indicated the new panel of 12 X-STR loci might be useful for forensic science application.

[Identification and analysis of the rare HLA-A/B/DRB1 allele genes of 10165 bone marrow registry donors in Shaanxi region].

This study was purposed to analyze the detected status of rare alleles from HLA-A/B/DRB1 typing of 10165 unrelated hematopoietic stem cell donors in Shaanxi region during 2009-2012. The rare allele distributions of HLA-A/B/DRB1 gene typing of 10165 unrelated-donors from Shaanxi sub-registry of Chinese National Marrow Donor Project (CMDP) were detected and analyzed by PCR-SBT. The results showed that there were 40 rare alleles from 48 donors identified by PCR-SBT in 10165 unrelated-donors of Shaanxi sub-registry. Among them, 10 rare alleles of A*02:04, B*07:10, B*27:09, B*35:11, B*44:29, DRB1*03:04, DRB1*08:18, DRB1*13:05, DRB1*13:14 and DRB1*14:11 from 15 donors were not included in the common alleles and well documented alleles (CWD) of China, but were included in the CWD of American Society for Histocompatibility and Immunogenetics (ASHI). The alleles of A*68:24, B*35:11, B*44:29, DRB1*03:04, DRB1*08:18 and DRB1*13:05 were confirmed in more than two samples. There were totally 21 novel HLA alleles identified by our laboratory and officially assigned by the WHO Nomenclature Committee from 2005 to 2012, and some of them were also detected from multiple donors in other HLA typing laboratories of China. Now the novel alleles of HLA-A*02:90, HLA-B*48:14 and HLA-DRB1*01:14 were added into the Chinese CWD list. It is concluded that to ensure the polymorphism integrity and accurate population distribution of HLA genes and its constant accumulations on CMDP, it is necessary to recognize and submit timely the potential novel alleles in our practical work, as well as to record and statistics the identified rare alleles, which can provide the basis for the modification of Chinese CWD. When CWD list is referred, it should be careful for ambiguous results containing the identified rare alleles in order to avoid the occurrence of false or undiscovered detection, and ensure that the patients carrying rare alleles could find a matching donor with the maximum opportunity.

Aluminum-induced programmed cell death promoted by AhSAG, a senescence-associated gene in Arachis hypoganea L.

Programmed cell death (PCD) is a foundational cellular process in plant development and elimination of damaged cells under environmental stresses. In this study, Al induced PCD in two peanut (Arachis hypoganea L.) cultivars Zhonghua 2 (Al-sensitive) and 99-1507 (Al-tolerant) using DNA ladder, TUNEL detection and electron microscopy. The concentration of Al-induced PCD was lower in Zhonghua 2 than in 99-1507. AhSAG, a senescence-associated gene was isolated from cDNA library of Al-stressed peanut with PCD. Open reading frame (ORF) of AhSAG was 474bp, encoding a SAG protein composed of 157 amino acids. Compared to the control and the antisense transgenic tobacco plants, the fast development and blossom of the sense transgenic plants happened to promote senescence. The ability of Al tolerance in sense transgenic tobacco was lower than in antisense transgenic tobacco according to root elongation and Al content analysis. The expression of AhSAG-GFP was higher in sense transgenic tobacco than in antisense transgenic tobacco. Altogether, these results indicated that there was a negative relationship between Al-induced PCD and Al-resistance in peanut, and the AhSAG could induce or promote the occurrence of PCD in plants.

[Analysis of gene polymorphism of HPA and HLA-I in Chinese Xi'an voluntary platelet donors].

To study the allele frequencies and their polymorphism characteristics of human platelet antigen (HPA) and human leucocyte antigen-I (HLA-I) in Chinese xi'an population, the types of HPA and HLA-I in 375 Chinese xi'an voluntary platelet donors were detected by PCR-SSP and PCR-SSO as well as flow cytometry with magnetic beads, and were analyzed. The results showed that there was no polymorphism in HPA-7-HPA-14, HPA-16 and HPA-17 which only expressed-aa type, the -bb type was only detected in HPA-3 and HPA-15, 9 out of 16 samples for the HPA-5ab phenotype simultaneously expressed HPA-15ab, the other 7 samples expressed HPA-15bb, no HPA-15aa phenotype was observed. Phenotypes detected in this study were HPA-1aa-17aa, HPA-1ab, -2ab, -3ab, -3bb, -4ab, -5ab, -6ab, -15ab and -15bb. Among 375 cases, HLA-A specificity of 16 species was observed, which accounted for 76% (16/21) of detectable phenotype specificity in this locus, moreover, 11 species showed frequency > 1%; HLA-B specificity of 36 species was observed which accounted for 84% (36/43) of detectable phenotype specificity in this locus, moreover 23 species showed frequency > 1%, these species were covered by common specific HLA-I in northern China, 264 species haplotype HLA-A-B were found in 375 cases, the frequency of 30 species was > 1%. It is concluded that the gene frequency distribution of HPA and HLA-I in Chinese Xi'an population is in accordance with population of northern China on the whole, but it has its own characteristics.